Abstract:
A crystalline compound of formula (I): 
     
       
                 
         
             
             
         
       
     
     The compound of formula (I) is a β-lactamase inhibitor and may be administered in combination with an antibacterial agent for prevention or treatment of bacterial infection.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to crystalline (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide, processes for the preparation thereof, pharmaceutical compositions comprising (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide and uses of the compound, including uses of compositions containing the compound, in particular use with an antibacterial agent in treatment or prevention of bacterial infection. 
       BACKGROUND OF THE INVENTION 
       [0002]    Emergence and dissemination of resistance is an inevitable consequence of the evolutionary dynamic set in motion by the introduction of antibiotics, irrespective of structural class or mode of action (Shapiro S. 2013. Speculative strategies for new antibacterials: all roads should not lead to Rome. J. Antibiot. 66: 371-386). Spread of resistance amongst clinically relevant pathogens has had an especially strong impact on the value of β-lactam antibiotics, heretofore regarded as very safe and efficacious therapies for serious bacterial infections. The appearance of new and aggressive β-lactamases, particularly extended spectrum β-lactamases (ESBLs) and other class A enzymes, has compromised the ability of β-lactams to combat infections, highlighting the need for development of new products (Fisher J F, Meroueh S O, Mobashery S. 2005. Bacterial resistance to β-lactam antibiotics: compelling opportunism, compelling opportunity. Chem. Rev. 105: 395-424). Whilst several β-lactamase inhibitors, which protect β-lactam antibiotics from hydrolysis, have been used in combination with some β-lactams, the capability of these β-lactamase inhibitors to preserve the antibacterial activity of β-lactams has eroded severely during the past decade, necessitating the search for new, more potent β-lactamase inhibitors to restore therapeutic utility of their β-lactam partners (Watkins R R, Papp-Wallace K M, Drawz S M, Bonomo R A. 2013. Novel β-lactamase inhibitors: a therapeutic hope against the scourge of multidrug resistance. Front. Microbiol. 4: 392). 
         [0003]    WO 2008/010048 discloses the β-lactamase inhibitor (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (formula I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0004]    WO 2008/010048 discloses formation of an amorphous compound of Formula (I) which is isolated by filtering and lyophilisation. 
         [0005]    The present inventors have found that the compound of formula (I) as prepared by the process of WO 2008/010048 is hygroscopic, and has limited stability when stored at room temperature. 
         [0006]    It is an object of the invention to provide a more stable form of the compound of formula (I). 
         [0007]    It is a further object of the invention to provide a form of the compound of formula (I) that is easy to purify. 
         [0008]    It is a further object of the invention to provide a form of the compound of formula (I) that is easy to handle. 
       SUMMARY OF THE INVENTION 
       [0009]    The present inventors have developed crystalline compounds of formula (I). The present inventors have surprisingly found that crystalline compounds of formula (I) have improved thermal stability, are less hygroscopic and easier to purify and handle than the compound of formula (I) in amorphous form. 
         [0010]    In a first aspect the invention provides a crystalline compound of formula (I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0011]    In a first embodiment of the first aspect there is provided a crystalline compound of formula (I, hereinafter “Form A”, characterised by an XRPD spectrum comprising four or more (preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably all ten) peaks selected from peaks with 2θ angles of: 8.82, 12.07, 14.43, 14.92, 16.26, 18.25, 19.06, 19.78, 20.82 and 23.51±0.1 degrees 2θ, optionally ±0.05 degrees 2θ. 
         [0012]    Preferably, the XRPD spectrum of Form A has one, two, three, four or all five peaks selected from peaks with 2θ angles of: 8.82, 12.07, 14.43, 18.25 and 19.78±0.1 degrees 2θ. 
         [0013]    Preferably, the XRPD spectrum of Form A has all ten peaks with 2θ angles of: 8.82, 12.07, 14.43, 14.92, 16.26, 18.25, 19.06, 19.78, 20.82 and 23.51±0.1 degrees 2θ, optionally ±0.05 degrees 2θ. 
         [0014]    Preferably, Form A has a XRPD spectrum substantially as shown in  FIG. 1 . 
         [0015]    Form A may be further characterised by its Thermo Gravimetric Analysis (TGA) curve showing an endothermic event at about 163° C.±2° C. The TGA curve may show a weight loss of about 6% up to 130° C.±2° C. due to water loss. 
         [0016]    Preferably, Form A has a TGA curve substantially as shown in  FIG. 9 . 
         [0017]    Form A may be further characterized by its differential scanning calorimetry (DSC) curve showing an endothermic event with a maximum at about 163° C.±2° C. The DSC curve may show an endothermic event starting at about 45° C.±2° C. due to water loss. 
         [0018]    Preferably, Form A has a DSC curve substantially as shown in  FIG. 5 . 
         [0019]    In a second embodiment of the first aspect there is provided a crystalline compound of formula (I), hereinafter “Form B”, characterised by an XRPD spectrum comprising four or more (preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably all ten) peaks selected from peaks with 2θ angles of: 9.37, 10.34, 12.59, 13.17, 15.00, 15.63, 18.51, 19.10, 20.79, 23.93±0.1 degrees 2θ, optionally ±0.05 degrees 2θ. 
         [0020]    Preferably, the XRPD spectrum of Form B has one, two, three, four or all five peaks selected from peaks with 2θ angles of: 10.34, 15.00, 15.63, 18.51 and 23.93±0.1 degrees 2θ. 
         [0021]    Preferably, the XRPD spectrum of Form B has all ten peaks with 2θ angles of: 9.37, 10.34, 12.59, 13.17, 15.00, 15.63, 18.51, 19.10, 20.79 and 23.93±0.1 degrees 2θ, optionally ±0.05 degrees 2θ. 
         [0022]    Preferably, Form B has a XRPD spectrum substantially as shown in  FIG. 2 . 
         [0023]    Form B may be further characterised by its Thermo Gravimetric Analysis (TGA) curve showing an endothermic event at about 155° C.±2° C. 
         [0024]    The TGA curve may show a weight loss of about 8% up to 120° C.±2° C. correlated with water desorption. 
         [0025]    Preferably, Form B has a TGA curve substantially as shown in  FIG. 10 . 
         [0026]    Form B may be further characterized by its differential scanning calorimetry (DSC) curve showing an endothermic event with a maximum at about 180° C.±2° C. The DSC curve may show an endothermic event starting at about 50° C.±2° C. due to water loss. 
         [0027]    Preferably, Form B has a DSC curve substantially as shown in  FIG. 6 . 
         [0028]    In a third embodiment of the first aspect there is provided a crystalline compound of formula (I), hereinafter “Form C”, characterised by an XRPD spectrum comprising four or more (preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably all ten) peaks selected from peaks with 2θ angles of: 9.33, 10.73, 14.85, 15.29, 15.77, 16.16, 18.60, 20.12, 21.00 and 23.22±0.1 degrees 2θ, optionally ±0.05 degrees 2θ. 
         [0029]    Preferably, the XRPD spectrum of Form C has one, two, three, four or all five peaks selected from peaks with 2θ angles of: 10.73, 14.85, 15.29, 20.12 and 23.22±0.1 degrees 2θ. 
         [0030]    Preferably, the XRPD spectrum of Form C has all ten peaks with 2θ angles of: 9.33, 10.73, 14.85, 15.29, 15.77, 16.16, 18.60, 20.12, 21.00 and 23.22±0.1 degrees 2θ, optionally ±0.05 degrees 2θ. 
         [0031]    Preferably, Form C has a XRPD spectrum substantially as shown in  FIG. 3  or  FIG. 20 . 
         [0032]    Form C may be further characterised by its Thermo Gravimetric Analysis (TGA) curve showing an endothermic event at about 149° C. 
         [0033]    The TGA curve may show a weight loss of about 3% up to 120° C.±2° C. correlated with water desorption. 
         [0034]    Preferably, Form C has a TGA curve substantially as shown in  FIG. 11 . 
         [0035]    Form C may be further characterized by its differential scanning calorimetry (DSC) curve showing an endothermic event with a maximum at about 185° C.±2° C. 
         [0036]    Preferably, Form C has a DSC curve substantially as shown in  FIG. 7 . 
         [0037]    In a fourth embodiment of the first aspect there is provided a crystalline compound of formula (I), hereinafter “Form D”, characterised by an XRPD spectrum comprising four or more peaks (preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably all ten peaks) selected from peaks with 2θ angles of: 6.78, 15.45, 16.39, 17.10, 20.06, 20.63, 23.23, 23.68, 26.18 and 32.47±0.05 degrees 2θ. 
         [0038]    Preferably, the XRPD spectrum of Form D has one, two, three, four or all five peaks selected from peaks with 2θ angles of: 6.78, 16.39, 17.10, 20.63 and 23.23, ±0.05 degrees 2θ. 
         [0039]    Preferably, the XRPD spectrum of Form D has all ten peaks with 2θ angles of 6.78, 15.45, 16.39, 17.10, 20.06, 20.63, 23.23, 23.68, 26.18 and 32.47±0.05 degrees 2θ. 
         [0040]    Preferably, Form D has an XRPD spectrum substantially as shown in  FIG. 25 . 
         [0041]    In a fifth embodiment of the first aspect there is provided a crystalline compound of formula (I), hereinafter “Form E”, characterised by an XRPD spectrum comprising four or more peaks (preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably all ten peaks) selected from peaks with 2θ angles of: 6.82, 15.04, 15.68, 16.47, 17.17, 18.44, 20.69, 23.34, 23.88 and 25.38±0.05 degrees 2θ. 
         [0042]    Preferably, the XRPD spectrum of Form E has one, two, three, four or all five peaks selected from peaks with 2θ angles of: 15.04, 15.68, 16.47, 20.69 and 23.88±0.05 degrees 2θ. 
         [0043]    Preferably, the XRPD spectrum of Form E has all ten peaks with 2θ angles of: 6.82, 15.04, 15.68, 16.47, 17.17, 18.44, 20.69, 23.34, 23.88 and 25.38±0.05 degrees 2θ. 
         [0044]    Preferably, Form E has an XRPD spectrum substantially as shown in  FIG. 27 . 
         [0045]    In a sixth embodiment of the first aspect there is provided a crystalline compound of formula (I), hereinafter “Form F”, characterised by an XRPD spectrum comprising four or more peaks (preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably ten or more, preferably all eleven peaks) selected from peaks with 2θ angles of: 12.73, 15.36, 15.95, 16.42, 18.12, 20.48, 22.85, 23.22, 27.04, 27.69 and 32.47±0.05 degrees 2θ. 
         [0046]    Preferably, the XRPD spectrum of Form F has one, two, three, four or all five peaks selected from peaks with 2θ angles of: 12.73, 15.36, 15.95, 16.42 and 20.48±0.5 degrees 2θ. 
         [0047]    Preferably, the XRPD spectrum of Form F has all eleven peaks with 2θ angles of: 12.73, 15.36, 15.95, 16.42, 18.12, 20.48, 22.85, 23.22, 27.04, 27.69 and 32.47±0.05 degrees 2θ. 
         [0048]    Preferably, Form F has an XRPD spectrum substantially as shown in  FIG. 29 . 
         [0049]    In a second aspect the invention provides a process for preparing crystalline compound of formula (I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    the process comprising the steps of:
 
forming a formulation by dissolving or suspending an amorphous compound of formula (I) in a solvent or solvent mixture; and
 
crystallising the compound of formula (I) from the formulation.
 
         [0050]    The amorphous compound of formula (I) in the formulation may substantially all be dissolved in the formulation; may substantially all be dispersed in the formulation; or may partly be dissolved and partly dispersed in the formulation. 
         [0051]    The quantity of the amorphous compound of formula (I) used in the process of the second aspect of the invention may be below a solubility limit of the amorphous compound in the solvent or solvent mixture, in which case the formulation is a solution, or may be above the solubility limit, in which case the formulation is a suspension. 
         [0052]    Solvents for dissolving the amorphous compound of formula (I) may be selected from solvents in which the amorphous compound of formula (I) has a solubility at 20° C. of greater than 200 mg/ml, optionally greater than 400 mg/ml. Solvents may be polar, protic or dipolar aprotic solvents. Exemplary polar, protic solvents are water, primary alcohols, preferably methanol, ethanol and 1-propanol. Further exemplary dipolar aprotic solvents are dimethylsulfoxide and N,N-dimethylformamide, N-methylpyrrolidone and the alike. Primary alcohols are preferred. Methanol and ethanol are particularly preferred. Water content of a primary alcohol solvent is preferably less than 4 wt %, more preferably less than 2 wt %. When the primary alcohol is methanol the water content is preferably less than 1%. 
         [0053]    Crystallisation of a crystalline compound of formula (I) may be induced by adding an antisolvent to a formulation containing dissolved amorphous compound of formula (I). Antisolvents may be solvents in which the amorphous compound of formula (I) has a solubility at 20° C. of less than 50 mg/ml, optionally less than 30 mg/ml. 
         [0054]    Antisolvents may be aprotic materials. Exemplary antisolvents are acetone, ethyl acetate, methyl-tert-butyl ether, heptane, 2-propanol, isopropyl acetate, diisopropyl ether, methylethyl ketone, tetrahydrofuran, anisole, and tert-butyl acetate. 
         [0055]    In another embodiment of the second aspect, the amorphous compound of formula (I) may have little or no solubility in the solvent or solvent mixture used to form the formulation, in which case the formulation is a suspension. 
         [0056]    A nucleating agent may be added to the formulation. The nucleating agent may be a crystalline seed of a compound of formula (I). 
         [0057]    The purity of the solvent may affect solubility of the compound of formula (I) in the solvent, either in its amorphous form or in one or more of its crystalline forms. 
         [0058]    The temperature of the formulation may be lowered following formation of the formulation. The solvent or solvent mixture may be heated during formation of the formulation, and may be cooled following formation of the formulation. 
         [0059]    In a third aspect the invention provides crystalline compounds of formula (I) prepared by a process according to the second aspect of the invention. 
         [0060]    The invention further provides crystalline compounds of formula (I) preparable by a process according to the second aspect of the invention. 
         [0061]    For pharmaceuticals in which the active ingredient can exist in more than one polymorphic form, problems in dissolution and/or bioavailability of pharmaceutical compositions containing the compound can result if the manufacturing process leads to a polymorph with varying degrees of polymorphic purity and/or where the process does not control polymorphic interconversion. 
         [0062]    If crystalline forms are made with polymorphic impurities, this may cause instability and it can accelerate significant interconversion to another polymorphic form. Therefore it is advantageous to produce crystalline forms with high polymorphic purity. 
         [0063]    Preferably the crystalline compound of formula (I) according to the first or third aspects of the invention comprises more than 90% of a single crystalline polymorph of the compound, preferably more than 95%, more preferably more than 99%, even more preferably more than 99.5% and most preferably more than 99.8% as measured by XRPD or DSC, preferably as measured by XRPD. Preferably, the single polymorph is one of Form A, Form B, Form C, Form D, Form E, and Form F. 
         [0064]    Preferably, the crystalline compound of formula (I) according to the first or third aspects of the present invention has a chemical purity of at least 95 wt %, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%, even more preferably at least 99.8%, and most preferably at least 99.9%, preferably as measured by HPLC. 
         [0065]    The crystalline compound of formula (I) may be suitable for reconstitution with a pharmaceutically acceptable vehicle for administration. 
         [0066]    In a fourth aspect of the present invention there is provided a pharmaceutical composition comprising an antibiotic and the crystalline compound of formula (I) according to the first or third aspects of the present invention. Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. 
         [0067]    In a fifth aspect the invention provides a pharmaceutical composition according to the fourth aspect for treatment of bacterial infection. 
         [0068]    In a sixth aspect the invention provides a method of treating a bacterial infection comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition according to the fourth aspect of the present invention. 
         [0069]    In a seventh aspect the invention provides a method of forming a pharmaceutical composition comprising a compound of formula (I), the method comprising the step of dissolving or dispersing the crystalline compound of formula (I) in a carrier liquid. Optionally the carrier liquid is a pharmaceutically acceptable vehicle for intravenous injections such as Dextrose, Sodium chloride &amp; Dextrose 5 mixture. Sodium chloride, Sodium lactate, etc. Optionally, the carrier liquid is an aqueous saline solution. 
         [0070]    The concentration of a compound of formula (I) in the pharmaceutical composition range from 1 mg/ml to 700 mg/ml, preferably from 100 to 500 mg/ml, more preferably from 150 to 250 mg/ml. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0071]    The invention will now be described in more detail with reference to the Figures in which: 
           [0072]      FIG. 1  is a X-ray powder diffraction pattern of Form A of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0073]      FIG. 2  is a X-ray powder diffraction pattern of Form B of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0074]      FIG. 3  is a X-ray powder diffraction pattern of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0075]      FIG. 4  is a X-ray powder diffraction pattern of amorphous form of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0076]      FIG. 5  is a differential scanning calorimetric thermogram of Form A of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0077]      FIG. 6  is a differential scanning calorimetric thermogram of Form B of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0078]      FIG. 7  is a differential scanning calorimetric thermogram of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0079]      FIG. 8  is a differential scanning calorimetric thermogram of amorphous form of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0080]      FIG. 9  is a thermogravimetric curve of Form A of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0081]      FIG. 10  is a thermogravimetric curve of Form B of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0082]      FIG. 11  is a thermogravimetric curve of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0083]      FIG. 12  is a plot of HPLC response area vs. concentration for solutions or suspensions of amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0084]      FIG. 13  is a 25× magnified optical microscope image of Form A of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0085]      FIG. 14  is a 25× magnified optical microscope image of Form B of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0086]      FIG. 15  is a 25× magnified optical microscope image of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0087]      FIG. 16  is a Raman spectrum of Form A of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0088]      FIG. 17  is a FT-RT spectrum of Form A of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0089]      FIG. 18  is a Raman spectrum of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0090]      FIG. 19  is a FT-RT spectrum of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0091]      FIG. 20  is a X-ray powder diffraction pattern of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide, obtained according to Example 13; 
           [0092]      FIG. 21  is a thermogravimetric curve of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide, obtained according to Example 13; 
           [0093]      FIG. 22  is a 25× magnified optical microscope image of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide, obtained according to Example 13; 
           [0094]      FIG. 23  is an  1 H-NMR spectrum of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0095]      FIG. 24  shows particle size distribution curves of Form C of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide, obtained according to Example 13; 
           [0096]      FIG. 25  is a X-ray powder diffraction pattern of Form D of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0097]      FIG. 26  is a Raman spectrum of Form D of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0098]      FIG. 27  is a X-ray powder diffraction pattern of Form E of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0099]      FIG. 28  is a Raman spectrum of Form E of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0100]      FIG. 29  is a X-ray powder diffraction pattern of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0101]      FIGS. 30 and 31  are Raman spectra of three bathes of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0102]      FIGS. 32-39  are scanning electron microscopy images of samples of a first batch of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0103]      FIGS. 40-46  are scanning electron microscopy images of samples of a second batch of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0104]      FIGS. 47-50  are scanning electron microscopy images of samples of a third batch of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0105]      FIG. 51  is a FT-RT spectrum of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
           [0106]      FIG. 52  is a differential scanning calorimetric thermogram of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0107]      FIG. 53  is a thermogravimetric curve of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0108]      FIG. 54  is a gas evolution image of Evolved Gas Analysis (EGA) of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; 
           [0109]      FIG. 55  is a plot of Dynamic Vapor Sorption (DVS) change in mass of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide; and 
           [0110]      FIG. 56  shows Dynamic Vapor Sorption (DVS) isotherm plots of Form F of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0111]    The present invention provides crystalline (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide which is non-hygroscopic, thermally stable and has beneficial properties that avoid problems associated with the prior art forms. 
         [0112]    The present invention further provides a process for forming crystalline (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. The process allows formation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide in high polymorphic purity. 
         [0113]    Suitable crystallization techniques for forming crystalline compounds of formula (I) include, without limitation, precipitation and re-crystallization (including antisolvent crystallization) processes, with or without seeding with nucleating agents. In a preferred embodiment, antisolvent crystallization processes are used. 
         [0114]    Diluted, saturated or super-saturated solutions may be used for crystallization. 
         [0115]    A solution of an amorphous compound of formula (I) may be cooled to promote crystallization of crystalline compounds of formula (I). 
         [0116]    An amorphous compound of formula (I) may be dissolved at a temperature in the range of 20-50° C. The solution may be cooled down to about 0° C. or about 10° C. to promote the crystallization. 
         [0117]    Methods of preparing crystalline forms of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide, include, without limitation, the following methods: 
       Form A Method 1: 
       [0000]    
       
         
           
             stirring a solution of amorphous compound of formula (I) in ethanol 96% at 20° C., 
             collecting the solid by filtration. 
           
         
       
     
       Form A Method 2: 
       [0000]    
       
         
           
             stirring a saturated solution of amorphous compound of formula (I) in ethanol 96% at 20° C., 
             adding methyl tert-butyl ether as antisolvent, 
             stirring the mixture at room temperature overnight, 
             collecting the solid by filtration. 
           
         
       
     
       Form A Method 3: 
       [0000]    
       
         
           
             stirring a saturated solution of amorphous compound of formula (I) in ethanol 96% at 20° C., 
             seeding with nucleating agent, 
             adding heptane as antisolvent, 
             stirring the mixture at room temperature overnight, 
             collecting the solid by filtration. 
           
         
       
     
       Form A Method 4 
       [0000]    
       
         
           
             stirring a saturated solution of amorphous compound of formula (I) in ethanol 96% at 20° C., 
             seeding with nucleating agent, 
             adding 2-propanol as antisolvent, 
             stirring the mixture at room temperature overnight, 
             collecting the solid by filtration. 
           
         
       
     
       Form A Method 5 
       [0000]    
       
         
           
             dissolving amorphous compound of formula (I) in ethanol 96% by heating to 35° C. 
             slowly adding (time: about 1 hour) methyl tert-butyl ether as antisolvent, 
             cooling the mixture to 10° C. 
             stirring the mixture at 10° C. overnight, 
             collecting the solid by filtration. 
           
         
       
     
       Form A Method 6 
       [0000]    
       
         
           
             stirring a saturated solution of amorphous compound of formula (I) in ethanol 96% at 35° C., 
             seeding the solution with nucleating agent, 
             slowly adding (time: about 20 min.) methyl tert-butyl ether as antisolvent at 20° C., 
             cooling the mixture to 20° C. overnight, 
             collecting the solid by filtration 
           
         
       
     
       Form A Method 7 
       [0000]    
       
         
           
             stirring a saturated solution of amorphous compound of formula (I) in ethanol 96% at 40° C., 
             seeding the solution with nucleating agent, 
             cooling the mixture to 20° C. over about 5 hours, 
             stirring the mixture at 20° C., 
             collecting the solid by filtration 
           
         
       
     
       Form B Method 1 
       [0000]    
       
         
           
             stirring a saturated solution of amorphous compound of formula (I) in acetone at 40° C., 
             collecting the solid by filtration. 
           
         
       
     
       Form C Method 1 
       [0000]    
       
         
           
             stirring a solution of amorphous compound of formula (I) in ethanol 99.8% at 40° C., 
             seeding the solution with nucleating agent at 36° C. 
             cooling the solution at 15° C., 
             stirring the mixture overnight 
           
         
       
     
         [0155]    Forms D, E and F may be formed by crystallization from dimethylformamide solution. The present inventors have found that Forms D and E may crystallize initially from DMF solution but do not form once form F has formed. Without wishing to be bound by any theory, this may be due to Form F having greater stability than either Form D or Form E. 
         [0156]    Surprisingly, the present inventors have found that one crystal form of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide can be used to form another crystal form of this compound. One of crystal forms A, B and C may be used as a seed in crystallisation of another of forms A, B and C. 
         [0157]    A pharmaceutical composition as described herein may be in an injectable form for intravenous injection. The composition may contain stabilizing agents. The composition may be in suitable sterile solid form ready for reconstitution to form an injectable solution. 
         [0158]    A pharmaceutical composition containing a crystalline compound of formula (I) as described herein may be administered either alone or may be co-administered with therapeutically effective amount of an antibiotic. 
         [0159]    A pharmaceutical composition as described herein may comprise an antibiotic and may comprise one or more conventional pharmaceutically acceptable excipient(s). 
         [0160]    Exemplary antibiotics are β-lactam antibiotics, in particular penicillins and cephalosporins and may be selected from Amoxicillin, Ampicillin, Apalcillin, Azlocillin, Bacampicillin, Carbenacillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Lenampicillin, Mecillinam, Methacillin, Mezlocillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Aztreonam, BAL30072, Carumonam, PTX2416, Tigemonam, Cefaclor, Cefadroxil, Cefalexin, Cefalotin, Cefamandole, Cefapirin, Cefazolin, Cefbuperazone, Cefdinir, Cefepime, Cefetamet, Cefixime, Cefmenoxime, Cefmetazole, Cefminox, Cefonicid, Cefoperazone, Cefotaxime, Cefotetan, Cefotiam, Ceftiofur, Cefovecin, Cefoxtin, Cefpodoxime, Cefprozil, Cefquinome, Cefradine, Cefminox, Cefsulodin, Ceftaroline, Ceftazidime, Ceftezole, Ceftibuten, Ceftizoxime, Ceftobiprole, Ceftolozane, Ceftriaxone, Cefuroxime, Cefuzoname, Cephalexin, Cephalotin, Flomoxef, Latamoxef, Loracarbef Imipenem, Meropenem, Doripenem, Ertapenem, Biapenem, Panipenem, Faropenem or derivatives thereof. 
         [0161]    The antibiotic may be selected from aminoglycosides: Amikacin, Arbekacin, Apramycin, Dibekacin, Gentamicin, Isepamicin, Kanamycin, Neomycin, Netilmicin, Plazomicin, Sisomicin, Spectinomyin, Streptomycin, Tobramycin or derivatives thereof. 
         [0162]    The antibiotic may be selected from quinolones: Cinoxacin, Ciprofloxacin, Enofloxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Oxafloxacin, or derivatives thereof. 
         [0163]    The antibiotic may be selected from antimicrobial peptides, for example Colistin, Polymyxin B or derivatives thereof. 
         [0164]    A pharmaceutical composition as described herein may comprise only one or more than one antibiotic. 
         [0165]    A pharmaceutical composition containing a crystalline compound of formula (I) may contain or be co-administered with bactericidal or permeability-increasing-g protein product (BPI) or efflux pump inhibitors to improve activity against gram negative bacteria and bacteria resistant to antimicrobial agents. Antiviral, antiparasitic, antifungal agents may also be administered in combination with the inhibitor compounds. 
         [0166]    The pharmaceutical composition may contain complexing agents or anticoagulants, antioxidants, stabilizers, aminoglycosides, pharmaceutically acceptable salts or the like or mixtures thereof. 
         [0167]    In particular the pharmaceutical composition may contain β-lactam antibiotics, preferably penicillins, cephalosporins, carbapenem, monobactams, more preferably piperacillin, cefepime; ceftriaxone; meropenem, aztreonam. 
         [0168]    The pharmaceutical composition may contain buffers, for example sodium citrate, sodium acetate, sodium tartrate, sodium carbonate, sodium bicarbonate, morpholinopropanesulfonic acid, other phosphate buffers and the like and chelating agents like ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, hydroxyethylenediaminetriacetic acid, nitrilotriacetic acid, 1,2-diaminocyclohexanetetraacetic acid, bis(2-aminoethyl)ethyleneglycoltetraacetic acid, 1,6-hexamethylenediaminetetraacetic acid and the like or pharmaceutically acceptable salts thereof. 
         [0169]    A pharmaceutical composition as described herein may be administered to a human or warm-blooded animal by any suitable method, and preferably by intravenous injection. 
       EXAMPLES 
       [0170]    All XRPD data described herein were acquired in transmission mode on an X&#39;pert Pro instrument with X&#39;celerator detector. The data were evaluated using the Highscore Plus software using copper as radiation source at a wavelength of 1.54 Å. 
         [0171]    DSC analyses were run on a TA Q2000 MDSC instrument. 
         [0172]    TGA analyses were run on a TA Q5000 instrument. The data were evaluated using Universal Analysis software. 
         [0173]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide, was prepared according to example 1 of WO 2008010048, the contents of which are incorporated herein by reference. 
       Example 1 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A 
       [0174]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (200 mg) was dissolved in ethanol 96% (0.5 mL). The solution was stirred at 20° C., after 30 minutes a solid was formed. The mixture was stirred for 4 hours at 20° C. and the solid was isolated by filtration and dried overnight at room temperature in a vacuum oven. The obtained product (30 mg) was crystalline Form A which was characterized by an XRPD pattern as shown in  FIG. 1  and summarized in Table 1. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                   
                 d-spacing 
               
               
                   
                 Angle [°2θ] 
                 [Å] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 8.8223 
                 10.01516 
               
               
                   
                 12.0725 
                 7.32517 
               
               
                   
                 14.4346 
                 6.13137 
               
               
                   
                 14.9183 
                 5.93364 
               
               
                   
                 16.2594 
                 5.44711 
               
               
                   
                 18.2478 
                 4.85778 
               
               
                   
                 19.0618 
                 4.65213 
               
               
                   
                 19.7798 
                 4.48485 
               
               
                   
                 20.8191 
                 4.26326 
               
               
                   
                 23.5119 
                 3.78074 
               
               
                   
               
             
          
         
       
     
         [0175]    DSC ( FIG. 5 ) showed the sample to have a melting endotherm with a maximum at 163° C. TGA thermal curve is shown in  FIG. 9 . 
         [0176]    An optical microscope image of Form A is shown in  FIG. 13 . 
       Example 2 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A 
       [0177]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (1 g) was suspended in ethanol 96% (3 mL). The resulting mixture was filtered through a syringe filter. The saturated solution was treated with methyl tert-butyl ether (0.5 mL) as antisolvent. The antisolvent addition results in a solid precipitation. The mixture was stirred at room temperature overnight and the solid was isolated by filtration and dried overnight at room temperature in a vacuum oven. The solid recovered was crystalline Form A characterized by XRPD concordant with XRPD pattern given in Example 1. 
       Example 3 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A 
       [0178]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (1 g) was suspended in ethanol 96% (5 mL). The resulting mixture was filtered through a syringe filter. A pinch of Form A material was added to the solution as seed. The seed was not dissolved and the saturated solution was treated with heptane (0.5 mL) as antisolvent. The antisolvent addition results in a solid precipitation. The mixture was stirred at room temperature overnight and the solid was isolated by filtration and dried overnight at room temperature in a vacuum oven. The solid recovered was crystalline Form A characterized by XRPD concordant with XRPD pattern given in Example 1. 
       Example 4 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A 
       [0179]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (1 g) was suspended in ethanol 96% (5 mL). The resulting mixture was filtered through a syringe filter. A pinch of Form A material was added to the solution as seed. The seed was not dissolved and the saturated solution was treated with 2-propanol (0.5 mL) as antisolvent. The antisolvent addition results in a solid precipitation. The mixture was stirred at room temperature overnight and the solid was isolated by filtration and dried overnight at room temperature in a vacuum oven. The solid recovered was crystalline Form A characterized by XRPD concordant with XRPD pattern given in Example 1. 
       Example 5 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A 
       [0180]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (4 g) was weighed in a multimax vessel equipped with an impeller stirrer. The solid was suspended in ethanol 96% (32 mL). The mixture was heated to 35° C. and stirred at 800 RPM. At 35° C. the starting material seemed to be dissolved but the solution appeared slightly opaque. Methyl tert-butyl ether (8 mL) as antisolvent was added to the opaque solution over 1 hour. The addition of the antisolvent resulted in a solid formation. The mixture was cooled down to 10° C. over 1 hour. During the cooling ramp the material became sticky and the majority of the material adhered to the vessel walls. The mixture was stirred overnight and the solid obtained was discharged from the vessel by mechanical removal of the sticky solid from the vessel wall. The obtained mixture was filtered under vacuum; the cake was dried at room temperature in a vacuum oven for 60 hours to afford 2.75 g of a white solid. The solid recovered was crystalline Form A characterized by XRPD concordant with XRPD pattern given in Example 1. 
       Example 6 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A 
       [0181]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (5 g) was weighed in a multimax vessel equipped with an impeller stirrer. The solid was suspended in ethanol 96% (30 mL). The mixture was heated to 35° C. and stirred at 800 RPM. At 35° C. the starting material seemed to be dissolved but the solution appeared slightly opaque. The opaque solution was filtered through a syringe filter to obtain a clear solution. A pinch of Form A material was added to the solution as seed; the seed was not dissolved and the mixture was cooled to 20° C. over 45 minutes. At this temperature methyl tert-butyl ether (10 mL) was added as antisolvent over 20 minutes. The addition of the antisolvent resulted in a sticky solid formation, the majority of the material adhered to the vessel walls. The mixture was stirred overnight and the solid obtained was discharged from the vessel by mechanical removal of the sticky solid from the vessel wall. The obtained mixture was filtered under vacuum; the cake was dried at room temperature in a vacuum oven for 60 hours to afford 3.61 g of a white solid. The solid recovered was crystalline Form A characterized by XRPD concordant with XRPD pattern given in Example 1. 
       Example 7 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A 
       [0182]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (7 g) was weighed in a multimax vessel equipped with an impeller stirrer. The solid was suspended in ethanol 96% (21 mL). The mixture was heated to 40° C. and stirred at 400 RPM. At 40° C. the starting material seemed to be dissolved but the solution appeared slightly opaque. The opaque solution was filtered through a syringe filter to obtain a clear solution. A pinch of Form A material was added to the solution as seed; the seed was not dissolved and the mixture was stirred at 40° C. for 1 hour. The mixture is then cooled to 10° C. over 5 hours and stirred for 60 hours. The obtained material adhered to the vessel walls and was discharged by mechanical removal of the sticky solid from the vessel wall. The obtained mixture was filtered under vacuum; the cake was dried at room temperature in a vacuum oven for 18 hours to afford 5.54 g of a white solid. The solid recovered was crystalline Form A characterized by XRPD concordant with XRPD pattern given in Example 1. 
       Example 8 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form B 
       [0183]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (200 mg) was suspended in acetone (0.5 mL) and the slurry was stirred for 4 hours at 40° C. The solid was isolated by filtration and dried overnight at room temperature in a vacuum oven. The obtained product (150 mg) was crystalline Form B which was characterized by an XRPD pattern as shown in  FIG. 2  and summarized in Table 2. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                 Angle 
                 d-spacing 
               
               
                   
                 [°2θ] 
                 [Å] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 9.3736 
                 9.42739 
               
               
                   
                 10.343 
                 8.54587 
               
               
                   
                 12.5922 
                 7.024 
               
               
                   
                 13.172 
                 6.71609 
               
               
                   
                 14.998 
                 5.90227 
               
               
                   
                 15.636 
                 5.66284 
               
               
                   
                 18.5083 
                 4.79001 
               
               
                   
                 19.1049 
                 4.64175 
               
               
                   
                 20.7935 
                 4.26845 
               
               
                   
                 23.9264 
                 3.71616 
               
               
                   
               
             
          
         
       
     
         [0184]    DSC ( FIG. 6 ) showed the sample to have a melting endotherm with a maximum at 180° C. TGA thermal curve is shown in  FIG. 10 . 
         [0185]    An optical microscope image of Form B is shown in  FIG. 14 . 
       Example 9 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form C 
       [0186]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (5 g) was weighed in a multimax vessel equipped with an impeller stirrer. The solid was suspended in ethanol HPLC grade 99.8% (20 mL). The mixture was heated to 40° C. and stirred at 500 RPM. At 40° C. the starting material seemed to be dissolved but the solution appeared slightly opaque. The opaque solution was filtered through a syringe filter to obtain a clear solution. The solution was cooled to 36° C. over 15 minutes and Form B material (30 mg) was added to the solution as seed; the seed was not dissolved and promoted the product crystallization. The mixture was stirred at 36° C. for 30 minutes and is then cooled to 15° C. over 3.5 hours. The slurry was aged overnight and then was filtered under vacuum; the cake was dried at room temperature in a vacuum oven for 18 hours to afford 3.7 g of a white solid. The obtained product was crystalline Form C which was characterized by an XRPD pattern as shown in  FIG. 3  and summarized in Table 3. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                 Angle 
                 d-spacing 
               
               
                   
                 [°2θ] 
                 [Å] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 9.331 
                 9.47026 
               
               
                   
                 10.7259 
                 8.24161 
               
               
                   
                 14.8509 
                 5.96039 
               
               
                   
                 15.2924 
                 5.7893 
               
               
                   
                 15.7717 
                 5.61443 
               
               
                   
                 16.1565 
                 5.48158 
               
               
                   
                 18.6025 
                 4.76595 
               
               
                   
                 20.1156 
                 4.41074 
               
               
                   
                 20.9959 
                 4.22776 
               
               
                   
                 23.2215 
                 3.82734 
               
               
                   
               
             
          
         
       
     
         [0187]    DSC ( FIG. 7 ) showed the sample to have a melting endotherm with a maximum at 185° C. 
         [0188]    TGA thermal curve is shown in  FIG. 11 . 
         [0189]    An optical microscope image of Form C is shown in  FIG. 15 . 
       Comparative Example 
       [0190]    The XRPD spectrum of amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide prepared as described in WO 2008/010048 is shown in  FIG. 4 . No crystalline character is detectable in this spectrum. 
       Solubility Evaluation 
       [0191]    Solubility values of solvents were calculated with respect to the HPLC response factor, set out in  FIG. 12 . 
         [0192]    HPLC response factor was calculated for the amorphous compound of formula (I) using samples dissolved in acetonitrile/water 9/1 with the following method: 
       Column: ZORBAX Eclipse XDB-C18 (150×4.6 mm, 5 μm) 
     Temperature: 25° C. 
       [0193]    Mobile phase: A: 0.05M Sodium ortophosphate/water, B: Acetonitrile
 
Gradient: from 5% of B to 95% of B in 10 min
 
Detector. UV λ=220 nm
 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                 Concentration 
                 HPLC 
               
               
                 Sample 
                 (mg/ml) 
                 area 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 0.62 
                 333.445 
               
               
                 2 
                 1.24 
                 660.935 
               
               
                 3 
                 1.68 
                 1219.92 
               
               
                 4 
                 2.25 
                 1643.32 
               
               
                 5 
                 2.30 
                 1940.44 
               
               
                 6 
                 3.10 
                 2830.31 
               
               
                   
               
             
          
         
       
     
         [0194]    Slurries of the amorphous compound of formula (I) in the selected solvents were prepared and stirred at 20° C. and 40° C. for 4 hours. 
         [0195]    Samples of each slurry were filtered and the mother liquors injected in HPLC. 
         [0196]    The solid residual were isolated and analyzed by XRPD. 
         [0197]    The results are summarized in the following Table 4. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Solubility of amorphous compound of formula (I) 
               
             
          
           
               
                   
                 Solubility (mg/ml) 
                   
               
             
          
           
               
                   
                 Solvent 
                 20° C. 
                 40° C. 
               
               
                   
               
             
          
           
               
                   
                 Acetone 
                 0 
                 0 
               
               
                   
                 Ethanol 
                 420 
                 &gt;420 
               
               
                   
                 Ethyl acetate 
                 0 
                 0 
               
               
                   
                 Methyl tert-butyl ether 
                 0 
                 0 
               
               
                   
                 Heptane 
                 0 
                 0 
               
               
                   
                 Water 
                 &gt;400 
                 &gt;400 
               
               
                   
                 2-propanol 
                 23 
                 28 
               
               
                   
                 Iso-propyl acetate 
                 0 
                 0 
               
               
                   
                 Di-isopropyl ether 
                 0 
                 0 
               
               
                   
                 Methanol 
                 &gt;400 
                 &gt;400 
               
               
                   
                 Methylethyl ketone 
                 0 
                 0 
               
               
                   
                 Tetraydrofurane 
                 0 
                 0 
               
               
                   
                 Anisolo 
                 0 
                 0 
               
               
                   
                 Tert-butyl acetate 
                 0 
                 0 
               
               
                   
                 Dimethylsulfoxide 
                 &gt;400 
                 &gt;400 
               
               
                   
                 1-propanol 
                 295 
                 &gt;400 
               
               
                   
                 1-butanol 
                 97 
                 167 
               
               
                   
                 Acetonitrile 
                 6 
                 n.a. 
               
               
                   
                 Chlorobenzene 
                 0 
                 n.a. 
               
               
                   
                 Dichloromethane 
                 0 
                 n.a. 
               
               
                   
                 1,4-dioxane 
                 0 
                 n.a. 
               
               
                   
                 Ethanol/methyl tert-butyl ether 20% 
                 52 
                 n.a. 
               
               
                   
                 Ethanol/methyl tert-butyl ether 40% 
                 16 
                 n.a. 
               
               
                   
                 Ethanol/acetone 20% 
                 &gt;300 
                 n.a. 
               
               
                   
                 Ethanol/acetone 40% 
                 &gt;300 
                 n.a. 
               
               
                   
               
             
          
         
       
     
       Form a Characterization by Raman Spectrum and Fourier Transform Infrared Spectroscopy (FT-IR) 
       [0198]    The Raman spectrum of Form A is shown in  FIG. 16  with the related peak bands list in Table 5. 
         [0199]    Peak List: 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                 Position 
                 Intensity 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 247.89 
                 4066.127 
               
               
                   
                 268.70 
                 4076.600 
               
               
                   
                 285.77 
                 5666.532 
               
               
                   
                 297.80 
                 7186.507 
               
               
                   
                 322.04 
                 4385.802 
               
               
                   
                 411.78 
                 3861.458 
               
               
                   
                 436.26 
                 2433.529 
               
               
                   
                 499.66 
                 2023.949 
               
               
                   
                 521.68 
                 4054.372 
               
               
                   
                 560.04 
                 2419.952 
               
               
                   
                 588.92 
                 1163.452 
               
               
                   
                 629.52 
                 6647.466 
               
               
                   
                 640.58 
                 4792.760 
               
               
                   
                 687.14 
                 1836.374 
               
               
                   
                 718.78 
                 1714.527 
               
               
                   
                 758.37 
                 1345.186 
               
               
                   
                 794.58 
                 2302.231 
               
               
                   
                 836.54 
                 1806.043 
               
               
                   
                 872.19 
                 5315.287 
               
               
                   
                 932.18 
                 1889.917 
               
               
                   
                 949.44 
                 2637.407 
               
               
                   
                 962.31 
                 2419.830 
               
               
                   
                 985.74 
                 2736.112 
               
               
                   
                 1049.63 
                 5534.104 
               
               
                   
                 1074.79 
                 2056.236 
               
               
                   
                 1097.28 
                 4171.412 
               
               
                   
                 1135.89 
                 5311.271 
               
               
                   
                 1148.59 
                 3581.329 
               
               
                   
                 1178.28 
                 2121.957 
               
               
                   
                 1215.25 
                 2643.923 
               
               
                   
                 1239.16 
                 3338.948 
               
               
                   
                 1266.18 
                 3677.753 
               
               
                   
                 1325.12 
                 8522.793 
               
               
                   
                 1368.61 
                 5404.136 
               
               
                   
                 1394.52 
                 6973.028 
               
               
                   
                 1425.05 
                 4802.836 
               
               
                   
                 1457.84 
                 5583.813 
               
               
                   
                 1534.20 
                 4855.332 
               
               
                   
                 1648.81 
                 3369.165 
               
               
                   
                 1773.12 
                 4261.622 
               
               
                   
                 2890.60 
                 6428.710 
               
               
                   
                 2962.11 
                 19794.615 
               
               
                   
                 2986.55 
                 7243.053 
               
               
                   
                 3015.84 
                 7382.472 
               
               
                   
                 3049.43 
                 4000.206 
               
               
                   
               
             
          
         
       
     
         [0200]      FIG. 17  shows the FT-IR spectrum of Form A with the related peak bands list in Table 6. 
         [0201]    Peak List 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Position 
                 Intensity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 673.84 
                 0.0846 
               
               
                   
                 686.51 
                 0.118 
               
               
                   
                 718.98 
                 0.111 
               
               
                   
                 756.67 
                 0.0942 
               
               
                   
                 781.58 
                 0.0916 
               
               
                   
                 797.38 
                 0.100 
               
               
                   
                 834.89 
                 0.0756 
               
               
                   
                 871.89 
                 0.0672 
               
               
                   
                 932.05 
                 0.0646 
               
               
                   
                 948.44 
                 0.0932 
               
               
                   
                 1025.16 
                 0.0712 
               
               
                   
                 1050.31 
                 0.0580 
               
               
                   
                 1075.14 
                 0.0752 
               
               
                   
                 1094.42 
                 0.113 
               
               
                   
                 1134.65 
                 0.124 
               
               
                   
                 1148.93 
                 0.106 
               
               
                   
                 1204.60 
                 0.0957 
               
               
                   
                 1240.06 
                 0.0661 
               
               
                   
                 1235.85 
                 0.0661 
               
               
                   
                 1309.76 
                 0.147 
               
               
                   
                 1363.83 
                 0.0819 
               
               
                   
                 1392.60 
                 0.0512 
               
               
                   
                 1425.57 
                 0.0468 
               
               
                   
                 1452.48 
                 0.0538 
               
               
                   
                 1533.83 
                 0.0601 
               
               
                   
                 1622.97 
                 0.119 
               
               
                   
                 1766.49 
                 0.109 
               
               
                   
                 2890.12 
                 0.0390 
               
               
                   
                 2964.73 
                 0.0446 
               
               
                   
                 3013.48 
                 0.0440 
               
               
                   
                 3049.64 
                 0.0414 
               
               
                   
                 3089.32 
                 0.0425 
               
               
                   
                 3343.53 
                 0.0427 
               
               
                   
                 3530.97 
                 0.0395 
               
               
                   
                   
               
             
          
         
       
     
       Form C Characterization by Raman Spectrum and FT-IR 
       [0202]    The Raman spectrum of Form C is shown in  FIG. 18  with the related peak bands list in Table 7. 
         [0203]    Peak List: 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Position 
                 Intensity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 240.20 
                 4128.340 
               
               
                   
                 278.20 
                 10739.558 
               
               
                   
                 299.77 
                 10722.921 
               
               
                   
                 316.97 
                 8908.874 
               
               
                   
                 389.49 
                 3492.405 
               
               
                   
                 403.91 
                 5676.352 
               
               
                   
                 419.31 
                 6378.482 
               
               
                   
                 438.01 
                 3159.695 
               
               
                   
                 514.23 
                 9161.536 
               
               
                   
                 540.24 
                 2881.736 
               
               
                   
                 560.59 
                 5050.867 
               
               
                   
                 624.85 
                 13700.852 
               
               
                   
                 640.80 
                 5770.215 
               
               
                   
                 692.53 
                 7222.112 
               
               
                   
                 715.48 
                 2197.299 
               
               
                   
                 753.71 
                 2920.133 
               
               
                   
                 800.11 
                 2731.873 
               
               
                   
                 839.41 
                 3232.516 
               
               
                   
                 868.99 
                 6613.900 
               
               
                   
                 938.91 
                 4443.281 
               
               
                   
                 967.79 
                 3605.101 
               
               
                   
                 985.96 
                 4480.407 
               
               
                   
                 1033.35 
                 5823.568 
               
               
                   
                 1049.82 
                 6638.105 
               
               
                   
                 1096.10 
                 10022.146 
               
               
                   
                 1141.01 
                 9717.918 
               
               
                   
                 1180.11 
                 4361.805 
               
               
                   
                 1197.40 
                 3267.057 
               
               
                   
                 1235.20 
                 3502.896 
               
               
                   
                 1317.60 
                 10464.665 
               
               
                   
                 1362.32 
                 6745.435 
               
               
                   
                 1395.94 
                 9937.875 
               
               
                   
                 1457.27 
                 6235.580 
               
               
                   
                 1535.79 
                 4771.901 
               
               
                   
                 1640.00 
                 4841.217 
               
               
                   
                 1775.78 
                 7336.955 
               
               
                   
                 2879.07 
                 5109.468 
               
               
                   
                 2909.71 
                 11865.885 
               
               
                   
                 2947.89 
                 19208.596 
               
               
                   
                 2958.72 
                 17883.816 
               
               
                   
                 2983.99 
                 21848.400 
               
               
                   
                 2999.93 
                 12395.464 
               
               
                   
                 3014.33 
                 15550.745 
               
               
                   
                 3084.97 
                 4124.013 
               
               
                   
                 3169.92 
                 8548.841 
               
               
                   
                   
               
             
          
         
       
     
         [0204]    The FT-IR spectrum of Form C is shown in  FIG. 19  with the related peak bands list in Table 8. 
         [0205]    Peak List: 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Position 
                 Intensity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 671.04 
                 0.103 
               
               
                   
                 691.08 
                 0.151 
               
               
                   
                 715.10 
                 0.126 
               
               
                   
                 752.68 
                 0.145 
               
               
                   
                 780.33 
                 0.117 
               
               
                   
                 790.47 
                 0.149 
               
               
                   
                 799.40 
                 0.136 
               
               
                   
                 838.87 
                 0.0751 
               
               
                   
                 868.41 
                 0.0772 
               
               
                   
                 939.45 
                 0.111 
               
               
                   
                 956.58 
                 0.106 
               
               
                   
                 985.83 
                 0.0629 
               
               
                   
                 1023.40 
                 0.101 
               
               
                   
                 1089.49 
                 0.135 
               
               
                   
                 1098.28 
                 0.145 
               
               
                   
                 1138.00 
                 0.213 
               
               
                   
                 1195.45 
                 0.166 
               
               
                   
                 1233.08 
                 0.0960 
               
               
                   
                 1269.19 
                 0.142 
               
               
                   
                 1309.02 
                 0.208 
               
               
                   
                 1361.03 
                 0.114 
               
               
                   
                 1387.96 
                 0.0675 
               
               
                   
                 1456.72 
                 0.0694 
               
               
                   
                 1536.21 
                 0.0645 
               
               
                   
                 1637.71 
                 0.174 
               
               
                   
                 1770.33 
                 0.174 
               
               
                   
                 2972.20 
                 0.0498 
               
               
                   
                 3015.04 
                 0.0553 
               
               
                   
                 3083.68 
                 0.0481 
               
               
                   
                 3168.20 
                 0.0456 
               
               
                   
                 3375.47 
                 0.0422 
               
               
                   
                   
               
             
          
         
       
     
         [0206]    The analyses performed on Form A and Form C, including the information collected on the influence of the water content during the crystallization, has supported the hypothesis that Form A is a hydrate form with a rapid water exchange with the ambient and Form C is a more stable anhydrous form. Therefore, Form C was selected for further optimisation and scale-up of the crystallization process, and assessments as described below. 
       Optimization of Form C Crystallization 
     Example 10 
     Crystallization Procedure Using a Form C Seed 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form C 
       [0207]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (5 g) was weighed in a 50 ml multimax vessel equipped with an impeller stirrer. The solid was suspended in 20 ml of ethanol HPLC grade 99.8%. The mixture was heated to 40° C. and stirred at 700 RPM. At 40° C. the starting material was dissolved. The solution was cooled to 36° C. over 15 minutes and Form C material (27 mg) was added to the solution as seed; the seed was not dissolved and promoted the product crystallization. The mixture was cooled to 15° C. over 3.5 hours. The slurry was aged overnight and then was filtered under vacuum; the cake was dried at 30° C. in a vacuum oven for 40 hours to afford 3.7 g of a white solid. The solid showed an XRPD pattern for Form C. 
         [0208]    The quality of the ethanol system was also investigated in the production of Form C material using 96% ethanol instead of ethanol HPLC grade 99.8% as described in Example 11. 
       Example 11 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A 
       [0209]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (5 g) was weighed in a 50 ml multimax vessel equipped with an impeller stirrer. The solid was suspended in 20 ml of ethanol 96%. The mixture was heated to 40° C. and stirred at 700 RPM. At 40° C. the starting material seemed to be dissolved but the solution appeared slightly opaque. The opaque solution was filtered through a syringe filter to obtain a clear solution. The solution was cooled to 35° C. over 15 minutes and Form C material (28 mg) was added to the solution as seed. After 10 minutes at 35° C. was dissolved. The temperature was lowered to 30° C. over 15 minutes and more Form C material (27 mg) was added as seed. The seed was dissolved after 15 minutes. The solution was heated up to 35° C. and a pinch of Form B material was added to the solution but was dissolved after few minutes. A pinch of Form A material was added as seed; this time the seed did not dissolve and promoted the product crystallization. The mixture was cooled to 15° C. over 3.5 hours. The slurry was aged overnight and then was filtered under vacuum; the cake was dried at 30° C. in a vacuum oven for 18 hours to afford 3.1 g of a white solid. The solid showed an XRPD pattern concordant to Form A. 
         [0210]    Examples 10 and 11 procedures demonstrate that the water content in the ethanol system can affect production of Forms A and C by a seeded approach. The formation of Form A material is possible in ethanol 96%, whereas the formation of Form C from a Form C crystal required use of ethanol HPLC grade 99.8%. 
       Example 12 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form C 
       [0211]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (8 g) was weighed in a 50 ml Multimax vessel equipped with an impeller stirrer. The solid was suspended in 20 ml of ethanol HPLC grade 99.8%. The mixture was heated to 40° C. and stirred at 800 RPM. At 40° C. the starting material was dissolved. The solution was cooled to 36° C. over 10 minutes and Form C material (24 mg) was added to the solution as seed; the seed was not dissolved and promoted the product crystallization. After 15 minutes stirring at 36° C. the mixture was cooled to 15° C. over 3.5 hours. The slurry was aged overnight and then was filtered under vacuum in nitrogen atmosphere (a funnel connected to a nitrogen flux was put over the filter). The cake was washed with 8 ml of ethanol HPLC grade 99.8%. The cake was dried inside the filter at 30° C. in a vacuum oven for 2 hours, after this time the product was transferred to a crystallizer and dried for further 16 hours. The product was analyzed by  1 H-NMR to check the solvent content and showed the presence of ˜1.3% w/w of ethanol. The cake was further dried at 35° C. in the vacuum oven for 6 hours. A new sample was taken and analyzed by  1 H-NMR for solvent content. The ethanol residual was comparable to the first sample. The product was stored at −20° C. for the week-end and then put in the vacuum oven at 40° C. for 24 hours to yield 6 g of the product. The solid showed an XRPD pattern concordant with Form C.  1 H-NMR confirmed the presence of ˜1.3% w/w of ethanol residual in the cake. 
         [0212]    The decrease of the seed loading did not have any negative impact on the product crystallization and was implemented in the scaled-up procedure as described in Example 13. 
       Example 13 
     Preparation scale up of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form C at 36 g scale 
       [0213]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (36.45 g) was weighed in a 250 ml multimax vessel equipped with an impeller stirrer. The solid was suspended in 146 ml of ethanol HPLC grade 99.8%. The mixture was heated to 40° C. over 20 minutes. After 15 minutes at 40° C. the starting material was completely dissolved and the solution was cooled to 36° C. over 10 minutes and Form C material (110 mg) was added to the solution as seed; the seed was not dissolved and promoted the product crystallization. After 10 minutes stirring at 36° C. the mixture was cooled to 15° C. over 3.5 hours. The obtained mixture was aged overnight and then was filtered under vacuum. The cake was washed with 40 ml of ethanol HPLC grade 99.8% and three times with 40 ml of methyl tert-butyl ether to remove residual ethanol from the cake. The cake was deliquored in nitrogen atmosphere (a funnel connected to a nitrogen flux was put over the filter) under vacuum. The cake was dried in a vacuum oven for 24 hours to yield 26.8 g of the final product as a white solid. 
         [0214]    The solid was analyzed by XRPD, TGA, optical microscopy (OM) and  1 H-NMR. 
         [0215]    The XRPD analysis of the product showed crystalline material with a pattern consistent with Form C ( FIG. 20 ). 
         [0216]    The TGA analysis for the product ( FIG. 21 ) shows a weight loss of circa 2% up to 120° C. probably due to adsorbed water and solvent residual. 
         [0217]    The OM analysis in  FIG. 22  shows Form C crystals. Birefringent particles using polarized light could be seen. 
         [0218]    The  1 H-NMR spectrum ( FIG. 23 ) is consistent with the structure of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. The ethanol residue was calculated comparing the ethanol signal at 1.06 ppm and the API signal at 1.40 ppm. Considering integrals values, number of protons and the molecular weight of the reference signals the estimated ethanol residue is equal to 0.4% w/w respect to the API. 
       Solubility Assessment in Saline Physiological Solution 
       [0219]    The Form C solubility was calculated by HPLC employing a dedicated walk-up method. The product obtained by the scaled up procedure described in Example 13 was used to perform the experiments. 
         [0220]    1.9 g of the product was suspended in 1 ml of commercial physiologic solution (0.9% of NaCl) at ambient temperature (˜20° C.). The suspension resulted slightly opaque and quite viscous after 30 min. After this time the suspension was sampled and the sample injected in HPLC to determine its concentration. After 2 hrs stirring the solid residue was completely dissolved. The addition of more solid was not performed to avoid the gelatinisation of the viscous solution. A sample was taken and injected in HPLC to determine its concentration. The solution was stirred other 3 hrs and sampled again. The 5 hrs sample was also injected in HPLC to determine its concentration. The HPLC traces did not show the formation of significant impurities. Table 9 shows the solubility results for the time-points selected. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Physiological 
                   
                 Solubility (mg/ml) at 
               
               
                 solution 
                 Timepoint 
                 ambient temperature 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 30 mins 
                 772 
               
               
                 2 
                 12 hours 
                 &gt;883 
               
               
                 3 
                  5 hours 
                 &gt;812 
               
               
                   
               
             
          
         
       
     
       Particle Size Distribution 
       [0221]    The particle size analysis was performed on the product obtained by the scaled up procedure described in Example 13 using the procedure described below. Three measurements for each suspension were recorded and the results are shown in  FIG. 24  and in Table 10. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Sample Name 
                 d (0.1) 
                 d (0.5) 
                 d (0.9) 
               
               
                   
                   
               
             
             
               
                   
                 Suspension 1, Measurement 1 
                 29.08 
                 129.34 
                 249.92 
               
               
                   
                 Suspension 1, Measurement 2 
                 28.94 
                 128.69 
                 246.31 
               
               
                   
                 Suspension 1, Measurement 3 
                 28.90 
                 128.42 
                 247.37 
               
               
                   
                 Suspension 2, Measurement 1 
                 28.26 
                 130.37 
                 251.74 
               
               
                   
                 Suspension 2, Measurement 2 
                 26.80 
                 125.95 
                 248.77 
               
               
                   
                 Suspension 2, Measurement 3 
                 25.40 
                 119.25 
                 239.11 
               
               
                   
                 Suspension 3, Measurement 1 
                 28.54 
                 133.06 
                 256.35 
               
               
                   
                 Suspension 3, Measurement 2 
                 26.85 
                 128.64 
                 249.66 
               
               
                   
                 Suspension 3, Measurement 3 
                 26.09 
                 126.42 
                 244.15 
               
               
                   
                 Average 
                 27.65 
                 127.79 
                 248.15 
               
               
                   
                   
               
             
          
         
       
     
       Example 14 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form D 
       [0222]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (30 g) was suspended in 200 mL of N,N-dimethylformamide, pre-heated to +20/25° C. After 5 minutes stirring a solution is obtained and after few minutes of stirring crystallization takes place. The suspension is stirred for about 2 hours. Then the suspension is cooled down to 0/+5° C. and stirred for about 2 hours. 
         [0223]    The obtained solid is filtered and washed with 50 mL of N,N-dimethylformamide pre-cooled to 0/+5° C. The wet product is then suspended in 300 mL of dichloromethane and the temperature is adjusted to +30/32° C. The suspension is stirred for 45 minutes then the solid is filtered and washed with 100 mL of dichloromethane pre-heated to +30/32° C. The product is dried under vacuum at +40° C. until constant weight is achieved. The obtained product (19.3 g) was crystalline form D which was characterized by an XRPD pattern as shown in  FIG. 25  and summarized in the following Table 11. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 d-spacing 
                   
                   
                   
                   
                   
               
               
                 No. 
                 Angle [°2θ] 
                 [Å] 
                 Height (cps) 
                 FWHM (deg) 
                 Int. 
                 deg 
                 Int. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 6.7824 
                 13.02204 
                 369.45 
                 0.2491 
                 119.22 
                 0.3227 
                 333.64 
               
               
                 2 
                 9.5032 
                 9.29904 
                 105.18 
                 0.2064 
                 26.05 
                 0.2477 
                 403.40 
               
               
                 3 
                 10.4510 
                 8.45774 
                 31.17 
                 0.2565 
                 10.15 
                 0.3256 
                 324.89 
               
               
                 4 
                 11.6074 
                 7.61762 
                 109.02 
                 0.2733 
                 31.72 
                 0.2910 
                 305.11 
               
               
                 5 
                 12.7850 
                 6.91847 
                 41.58 
                 0.2692 
                 11.91 
                 0.2865 
                 310.18 
               
               
                 6 
                 13.4325 
                 6.58642 
                 64.83 
                 0.2025 
                 13.98 
                 0.2156 
                 412.47 
               
               
                 7 
                 14.2560 
                 6.20776 
                 275.43 
                 0.2923 
                 86.51 
                 0.3141 
                 286.05 
               
               
                 8 
                 15.4567 
                 5.72810 
                 77.92 
                 1.8085 
                 152.28 
                 1.9543 
                 46.30 
               
               
                 9 
                 16.3961 
                 5.40199 
                 835.69 
                 0.4340 
                 388.58 
                 0.4650 
                 193.15 
               
               
                 10 
                 17.1082 
                 5.17871 
                 522.62 
                 0.3370 
                 188.77 
                 0.3612 
                 249.00 
               
               
                 11 
                 18.2742 
                 4.85081 
                 148.14 
                 0.3388 
                 53.91 
                 0.3639 
                 248.02 
               
               
                 12 
                 20.0651 
                 4.42173 
                 194.88 
                 0.5228 
                 109.82 
                 0.5635 
                 161.19 
               
               
                 13 
                 20.6373 
                 4.30040 
                 624.11 
                 0.3160 
                 211.90 
                 0.3395 
                 266.91 
               
               
                 14 
                 22.7520 
                 3.90524 
                 167.10 
                 0.2473 
                 44.02 
                 0.2635 
                 342.21 
               
               
                 15 
                 23.2376 
                 3.82472 
                 236.56 
                 0.6238 
                 157.13 
                 0.6642 
                 135.79 
               
               
                 16 
                 23.6811 
                 3.75409 
                 198.42 
                 0.5077 
                 107.27 
                 0.5406 
                 167.00 
               
               
                 17 
                 25.6817 
                 3.46600 
                 163.48 
                 0.4133 
                 71.93 
                 0.4400 
                 205.89 
               
               
                 18 
                 26.1802 
                 3.40112 
                 205.89 
                 0.5004 
                 109.66 
                 0.5326 
                 170.25 
               
               
                 19 
                 26.9957 
                 3.30020 
                 138.32 
                 0.4481 
                 65.98 
                 0.4770 
                 190.41 
               
               
                 20 
                 27.7606 
                 3.21098 
                 221.24 
                 0.3671 
                 86.44 
                 0.3907 
                 232.85 
               
               
                 21 
                 28.7686 
                 3.10073 
                 75.61 
                 0.2697 
                 21.70 
                 0.2870 
                 317.65 
               
               
                 22 
                 30.4020 
                 2.93775 
                 125.52 
                 0.3451 
                 46.72 
                 0.3722 
                 249.17 
               
               
                 23 
                 31.4633 
                 2.84104 
                 102.42 
                 0.6496 
                 72.49 
                 0.7078 
                 132.70 
               
               
                 24 
                 32.4753 
                 2.75478 
                 268.97 
                 0.4635 
                 134.78 
                 0.5011 
                 186.43 
               
               
                 25 
                 34.4252 
                 2.60307 
                 21.60 
                 0.6492 
                 14.92 
                 0.6911 
                 133.80 
               
               
                 26 
                 34.9492 
                 2.56524 
                 12.83 
                 0.4399 
                 6.01 
                 0.4682 
                 197.76 
               
               
                 27 
                 36.0489 
                 2.48946 
                 80.97 
                 0.3230 
                 27.84 
                 0.3438 
                 270.15 
               
               
                 28 
                 38.4794 
                 2.33762 
                 119.18 
                 0.6568 
                 83.33 
                 0.6992 
                 133.80 
               
               
                 29 
                 40.2292 
                 2.23989 
                 27.09 
                 0.9554 
                 27.55 
                 1.0170 
                 92.49 
               
               
                 30 
                 42.6703 
                 2.11723 
                 63.61 
                 0.5331 
                 36.10 
                 0.5675 
                 167.09 
               
               
                 31 
                 43.9731 
                 2.05748 
                 22.45 
                 0.4622 
                 11.29 
                 0.5030 
                 193.59 
               
               
                 32 
                 53.8897 
                 1.69994 
                 16.28 
                 0.5850 
                 11.95 
                 0.7339 
                 159.10 
               
               
                   
               
             
          
         
       
     
         [0224]    The Raman spectrum of Form D is shown in  FIG. 26  with the related peak band list in the following Table 12 (using Raman Jasco RFT-600 instrument, light source Nd-YAG, 1064 nm: exciting wavelength). 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Wave number 
                 Y value 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 3157.83 
                 0.0281958 
               
               
                 2 
                 3009.34 
                 0.072899 
               
               
                 3 
                 2974.63 
                 0.100304 
               
               
                 4 
                 2904.24 
                 0.0444922 
               
               
                 5 
                 1772.23 
                 0.0339617 
               
               
                 6 
                 1663.27 
                 0.0258104 
               
               
                 7 
                 1474.28 
                 0.0302334 
               
               
                 8 
                 1458.85 
                 0.0264177 
               
               
                 9 
                 1437.64 
                 0.0373852 
               
               
                 10 
                 1400.04 
                 0.0571817 
               
               
                 11 
                 1352.79 
                 0.0302512 
               
               
                 12 
                 1302.65 
                 0.0862235 
               
               
                 13 
                 1195.62 
                 0.0247634 
               
               
                 14 
                 1175.37 
                 0.0330307 
               
               
                 15 
                 1138.73 
                 0.0701386 
               
               
                 16 
                 1092.45 
                 0.11397 
               
               
                 17 
                 1031.7 
                 0.0433419 
               
               
                 18 
                 993.13 
                 0.0292598 
               
               
                 19 
                 947.811 
                 0.0372661 
               
               
                 20 
                 874.529 
                 0.0711543 
               
               
                 21 
                 838.853 
                 0.028534 
               
               
                 22 
                 783.892 
                 0.0241906 
               
               
                 23 
                 688.432 
                 0.0231856 
               
               
                 24 
                 661.434 
                 0.0387182 
               
               
                 25 
                 624.793 
                 0.134281 
               
               
                 26 
                 556.332 
                 0.0499649 
               
               
                 27 
                 514.87 
                 0.0831978 
               
               
                 28 
                 433.875 
                 0.0597285 
               
               
                 29 
                 414.59 
                 0.0366139 
               
               
                 30 
                 340.344 
                 0.0295258 
               
               
                 31 
                 324.916 
                 0.058052 
               
               
                 32 
                 287.311 
                 0.0680197 
               
               
                 33 
                 249.706 
                 0.0476452 
               
               
                   
               
             
          
         
       
     
       Example 15 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form E 
       [0225]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (5 g) was suspended in 30 mL of N,N-dimethylformamide, pre-heated to +20/25° C. After 5 minutes of stirring, a solution is obtained and after few minutes a crystallization takes place. The suspension is stirred for about 2 hours. 
         [0226]    The obtained solid is filtered and washed with 12.5 mL of N,N-dimethylformamide. The wet product is then suspended in 100 mL of ethyl acetate and the temperature is adjusted to +40/45° C. The suspension is stirred for 60 minutes then the solid is filtered and washed with 50 mL of ethyl acetate pre-heated to +40/45° C. 
         [0227]    Finally the product is dried under vacuum at +40° C. till constant weight is achieved. 
         [0228]    The obtained product (2.4 g) was crystalline form E which was characterized by an XRPD pattern as shown in  FIG. 27  and summarized in the following Table 13. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 d-spacing 
                   
                   
                   
                   
                   
               
               
                 No. 
                 Angle [°2θ] 
                 [Å] 
                 Height (cps) 
                 FWHM (deg) 
                 Int. 
                 deg) 
                 Int. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 6.8269 
                 12.93732 
                 260.98 
                 0.2205 
                 68.42 
                 0.2622 
                 376.99 
               
               
                 2 
                 9.5377 
                 9.26545 
                 102.12 
                 0.2256 
                 29.95 
                 0.2933 
                 369.14 
               
               
                 3 
                 10.4196 
                 8.48314 
                 117.20 
                 0.2867 
                 38.29 
                 0.3267 
                 290.56 
               
               
                 4 
                 11.6525 
                 7.58825 
                 82.26 
                 0.2299 
                 20.13 
                 0.2447 
                 362.76 
               
               
                 5 
                 12.6274 
                 7.00451 
                 83.34 
                 0.3681 
                 32.66 
                 0.3919 
                 226.76 
               
               
                 6 
                 13.3413 
                 6.63125 
                 95.57 
                 0.3884 
                 39.52 
                 0.4135 
                 215.07 
               
               
                 7 
                 14.2802 
                 6.19726 
                 104.47 
                 0.2212 
                 26.92 
                 0.2577 
                 378.06 
               
               
                 8 
                 15.0475 
                 5.88296 
                 494.41 
                 0.3508 
                 199.68 
                 0.4039 
                 238.55 
               
               
                 9 
                 15.6848 
                 5.64531 
                 378.40 
                 0.3968 
                 173.35 
                 0.4581 
                 211.11 
               
               
                 10 
                 16.4735 
                 5.37678 
                 557.04 
                 0.3770 
                 234.97 
                 0.4218 
                 222.38 
               
               
                 11 
                 17.1773 
                 5.15801 
                 229.03 
                 0.3175 
                 81.31 
                 0.3550 
                 264.28 
               
               
                 12 
                 18.4488 
                 4.80530 
                 297.04 
                 0.3867 
                 122.28 
                 0.4117 
                 217.36 
               
               
                 13 
                 19.0164 
                 4.66312 
                 93.86 
                 0.2902 
                 28.99 
                 0.3089 
                 289.90 
               
               
                 14 
                 20.0808 
                 4.41830 
                 143.91 
                 0.4955 
                 75.93 
                 0.5276 
                 170.05 
               
               
                 15 
                 20.6999 
                 4.28752 
                 421.62 
                 0.3301 
                 148.17 
                 0.3514 
                 255.52 
               
               
                 16 
                 22.2167 
                 3.99811 
                 90.66 
                 0.6225 
                 62.16 
                 0.6857 
                 135.85 
               
               
                 17 
                 22.7863 
                 3.89944 
                 130.42 
                 0.4242 
                 61.57 
                 0.4721 
                 199.52 
               
               
                 18 
                 23.3436 
                 3.80760 
                 273.25 
                 0.4007 
                 120.48 
                 0.4409 
                 211.43 
               
               
                 19 
                 23.8843 
                 3.72261 
                 447.75 
                 0.4942 
                 242.53 
                 0.5417 
                 171.61 
               
               
                 20 
                 25.3818 
                 3.50627 
                 95.40 
                 1.0693 
                 109.22 
                 1.1449 
                 79.54 
               
               
                 21 
                 26.2231 
                 3.39566 
                 113.17 
                 0.5204 
                 63.45 
                 0.5606 
                 163.70 
               
               
                 22 
                 27.8574 
                 3.20005 
                 112.17 
                 0.2916 
                 35.41 
                 0.3157 
                 293.14 
               
               
                 23 
                 29.9383 
                 2.98219 
                 52.64 
                 0.4091 
                 38.32 
                 0.7279 
                 209.96 
               
               
                 24 
                 31.3100 
                 2.85459 
                 70.72 
                 0.3247 
                 40.45 
                 0.5720 
                 265.41 
               
               
                 25 
                 33.3041 
                 2.68809 
                 41.89 
                 0.3114 
                 17.99 
                 0.4295 
                 278.15 
               
               
                 26 
                 38.5117 
                 2.33574 
                 15.66 
                 1.2693 
                 21.16 
                 1.3512 
                 69.24 
               
               
                 27 
                 41.1953 
                 2.18957 
                 21.01 
                 1.1036 
                 24.69 
                 1.1748 
                 80.32 
               
               
                 28 
                 49.2559 
                 1.84846 
                 16.52 
                 0.9722 
                 17.90 
                 1.0835 
                 93.88 
               
               
                   
               
             
          
         
       
     
         [0229]    The Raman spectrum of Form E is shown in  FIG. 28  with the related peak band list in the following Table 14 (using Raman Jasco RFT-600 instrument, light source Nd-YAG, 1064 nm: exciting wavelength). 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Peak 
                 Wave number 
                 Y value 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 3158.8 
                 0.0221892 
               
               
                 2 
                 3051.77 
                 0.0289691 
               
               
                 3 
                 3011.27 
                 0.0722239 
               
               
                 4 
                 2973.67 
                 0.168653 
               
               
                 5 
                 2900.38 
                 0.0707581 
               
               
                 6 
                 1772.23 
                 0.0829712 
               
               
                 7 
                 1482 
                 0.036189 
               
               
                 8 
                 1438.61 
                 0.0318463 
               
               
                 9 
                 1397.14 
                 0.0563706 
               
               
                 10 
                 1352.79 
                 0.0498639 
               
               
                 11 
                 1302.65 
                 0.120509 
               
               
                 12 
                 1266.01 
                 0.0335002 
               
               
                 13 
                 1202.37 
                 0.0466914 
               
               
                 14 
                 1185.01 
                 0.0332323 
               
               
                 15 
                 1139.69 
                 0.0742465 
               
               
                 16 
                 1092.45 
                 0.128341 
               
               
                 17 
                 1031.7 
                 0.0532132 
               
               
                 18 
                 989.273 
                 0.0474664 
               
               
                 19 
                 949.74 
                 0.0622083 
               
               
                 20 
                 873.565 
                 0.0993489 
               
               
                 21 
                 836.924 
                 0.0338838 
               
               
                 22 
                 782.927 
                 0.0376849 
               
               
                 23 
                 715.431 
                 0.0287148 
               
               
                 24 
                 589.397 
                 0.028656 
               
               
                 25 
                 646.006 
                 0.0458421 
               
               
                 26 
                 624.793 
                 0.177092 
               
               
                 27 
                 556.332 
                 0.0561384 
               
               
                 28 
                 613.906 
                 0.109643 
               
               
                 29 
                 433.875 
                 0.0568177 
               
               
                 30 
                 407.84 
                 0.0759362 
               
               
                 31 
                 325.881 
                 0.68886 
               
               
                 32 
                 288.276 
                 0.0859623 
               
               
                 33 
                 254.527 
                 0.050626 
               
               
                 34 
                 216.922 
                 0.0240766 
               
               
                   
               
             
          
         
       
     
       Example 16 
     Preparation of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form F 
       [0230]    Amorphous (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide (130 g) was suspended in 800 mL of N,N-dimethylformamide, pre-heated to +20/25° C. 100 mL of N,N-dimethylformamide was added to wash the walls of the flask. After 5 minutes stirring a solution is obtained and after few minutes of stirring crystallization takes place. The suspension is stirred for about 3 hours. Then the suspension is cooled down to 0/+5° C. and stirred for about 3 hours. 
         [0231]    The obtained solid is filtered and washed with 300 mL of N,N-dimethylformamide pre-cooled to 0/+5° C. The wet product is then suspended in 700 mL of ethyl acetate and the temperature is adjusted to +40/45° C. The suspension is stirred for 30 minutes then the solid is filtered and washed with 150 mL of ethyl acetate pre-heated to +40/45° C. The procedure with the suspension in Ethyl acetate is repeated twice. Finally the product is dried under vacuum at +40° C. till constant weight is achieved. 
         [0232]    The obtained product (65-66 g, molar yield about 76%, with an assay of 98-99% was crystalline form F, which was characterized by an XRPD pattern as shown in  FIG. 29  and summarized in the following Table 15. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Angle 
                 d-spacing 
                   
                   
                   
                   
                   
               
               
                 No. 
                 [°2θ] 
                 [Å] 
                 Height (cps) 
                 FWHM (deg) 
                 Int. 
                 deg) 
                 Int. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 8.5718 
                 10.30725 
                 116.72 
                 0.1981 
                 26.19 
                 0.2244 
                 419.96 
               
               
                 2 
                 10.3165 
                 8.56773 
                 182.16 
                 0.2142 
                 42.67 
                 0.2343 
                 388.97 
               
               
                 3 
                 12.7398 
                 6.94292 
                 420.49 
                 0.2216 
                 103.83 
                 0.2469 
                 376.75 
               
               
                 4 
                 15.3615 
                 5.76339 
                 870.60 
                 0.2471 
                 241.26 
                 0.2771 
                 338.84 
               
               
                 5 
                 15.9547 
                 5.55042 
                 1374.98 
                 0.2605 
                 400.47 
                 0.2913 
                 321.60 
               
               
                 6 
                 16.4290 
                 5.39123 
                 1343.96 
                 0.2344 
                 352.88 
                 0.2626 
                 357.69 
               
               
                 7 
                 17.1990 
                 5.15158 
                 477.25 
                 0.2281 
                 118.86 
                 0.2490 
                 367.89 
               
               
                 8 
                 18.1207 
                 4.89155 
                 531.20 
                 0.2398 
                 146.12 
                 0.2751 
                 350.36 
               
               
                 9 
                 20.4870 
                 4.33160 
                 915.19 
                 0.2443 
                 275.10 
                 0.3006 
                 345.16 
               
               
                 10 
                 21.4040 
                 4.14805 
                 37.20 
                 0.1769 
                 7.01 
                 0.1884 
                 477.23 
               
               
                 11 
                 22.8548 
                 3.88791 
                 528.69 
                 0.2904 
                 164.14 
                 0.3105 
                 291.53 
               
               
                 12 
                 23.2204 
                 3.82751 
                 502.41 
                 0.3500 
                 188.64 
                 0.3755 
                 242.00 
               
               
                 13 
                 23.4688 
                 3.78756 
                 292.42 
                 0.1501 
                 47.04 
                 0.1609 
                 564.73 
               
               
                 14 
                 24.4199 
                 3.64215 
                 132.35 
                 0.2404 
                 34.95 
                 0.2641 
                 353.09 
               
               
                 15 
                 25.6394 
                 3.47163 
                 359.02 
                 0.2563 
                 104.03 
                 0.2897 
                 331.96 
               
               
                 16 
                 25.9983 
                 3.42450 
                 94.56 
                 0.2531 
                 27.13 
                 0.2869 
                 336.47 
               
               
                 17 
                 26.2914 
                 3.38699 
                 134.69 
                 0.2951 
                 45.04 
                 0.3344 
                 288.79 
               
               
                 18 
                 27.0457 
                 3.29421 
                 387.38 
                 0.3463 
                 151.47 
                 0.3910 
                 246.47 
               
               
                 19 
                 27.6934 
                 3.21862 
                 412.53 
                 0.2941 
                 136.95 
                 0.3320 
                 290.62 
               
               
                 20 
                 28.7394 
                 3.10381 
                 190.86 
                 0.2739 
                 56.91 
                 0.2982 
                 312.74 
               
               
                 21 
                 29.7603 
                 2.99962 
                 32.77 
                 0.2736 
                 9.54 
                 0.2913 
                 313.76 
               
               
                 22 
                 30.3078 
                 2.94667 
                 222.03 
                 0.2854 
                 67.46 
                 0.3038 
                 301.19 
               
               
                 23 
                 31.4660 
                 2.84080 
                 125.87 
                 0.5371 
                 71.97 
                 0.5717 
                 160.49 
               
               
                 24 
                 32.3054 
                 2.76888 
                 98.55 
                 0.2002 
                 21.00 
                 0.2131 
                 431.51 
               
               
                 25 
                 32.4785 
                 2.75451 
                 363.46 
                 0.4069 
                 157.43 
                 0.4331 
                 212.38 
               
               
                 26 
                 33.1981 
                 2.69643 
                 37.54 
                 0.2403 
                 9.60 
                 0.2558 
                 360.31 
               
               
                 27 
                 33.7446 
                 2.65401 
                 15.05 
                 0.5057 
                 8.10 
                 0.5383 
                 171.46 
               
               
                 28 
                 34.3283 
                 2.61020 
                 55.64 
                 0.1955 
                 11.58 
                 0.2081 
                 444.20 
               
               
                 29 
                 35.0200 
                 2.56021 
                 21.77 
                 0.6046 
                 14.01 
                 0.6435 
                 143.92 
               
               
                 30 
                 35.9880 
                 2.49354 
                 133.13 
                 0.2751 
                 38.98 
                 0.2928 
                 317.16 
               
               
                 31 
                 38.4256 
                 2.34077 
                 142.45 
                 0.6826 
                 103.50 
                 0.7266 
                 128.73 
               
               
                 32 
                 40.2911 
                 2.23659 
                 56.34 
                 0.4183 
                 25.09 
                 0.4453 
                 211.28 
               
               
                 33 
                 40.8969 
                 2.20485 
                 33.86 
                 0.3473 
                 12.52 
                 0.3697 
                 254.95 
               
               
                 34 
                 42.6047 
                 2.12034 
                 130.78 
                 0.2718 
                 59.44 
                 0.4545 
                 327.66 
               
               
                 35 
                 43.7327 
                 2.06823 
                 39.36 
                 0.5339 
                 22.37 
                 0.5684 
                 167.46 
               
               
                 36 
                 44.8088 
                 2.02103 
                 29.53 
                 0.2009 
                 6.31 
                 0.2138 
                 446.84 
               
               
                 37 
                 53.9562 
                 1.69800 
                 23.47 
                 0.6255 
                 15.68 
                 0.6680 
                 148.86 
               
               
                   
               
             
          
         
       
     
         [0233]    Raman spectra for three bathes of Form F are shown in  FIGS. 30 and 31 . 
         [0234]    Both XRPD and Raman spectra acquired for different batches of Form F product are overlapping. 
         [0235]    Scanning electron microscopy images of samples of the three batches of Form F are shown in  FIGS. 32-50 . The SEM images of the samples were obtained using a JEOL JSM 5500 LV scanning electron microscope, operating at 30 kV in low vacuum (30 Pa) with the backscattered electron technique. 
       Form F Characterization by FT-IR, DSC, TGA, EGA 
       [0236]      FIG. 51  shows the FT-IR spectrum of Form F with the related peak bands list in Table 16. 
         [0237]    Peak List: 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Position 
                 Intensity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 502 
                 50.668 
               
               
                   
                 514 
                 59.193 
               
               
                   
                 538 
                 66.311 
               
               
                   
                 554 
                 48.279 
               
               
                   
                 586 
                 76.021 
               
               
                   
                 623 
                 60.523 
               
               
                   
                 635 
                 58.506 
               
               
                   
                 675 
                 73.819 
               
               
                   
                 688 
                 65.213 
               
               
                   
                 711 
                 63.330 
               
               
                   
                 752 
                 53.517 
               
               
                   
                 783 
                 68.207 
               
               
                   
                 808 
                 55.605 
               
               
                   
                 826 
                 52.413 
               
               
                   
                 872 
                 72.360 
               
               
                   
                 908 
                 81.158 
               
               
                   
                 928 
                 78.947 
               
               
                   
                 948 
                 62.908 
               
               
                   
                 953 
                 63.041 
               
               
                   
                 989 
                 78.973 
               
               
                   
                 1020 
                 62.785 
               
               
                   
                 1067 
                 55.907 
               
               
                   
                 1088 
                 52.453 
               
               
                   
                 1102 
                 46.426 
               
               
                   
                 1136 
                 35.517 
               
               
                   
                 1186 
                 50.232 
               
               
                   
                 1199 
                 50.943 
               
               
                   
                 1228 
                 75.847 
               
               
                   
                 1266 
                 64.974 
               
               
                   
                 1300 
                 44.572 
               
               
                   
                 1307 
                 44.644 
               
               
                   
                 1351 
                 62.003 
               
               
                   
                 1396 
                 78.685 
               
               
                   
                 1472 
                 75.504 
               
               
                   
                 1525 
                 78.318 
               
               
                   
                 1637 
                 36.877 
               
               
                   
                 1735 
                 80.927 
               
               
                   
                 1771 
                 48.478 
               
               
                   
                 1783 
                 51.962 
               
               
                   
                 2898 
                 88.274 
               
               
                   
                 2972 
                 84.793 
               
               
                   
                 3017 
                 86.781 
               
               
                   
                 3051 
                 88.751 
               
               
                   
                 3156 
                 84.061 
               
               
                   
                   
               
             
          
         
       
     
         [0238]    The DSC profile of form F is presented in  FIG. 52 . The DSC profile shows an exothermic peak at approximately 184° C. (Onset 175° C.) associated with the degradation of the sample. 
         [0239]    The Thermo Gravimetric Analysis (TGA) profile of Form F presented on  FIG. 53  shows a significant weight loss after approximately 160° C. associated with the degradation of the sample. That is further confirmed by an Evolved Gas Analysis (EGA) shown in  FIG. 54 . The EGA evidences that the event observed in TGA analysis is caused by the loss of degradation products (e.g. carbon dioxide, sulphur dioxide, etc). 
       Form F Characterization by Dynamic Vapor Sorption (DVS) 
       [0240]    Kinetic moisture sorption measurements were performed at 25° C. and at relative humidity (RH % target as follows:
       From 40% RH to 90% RH   Form 90% RH to 0% RH   From 0% RH to 90% RH   From 90% RH to 0% RH       
 
         [0245]    The obtained results are presented in  FIG. 55 , wherein the red line traces the percentage changes in mass as function of the time, while the blue line traces the relative humidity changes as function of the time. 
         [0246]    DVS isotherms plots are reported in  FIG. 56 , wherein the red line depicts the first sorption phase, the blue line depicts the first desorption phase, the green line depicts the second sorption phase and the pink line depicts the second desorption phase. 
         [0247]    The DVS analyses show that Form F is stable at up to approximately 50% RH and that at 90% RH, the sample showed a weight increase that is greater than 50% w/w. After this event the sample releases and takes water reversibly. 
       Stability of Form F 
       [0248]    The sample becomes a viscous liquid after a day at 25° C. and 60% RH and after a day at 60° C. and 75% RH. 
       Hygroscopicity of Form F 
       [0249]    The hygroscopicity was calculated using the following equation: 
         [0000]      % Weight Change=[( W 2− W 1)/ W 1]*100
 
         [0000]    wherein,
 
W1 is weight of sample at the start of the experiment; and
 
W2 is weight of sample at 25° C. and 80% RH in the first absorption cycle.
 
         [0250]    Obtained results show that the sample is very hygroscopic, with a mass increase that is greater than 15%, and becomes a viscous liquid at high humidity. 
         [0251]    The analytical methods used for the product assessment are performed as described below. 
       Analytical Methods 
     HPLC Method 
       [0252]    Column: ZORBAX Eclipse XDB-C18 (150×4.6 mm, 5 μm); column temperature 25° C.
 
Mobile phase: A: Sodium dihydrogen orthophosphate dihydrate 0.05 M; B: Acetonitrile
 
       Gradient: 
       [0253]      
         [0000]                                                  Time (min)   % A   % B                                0   95   5       10   5   95       10.2   95   5       12   95   5                    
Flow: 1.0 mL/min
 
Detector. UV DAD@220 nm
 
         [0254]    The obtained crystalline products of (2S,3S,5R)-3-methyl-3-((3-methyl-1H-1,2,3-triazol-3-ium-1-yl)methyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide Form A, B, C, D, E and F have an HPLC purity of at least 98%, preferably at least 99%, preferably at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%. 
         [0255]    NMR 
         [0256]    The samples for NMR analysis were prepared by complete dissolution of an appropriate amount of material in approximately 0.75 ml of NMR solvent (DMSO-d6) 
         [0257]      1 H NMR spectra were recorded at 25° C. using an either a Varian INOVA 400 MHz NMR Spectrometer equipped with a Varian ATB probe. 
         [0258]    Variable number of scans (16-256) was applied, using standard acquisition parameters. The pre-acquisition delay was set to 10 sec whenever NMR quantification was carried out. Appropriate phasing and baseline corrections were applied in processing the spectra. 
       XRPD 
       [0259]    The XRPD spectra were collected in transmission mode on an analytical X&#39;pert Pro instrument with X&#39;celerator detector using a standard Aptuit method. The data were evaluated using the HighScore Plus software. The instrumental parameters used are listed below. 
         [0000]    
       
         
               
               
             
           
               
                   
               
               
                 Instrumental parameter 
                 Value 
               
               
                   
               
             
             
               
                 2-theta range 
                 2-45 
               
               
                 Step size [°2-theta] 
                 0.0170 
               
               
                 Time per step [sec] 
                 60.7285 sec 
               
               
                 Wavelength [nm] 
                 0.154060 (Cu K-Alpha1) 
               
               
                 Rotation [Yes/No] 
                 Yes 
               
               
                 Slits divergence/ 
                 Incident Mask fixed 10 mm; Divergence slits 
               
               
                 antiscatter. 
                 ½, Antiscat.slits ½ on incident beam; 
               
               
                   
                  1/32 on diffracted 
               
               
                 X-ray Mirror 
                 Inc. Beam Cu W/Si focusing MPD, Acceptance 
               
               
                   
                 Angle 0.8°, Length 55.3 mm 
               
               
                 Temperature 
                 Room temperature 
               
               
                 Humidity values [% RH] 
                 Ambient 
               
               
                 Fixed Slits 
                 0.02 rad fixed Soller slits on incident and 
               
               
                   
                 diffracted beam 
               
               
                 Monochromator 
                 None 
               
               
                 Detector type 
                 X′celerator (active length 2.122 2 theta degree), 
               
               
                   
                 scanning mode 
               
               
                 Sample holder 
                 Transmission sample holder. Use Insert to keep 
               
               
                   
                 thickness at 1 mm, 5 mm diameter 
               
               
                 Configuration 
                 Transmission 
               
               
                 Generator voltage/current 
                 40 KV/40 mA 
               
               
                   
               
             
          
         
       
     
       Optical Microscopy 
       [0260]    Optical microscopy analyses were run on the Leica DM microscope equipped with a double polarizer and digital camera. The method parameters are listed below. 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                   
                   
               
               
                   
                 Value 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Polarized light [Y/N] 
                 Yes 
               
               
                 Magnification [eyepiece] 
                 10x 
               
               
                 Objective 
                 Typically 5x, 10x, 20x, 40x 
               
               
                 Filter slider 
                 Use the best filter to optimize the image 
               
               
                   
               
             
          
         
       
     
       TGA and DSC 
       [0261]    The TGA analyses were run on a TA Q5000 instrument or on Mettler Toledo Star System (Form F analysis). The DSC analyses were run on the TA Q2000 MDSC or on the DSC 200 F3 Maia (Form F analysis) instruments. DSC and TGA method details are listed below: 
         [0000]    
       
         
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                   
               
               
                 Instrumental parameter 
                 Value 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 TGA 
               
             
          
           
               
                 Balance purge gas [mL/min] 
                 10 
               
               
                 Sample purge gas [mL/min] 
                 25 
               
               
                 Gas 
                 Nitrogen 
               
               
                 Temperature-Time-Rate 
                 Typically from room temperature to 
               
               
                   
                 250/350° C. at 10° C./min (TA Q5000 
               
               
                   
                 instrument); or to 450° C. at 10°K/min 
               
               
                   
                 (Mettler Toledo Star System) 
               
               
                 Typical sample amount [mg] 
                 Usually from 2 mg to 20 mg 
               
               
                 Pan [Pt/Al] 
                 Hermetically sealed Al (punched) 
               
             
          
           
               
                 DSC 
               
             
          
           
               
                 Cooling [ON/OFF] 
                 ON 
               
               
                 Gas 
                 Nitrogen 
               
               
                 Temperature-Time-Rate 
                 From 0° C. to ~160° C. Ramp at 
               
               
                   
                 10° C./min (TA Q2000 MDSC); or 
               
               
                   
                 from 25° C. to ~350° C. Ramp at 
               
               
                   
                 10°K/min (DSC 200 F3 Maia). 
               
               
                 Typical sample amount [mg] 
                 Usually from 0.5 mg to 2.5 mg 
               
               
                 Pan 
                 Not hermetic Al (TA Q2000 MDSC); or 
               
               
                   
                 hermetically sealed Al ((DSC 200 F3 
               
               
                   
                 Maia) 
               
               
                   
               
             
          
         
       
     
       Raman 
       [0262]    Raman analyses were performed with a Keiser Optical Systems RXN1 MicroRaman with Leica Microscope and digital camera 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Instrumental Parameter 
                 Value 
               
               
                   
                   
               
             
             
               
                   
                 Probe 
                 N 
               
               
                   
                 Objective 
                 50x, 50x LWD, 10x 
               
               
                   
                 Exposure [sec] 
                 Typically 0.5-1 
               
               
                   
                 Laser Power [mW] 
                 50-400 
               
               
                   
                 Autofocus [Y/N] 
                 Typically N 
               
               
                   
                 Accumulation 
                 Typically 10 
               
               
                   
                 Cosmic ray filter [Y/N] 
                 Y 
               
               
                   
                 Intensity calibration [Y/N] 
                 Y 
               
               
                   
                 Dark subtract [Y/N] 
                 Y 
               
               
                   
                   
               
             
          
         
       
     
       FT-IR 
       [0263]    FT-IR analyses were performed with a Thermo Nicolet Nexus 470 FT-IR or with a Thermo Nicolet 6700 FT-IR (Form F analyses). 
         [0000]    
       
         
               
               
             
           
               
                   
               
               
                 Instrumental Parameter 
                 Value 
               
               
                   
               
             
             
               
                 Accessory 
                 Attenuated Total Reflectance (ATR) - ZnSe 
               
               
                   
                 Crystal 
               
               
                 # of scans 
                 64 
               
               
                 Resolution [cm −1 ] 
                  4 
               
               
                 Gain 
                 Autogain 
               
               
                 Detector 
                 DTGS KBr 
               
               
                 Spectral Range [cm −1 ] 
                 4000-650 
               
               
                   
               
             
          
         
       
     
       Particle Size Distribution 
       [0264]    Particle Size Distribution by laser light scattering was performed after developing a wet dispersion method using Malvern Mastersizer 2000 instrument. The method parameters are listed below. 
         [0000]    
       
         
               
               
             
           
               
                   
               
             
             
               
                 Instrument 
                 Malvern Mastersizer 2000 
               
               
                 Accessory 
                 Hydro2000S+ 
               
               
                 Parameter 
                 Value 
               
               
                 Stirring speed 
                 1750 rpm 
               
               
                 Dispersant 
                 0.1% w/v Span85/Cyclohexane 
               
               
                 Sample Quantity 
                 Around 100 mg suspended in 5 mL of dispersant 
               
               
                 Calculation Model 
                 General Purpose - Irregular 
               
               
                 Optical Model 
                 Fraunhofer with 1.426 refractive index for 
               
               
                   
                 the dispersant 
               
               
                 Sweeps number 
                 15000 background/15000 sample 
               
               
                 Laser Obscuration [%] 
                 between 5 and 20% (typically 8-12%) 
               
               
                   
               
             
          
         
       
     
         [0265]    The experiments were conducted using the following sample preparation:
       (i) 100 mg of material were weighted in a 10 ml vial and they were suspended in 5 mL of dispersant;   (ii) once the material was all wetted the suspension was added into the cell and the vial was washed using additional 5 mL of the dispersant;   (iii) the suspension was measured immediately.       
 
       EGA 
       [0269]    The EGA analysis was carried out on the gas produced during the TGA analysis. 
       DVS Analyses 
     Instrument Details 
       [0270]    Temperature range: 20-40° C. (standard)
 
Maximum sample mass: (low/high mass instrument) 1 g/4 g
 
Mass change: +/−150 mg
 
Stability (24 hours @25° C. and 0% RH)&lt;5 μg
 
Mass resolution: +/−0.1 μg
 
       Humidity Range: 0-98% RH 
     RH Accuracy: +/−1% RH 
       [0271]    Temperature stability: +/−0.1° C.
 
Typical gas flow rate: 100/200 sccm
 
Sample chamber: 40 mm wide×50 mm deep×50 mm high
 
Reservoir volume: 100 ml reservoir capacity
 
Heating system: Peltier+Cartridges
 
         [0272]    The kinetic moisture sorption measurement was performed at 25° C. and in a RH % range described in the following: 
       From 40% RH to 90% RH 
     Form 90% RH to 0% RH 
     From 0% RH to 90% RH 
     From 90% RH to 0% RH 
       [0273]    The experiment is performed on 10-15 mg of sample and the equilibrium criterion is set as dm/dt&lt;0.002% w/w in 10 min with a maximum step time of 240 min. 
       Stability Tests 
       [0274]    The sample was positioned on the sample holder and stored in the following conditions: 
         [0000]    25° C. and 60% RH for 7 days
 
60° C. and 75% RH for 3 days
 
         [0275]    The samples were analyzed after the test by XRPD. 
       Hygroscopicity 
       [0276]    The hygroscopicity of the sample was determined using the method reported in the academic article “ Efficient throughput method for hygroscopicity classification of an active and inactive pharmaceutical ingredients by water vapor sorption analysis ” V. Murikipudi et al.,  Pharmaceutical Development and Technology,  2013, 18(2): 348-358. 
         [0277]    The hygroscopicity was calculated using the following equation: 
         [0000]      % Weight Change=[( W 2− W 1)/ W 1]*100; wherein
 
         [0000]    W1 is a weight of sample at the start of the experiment; and
 
W2 is a weight of sample at 25° C. and 80% RH in the first absorption cycle.
 
       Classification Criteria 
       [0278]    Non hygroscopic: increase in mass is less than 0.2%;
 
Slightly hygroscopic: increase in mass is less than 2% and equal to or greater than 0.2%;
 
Hygroscopic: increase in mass is less than 15% and equal to or greater than 2%;
 
Very Hygroscopic: increase in mass is equal to or greater than 15%; and
 
Deliquescent: sufficient water is absorbed to form a liquid.
 
         [0279]    Although the present invention has been described in terms of specific exemplary embodiments, it will be appreciated that various modifications, alterations and/or combinations of features disclosed herein will be apparent to those skilled in the art without departing from the scope of the invention as set forth in the following claims.