Methods of sterilizing polymers

Methods of heat sterilizing polymers are disclosed. The methods involve the preparation of a polymer suspension in an water-miscible organic liquid and subsequent application of moderate heat for a period of time sufficient to sterilize the polymer.

BACKGROUND OF THE INVENTION 
The present invention relates to the sterile preparation of polymers. More 
specifically, the present invention relates to moderate heat sterilization 
of polymers, and preferably, heat-labile polymers. The methods of the 
present invention involve the formation of suspension comprising a 
water-miscible organic liquid and a polymer, followed by subsequent heat 
sterilization. 
Polymers are used extensively in the preparation of food, medical and 
pharmaceutical compositions. The type of polymer used will depend on the 
functional needs of a given composition. For example, if viscosity 
enhancement is required, polymers providing viscosity enhancing effects, 
such as polyvinyl alcohol, polyvinyl pyrrolidone, various cellulosic 
polymers such as carboxymethyl cellulose or hydroxypropylmethyl cellulose, 
may be used. If a surfactant is required, surfactants such as polaxamines, 
polaxamers, alkyl ethoxylates or others may be used. If a gelling polymer 
is required, polymers such as gellan, carageenan or carbomers may be used. 
Certain preparations of food, medical or pharmaceutical compositions 
require the employment of sterilization methods in order to eliminate 
microbial contamination. Various methods of polymer sterilization have 
been available in the art. For example, techniques of filter sterilization 
can be used. Such methods usually involve the filtration of the 
composition, partial formulation or individual ingredients of the 
compositions, wherein such components are passed through a filter with a 
pore size too small for microbes to pass through. Autoclaving, another 
method of sterilization, involves the steam heat and pressure treatment of 
a composition, or individual components of the composition, for a given 
time to effect the elimination of microbes. Other methods of sterilization 
involve the irradiation of the composition, or individual components, with 
particle/energy rays such as gamma rays or electron beams. Still other 
methods of sterilization involve the dry heat treatment of the polymers. 
Although the foregoing methods of sterilization are useful for numerous 
polymers, certain polymers require more complicated steps of 
sterilization. For example, if certain composition or components are too 
viscous or comprise polymers or particles that are too large to pass 
through the pores of a filter, then filter sterilization will not be 
useful. If certain compositions or individual components are 
hydrolytically unstable, then autoclaving methods will not be effective. 
Although dry heat sterilization techniques may provide an alternative to 
polymers susceptible to autoclaving hydrolysis, there are still some 
polymers which degredate or otherwise lose cross-linking ability during 
dry heat treatments. Additionally, some compositions may comprise 
components that present a variety of sterilization problems which make the 
sterile preparation of such compositions possible, using conventional 
techniques, but labor and cost prohibitive. Therefore, what is needed are 
new methods of sterilization which provide effective sterilization of 
polymers, and especially, difficult to sterilize polymers, thereby 
providing a labor/cost improvement over the prior art methods. 
SUMMARY OF THE INVENTION 
The present invention relates to the preparation of sterile polymers. More 
specifically, the present invention relates to moderate heat sterilization 
methods of polymers and especially heat-labile polymers. The methods 
involve the dispersion of the polymers in a water-miscible organic liquid 
and subsequent moderate heat treatment of the suspension to sterilize the 
polymer. 
The addition of such organic liquids provides a more homogeneous and 
effective heat transfer to the polymer suspension. Additionally, less 
oxidation of the polymer occurs due to the limited supply and access of 
oxygen to the polymer, as compared to dry heat sterilization. Thus, one 
object of the present invention is to provide alternative methods of 
sterilization which allow for the moderate heat sterilization of otherwise 
heat-labile polymers, with minimal effects on polymer properties. 
DETAILED DESCRIPTION OF THE INVENTION 
The methods of the present invention involve the suspension of a polymer in 
a water-miscible organic liquid and the subsequent heat sterilization of 
the mixture. As used herein, "water-miscible organic liquid," "organic 
liquid" or "stabilizer" refers to a pharmaceutically acceptable organic 
compound that forms one liquid phase with water when added to water. As 
stated above, the addition of the organic molecule to the polymer effects 
greater heat transfer and protection of the polymer from oxidation. The 
particular structure of the organic molecule is generally not an important 
factor in effective sterilization of the polymer. It is rather the ability 
of the organic liquid to form one phase with water, and the safety of the 
organic liquid, that determines its stabilizing utility. Additionally, the 
stabilizers must also be suitable for consumer use and will thus exhibit 
minimal adverse effects with consumer use. Preferably, the stabilizers 
will be compatible for ophthalmic use. For example, the stabilizer will 
not contribute to ocular irritation/toxicity or interfere with the 
anti-microbial efficacy of an anti-microbial agent. Given the above 
criteria, various and numerous molecules may be used in the present 
invention to stabilize the polymer during sterilization methods of the 
present invention. 
As used herein, "heat-labile polymer" refers to polymers that undergo 
hydrolysis or and/or oxidation following standard autoclaving or dry heat 
sterilization temperatures. Examples of heat-labile polymers are those 
which do not exhibit a cloud point. As used herein "sterilization" means 
the effective inactivation or kill of microbes contained in the polymer 
powder, mixture, suspension or solution. The level of inactivation or kill 
may vary, but it will be in amount acceptable by the applicable commercial 
and/or FDA standards for the intended product. 
Preferred water-miscible organic liquids useful in the methods of the 
present invention include polyethylene glycols (PEGs), such as PEG 200, 
PEG 400, PEG 600; and glycerol, propylene glycol or mixtures thereof. The 
most preferred organic liquids are the PEGs, and most preferably, PEG 400. 
The methods of the present involve the addition of the organic liquid to 
the polymer powder and subsequent heat sterilization of the suspension. In 
general, a 1:0.1 to 1:5 suspension of the organic liquid to the polymer is 
first prepared. The particular ratio will depend on various factors, such 
as the chemical nature of the polymer and the organic liquid. In general, 
such suspension preparation involves the dispersion of a polymer powder in 
an organic liquid with subsequent mixing. Particular parameters, i.e., 
mixing conditions, time and temperature, will vary depending on the 
polymer and organic liquid employed. In general, however, the polymer 
suspensions will typically be mixed for 15 minutes at room temperature. 
The conditions for moderate heat sterilization will vary. In general, a 
temperature of about 110.degree. to 150.degree. C. and for a duration of 
about 0.5 to 8 hours will be employed. Preferred methods will employ 
temperatures of 125.degree. C. and for a duration of 1 hour. Unlike 
autoclaving, the methods of the present invention do not involve the use 
of direct moisture and pressure application to the polymer. Rather, the 
methods of the present invention only employ moderate heat to a sealed, 
water vapor-proof vessel. Preferred methods involve the use of an 
autoclaving device to heat the sealed vessel. Following sterilization, the 
polymer mixture is then set aside for final mixing with any additional 
sterilized components. 
Although the methods of the present invention can be used with any number 
of polymers, the methods are particularly suited for heat-labile polymers 
exhibiting high cloud point or no cloud point, such as galactomannan 
polysaccharides. Thus, methods of the present invention are most 
preferably employed to sterilize heat-labile polymers including guar gum, 
locust bean gum and tara gum. 
The types of galactomannans that may be sterilized by the methods of the 
present invention are typically derived from guar gum, locust bean gum and 
tara gum. As used herein, the term "galactomannan" refers to 
polysaccharides derived from the above natural gums or similar natural or 
synthetic gums containing mannose or galactose moieties, or both groups, 
as the main structural components. Of particular interest are 
galactomannans made up of linear chains of (1-4)-.beta.-D-mannopyranosyl 
units with .alpha.-D-galactopyranosyl units attached by (1-6) linkages. 
The ratio of D-galactose to D-mannose varies, but generally will be from 
about 1:2 to 1:4. Galactomannans having a D-galactose:D-mannose ratio of 
about 1:2 are of greatest interest. Additionally, other chemically 
modified variations of the polysaccharides are also included in the 
"galactomannan" definition. For example, hydroxyethyl, hydroxypropyl and 
carboxymethylhydroxypropyl substitutions may be made to the galactomannans 
prior to sterilization. Still other non-ionic variations to the 
galactomannans, such as those containing alkoxy and alkyl (C1-C6) groups 
(e.g., hydroxylpropyl substitutions) may be sterilized by methods of the 
present invention. An example of non-ionic substitution of a galactomannan 
of the present invention is hydroxypropyl guar, which is preferably 
substituted up to about a 0.4 molar ratio. 
The galactomannans may be obtained from numerous sources. Such sources 
include guar gum, locust bean gum and tara gum, as further described 
below. Additionally, the galactomannans may also be obtained by classical 
synthetic routes or may be obtained by chemical modification of naturally 
occurring galactomannans. 
Guar gum is the ground endosperm of Cyamopisis tetragonolobus (L.) Taub. 
The water soluble fraction (85%) is called "guaran" (molecular weight of 
220,000), which consists of linear chains of (1-4)-.beta.-D mannopyranosyl 
units with .alpha.-D-galactopyranosyl units attached by (1-6) linkages. 
The ratio of D-galactose to D-mannose in guaran is about 1:2. The gum has 
been cultivated in Asia for centuries and is primarily used in food and 
personal care products for its thickening property. It has five to eight 
times the thickening power of starch. Its derivatives, such as those 
containing hydroxypropyl or hydroxypropyltrimonium chloride substitutions, 
have been commercially available for over a decade. Guar gum can be 
obtained, for example, from Rhone-Polulenc (Cranbury, N.J.), Hercules, 
Inc. (Wilmington, Del.) and TIC Gum, Inc. (Belcamp, Md.). 
Locust bean gum or carob bean gum is the refined endosperm of the seed of 
the carob tree, ceratonia siliqua. The ratio of galactose to mannose for 
this type of gum is about 1:4. Cultivation of the carob tree is old and 
well known in the art. This type of gum is commercially available and may 
be obtained from TIC Gum, Inc. (Bekamp, Md.) and Rhone-Polulenc (Cranbury, 
N.J.). 
Tara gum is derived from the refined seed gum of the tara tree. The ratio 
of galactose to mannose is about 1:3. Tara gum is not produced in the 
United States commercially, but the gum may be obtained from various 
sources outside the United States. 
Modified galactomannans of various degree of substitution are commercially 
available from Rhone-Poulenc (Cranbury, N.J.). Hydroxypropyl guar with low 
molar substitution (e.g., less than 0.6) are of particular interest. 
The polymer compositions to be sterilized by the methods of the present 
invention may contain other ingredients. Such ingredients include 
pharmaceuticals, carriers, antimicrobial/preservative agents, tonicity 
adjusting agents, buffers and chelating agents. Tonicity adjusting agents 
useful in the compositions of the present invention may include salts such 
as sodium chloride, potassium chloride and calcium chloride; non-ionic 
tonicity agents may include propylene glycol and glycerol; chelating 
agents may include EDTA and its salts; and pH adjusting agents may include 
hydrochloric acid, Tris, triethanolamine and sodium hydroxide. Suitable 
anti-microbial agents/preservatives are discussed more fully below. The 
above listing of examples is given for illustrative purposes and is not 
intended to be exhaustive. As stated above, the use of the methods of the 
presentation in the preparation of compositions for ophthalmic 
applications is of particular interest to the inventor. Thus, examples of 
other agents useful for the foregoing purposes are well known in contact 
lens care formulation and are contemplated by the present invention. 
Disinfecting compositions to be sterilized by methods of the present 
invention will contain an antimicrobial agent. Antimicrobial agents may be 
either monomeric or polymeric antimicrobial agents which derive their 
antimicrobial activity through a chemical or physicochemical interaction 
with the organisms. As used in the present specification, the term 
"polymeric antimicrobial agent" refers to any nitrogen-containing polymer 
or co-polymer which has antimicrobial activity. Preferred polymeric 
antimicrobial agents include: polyquaternium-1, which is a polymeric 
quaternary ammonium compound; and polyhexamethylene biguanide ("PHMB") or 
polyaminopropyl biguanide ("PAPB"), which are polymeric biguanides. These 
preferred antimicrobial agents are disclosed in U.S. Pat. Nos. 4,407,791 
and 4,525,346, issued to Stark, and 4,758,595 and 4,836,986, issued to 
Ogunbiyi, respectively. The entire contents of the foregoing publications 
are hereby incorporated in the present specification by reference. Other 
antimicrobial agents suitable in the compositions and methods of the 
present invention include: other quaternary ammonium compounds, such as 
benzalkonium halides, and other biguanides, such as chlorhexidine. The 
antimicrobial agents used herein are preferably employed in the absence of 
mercury-containing compounds such as thimerosal. Particularly preferred 
antimicrobial agents of the present invention are polymeric quaternary 
ammonium compounds of the structure: 
##STR1## 
wherein: R.sub.1 and R.sub.2 can be the same or different and are selected 
from: 
N.sup.+ (CH.sub.2 CH.sub.2 OH).sub.3 X.sup.-, 
N(CH.sub.3).sub.2 or OH; 
X is a pharmaceutically acceptable anion, preferably chloride; and 
n=integer from 1 to 50. The most preferred compounds of this structure is 
polyquaternium-1, which is also known as Onamer M.TM. (registered 
trademark of Onyx Chemical Corporation) or as Polyquad.RTM. (registered 
trademark of Alcon Laboratories, Inc.). Polyquaternium-1 is a mixture of 
the above referenced compounds, wherein X is chloride and R.sub.1, R.sub.2 
and n are as defined above. 
The polymers may be sterilized alone (that is, in the presence of the 
organic liquid) or in combination with any of the ingredients described 
above.

EXAMPLE 1 
This example demonstrates the degradation susceptibility of galactomannans 
sterilized by typical autoclave cycles. A 0.5% weight/volume ("w/v") Guar 
gum was prepared and polish filtered to remove insoluble materials. The 
polymer solution was subjected to a 20, 30 or a 60 minute autoclave cycle. 
Following the autoclave cycle, the viscosity of the solution was measured 
and compared to the non-autoclaved solution. The effects of autoclaving 
the unprotected polymer are illustrated in Table 1: 
TABLE 1 
______________________________________ 
Treatment Viscosity (CPS) 
______________________________________ 
Control 399 
Post-autoclave 20 min. cycle 39 
Post-autoclave 30 min. cycle 18 
Post-autoclave 60 min. cycle 5.9 
______________________________________ 
Significant reduction in viscosity is observed even for a 20 minute cycle 
and the effect increases with longer cycle times. 
EXAMPLE 2 
This example demonstrates the sterilization efficacy of a method of the 
present invention as compared to other methods of sterilization. The 
comparison was made with Hydroxypropyl guar, a heat-labile polymer. 
Viscosity measurement were taken from a 0.5% w/v polymer solution which 
was prepared following each treatment. The preparation of the polymer for 
the present invention method (No. 1) and method No. 2 involved the 
addition of 1 gram of HP guar gum to 2 grams of PEG-400. All other methods 
used a standard dry powder of HP guar gum. The results are illustrated in 
Table 2: 
TABLE 2 
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HP-Guar Polymer Sterilized with Various Methods 
Viscosity 
No. Treatment Appearance (CPS) 
______________________________________ 
1 Suspension of Gum in 
Light Yellow powder in 
255.6, 276.4 
PEG (Present clear Solution 
Invention) 
2 Suspension of Gum Light Brown in Slightly 115.6, 114.8 
in PBG Dry Yellow Solution 
Heat, in Open Container 
3 Vacuum Dried followed Dark Brown Powder 22.6, 21.7 
by dry heat 
4 Dry Heat Dark Brown Powder 48.7, 49.0 
5 Dry Heat under N.sub.2 Dark Brown Powder 21.2, 22.9 
6 Unsterilized Gum Light Yellow Powder 278.2, 278.4 
(control) 
______________________________________ 
Minimal depolymerization was observed with the present invention method 
(No. 1), as compared to the other methods employed. Additionally, there 
was no change in physical appearance of the polymer using the method of 
present invention, in contrast to the prior art methods. This observation 
may show that limited oxidation occurred through the present invention 
method. 
EXAMPLE 3 
This example illustrates a method of the sterile preparation a 
multi-purpose, contact lens care solution using a method of the present 
invention: 
______________________________________ 
Ingredient Concentration (% W/V) 
Amount per 20 liters 
______________________________________ 
Hydroxypropyl 
0.20 40 g 
Guar Gum 
Polyethylene Glycol 0.40 80 g 
400 
Tetronic 1304 0.25 50 g 
Boric Acid 1.00 200 g 
Propylene Glycol 0.90 180 g 
Disodium Edetate 0.01 2 g 
Polyquaternium-1 0.001 + 50% Excess 0.3 g (100%) 
Sodium Hydroxide adjust pH (6.8-7.2) n/a 
and/or Hydrochloric (target 7.0) 
Acid 
Purified Water QS 100% QS 20 liters or 20.06 kg 
______________________________________ 
Preliminarily, a compounding vessel (20 L stainless steel pressure can), a 
0.2 micron sterilizing filter, a receiving vessel (20 L carboy), a 4.5 
micron polishing filter, a 0.2 micron sterilizing filter, a vent filter, 
and the filling equipment are sterilized by autoclaving. 
In a beaker equipped with an overhead agitator, add the weighed amount of 
polyethylene glycol 400 (200 g). While mixing slowly disperse the weighed 
amount of hydroxypropyl ("HP") Guar gum (100 g). Mix until completely 
homogeneous. In a 500 ml Schott bottle, equipped with a magnetic stir bar, 
weigh exactly 120.0 g of the HPGuar gum/PEG-400 dispersion. Prepare to 
sterilize by autoclaving. In a second identical 500 ml Schott bottle weigh 
exactly 120.0 g of the same dispersion. Prepare to use as a dummy during 
the autoclaving cycle. To both bottles add 1.3 ml of purified water 
(amount equivalent, by volume, of the microorganism suspension used to 
inoculate the bottles during the validation study). Mix both bottles for 
10 minutes using a magnetic stir plate. Autoclave the HPGuar gum/PEG-400 
dispersion using the validated time-temperature cycle of 80 minutes at 
125.degree. C. 
In a vessel equipped with an overhead agitator, add purified water 
equivalent to approximately 70% of the theoretical batch weight 
(approximately 14 Kg). While mixing at moderate speed, slowly add the 
other ingredients desired: Tetronic 1304, Boric Acid, Propylene Glycol, 
and Disodium Edetate. Mix for a minimum of 60 minutes, or until completely 
homogeneous. Check the temperature and, if necessary, cool to 35.degree. 
C. or below. While mixing at low speed slowly add the Polyquaternium-1. 
Mix for a minimum of 15 minutes, or until completely homogeneous. Transfer 
into a pre-sterilized compounding vessel equipped with an agitator through 
a 0.2 micron sterilizing filter (the recommended compounding vessel is a 
pressure vessel and recommended agitator is an overhead mixer that can be 
used in sterile compounding area). Rinse the vessel and filter assembly 
with room temperature WFI. 
Aseptically transfer the sterilized HPGuar gun/PEG-400 dispersion into the 
pre-sterilized compounding vessel. Rinse the bottle content with 
sterilized purified water. Bring the content of the compounding vessel to 
exactly 95% of the theoretical batch weight (19.0 liters or 19.06 Kg) 
using sterile room temperature purified water. Allow the HPGuar gum/PEG 
slurry to hydrate while mixing, at moderate speed, in the compounding 
vessel for a minimum of 2 hours. Transfer the contents of the compounding 
vessel through a 4.5 micron pre-sterilized polishing filter into the 
pre-sterilized receiving vessel equipped with a stir bar. There will be 
some loss of the contents due to the product held in filter housing and 
filter cartridge. Check and adjust pH, if necessary, to 6.9-7.1 (target 
7.0) using 1N NaOH or 1N HCl. Approximately 3-4 ml of 1N NaOH per 1 liter 
of final batch weight is needed to achieve the desired pH. QS to final 
batch weight using sterile purified water. Mix at low speed for a minimum 
of 30 minutes. 
EXAMPLE 4 
The sterilization efficacy of the methods of the present invention were 
tested in the following microbial challenge assay: 
The following stock solutions and suspensions were prepared: 
1. Three 0.12 kg suspensions of HP guar comprised of 40 g of HP guar and 80 
g of PEG-400 were prepared by dispersing dry polymer powder into PEG-400 
and mixing to prepare a homogeneous suspension for each of the studies 
below. The mixing time was about 15-30 minutes. 
Study 1 and 2: 
2. Spore suspension (40%) ethanol--Bacillus stearothermophilus ATCC 12980 
(AMSCO SPORDEX.RTM.) label count 1.3.times.10.sup.7 CFU/0.1 mL and a 
D.sub.121 Value of 1.7 minutes. 
3. Spore suspension (40%) ethanol--Bacillus subtilis var. niger ATCC 9372 
(AMSCO SPORDEX.RTM.) label count 1.2.times.10.sup.7 CFU/0.1 mL and a D121 
Value of 1.3 minutes. 
Study 3: 
4. Spore suspension (40%) ethanol--Bacillus stearothermophilus ATCC 12980 
(AMSCO SPORDEX(.RTM.) label count 1.4.times.10.sup.7 CFU/0.1 mL and a 
D.sub.121 Value of 1.6 minutes. 
5. Spore suspension (40%) ethanol--Bacillus subtilis var. niger ATCC 9372 
(AMSCO SPORDEX.RTM.) label count 1.1.times.10.sup.7 CFU/0.1 mL and a D121 
Value of 1.3 minutes. 
120 gram aliquots of the HP guar/PEG-400 suspension were placed in 500 mL 
screw cap media bottles with a stir bar. Each of two test suspensions were 
inoculated with Bacillus stearothermophilus spores to attain approximately 
10.sup.6 CFU/mL of test suspension. Two additional containers of test 
suspension were inoculated with Bacillus subtilis var. niger spores to 
attain approximately 10.sup.6 CFU/mL of test suspension. (Study 1: 
Bacillus stearothermophilus, 1.9.times.10.sup.6 CFU/mL of test suspension 
or 1.6.times.10.sup.8 CFU/bottle; Bacillus subtilis var. niger, 
2.6.times.10.sup.6 CFU/mL of test suspension or 2.6.times.10.sup.8 
CFU/bottle; Study 2: Bacillus stearothermophilus, 1.7.times.10.sup.6 
CFU/mL of test suspension or 1.5.times.10.sup.8 CFU/bottle; Bacillus 
subtilis var. niger, 3.2.times.10.sup.6 CFU/mL of test suspension or 
3.0.times.10.sup.8 CFU/bottle; Study 3: Bacillus stearothermophilus, 
1.8.times.10.sup.6 CFU/mL of test suspension or 1.7.times.10.sup.8 
CFU/bottle; Bacillus subtilis var. niger, 4.3.times.10.sup.6 CFU/mL of 
test suspension or 3.9.times.10.sup.8 CFU/bottle.) 
Samples were placed on magnetic stir plates and the contents allowed to 
stir for a minimum of 10 minutes. One container inoculated with Bacillus 
stearothermophilus and one container inoculated with Bacillus subtilis 
var. niger were then placed in an autoclave device for an autoclave cycle 
of 40 minutes at 125.degree. C. One container inoculated with Bacillus 
stearothermophilus and one container inoculated with Bacillus subtilis 
var. niger were maintained as controls. Following the treatment (or 
control) cycle, aliquots of each set of suspensions were serially diluted 
and then mixed with trypticase soy agar (TSA). The Bacillus 
stearothermophilus plates were wrapped in parafilm to prevent desiccation 
and incubated for 55.degree.-60.degree. C. for 48-72 hours (controls) or 7 
days (experimentals). The Bacillus subtilis var. niger were incubated at 
30.degree.-35.degree. C. for 48-72 hours (controls) or 7 days 
(experimentals). Following the incubation period, each plate was examined 
microscopically for growth. Table 3 illustrates the total kill effect of 
the heat sterilization methods of the present invention on the test HP 
guar suspension: 
TABLE 3 
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Log Reduction 
Study Bacillus stearothermophilus 
Bacillus subtilis var. niger 
No. per mL per bottle per mL 
per bottle 
______________________________________ 
1 6.3 8.2 6.4 8.4 
2 6.2 8.2 6.5 8.5 
3 6.3 8.2 6.6 8.6 
______________________________________