METHOD FOR THE PURIFICATION AND ANALYSIS OF INTERFERON

Abstract: Method for the purification of interferon from raw interferon sample prior to its analysis for the purity and structure of interferon in the raw interferon sample.

Description: METHOD FOR THE PURIFICATION AND ANALYSIS OF INTERFERON Technical Field This invention relates to the use of multiplexing techni- ques (computers) and sonication factors (wave energy) for the large scale production, purification and rapid analysis of the Interferons. Wave energy is injected into medium in containers ' (electronic electrochemical monitoring and con¬ trolling devices) equipped with associated electronics con- trolled by computers, thus affording the opportunity to standardize procedures for the dissociation of interferon from cells and other debris, while facilitating the rapid anlysis of said interferon. The recent availability of sufficient quantities of human interferon for clinical trials has marie possible the demon- • stration that interferon is effective in the treatment of Hepatitis B, respirator virus infections, Varicella-Zoster and other herpes virus infections, and perhaps human cancer_. The quantum and wave mechanics of the induction of the three translation regulatory enzymes by the interferons, provide a convenient model system to compute and to measure the heter- ogeniety of interferon induced activity. The dosage neces¬ sary for the induction of protein kinase (P I) molecular weight 67,000, is shown to be similar to that for the devel- opment of the antiviral state. The end point of the assay can be measured directly by a reduction in the 3'iel in in¬ fectivit to a significant level. In ad d ition to its basic functions of inhibiti ~ virus growth, a number cf other ef¬ fects of interferes have been described, these include: (1) effects en the ir.rune system, such as inhibition and stimulation o^ antibody formation, mεcroprare activation, and inhibitor;/ effects on cell mediated immunity, (2) effects on cell surface anti:ens, (3) enhanced toxicity of double stranded RITA, (4) antiturnor effects in-vivo and in-vitro arid —C— (5 ' )anticellular effects most notably, inhibition of cell growth. Background Art. In accordance with the present state of interferon tech¬ nology our invention (process) consisting o^ a combination of manual and automatic m.anuevers, is designed to produce, purif.y and rabidly analyze the interferons. The use of multiplexing techniques (computers) as an integral .part of the production, puri f ication and ranid analysis o^ interfer¬ on offers advantages that v/ere heretofore nonexistent. Disclosure of Invention Spectrophotometric digital computers may be interfaced for routine samule analysis, for data acquisition, and on¬ line control; A computerized operation was designed to prove the principles of measurement and controls by conduc- tivity determinations. A critical parameter for the separ¬ ation cells is the position of the interface between the . cells. The position of the interface is sensed optically. Fiber optic rods for instance can carry light through a de¬ signated area of a container. The light is picked up by an- other optic device * positioned near by, and then deteched by a photodiode. An electric circuit detects the light pulse and produces a d.c. voltage is measured by the interface position and is used as a control signal- Digital analytical accessories are currently being used in chromosomal studies. Rapid analysis can then be achieved with the interface of a 3 ^ - thermolu finescent device. In addition to the computer, a scanning monochromator and a highly sophisticated temperature programmer and controller are necessary to fully utlize the spectra o J the emitted light. Between 50,000-100,000 data points are plotted on each sample. The data is then automatically analyzed and prepared for plotting out as a function of wave length and sample temperature. They are presented graphically end facilitate rapid and thorough analysis of the data * The details of the components of the process will be de¬ scribed in connection with the accompanying drawing, in which fig. 1, shows a flow chart for the production, puri¬ fication and rapid analysis of interferon. As is apparent, the equipment is arranged so that their constant monitoring and control is performed by a spectro- photo etric (digital) computer. Purification to apparent homogeneity may be accomplished by liquid and gas chroma- tography.- Final confirmation of the contaminant levels should be determined by 3-D thermoluminescence procedures. Descript.ion of the drawing; (a) placing crude interferon in a leukochronometer, said crude interferon containing interferon and debris.... (b) quantizing said crude interferon.... (c) moving said crude into a container equipped wit wave energy injected, with a predetermined broth medium.... (d) inject said wave energy into said container to promote medium agitation by dissociation so as to isolate the inter¬ feron from its debris and cells.... (e) agitating said broth medium and crude interferon for a predetermined time.... (f) ' removal of cells and debris by low speed centrifuga- tion. (g) adding TCA (trichloroacetic acid) and other reagents • • • • (h) freezing mixture for a predetermined time and repeat¬ ing step (f) .... (i) thawing said mixture and repeating step (f).... (j) waiting a predetermined time then repeating step (f) .... (k) redissolving in aECC and repeating step (f) .... (1) controlling, steps ε-1 by means of a snecial purpose digital spectrophotometric computer.... The best mode for carrying out this process is an follows: "Thite blood cells (buffy coats) obtained fror normal donors may be lεced in a leukochronometer-^- for quantita- ticn. The crude interferon is then nlεcefϊ in containers BAD (electronic electrochemical monitoring and controlling de- vice (s) equipped with associated electronics that util¬ ize combined or separate A.C.D.C. audio and radio frequen— cy (wave energy) to promote medium agitation (oarticle separation) by dissociation of the elements or molecules (cells, debris) in suspension solution. The me i m is then innoculated with viruses, mitogens (staphlocccccus antero- toxin A or other interferon inducers. Casein is then added to rpcvide a si plier ileu from which to isolate the inter¬ feron. After agitation for 35 minutes the broth is incuba¬ ted for approx., Iδ hours. Pilot experiments have jrielded titers of 10,000 to 20,000 units/ml. Concentration and in- tital fractionation processes should be carried out at temperatures of 0-4°C. After incubation, cells and debris can be removed by low- speed centrifugation, a task easily performed by commercial <_> celltrifuges , for approx. 15 minutes @ 500 x g. The case¬ in can be precipitated bj" acidification v.ith HCL to ph 4. 0. The mixture can then be centrifuged for 10 mih @ 12,000 x g discarding the debris. The supernatant can be adjusted per centage wise (wt/vol) by trichloroacetric acid.. After one hour the precipitatant can then be collect¬ ed by centrifugation 10 minutes 12,000 x g and received in 50 ml of 0.1-E ITaHC0 3 . riton x-100 (0,5g) is then added and then acetic acid (1.5 ml) is added drop-wise with stir¬ ring. The mixture is -then stored at 0°C for 1 hour, and then for 15 hours {_? -20°C. It is then thawed and centri¬ fuged (10 in. 17,000 x g) . -The pellets are then discard- ed and the supernatant adjusted to 4f= trichloroacetic acid. After 1 hour the mixture is then centri f uged 10 minutes 12,000 x g. The precipitate can then be collected and re- dissolved in 5 ml o^ 0.5 EaHCCs. After the initial procedures, urea (1.5g) is added to the interferon concentrate and then the solution can be ap¬ plied to a column c^ se hadex G-103 fine (2.5 x 90 cm). The columns can be εluted at ph.7.5 εt rocm temperatures at a flow rate c^ 0.5 ml/minute (apnrox) . Eig performance liquid chromεtography techniques may be employed to obtain purified interferon. The analysis can be performed on a chromatographic mass spectrometer on-line with a computer. Chromatography can be performed on coiled columns. 5.7ass spectrometer opera¬ tional parameters include: molecular- sparator, 230°C; ion source, 260°C; electron voltage, 22.5 eV; scans may be taken every 5s from mass 0 to 410. The relative la¬ belled to unlabelled ion intensity (^ /IH " rόtio) for each amino acid can be determined in the following way: the maximum intensity of a significant ion from the unla- belled amino acid is found; the intensities above a spec¬ ified background value of this spectral line are integra¬ ted. Isotope ratio determined can be used as the quantitat- ing technique via multiple internal standards. The exact compostition of a deuterated amino acid mixture can be determined against a standard amino acid calibration mix¬ ture and in turn the protein amino acid composition is determined against the deuterated amino acid mixture. The mixture ~ of deuterated amino acids with known concen- " -trations can be used to measure the relative concentra¬ tion of unlabelled interferon amino acid. A reference is needed for use in collaborative studies on interferon standards in which several laboratories participate and where a common assay method must be used. The assay must be reproducible and the end products ob¬ jective and unequivocal. The end point of the assay should be measured directly by a reduction in yield of virus infectivity to a significant level, between 0.5 log 0 and 1.0 lo ^ Q compared to control cultures not treated with interferon. Other objective measurements of endpoints are available in a number of methods; (1) those which pro¬ vide for obtaining continuous, whole-number observations, including per centage reduction methods (the number of lytic plaques, flcrescent or transferred foci, amount of dye uptake enzyme yiel* 3 , rs J icl?beled urecursor incorpor- — st¬ ation and quεntitive haemadsorption) and (2) these which provide for obtaining discontinuous observations, such as reduction in logεrhythms of haemagglutin yield. The large number and- variety of assays reflect the many possible combinations of cells and challenge viruses an^ radiation that can be employed and their various types of induced cellular effects and components or properties as well as the direct approaches that can be taken tc their measure¬ ment. The Robinson/Eurtt, glycolytic/interferor. bioεssay, has been designed to measure disease dependent gl colytic activity, whose presence and titers are alterably controll¬ ed by interferons. By monitoring the rate of glycolysis in standard calibration medium on . hould be able tc measure the rate of glucose/interfercn activit;/, while elucidating the independence of the carbohydrate and the fεtoxidizing systems. Since numerous additions and changes may ba made in the above described interferon procedures for p ri f ication and rapid analysis, and still embodiments of this invention maybe made without departing from the spirit and scope thereof. Tt is intended that all matter contained in the foregoing description or shewn in the accompanying draw¬ ing shall be interpreted as illustrative and not in a limiting sense. BAD ORIGINAL OftlPI

Claims: Claims I- The Robinson-Hurtt, glycolytic/interferon bio-assa t a method designed to monitor the rate of degradation of a controlled substance, in calibrated standard medium, thus determining the proportional influence of inter¬ feron titers to glycolytic activity, K S - --= 0.. 2. The use of computed chromatographic mass spectrometric analysis methods employing isotope (optional) compari¬ son techniques that can be verified by nuclear magnetic and electron spin resonance spectrosccpy^, to determine the protein amino acid composition of the interferons, e.g. 1S C-M_R, lE-SSR. 3. The electronic electrochemical monitoring and control¬ ling device whose associated electronic equiptment can be optimized (adjusted) to oscillate at amplitudes that coincide with DI-TA breathing (melting), thus using computed sonication activity (factors) for the destruc¬ tion of etiological agents, or in the case of radioac¬ tive byproducts (radioactive labels), said equiptment can be used to dissociate particulεtεs (fisso-chemis- try) , according to their individual resonances (A.C radio frequency ranges (RF) for ultrasound; 50,000-70 x 10 vns.). Each odd nucleon has an intrinsic angular momentum, referred to as spin, " when the odd neutron and the odd proton interact (couple) in the nucleus, their spins can either add to or subtract from each other, resulting in two states, ore of which will be the ground state and the other of which will be an excited, state. If the two states differ widely in their spins (four or more units) , then the decay o^ one state into the other will be highly hindered and the states can exist separately as iscmers. (isomεrs-two nuclear stat es of the same ήuclide of different energies with me - ' '* surable half -lives) . BAD ORIGINAL _____________ 4. The use *J_.-D thermcluminezence techniques to analyze hybridized genetic activity expressed in prokaryotic or cell free systems, using computed codes, e.g. CO. R.I., H.ϋ.R.C.C, etc. BADOHieiNAL

Inventors: HURTT J, ROBINSON W
Applicants: HURTT J, ROBINSON W, UNITED STATES RES CORP
Application year: 1980
Main classification: C12Q1
IPC codes: C07K14, G01N33