METHOD FOR THE PURIFICATION AND ANALYSIS OF INTERFERON
Technical Field
This invention relates to the use of multiplexing techniques (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 controlling devices) equipped with associated electronics controlled 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 demonstration that interferon is effective in the treatment of Hepatitis B, respiratory 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 heterogeniety of interferon induced activity. The dosage necessary for the induction of protein kinase (PKI) molecular weight 67,000, is shown to be similar to that for the development of the antiviral state. The end point of the assay can be measured directly by a reduction in the 3'ield in infectivity to a significant level. In addition to its basic functions of inhibitin virus growth, a number cf other effects of interferes have been described, these include: (1) effects en the ir.rune system, such as inhibition and stimulation of antibody formation, mεcroprare activation, and inhibitory effects on cell mediated immunity, (2) effects on cell surface antirens, (3) enhanced toxicity of double stranded RNA, (4) antitumor effects in-vivo and in-vitro and
(5)anticellular effects most notably, inhibition of cell growth.
Background Art.
In accordance with the present state of interferon technology our invention (process) consisting of a combination of manual and automatic m.anuevers, is designed to produce, purify and rabidly analyze the interferons. The use of multiplexing techniques (computers) as an integral part of the production, purification and rapid analysis of interferon offers advantages that were heretofore nonexistent.
Disclosure of Invention
Spectrophotometric digital computers may be interfaced for routine sample analysis, for data acquisition, and online control; A computerized operation was designed to prove the principles of measurement and controls by conductivity determinations. A critical parameter for the separation 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 designated area of a container. The light is picked up by another 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-D thermolumfinescent device. In addition to the computer, a scanning monochromator and a highly sophisticated temperature programmer and controller are necessary to fully utlize the spectra of 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 described in connection with the accompanying drawing, in which fig. 1, shows a flow chart for the production, purification and rapid analysis of interferon.
As is apparent, the equipment is arranged so that their constant monitoring and control is performed by a spectrophotometric (digital) computer. Purification to apparent homogeneity may be accomplished by liquid and gas chromatography. 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 with 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 interferon 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 centrifugation.
(g) adding TCA (trichloroacetic acid) and other reagents
.... (h) freezing mixture for a predetermined time and repeating step (f) ....
(i) thawing said mixture and repeating step (f)....
(j) waiting a predetermined time then repeating step (f) .... (k) redissolving in NaHCO3 and repeating step (f) ....
(l) controlling, steps a-1 by means of a special purpose digital spectrophotometric computer....
The best mode for carrying out this process is an follows: White blood cells (buffy coats) obtained fror normal donors may be placed in a leukochronometer1 for quantitation. The crude interferon is then placed in containers
(electronic electrochemical monitoring and controlling device (s) equipped with associated electronics that utilize combined or separate A.C.D.C. audio and radio frequency (wave energy) to promote medium agitation (oarticle separation) by dissociation of the elements or molecules (cells , debris ) in suspension solution. The medium is then innoculated with viruses, mitogens (staphlocccccus anterotoxin A or other interferon inducers. Casein is then added to rpcvide a simplier mileu from which to isolate the interferon. After agitation for 35 minutes the broth is incubated for approx., 16 hours. Pilot experiments have yielded titers of 10,000 to 20,000 units/ml. Concentration and intital fractionation processes should be carried out at temperatures of 0-4°C.
After incubation, cells and debris can be removed by lowspeed centrifugation, a task easily performed by commercial celltrifuges3, for approx. 15 minutes @ 500 × g. The casein can be precipitated by acidification with HCL to ph 4. 0. The mixture can then be centrifuged for 10 mih @
12,000 × 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 collected by centrifugation 10 minutes 12,000 × g and received in 50 ml of 0.1 M NaHCO3. Triton x-100 (0,5g) is then added and then acetic acid (1.5 ml) is added drop-wise with stirring. The mixture is then stored at 0°C for 1 hour, and then for 15 hours @ -20°C. It is then thawed and centrifuged (10 min. 17,000 × g). The pellets are then discarded and the supernatant adjusted to 4% trichloroacetic acid. After 1 hour the mixture is then centrifuged 10 minutes 12,000 × g. The precipitate can then be collected and redissolved in 5 ml of 0.5 EaHCC3.
After the initial procedures, urea (1.5g) is added to the interferon concentrate and then the solution can be applied to a column of senhadex G-103 fine (2.5 × 90 cm).
The columns can be εluted at ph.7.5 at room temperatures at a flow rate of 0.5 ml/minute (apnrox).
High performance liquid chromatography 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. Mass spectrometer operational 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 labelled to unlabelled ion intensity (2H/1H rotio) for each amino acid can be determined in the following way: the maximum intensity of a significant ion from the unlabelled amino acid is found; the intensities above a specified background value of this spectral line are integrated.
Isotope ratio determined can be used as the quantitating technique via multiple internal standards. The exact compostition of a deuterated amino acid mixture can be determined against a standard amino acid calibration mixture and in turn the protein amino acid composition is determined against the deuterated amino acid mixture. The mixture of deuterated amino acids with known concentrations can be used to measure the relative concentration 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 objective 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 log10 and 1.0 log10 compared to control cultures not treated with interferon. Other objective measurements of endpoints are available in a number of methods; (1) those which provide 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 yield, radiolabeled precursor incorpor
ation and quantitive haemadsorption) and (2) these which provide for obtaining discontinuous observations, such as reduction in logarhythms of haemagglutin yield. The large number and variety of assays reflect the many possible combinations of cells and challenge viruses and 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 measurement. The Robinson/Hurtt, glycolytic/interferor. bioεssay, has been designed to measure disease dependent glycolytic activity, whose presence and titers are alterably controlled by interferons. By monitoring the rate of glycolysis in standard calibration medium one should be able to measure the rate of glucose/interfercn activity, while elucidating the independence of the carbohydrate and the fatoxidizing systems.
Since numerous additions and changes may ba made in the above described interferon procedures for purification and rapid analysis, and still embodiments of this invention maybe made without departing from the spirit and scope thereof. It is intended that all matter contained in the foregoing description or shewn in the accompanying drawing shall be interpreted as illustrative and not in a limiting sense.