PROCESS FOR THE PURIFICATION AND REFOLDING OF HUMAN RESPIRATORY SYNCYTIAL VIRUS FG GLYCOPROTEIN

Abstract: The present invention comprises a process for the purification of FG glycoprotein and its refolding into its proper conformation.

Description: PROCESS FOR THE PURIFICATION AND REFOLDING OF HUMAN RESPIRATORY SYNCYTIAL VIRUS FG GLYCOPROTEIN BACKGROUND OF THE INVENTION Human respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of viruses, is the predominant cause of severe lower respiratory tract illness in infants and young children. Kim et. al., American Journal of Epidemiology Vol. 98, pp. 216-225 (1973). Numerous attempts to produce a safe and effective vaccine against RSV had failed. Olmsted et al., Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 7462-7466, (1986). In 1987, Wertz, at the University of North Carolina, published a World patent application that disclosed a vaccine for RSV that was composed of native structural viral proteins and their immunogenic derivatives. The preferred proteins disclosed were F and G glycoproteins. WO/87/04185, published 16 July 1987. RSV contains a single strand of RNA which is transcribed into 10 predominant messengers. The messengers have been translated in vitro and the protein products characterized. There are two envelope glycoproteins, the fusion glycoprotein F; MW ca. 68,000 to 70,000 and a second methionine poor glycoprotein G, MW ca. 84,000 to 90,000. Collins, et al.. Journal of Virology, Vol. 49 pp. 572-578 (1984). In 1989, Wathen, Brideau, Thomsen and Murphy reported the characterization of a novel chimeric glycoprotein they named, FG. Wathen et al., Journal of General Virology Vol. 70, pp. 2625-2635 (1989). In a companion paper, Brideau et al., reported on the effectiveness of FG when used as an RSV vaccine. Brideau et al., Journal of General Virology Vol. 70, pp. 2637-2644 (1989). FG, a novel chimeric glycoprotein, contains the extracellular regions of the RSV F (fusion) glycoprotein linked to the extracellular region of the RSV G (attachment) glycoprotein. The FG glycoprotein can be expressed in insect cells using a baculovirus vector. Chinese Hamster Ovary (CHO) cells, COS cells (African green monkey kidney cells) or other suitable eukaryotic cell lines or expression systems can also express the FG glycoprotein. The FG glycoprotein is heterogeneous with respect to both size and isoelectric point, and appears to be antigenically similar to the native RSV F and G glycoproteins. Wathen et. al (1989). Vaccination of the cotton rat model of RSV with FG resulted in induction of neutralizing antibody and was correlated with protection of lung tissue from RSV challenge against both serogroup A and B virus strains. Brideau et al., (1989). This invention now discloses an improved process for the isolation, purification and refolding of FG Glycoprotein produced from baculovirus/Sf (Spodoptera frugiperda) insect cell culture harvests, from Chinese Hamster Ovary (CHO) cells, from COS cells or from other suitable expression systems. The invention comprises the purification of FG glycoprotein utilizing guanidine in a refolding process that restores the proper conformation of the FG glycoprotein. The purification process produces FG glycoprotein that is correctly folded and immunogenically active. INFORMATION DISCLOSURE STATEMENT Brideau et al. J. Gen. Virology 70:2637-2644 (1989) describes the use of canon exchange chromatography in combination with MAb affinity chromatography to purify FG glycoprotein. Walsh et al.. J Infectious Disease 155: 1 198-1204 (1987) describe the use of MAb affinity chromatography to purify the F or G glycoproteins from RSV infected cells. SUMMARY OF INVENTION The present invention comprises a process for restoring the proper conformation to FG glycoprotein comprising treating a solution containing denatured FG glycoprotein with the following, (a) guanidine to raise the concentration of the solution to between 5.0 M - 8.0 M guanidine, (b) a buffering agent with a pK value between 5 and 10, (c) a suitable nonionic or zwitterionic detergent, and (d) a basic solution to establish a pH between 5 - 10. followed by the removal of guanidine by dialysis, diafiltration or gel filtration. The final pH may be in the range of 6-9, 6-8, or about 8. Suitable buffering agents include TRIS, MES, HEPES and "Good" buffers. If TRIS is used the concentration may vary, ranges include 100-300 mM and about 150 mM is preferred. The detergents may be non-ionic or zwitterionic. Non-ionic detergents are preferred such as Tween. If Tween is used the concentration may be between .001 and 20 %, a solution of 5 % is preferred. The invention also comprises the use of Reversed Phase High Pressure Liquid Chromatography (RP-HPLC) to purify the FG glycoprotein prior to the restoration of the proper conformation of the protein. The RP-HPLC aspect of the invention comprises, a process for the purification of FG glycoprotein to a level of greater than 90 or 95 percent purity comprising, I) purifying FG glycoprotein with reversed phase chromatography comprising reversed phase packing and a mobile phase A and mobile phase B. Mobile phase A is a dilute acid solution that may be trifluoroacetic acid with a concentration of 0.01 - 1 percent. Mobile phase B is an organic solvent and II) restoring proper conformation to the denatured FG glycoprotein comprising treating a solution containing denatured FG glycoprotein by adding the following; (a) guanidine to raise the concentration of the solution to between 5.0 M - 8.0 M guanidine, (b) a suitable buffering agent, (c) a suitable detergent, (d) a basic solution to establish a final pH between 5 - 10, followed by the removal of guanidine by dialysis, diafiltration or gel filtration. The solutions used in the refolding process above are also used in the RP-HPLC purification process. DETAILED DESCRIPTION OF THE INVENTION This invention describes a unique process for the refolding of a glycoprotein. Because of the surprising and unexpected refolding step the FG glycoprotein can be obtained in its proper conformation in a high degree of purity. One embodiment of the invention describes a multi-step process with the steps arbitrarily divided into four Phases. In Phase I, the cell culture harvest is collected and processed resulting in a partially purified eluate containing FG glycoprotein. In Phase II, the partially purified eluate containing the FG glycoprotein is further purified with Reversed Phase High Pressure Liquid Chromatography (RP-HPLC). In Phase III, the denatured glycoprotein is refolded to produce the protein in its proper conformation. In Phase IV, the purified FG glycoprotein is transferred into solution for storage. Detailed descriptions of specific embodiments describing the various Phases are provided. The invention is not limited to the detailed descriptions of the embodiments, rather the embodiments are intended merely as examples of procedures that may be used by one reasonably skilled in the art. In one preferred embodiment of this invention, reversed phase high pressure liquid chromatography (RP-HPLC) is coupled with a refolding procedure to produce a highly purified glycoprotein that is in its proper conformation. Ordinarily RP-HPLC inactivates glycoproteins that are similar to the FG glycoprotein and it is surprising and unexpected that the procedures described herein result in a glycoprotein with proper conformation. The RP-HPLC steps are described in Phase II. PHASE I Phase one is characterized by the following steps: Step 1, Precipitation of the Cell Culture Harvest; Step 2, Filtration of the Supernatant After Precipitation; Step 3, Cation Exchange Chromatography and Step 4, Immobilized Metal Affinity Chromatography. Phase I, Step 1. Precipitation of the Cell Culture Harvest. Baculovirus/Sf9 cell culture harvests (approximately 48 hours post-baculovirus infection) are obtained and the cells are removed by low speed centrifugation to produce cell-free supernatant. The pH of the cell-free supernatant is adjusted to pH 8.0 using 50% w/v NaOH with stirring. The precipitate which forms in the cell-free supernatant is allowed to settle under unit gravity for 2 hours at 4°C. Phase I, Step 2. Filtration of the Supernatant After Precipitation. The supernatant from the settling step is removed using a peristaltic pump and passed through a 1.0 μm TDC capsule prefilter and a 0.22 μm Millipak 60 filter. The conductivity of the filtered pH 8.0 cell culture harvest is adjusted to 10.4 mMho/cm using 25 mM HEPES buffer, pH 8.0. Phase I, Step 3. Cation Exchange Chromatography. A glass or acrylic column is packed with Pharmacia S Sepharose FF resin. The S Sepharose column is equilibrated with 25 mM HEPES, 90 mM NaCl, pH 8.0, conductivity 10.4 mMho/cm (S Sepharose A Buffer). The filtered pH 8.0 supernatant is loaded onto the S Sepharose FF column at a linear velocity of 55 cm/hour. After loading, the column is washed with S Sepharose A buffer until the absorbance at 280 n returns to baseline. The column is eluted using a NaCl gradient consisting of 136 mM NaCl to 636 mM NaCl in 25 mM HEPES buffer pH 8.0. After each run, the column is sanitized with 0.5 N NaOH and stored in a 0.01 N NaOH solution. The 0.01 N NaOH storage solution is displaced by S Sepharose A buffer during pre-equilibration of the column prior to the next run. Preferably, all liquid chromatography operations are carried out at 4-7°C. The desired eluate is measured by UV absorbance, collected and pooled. Phase I, Step 4. Immobilized Metal Affinity Chromatography. A glass or acrylic column is packed with Pharmacia Chelating Sepharose FF resin. The Chelating Sepharose column is washed extensively with H 2 0 and charged with 2% CuS0 4 in H 2 0 (2.3 bed volumes). After charging, the column is washed extensively with H 2 0 (>15 bed volumes) and pre-equilibrated with Cu 2+ IMAC A Buffer (25 mM HEPES, 0.5 M NaCl, 46 mMho/cm, pH 7.5) at a linear velocity 90 cm/hour. Eluate from the S Sepharose column, see Step 3 above, is adjusted to pH 7.5 using 2 M HCl. The conductivity of the S Sepharose eluate is adjusted to 44-46 mMho/cm using 25 mM HEPES, 4 M NaCl, pH 7.5. The S Sepharose eluate at pH 7.5 is loaded onto the pre- equilibrated Cu 2+ IMAC column at a linear velocity of 45 cm/hour. The column is washed with Cu 2+ IMAC A Buffer (15 bed volumes) and eluted with a series of step pH gradients consisting of 25 mM MES, 0.2 M NaCl, pH 5.5 (3.3 bed volumes); 25 mM MES, 0.2 M NaCl, pH 4.9 (26.0 bed volumes); and 0.10 M Na acetate, 0.45 M acetic acid, pH 4.0 (7.3 bed volumes). After the pH step gradient, the column is eluted with 70 mM imidazole in 0.10 M Na acetate, 0.66 M acetic acid, pH 4.1 (9.0 bed volumes) and stripped of Cu 2+ using 100 mM EDTA, pH 7.5 (5.6 bed volumes). The column is washed with H 2 0 (12.4 bed volumes) to remove residual EDTA. A flow rate of a linear velocity of 90 cm/hour is used for the pH step gradient, imidazole, and EDTA elution. Partially purified FG glycoprotein elutes at pH 5.5 and at pH 4.9 steps of the step gradient. Phase I, Analysis of Eluates. One method of analysis of the S Sepharose and Cu IMAC eluates is described. A Particle Concentration Fluorescence Immunoassay (PCFIA) was developed using commercially available anti-RSV (respiratory syncytial virus) antibodies. Goat anti-RSV biotin conjugate is coupled to avidin polystyrene beads and used as a capture reagent. The polystyrene beads with the goat anti-RSV antibody are pipetted into a Fluoricon 96-well assay plate. Samples of column eluates and dilutions of a FG glycoprotein standard are pipetted into the wells. The plate is incubated at room temperature for 15 minutes. Detection antibody consisting of goat anti-RSV FITC conjugate is pipetted into the wells and the plate is incubated in the dark for an additional 15 minutes. The PCFIA plate is evacuated at 20 inches Hg vacuum using an Idexx FCA (Fluorescence Concentration Analyzer) for one minute or until the wells are dry. The Idexx FCA is used to wash the wells with phosphate buffered saline (PBS) pH 7.2 containing 0.05% Nonidet P-40 detergent. After washing, the wells are evacuated and the plate is read at 485/535 nm using the Idexx FCA. Data from wells containing dilutions of the FG glycoprotein standard is analyzed using linear regression. The standard curve is linear between 0.03 μg/ml and 0.50 μg/ml FG glycoprotein. The FG glycoprotein concentration in the unknown samples is calculated using the linear regression equation, y = mx + b, where y is PCFIA Fluorescence Intensity Units and x is the FG glycoprotein concentration. PHASE II In Phase II, eluate containing partially purified FG glycoprotein is treated with Reversed Phase High Pressure Liquid Chromatography (RP-HPLC) to provide a highly purified glycoprotein in a denatured state. Phase II, Equipment. The HPLC system utilized to carry out the reversed phase (RP) step may consist of two LKB 2150 HPLC pumps, a Pharmacia P-500 pump, LKB 2152 LC controller, and LKB 2238 Uvicord SII UV but any suitable HPLC pumps and detectors may be used. Phase II, Mobile Phases. The mobile phases for the separation may be: Mobile Phase A (A) Milli-Q water/0.1% TFA and Mobile Phase B (B) UV grade acetonitrile/0.1% TFA. Samples from the Cu 2+ IMAC step in Phase I may be treated with an equal volume of 70% acetonitrile/30% water/0.1 % TFA to make the sample 35% in acetonitrile for loading. Similar mobile phases are, A: 0.1 % aqueous TFA and B: wateπacetonitrile (10:90) plus 0.085% TFA. Phase II, Step 1. Column Loading. The acetonitrile diluted samples are loaded onto a Vydac C-4 column, which is previously equilibrated in 35% B via the Pharmacia P-500 pump. Following loading, the P-500 pump and C-4 column are washed with 35% B until the UV absorbance (206 nm) returns to baseline. Phase II, Step 2. Column Elution. The Vydac C-4 column from Step 1 is then connected to the LKB pumps for the gradient elution. The gradient is increased linearly from 35% to 43% B over 1.5 column volumes, and then from 43% to 52% B over the next 10.8 column volumes. The flow rate during the gradient elution is 79 cm/hour and fractions are collected by visual observation of the elution profile. Alternative elution times and solutions may be used depending upon the specific column and pump used. PHASE III The Phase III refolding steps use a guanidine solution with a suitable detergent to restore the protein to its proper conformation. Phase III, Step 1. The rich pool from the Reversed Phase step is made 6M in guanidine hydrochloride (GdnHCl) by addition of 1 gm of reagent grade GdnHCl per ml of sample. After the GdnHCl is dissolved, the pool is treated with 0.36 ml of 750 mM Tris, 25% v/v Tween-80, pH 8.0 buffer per ml of starting volume (before GdnHCl addition). This gives a final Tris concentration of approximately 150 mM and Tween-80 concentration of 5% v/v. Phase III, Step 2. The sample from Step 1 is then placed in Spectrapor 1 dialysis tubing (6,000-8,000 MW cut-off). The samples are dialyzed against 100 mM Tris, 0.025% Tween-80, pH 8.0 at 4°C. After 12 hours, the samples are transferred to a second tank containing a fresh 100 mM Tris/0.025% Tween-80, pH 8.0 and dialysis continues at 4°C. Phase III, Role of Guanidine and a non-ionic detergent such as Tween-80 in Refolding. It is a surprising and unexpected finding that by using guanidine and a suitable detergent in this process the protein could be refolded and still retain its normal conformation. Other chaotropic agents such as urea were found to be unsuitable. Guanidine and a suitable detergent are thus essential in the process of this invention. Tween-80 apparently plays an important role in the refolding process. A study was undertaken in which lower levels of Tween-80 were utilized in the refolding buffer and the results of this study are presented in Table 1. The results show little, if any, decrease in the amount of FG recovered as the Tween-80 concentration is lowered from 0.25 to 0.001%. This indicates that Tween-80 concentration can be lowered substantially, even below the Critical Micelle Concentration, without sacrificing yield. One sample had been included in the study with a higher Tween-80 concentration (1%) and the recovery of FG activity from this sample was greater than a sample of 0.25% Tween-80. or any of the lower Tween-80 concentrations. Samples were also refolded in 1%, 2% and 5% Tween-80. See Table 2. The sample refolded in 1% Tween-80 yielded significantly more FG than the sample refolded in 0.025% Tween-80. Two samples were refolded in 1% Tween-80, one dialyzed against 1% Tween-80 and the other dialyzed against 0.025% Tween-80. The sample dialyzed against 1% Tween-80, yielded measurably more FG than the sample dialyzed against 0.025% Tween-80. The samples refolded in 2% and 5% Tween-80 were dialyzed against 0.025% Tween-80 and yielded greater amounts of FG than the 1% sample that had been dialyzed against 0.025% Tween-80. The sample refolded in 5% Tween-80 yielded the greatest amount of activity. Three alternative detergents were evaluated along with Tween-80 for use during the refolding steps. These detergents, CHAPS, CHAPSO, and β-D-glucopyranoside (BOG) may also be used but they were found to yield only one-third to one-half of the FG obtained with the lower Tween-80 concentrations (Table 2). PHASE IV In Phase IV, the refolded glycoprotein is placed into an appropriate delivery buffer, the glycoprotein is analyzed and then the glycoprotein can be diluted and stored. Phase IV, Step 1. Concentration/Buffer Exchange using S Sepharose FF Chromatography. After dialysis, the refolded protein is applied to a small S Sepharose FF column to concentrate the FG Glycoprotein and exchange the protein into an appropriate delivery buffer. In one embodiment the Tween-80 concentration is lowered from 5% to 0.025% Tween- 80 by the S Sepharose chromatography step described below. Columns 1.5 cm in diameter containing 6 to 9 ml of S Sepharose FF or columns 2.5 cm in diameter containing 15 ml of S Sepharose or other appropriate columns may be utilized. The columns are initially washed with 0.75 N NaOH and then treated with 200 mM NaH 2 P0 4 , pH 7.4 until the pH of the column effluent is below pH 8.0. The columns are then equilibrated in 20 mM NaH 2 P0 , 100 mM NaCl, 0.025% Tween-80, pH 7.4. The dialyzed samples are loaded on the equilibrated columns and following loading, the columns are washed with the equilibration buffer until the absorbance (206 nm) drops back to baseline. The columns are eluted with a step gradient of 20 mM NaH 2 P0 4 , 700 mM NaCl, 0.025% Tween-80, pH 7.4 and the rich pool was collected by visual observation of the elution profile. The S Sepharose procedures may be carried out at room temperature. Phase IV, Step 2. Evaluation of the FG Glycoprotein. The conformation of FG glycoprotein produced by this process is assessed using a broad panel of monoclonal antibodies (MAb) against RSV F and G glycoproteins. The concentration of FG glycoprotein in the samples is quantitated by amino acid analysis. Binding of the MAb to FG glycoprotein is assayed by ELISA. For each antibody in the panel, the ELISA units per μg FG glycoprotein are calculated and the results expressed as a percent of the reactivity of the antibody relative to a denatured FG glycoprotein standard. Phase IV, Step 3. Dilution and Storage of the glycoprotein The FG glycoprotein may be diluted 1/2 using 20 mM Na phosphate buffer, pH 7.3 (2.68 gm Na phosphate monobasic, monohydrate per liter; pH adjusted with 50% w/v NaOH). This dilution reduces the concentration of NaCl to 0.35 M and reduces the concentration of Tween-80 to 0.0125% v/v. The FG glycoprotein produced by this procedure does not have to be diluted. FG Glycoprotein may remain in the final S Sepharose elution buffer (20 mM Na Phosphate buffer. 0.7 M NaCl, 0.025% Tween-80, pH 7.4). The compound may be stored at -70°C or at 4°C. DEFINITIONS It is believed that one reasonably skilled in the art would understand the terms and expressions used above. The following terms and expressions, commonly used by those reasonably skilled in the art, are further defined with typical examples of specific materials that may be used with this invention. Avidin polystyrene beads are manufactured by Idexx, Westbrook, Maine. Buffering agents are solutions prepared to minimize changes in pH (the hydrogen ion concentration). Appropriate agents for this invention include substances with pK values between 5 and 10 such as "Good" buffers, MES, HEPES, Sodium bicarbonate, Sodium phosphate, and Tris. "Good" buffers are referenced at Good, N.E., et al., Biochem., 15:467-477 (1966), MES is 2-(N-Moιpholino)eth__nesulfonic acid, HEPES is N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid), Tris is Tris(hydroxymethyl)aminomethane ^H^NO (Trizma ). Buffers of this type are available from chemical supply companies. Detergents suitable, are non-ionic detergents that contain no charged functional groups and zwitterionic detergents. Non-ionic detergents are usually of the following types, n-octyl glucoside (β-octyl glucoside), polyoxyethylene ethers (BRIJ), polyoxyethylene sorbitan (Tween), Sorbitan (Span), Tergitol and Triton. Various types are readily available from chemical suppliers such as Sigma. For example Sigma carries Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85. Tween 80 is a monooleate polyoxyethelenesorbitan with a fatty acid composition of oleic acid approximately 75% and the balance primarily linoleic, palmitic and stearic acids. Zwitterionic detergents are also available from chemical suppliers. Examples of zwitterionic detergents are CHAPS, CHAPSO, Lauryl sulfobetaine (SB- 12) and Pamityl sulfobetaine (SB- 16). CHAPS is 3-((3-cholamidopropyl)-dimethylammomo)-l-propanesulfonate and CHAPSO is 3-((3-cholamidopropyl)-dimethylammonio)-2-hydroxy-l-propanesulfonate. Dilute acids include but are not limited to acids such as acetic acid, formic acid, phosphoric acid, and trifluoroacetic acid. EDTA is ethylenediaminetetraacetic acid and is available from chemical supply companies. ELISA is Enzyme Linked Immunosorbent Assay. Fluoricon 96-well assay plates are manufactured by Idexx, Westbrook, Maine. Goat anti-RSV. biotin and FITC. conjugates are manufactured by Virostat, Portland, Maine. Guanidine is aminomethanamidine and may be supplied as the carbonate, hydrochloride, or thiocyanate salt. Idexx FAC (Fluorescence Concentration Analyzers) are manufactured by Idexx, Westbrook, Maine. LKB pumps refer to two 2150 high pressure liquid chromatography pumps available from Pharmacia LKB Biotechnology, Picsataway, New Jersey. Millipak filters are available from the Millipore Company, Bedford, Massachusetts. Mho is a unit of conductivity. Organic solvents include but are not limited to solvents such as acetonitrile, and organic alcohols such as isopropanol, ethanol and methanol. PCFIA is Particle Concentration Fluorescence Immunoassay. Reversed Phase Chromatographv is chromatography in which the stationary phase is non-polar, such as C-2, C-3, C-4, C-8, C-18, phenyl, CN, and the mobile phase is polar. The non-polar stationary phase is bonded onto a stationary matrix such as a silica or polymer matrix. Spectrapor dialysis membranes are manufactured by Spectrum Medical Industries Inc., Houston, Texas. S Sepharose FF or S Sepharose Fast Flow refers to a cation exchanger available from Pharmacia LKB Biotechnology, Piscataway, New Jersey. TDC capsule prefilters are available from Gelman, Ann Arbor, Michigan. TABLES Table 1 Tween-80 Concentration Studies A Monoclonal antibody 2-27 is against a G glycoprotein epitope. Monoclonal antibodies 1269, 1121, and 1243 are directed against B10-F, A5-F, and Cl l-F epitopes respectively on F glycoprotein. Wathen et al., Journal of General Virology Vol. 70, pp. 2625-2635 (1989). The activity number reported for each monoclonal antibody is the reciprocal of the dilution of the experimental FG glycoprotein preparation multiplied by the optical density at that dilution. Table 2 Tween-80 Concentrations and Alternative Detergents 10 15 A - Dialyzed against 1.0% Tween-80 - Dialyzed against 0.025% Tween-80

Claims: CLAIMS 1. A process for restoring proper conformation to a FG glycoprotein comprising treating a solution containing denatured FG glycoprotein with the following, (a) guanidine to raise the concentration of the solution to between 5.0 M - 8.0 M guanidine (b) a buffering agent with a pK value between 5 and 10 (c) a suitable nonionic or zwitterionic detergent (d) a basic solution to establish a pH between 5 and 10 followed by the removal of guanidine by dialysis, diafiltration or gel filtration. 2. The process of claim 1 where the buffering agent is Tris, MES, HEPES or a Good buffer and the suitable detergent is a non-ionic detergent. 3. The process of claim 2 where the buffering agent is Tris and the non-ionic detergent is n-octyl glucoside (β-octyl glucoside), polyoxyethylene ethers (Brij), polyoxyethylene sorbitan (Tween) or sorbitan (Span). 4. The process of claim 3 where the buffering agent is Tris, the Tris concentration is between 100 - 300 mM, and the non-ionic detergent is Tween and the Tween concentration is .001 - 20 percent v/v and the final pH is between 6 and 9. 5. The process of claim 1 where the final pH is between 6 and 9. 6. The process of claim 2 where the final pH is between 6 and 9. 7. The process of claim 4 where the Tris concentration is about 150 mM, the Tween concentration is about 5.0 percent v/v and the final pH is between 6 and 8. 8. A process for the purification of FG glycoprotein to a level of greater than 90 percent purity comprising I) treating a solution of FG glycoprotein with reversed phase chromatography comprising reversed phase packing and a mobile phase A and mobile phase B where mobile phase A is a dilute acid and mobile phase B is an organic solvent and II) restoring proper conformation to the denatured FG glycoprotein comprising treating a solution containing denatured FG glycoprotein by adding the following; (a) guanidine to raise the concentration of the solution to between 5.0 M and 8.0 M guanidine (b) a suitable buffering agent (c) a suitable detergent (d) a basic solution to establish a final pH between 5 and 10 followed by the removal of guanidine by dialysis, diafiltration or gel filtration. 9. The process of claim 8 where the mobile phase A is trifluoroacetic acid and mobile phase B is acetonitrile. 10. The process of claim 9 where the buffering agent is Tris, the suitable detergent is Tween, the final pH is between 6 -9, the mobile phase A is trifluoroacetic acid with a concentration of .01 to 1 percent. 11. The process of claim 10 where the concentration of Tris is between 100 to 300 mM. the Tween concentration is between .001 - 20 percent v/v, and the pH is between 6 and 9. 12. The process of claim 11 where the concentration of Tris is about 150 mM, the Tween concentration is about 5 percent v/v and the pH is between 7 and 8. 13. The process of claim 8 where the FG glycoprotein is purified to a level greater than 95 percent. 14. The process of claim 9 where the FG glycoprotein is purified to a level greater than 95 percent. 15. The process of claim 10 where the FG glycoprotein is purified to a level greater than 95 percent. 16. The process of claim 11 where the FG glycoprotein is purified to a level greater than 95 percent. 17. The process of claim 12 where the FG glycoprotein is purified to a level greater than 95 percent. 18. The glycoprotein prepared by the process of claim 1. 19. The glycoprotein prepared by the process of claim 8.

Inventors: GARLICK ROBERT L, LYLE STEPHEN B, WATHEN MICHAEL, WELLS PETER A, GARLICK, ROBERT, L., LYLE, STEPHEN, B., WATHEN, MICHAEL, WELLS, PETER, A.
Applicants: GARLICK ROBERT L, LYLE STEPHEN B, UPJOHN CO, WATHEN MICHAEL, WELLS PETER A, GARLICK, ROBERT, L., LYLE, STEPHEN, B., THE UPJOHN COMPANY, WATHEN, MICHAEL, WELLS, PETER, A.
Application year: 1993
Main classification: C07K15
IPC codes: C07K1, C07K14, C07K19