Methods of purification and/or viral inactivation

Abstract

Methods of purification and/or viral deactivation of a protein (e.g. glycoprotein) comprising a step of treating the protein (e.g. glycoprotein) with a combination of caprylic acid and ethanol.

Claims

1. A method of purification of a glycoprotein, the method comprising a step of treating the glycoprotein with a combination of caprylic acid and ethanol, wherein the glycoprotein is FSH, hCG, or LH.

2. A method according to claim 1, which comprises treating a solution of the glycoprotein with a combination of caprylic acid and ethanol.

3. A method according to claim 2, further comprising concentrating the glycoprotein solution.

4. A method according to claim 1, wherein the glycoprotein is recombinant FSH, hCG, or LH.

5. A method according to claim 1, wherein the glycoprotein is a recombinant FSH, hCG, or LH produced in a cell by a method comprising culturing the cell in a suitable medium and harvesting the recombinant FSH, hCG, or LH from said cell and/or said medium.

6. A method according to claim 5, wherein the cell is a mammalian cell.

7. A method according to claim 1, wherein treating the glycoprotein with a combination of caprylic acid and ethanol takes place at pH 2 to pH 6.5.

8. A method according to claim 7, wherein treating the glycoprotein with a combination of caprylic acid and ethanol takes place at pH 3 to pH 6.5.

9. A method according to claim 7, wherein treating the glycoprotein with a combination of caprylic acid and ethanol takes place at pH 4 to pH 6.

10. A method according to claim 7, wherein treating the glycoprotein with a combination of caprylic acid and ethanol takes place at pH 4.5 to pH 5.5.

11. A method according to claim 1, wherein the caprylic acid concentration is from 10 mM to 30 mM caprylic acid.

12. A method according to claim 1 which comprises treating the glycoprotein with ethanol and caprylic acid for an incubation time of from 1 minute to 6 hours at a temperature of 232 C. with stirring.

13. A method according to claim 1, further comprising filtering the glycoprotein following the treatment with caprylic acid and ethanol.

14. A method according to claim 13, wherein the glycoprotein is filtered using a glass fiber filter.

15. A method according to claim 1, wherein the ethanol concentration is from 20% to 50% (v/v).

16. A method according to claim 1 which comprises treating the glycoprotein with ethanol and caprylic acid at a temperature of 4-8 C. for an incubation time of from 1 minute to 32 hours, without stirring.

17. A method according to claim 1 which comprises treating the glycoprotein with ethanol and caprylic acid for an incubation time of from 0.5 hours to 1 hour at a temperature of 232 C. with stirring, followed by reducing the temperature to a temperature of 4-8 C. and subsequent incubation a temperature of 4-8 C. for 14 hours to 18 hours, without stirring.

18. A method according to claim 1 which comprises treating the glycoprotein with ethanol and caprylic acid for a duration of 1 hour10 minutes at a pH of 5.50.1 and a temperature of 232 C.

19. A method of viral inactivation in a glycoprotein, the method comprising a step of treating the glycoprotein with a combination of caprylic acid and ethanol, wherein the glycoprotein is FSH, hCG, or LH.

Description

(1) The present invention will now be described with reference to the attached drawings in which:

(2) FIG. 1 shows a scheme of the recombinant FSH purification (viral deactivation) process according to an embodiment of the present invention;

(3) FIG. 2 shows the gel pattern following qualitative estimation of the impurities removal for various treated UF2-AD (Ultrafiltration after dialysis) FSH samples run on 12% reduced PAGE (polyacrylamide gel electrophoresis), wherein lane 1 LMW (low molecular weight) markers, lane 2 is Sample Buffer, lane 3 is UF2-AD, lane 4 is UF2-AD+10 mM CA, pH 5.5, lane 5 is UF2-AD+10 mM CA, 30% Ethanol, pH 5.5, lane 6 is Sample Buffer, lane 7 is low range MW, lane 8 is UF2-AD, lane 9 is UF2-AD+20 mM CA, pH 5.5 and lane 10 is UF2-AD+20 mM CA, 30% Ethanol pH 5.5 (see Example 3); and

(4) FIG. 3 shows the gel pattern following qualitative estimation of the impurities removal for various treated hCG samples run on 12% reduced PAGE (polyacrylamide gel electrophoresis), wherein lane 1 is LMW (low molecular weight) markers, lane 2 is hCG containing Harvest-AD, lane 3 is NRNot Relevant, lane 4 is supernatant of [Harvest-AD+15 mM C.A pH 5.0], and lane 5 is supernatant of [Harvest-AD+20 mM C.A+10% Ethanol pH 5.0], see Example 4.

EXAMPLE 1

(5) FIG. 1 shows an overview of the whole recombinant FSH purification process. As can be seen in FIG. 1, recombinant FSH is expressed in a PER.C6 cell line engineered by the methods disclosed in WO2013/020996 and WO2009/127826A using a bioreactor.

(6) The bioreactor is seeded and cell growth promoted by methods known to those skilled in the art, and the bioreactor run in perfusion mode to produce and continuously harvest recombinant FSH, using a Hollofibre (ATF4) system available from Repligen. The seeding of at least 110.sup.6 cells/mL in a total volume of 41 Litre takes place in 6GRO medium. The production takes place in the ProPer-1 medium, and the bioreactor harvest is collected into polyethylene bags. Thus, in this example the protein (glycoprotein) is a recombinant glycoprotein (recombinant FSH) produced in a PER.C6 cell line by a method comprising culturally the cell line in a suitable medium (ProPer-1 medium) and harvesting the recombinant glycoprotein from the medium (by harvesting the recombinant protein from the cell culture supernatant).

(7) The bioreactor harvest is pooled and subjected to 10 kDa ultrafiltration/diafiltration step (UF1) which, reduces the concentration of the harvest, conditions the harvest (in terms of pH and conductivity) for Capto-Q chromatography, and removes pigments and low molecular weight components of the culture medium. The retentate is filtered through a glass fibre filter of 0.8+0.65 m to clarify the process solution (see FIG. 1), and subsequently subjected to 0.2 m filtration as a bioburden control. Capto-Q anion exchange chromatography is then used, by methods well known in the art, to capture the recombinant FSH, and to remove DNA, endotoxin, host cell proteins and process related impurities. A further 0.2 m filtration is performed as a bioburden control.

(8) The filtered Capto-Q eluate is subjected to a further 10 kDa ultrafiltration/diafiltration (UF2) with the purpose of desalting and volume reduction prior to purification/viral inactivation, by methods well known in the art.

(9) CA/EtOH Step

(10) The retentate of UF2 is a solution of recombinant FSH in a buffer (100 mM ammonium acetate, 30 mM NaCl, pH 9.3-pH 9.7). The pH of the protein solution is first reduced from pH 9.3-pH 9.7 to pH 6.30.3 at 232 C. with stirring, then treated with a combination of 20 mM caprylic acid/30% ethanol (CA/EtOH) followed by further adjustment of the pH to 4.5-5.6 (the pH is adjusted by 1 M HCl). After this treatment the protein solution remains for 0.5 h to 1 h incubation at 232 with stirring (during this time viral inactivation takes place and white precipitation flakes-like are observed). At the end of 0.5 h-1 h, the temperature of the protein solution is reduced to 4-8 C., and the solution incubated for a further 14 h to 18 h, e.g. 14 to 16 h, without stirring (this allows the precipitation of HCP and non-enveloped viruses to continue). The CA/ETOH treatment step has a triple activity: (i) inactivation of enveloped virus; (ii) clearance of non-enveloped virus by precipitation followed by clarification step aimed to remove the precipitate; and (iii) host-related protein removal by precipitation.

(11) The precipitated impurities (non enveloped virus and host related protein) are removed by a step of filtration through a glass fibre filter of 0.8+0.65 micrometres, a filtration step which also clarifies the recombinant FSH solution.

(12) A further 0.2 m filtration is performed as a bioburden control.

(13) The solution is then subject to Sulfopropyl-Sepharose cation-exchange chromatography (SP-FF) by methods well known in the art to remove the caprylic acid, ethanol and further host cell proteins. A further 0.2 m filtration is performed as a bioburden control, followed by additional step of purification using Phenyl-Sepharose hydrophobic interaction chromatography (PS-FF) to remove free recombinant FSH sub units and host cell proteins.

(14) The PS eluate is subjected to a third 10 kDa ultrafiltration/diafiltration step (UF3) to remove the salt and condition the solution for the next step. A further 0.2 m filtration is performed as a bioburden control, prior to hydroxyapatite adsorption chromatography (HyA), aimed to remove dissociated FSH sub units and basic heterodimers, followed by a further 0.2 m filtration performed as a bioburden control.

(15) A Q-sepharose anion-exchange chromatography (QS-FF) polishing step is performed in bind/elute mode to remove host proteins, DNA, endotoxins and potential viruses. A further bioburden control 0.2 m filtration then takes place, prior to pooling of QS MPs and nanofiltration to remove potential viruses. The supernatant is subjected to a fourth 10 kDa ultrafiltration/diafiltration step (UF4) to concentrate the recombinant FSH to 0.5 to 1.1 mg/ml, followed by the addition of polysorbate 20 to final concentration of 0.005 mg/ml. These are dialysis and buffer adjustment steps, which are well known in the art. The recombinant FSH is subject to a final 0.2 m filtration step prior to aliquoting into primary packaging and storing at 20 C. until shipment.

(16) The applicants have found that the treatment with caprylic acid/ethanol is capable of markedly reducing the host-related impurities. According to various manufacturing runs (data not shown) 64 to 79% of the host related impurities are removed by the CA/EtOH step (and subsequent glass fibre filtration) described above, while a high yield of approximately 90 to 95% of the FSH was recovered. This is a significant reduction in host-related impurities which is provided by a simple process, with minimal loss of product protein (product glycoprotein).

(17) The remarkable viral clearance efficiency of the this step is summarized in Table 1, which shows a summary of the log.sub.10 reduction factors, which are in the region of 4.25 to 5.41. A log-reduction factor (LRF)4 Log.sub.10 (e.g. up to 8 Log.sub.10 or greater) is generally considered high, robust and effective.

(18) TABLE-US-00001 TABLE 1 Summary of the log.sub.10 reduction factors (rFSH) Nonenveloped Study Enveloped Viruses Virus Process Step number MuLV PRV EMCV.sup.(1) Caprylic acid/Ethanol treatment K1/B28/12 4.88/5.12 5.29/5.41 4.42/4.47 (protein conc. 2 mg/mL) K1/B28/13.sup.(1) Caprylic acid/Ethanol treatment K1/B28/12 4.94/4.58 5.11/5.29 4.25/4.36 (protein conc. 6 mg/mL) K1/B28/13.sup.(1) .sup.(1)Study with EMCV contains glass fiber filtration after the treatment

(19) The Example above relates to rFSH, but those skilled in the art will appreciate that process [e.g. the CA/EtOH step (and subsequent glass fibre filtration)] described above is readily applicable to purification/viral inactivation of other proteins (e.g. glycoproteins), for example rhCG [e.g. rhCG produced in a PER.C6 cell line by the method of in PCT/GB2010/001854 (published as WO2011/042688)].

EXAMPLE 2

(20) In another example of a method of the invention, there is provided an hCG purification/viral inactivation process. A method similar to Example 1 above (minus the Sulfopropyl-Sepharose cation-exchange chromatography (SP-FF) step) was used for purification/viral inactivation of rhCG produced in a PER.C6 cell line by the method of in PCT/GB2010/001854 (published as WO2011/042688). The applicants found that chemical inactivation by caprylic acid and ethanol at acidic pH, i.e. using the CA/EtOH step described in Example 1, removed approximately 65% of non hCG impurities (by precipitation), while a high yield of approximately 90% of the hCG was recovered.

(21) The excellent viral clearance efficiency of the CA/EtOH step in the Example is summarized in Table 2. Viral inactivation of MuLV was effective as determined by the high LRF obtained, in a range 5.31 to 5.48. LRFs between 2.03 and 2.74 were obtained for the CA/EtOH treatment and precipitate removal.

(22) TABLE-US-00002 TABLE 2 Summary of the log.sub.10 reduction factors (rhCG) LRFs Enveloped Nonenveloped Study Virus Virus Process Step Number (MuLV) (PPV) Caprylic acid/Ethanol KOP1-B01-15 5.43/5.31 2.03/2.21 treatment (protein conc. 2 mg/mL) Caprylic acid/Ethanol KOP1-B01-15 5.31/5.48 2.68/2.74 treatment (protein conc. 6 mg/mL)

EXAMPLE 3: EVALUATION OF PRECIPITATION CONDITIONS FOR IMPURITIES REMOVAL IN RFSH-CONTAINING SOLUTION

(23) Experiment Description:

(24) The UF2-AD (Ultrafiltration after dialysis) intermediate obtained using the rFSH purification process of Example 1 (up to the CA/EtOH step) contained 100 mM Ammonium Acetate+30 mM NaCl pH 9.500.20, 11.000.50 mS/cm. This was aliquoted and each aliquot was subjected to different precipitation conditions for impurities removal (rFSH is soluble in the solution). The tested precipitation conditions are as follows:

(25) 1. UF2-AD without treatment (control)

(26) 2. UF2-AD+10 mM Caprylic acid (CA), pH 5.5

(27) 3. UF2-AD+10 mM CA, 30% Ethanol, pH 5.5

(28) 4. UF2-AD+20 mM CA, pH 5.5

(29) 5. UF2-AD+20 mM CA, 30% Ethanol, pH 5.5

(30) All treated samples were incubated for 30 min at RT with stirring followed by additional incubation of 30 min without stirring. Further incubation was performed at 2-8 C. for 16-20 hr. The generated precipitants were removed by centrifugation while supernatants were collected.

(31) Performance Parameters of the Precipitation Process:

(32) rFSH Recovery

(33) rFSH concentration in all the samples were determined by FSH ELISA and the yields of each sample after treatment were calculated and are set out in Table 3.

(34) As seen in Table 3, 96%, 106%, 106% and 98% of the rFSH in the UF2-ADs process solutions was recovered following precipitation with 10 mM CA, 10 mM CA+30% Ethanol, 20 mM CA and 20 mM CA+30% Ethanol, respectively. This indicates that the rFSH is not precipitated and stays soluble in solution.

(35) Impurities Removal

(36) The absorbance at 280 nm in the un-treated UF2-AD and in each of the supernatants of the treated UF2-ADs was measured and impurities removal was calculated, as set out in Table 3. 33.9%, 64.9%, 65.9% and 68.8% of the total A.sub.280 impurity in the UF2-AD was removed after precipitation with 10 mM CA, 10 mM CA+30% Ethanol, 20 mM CA and 20 mM CA+30% Ethanol, respectively.

(37) The un-treated UF2-AD and the supernatants of the various treated UF2-ADs were run on 12% reduced PAGE (polyacrylamide gel electrophoresis) for qualitative estimation of the impurities removal. The gel pattern is shown in FIG. 2. The supernatant obtained following precipitation by 10 mM CA+30% Ethanol (lane 5) and 20 mM CA+30% Ethanol pH 5.5 (lane 10) is substantially purer relative to the supernatant obtained following precipitation by 10 mM CA pH 5.5 (lane 4) and 20 mM CA (lane 9), respectively. This shows the synergistic effect of the addition of ethanol to Caprylic acid in the precipitation step.

(38) Experiment Conclusions:

(39) 1. Precipitation of UF2-AD by Ethanol and Caprylic acid resulted in purer product compared to that obtained using Caprylic acid only.

(40) 2. The combination of Ethanol and Caprylic acid does not precipitate rFSH which stays soluble in solution, meaning this is a high yield purification and viral inactivation step.

(41) TABLE-US-00003 TABLE 3 Results Obtained from Different Precipitation Conditions for Impurities Removal in intermediate rFSH solution Absorbance at 280 nm rFSH (by ELISA) total Removal total Recov Supernatant of A.sub.280 A.sub.280 (%) g/ml g (%) 1. UF2-AD 3.265 32.650 677 6,770 2. UF2-AD + 10 mM CA, pH 5.5 2.066 21.571 33.9 207.1 6,487 96 3. UF2-AD + 10 mM CA, 30% 0.760 11.457 64.9 158.6 7,173 106 Ethanol, pH 5.5 4. UF2-AD + 20 mM CA, pH 5.5 1.060 11.122 65.9 227.80 7,170 106 5. UF2-AD + 20 mM CA, 30% Ethanol 0.678 10.197 68.8 146.5 6,610 98 pH 5.5

EXAMPLE 4: EVALUATION OF PRECIPITATION CONDITIONS FOR REMOVAL OF IMPURITIES IN HCG-CONTAINING HARVEST

(42) Experiment Description:

(43) hCG-containing harvest was concentrated 40 fold and dialyzed with 100 mM glycine, 50 mM NaCl pH 9.0, 6 mS/cm buffer by 10 kDa ultrafiltration (UF) system. At the end of the UF, the recovered protein solution, Harvest-AD (After dialysis) material, was aliquoted and each aliquot was subjected to different precipitation conditions for removal of impurities (while hCG is soluble in the solution). The tested precipitation conditions are as follows (Table 4):

(44) 1. Harvest-AD without treatment as a control;

(45) 2. Harvest-AD+15 mM CA, pH 5.0

(46) 3. Harvest-AD+20 mM CA, pH 5.0

(47) 4. Harvest-AD+20 mM CA+10% Ethanol, pH 5.0.

(48) All treated samples were incubated for 1 hr at R.T with stirring. The generated precipitants were removed by centrifugation while supernatants were collected.

(49) Performance Parameters of the Precipitation Process:

(50) hCG Recovery

(51) The hCG concentration in the un-treated harvest and in each of the supernatants of the treated Harvest ADs were determined and recoveries were calculated as detailed in Table 4.

(52) Impurities Removal

(53) The absorbance at 280 nm (A.sub.280) in the un-treated harvest and in each of the supernatants of the treated Harvest ADs were determined and impurities removals were calculated as detailed in Table 4.

(54) The un-treated harvest and the supernatants of the various treated Harvest ADs were run on 12% reduced PAGE (polyacrylamide gel electrophoresis) for qualitative estimation of the impurities removal. The gel pattern is shown in FIG. 3.

(55) Discussion:

(56) 1. hCG Recovery91%, 86% and 83% of the hCG in the Harvest-AD were recovered after precipitation with 15 mM CA, 20 mM CA and 20 mM CA+10% Ethanol respectively.

(57) 2. Impurities Removal by A.sub.28056.6%, 55.8% and 63.0% of the total A.sub.280 in the Harvest-AD were removed after precipitation with 15 mM CA, 20 mM CA and 20 mM CA+10% Ethanol respectively.

(58) 3. Impurities Removal by gelthe supernatant obtained following precipitation by 20 mM CA+10% Ethanol pH 5.0 (lane 5) is substantially purer relative to the supernatant obtained following precipitation by 15 mM CA pH 5.0 (lane 4)

(59) Conclusions:

(60) 1. Precipitation of hCG-containing Harvest added with Ethanol and Caprylic acid resulted in a purer product compared to that obtained using only Caprylic acid.

(61) TABLE-US-00004 TABLE 4 Results Obtained from Different Precipitation Conditions for Impurities Removal in hCG Containing Harvest Absorbance at 280 nm rFSH (by ELISA) total Removal total Recov Supernatant of A.sub.280 A.sub.280 (%) g/ml g (%) 1. Harvest AD 4.295 107.375 757.14 18,929 2. Harvest AD + 15 mM CA, pH 5.0 1.738 46.578 56.6% 639.72 17,144 91% 3. Harvest AD + 20 mM CA, pH 5.0 1.792 47.488 55.8% 612.24 16,224 86% 4. Harvest AD + 20 mM CA, 10% 1.369 39.701 63.0% 543.8 15,770 83% Ethanol, pH 5.0