DESC:
FIELD OF THE INVENTION
The present invention relates to the field of protein purification. In particular, the invention discloses a process for purification of darbepoetin.
BACK GROUND OF THE INVENTION
During the manufacturing of therapeutic proteins, numerous process-related impurities, may arise, and if not removed during subsequent purification steps, may remain in the final purified fraction. One such types of impurities are “cell substrate-derived impurities”.
Apart from the protein of interest itself, recombinant host cells also secrete endogenous cell components into the extracellular media, (hence the name “cell-substrate derived impurities). These include, predominantly, host cell proteins (HCP) and host cell DNA (HCD). These impurities, if present in the final protein solution, can be highly immunogenic, causing adverse reactions. HCPs, in particular the proteases component, may also degrade the protein of interest, thereby effecting its stability during storage. Hence, drug approval agencies mandate the levels of HCP and HCD in a therapeutic product to be less than <100 ppm and <100 pg respectively (Wang X et al., 2009 Biotechnology and Bioengineering, Vol. 103, No. 3, 446-458; http://www.bioprocessintl.com/analytical/downstream-validation/host-cellular-protein-quantification-326656/).
However, removal of cell-substrate derived impurities is challenging as they may tend to co-purify with the protein of interest. Another impediment to designing a purification scheme for therapeutic proteins is restrictions in the number of chromatography steps that may be productively used in purifying a protein, since each additional step would lead to loss in yield (Sung Kwan Yoon et al., 2001).
Hence purification methods need to be designed so that target protein solutions of high purity are achieved, without compromising yield.
The present invention describes a purification process for removal of cell-substrate derived impurities, particularly proteases and HCD, in a therapeutic protein product.
SUMMARY OF THE INVENTION
The present invention discloses a purification process for darbepoetin. Specifically, the invention discloses a purification process for removal of cell-substrate derived impurities, such as proteases and host cell DNA (HCD), from darbepoetin compositions, the process comprising an immobilized metal ion chromatography step operated in flow-through mode. The instant process also improves the yield of the protein in the final composition.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Demonstrates reverse phase analytical chromatogram for fraction-2A
Figure 2: Demonstrates reverse phase analytical chromatogram for fraction-3A.
Figure 3: Demonstrates reverse phase analytical chromatogram for fraction-3B.
Figure 4: Demonstrates reverse phase analytical chromatogram for fraction-3C.
Figure 5: Demonstrates reverse phase analytical chromatogram for fraction-4B.
Figure 6: Demonstrates reverse phase analytical chromatogram for fraction-4C-I.
Figure 7: Demonstrates reverse phase analytical chromatogram for fraction-4C-II.
Figure 8: Demonstrates reverse phase analytical chromatogram for fraction-4D.
Figure 9: Demonstrates reverse phase analytical chromatogram for fraction-4E.

DETAILED DESCRIPTION OF THE INVENTION
Definitions
“Flow-through mode” refers to the operational mode of a chromatographic purification process wherein, the desired protein is not bound to the chromatographic column and is obtained in the flow-through solution during loading or washing of the column.
“Bind-elute mode” refers to the operational mode of a chromatographic purification process wherein, the desired protein binds to the chromatographic column during loading and is subsequently eluted from the column using an elution buffer.
“Host cell proteins” or HCP’s are proteins produced or encoded by the host organisms used to produce recombinant therapeutic proteins. HCPs include proteases that may degrade the protein of interest.
Detailed description of the embodiments
The present invention discloses a process for removal of cell substrate-derived impurities from a darbepoetin composition, the process comprising an immobilized metal affinity chromatography (IMAC) used in flow-through mode, for removal of impurities from the said composition.
One embodiment of the invention discloses a process for removal of proteases from a darbepoetin composition, the process comprising an immobilized metal affinity chromatography (IMAC) used in flow-through mode, for removal of proteases from the said composition.
Another embodiment of the invention discloses a process for removal of host cell DNA (HCD) from a darbepoetin composition, the process comprising an immobilized metal affinity chromatography (IMAC) used in flow-through mode, for removal of HCD from the said composition.
In any of the above mentioned embodiments of the invention, the IMAC step results in darbepoetin compositions wherein, there is no protease mediated degradation in acidified darbepoetin samples even after 24 hours of incubation.
In any of the above mentioned embodiments of the invention, the process results in removal of HCD below detectable levels.
In any of the above mentioned embodiments of the invention, the increase in yield of the darbepoetin composition is by about 5 times as compared to yield of darbepoetin composition obtained using a process not comprising an IMAC step.
In any of the above mentioned embodiments of the invention, the equilibration buffer used in IMAC comprises of Tris buffer and salt at concentration greater than 0.5 molar.
In any of the above mentioned embodiments of the invention, the chelated transitional metal ions used in IMAC is copper ion.
In any of the above mentioned embodiments of the invention, the IMAC matrix used is Chelating Sepharose™ Fast Flow (GE Healthcare) with Iminodiacetic acid.
In any of the above mentioned embodiments of the invention, darbepoetin composition is produced by culturing mammalian cells, more particularly Chinese Hamster Ovary (CHO) cells.
In any of the above mentioned embodiments of the invention, the process comprise other chromatographic steps preceding or following IMAC step. Examples of such chromatography may be ion exchange chromatography, in particular anion exchange chromatography (Q Sepharose ™ Fast Flow, GE Healthcare), cation exchange chromatography (CM Sepharose™ Fast Flow, GE Healthcare), multimodal chromatography step (Capto™ Adhere, GE Healthcare), hydrophobic interaction chromatography, affinity chromatography.
The embodiments mentioned herein may optionally further comprise any of tangential flow filtration, concentration, diafiltration or ultrafiltration steps, between chromatographic steps.
The embodiments mentioned herein may include one or more viral inactivation steps or sterile filtration or nanofiltration steps.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples, being provided only for purposes of illustration. These examples should not be construed as limiting the scope of the invention in any manner.
EXAMPLES
Example 1:
Expression and harvest of darbepoetin:
Expression of darbepoetin in mammalian cells was accomplished as described in EP2456871 (A2), which is incorporated herein as reference. Harvested cell culture broth (CCB) was clarified by centrifugation to obtain clarified cell culture broth (CCCB). Thereafter, CCCB was loaded onto an anion exchange chromatography column. The eluate from the anion exchange chromatography (Q Sepharose ™ Fast Flow, GE Healthcare), contained darbepoetin, and was used in Example 2 to 4.
Example 2: Evaluation of darbepoetin from ion-exchange chromatography
The eluate, from the anion-exchange chromatography (Example-I), was loaded onto one or more cation exchange resin (CM Sepharose™ Fast Flow, GE Healthcare) operated in flow-through mode. The flow-through fractions from the cation exchange resin/s, containing darbepoetin, were collected, pooled and named 2A. Sample from pooled fraction 2A was analyzed as per Example 5 and its protease mediated darbepoetin degradation profile is represented in figure 1.
The fraction 2A was subjected to tangential flow filtration. The concentrate generated from the filtration step was loaded onto another cation exchange step, also operated in flow through mode. The flow-through fraction collected from this cation exchange step was thereafter loaded onto a mixed mode chromatography (Capto™ Adhere, GE Healthcare) operated in bind-elute mode. The eluate from the mixed mode chromatography containing purified darbepoetin composition was analysed for yield i.e., milligram of darbepoetin per litre of CCCB, and HCD values, using RT-PCR. The values are represented as “Darbepoetin composition – I” in Table-II.

Example 3: Evaluation of darbepoetin from Capto blue resin chromatography
The eluate, from anion-exchange chromatography (Example 1), was buffer exchanged with the equilibration buffer to prepare the load for Capto blue chromatographic resin (Capto ™ Blue, GE Healthcare). Three different equilibration buffers, EQB-I-III, were evaluated for Capto blue chromatography. The composition of EQB-I-III is given below in Table-I.
The prepared load was introduced onto Capto blue chromatographic resin using either of the equilibration buffers-EQB-I-III. Darbepoetin was eluted using an elution buffer (50mM Tris + 2M NaCl, pH of 7.4). The eluate, containing darbepoetin, were named as 3A, 3B, and 3C, corresponding to the use of different equilibration buffers EQB-I-III respectively. The eluates 3A, 3B and 3C were evaluated as described in Example 5 and their protease mediated darbepoetin degradation profile is represented in figure 2, 3, and 4 respectively.
Name of Equilibration buffer Composition of equilibration buffer
EQB-I 50 mM sodium acetate + 5mM CaCl2, pH 6.0
EQB-II 50 mM sodium acetate + 5mM CaCl2, pH 6.5
EQB-III 50 mM Tris + 0.2M NaCl, pH 7.4
Table-I: Details of equilibration buffers as evaluated in Example 3

Example 4: Evaluation of darbepoetin from IMAC chromatography
Example 4A: Run conditions for IMAC
The column used for IMAC chromatography was Chelating Sepharose™ Fast Flow (GE Healthcare) and Iminodiacetic acid (IDA) was the ligand. The column was subjected to metal charging with 5 column volume (CV) of 0.025 M CuSO4.5 H2O solution. This was followed by removal of unbound metal ions using 5CV of 0.05 M Sodium acetate and 0.5 M NaCl of pH 4.0. Post loading of the sample onto IMAC column, 5CV of equilibration buffer was passed through the column. The elution was done with 5CV of elution buffer. The last step was removal of metal ions and regeneration of column which was done by passing 3CV of 0.1 M EDTA.
Example 4B
The eluate from the anion-exchange chromatography (Example-I), was buffer exchanged with the equilibration buffer, EQB-IV (83.4 mM NaOAc, 0.2 M NaCl, pH 4.8,) to prepare load for IMAC. The prepared load was introduced onto an IMAC column using equilibration buffer-EQB-IV. The run conditions are as disclosed in Example 4A. The column was washed using equilibration buffer-EQB-IV. Following the wash, the column was eluted for remnants using an elution buffer, 50mM sodium acetate, 0.2 M NaCl having a pH of 4.0. Fractions containing darbepoetin were obtained during load and post load wash, and these fractions were pooled and named as 4B. The pooled fraction 4B was analyzed as per example 5 and its protease mediated darbepoetin degradation profile is represented in figure 5.

Example 4C
The eluate from the anion-exchange chromatography (Example-I), was buffer exchanged with equilibration buffer, EQB-V (50 mM Tris, 0.2 M NaCl, pH 7.4) to prepared load for IMAC. Alternatively, the eluate from the anion-exchange chromatography (Example-I), was neutralized to pH of 7.4 using 1 M Tris buffer having a pH of 9.0 to prepare load or IMAC. Each of the prepared load was individually introduced onto IMAC column using EQB-V. The run conditions are as disclosed in Example 4A. The column was washed using equilibration buffer EQB-V. Following the wash, the column was eluted for remnants using an elution buffer, 50mM sodium acetate, 0.2 M NaCl having a pH of 4.0. Fractions containing darbepoetin were obtained during load and post load wash, and these fractions were pooled and named as 4C-I-II, corresponding to buffer exchanged load and neutralized load respectively. The pooled fractions 4C-I and 4C-II were analyzed as per example 5 and their protease mediated darbepoetin degradation profile is represented in figure 6 and 7 respectively.
Example 4D
The eluate, from the anion-exchange chromatography (Example 1), was neutralized to a pH of 7.4 with 1M Tris and its conductivity adjusted in the range of 45 to 52 mS/cm using 2M NaCl, to prepare load for IMAC. The prepared load was loaded introduced onto an IMAC column using equilibration buffer-EQB-VI (50 mM Tris-HCl +0.5M NaCl). The run conditions are as disclosed in Example 4A. The column was washed using equilibration buffer EQB-VI. Following the wash, the column was eluted for remnants using an elution buffer, 50mM sodium acetate 50mM sodium acetate + 0.5 M NaCl having a pH of 4.0. Fractions containing darbepoetin were obtained during load and post load wash, and these fractions were pooled and named as 4D. The pooled fractions 4D was analyzed as per example 5 and its protease mediated darbepoetin degradation profile is represented in figure 8.

Example 4E
The eluate from the anion-exchange chromatography (example 1) was loaded onto a cation exchange chromatography and the flow-through fractions were collected. The flow-through fractions obtained from cation exchange chromatography were subjected to tangential flow filtration using equilibration buffer EQB VI (50 mM Tris-HCl +0.5M NaCl having a pH of 7.4) to prepared IMAC load. The prepared load was thereafter introduced onto an IMAC column using EQB-VI. The run conditions are as disclosed in Example 4A. The column was washed using equilibration buffer EQB VI. Following the wash, the column was eluted for remnants using an elution buffer, 50mM sodium acetate 50mM sodium acetate + 0.5 M NaCl having a pH of 4.0. Fractions containing darbepoetin were obtained during load and post load wash, and these fractions were pooled and named as 4E. The pooled fraction 4E was analyzed as per example 5 and its protease mediated darbepoetin degradation profile is represented in figure 9.
Example 4F
The pooled fractions obtained from Example 4D and Example 4E were separately processed to further purification scheme. The fraction was subjected to tangential flow filtration. The concentrate so generated from the filtration step was then loaded onto a cation exchange step operated in flow through mode. The flow-through fraction collected from the cation exchange step is thereafter loaded onto a mixed mode chromatography operated in bind-elute mode. The eluate from the mixed mode chromatography containing purified darbepoetin composition was analysed for yield i.e., milligram of darbepoetin per litre of CCCB, and HCD values, using RT-PCR. The values are represented as “Darbepoetin composition – II” and “Darbepoetin composition – III”, corresponding to fraction 4D and 4E respectively, in Table-II.

Darbepoetin composition name Yield
(mg/L of CCCB) HCD (pg/mg)
I 1.1 89
II 5.5 Not detected
III 4.8 Not detected
Table-II
Example 5: Analysis of residual protease activity
The flow-through or eluted fractions containing darbepoetin, from Examples 2 to 4, were evaluated for residual protease activity. The proteases, if present, get activated under acidic pH and degrade the darbepoetin. The degradants of darbepoetin, so generated due to proteases, elute earlier than intact darbepoetin when analysed by reverse phase chromatography.
The fractions, from Examples 2 to 4, were buffer exchanged with 83.4 mM sodium acetate pH 3.3; using 10 kDa cut off membrane Amicon centrifugal filter. The buffer exchanged fractions were incubated at 37 °C for different time i.e., 0 h, 3 h, 24 h, 48 h, to study the kinetics of protease mediated degradation of darbepoetin. Reverse phase chromatography was then performed corresponding to each incubation time.
,CLAIMS:CLAIMS
We claim:
1. A process for removal of cell substrate-derived impurities from a darbepoetin composition, the process comprising an immobilized metal affinity chromatography (IMAC) used in flow-through mode.
2. The process according to claim 1, wherein the cell substrate-derived impurities comprises of host cell protein (HCP) and/or host cell DNA (HCD).
3. The process according to claim 1, wherein the cell substrate-derived impurities comprises of proteases and/or host cell DNA (HCD)
4. The process according to claim 1, wherein the IMAC step results in darbepoetin compositions wherein, there is no protease mediated degradation in acidified darbepoetin samples even after 24 hours of incubation.
5. The process according to claim 1, wherein the process results in removal of HCD below detectable levels.
6. The process according to claim 1, wherein darbepoetin composition yield is increased by about 5 times as compared to yield of darbepoetin composition obtained using a process not comprising an IMAC.
7. The process according to claim 1, wherein the IMAC comprises use of equilibration buffer which comprises of Tris buffer and salt at concentration greater than 0.5 molar.
8. The process according to claim 1, wherein the IMAC comprises chelated transitional metal ion as copper ion.
9. The process according to claim 1, wherein the process comprise other chromatographic steps or filtration steps preceding or following IMAC.
10. The process according to claim 1, wherein darbepoetin composition is produced by Chinese Hamster Ovary (CHO) cells.