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Viral Clearance Using Traditional, Well-Understood Unit Operations (Session I): Anion Exchange Chromatography (AEX) David Roush
PDA J Pharm Sci and Tech 2014, 68 23-29 Access the most recent version at doi:10.5731/pdajpst.2014.00963
Downloaded from journal.pda.org on November 20, 2015
CONFERENCE PROCEEDING – ARTICLE
Viral Clearance Using Traditional, Well-Understood Unit Operations (Session I): Anion Exchange Chromatography (AEX) DAVID ROUSH, Ph.D. Merck, Sharp, and Dohme, BioProcess Development, 2015 Galloping Hill Road, Mailstop K15-2-H206, Kenilworth, New Jersey 07033 ©PDA, Inc. 2014 Background Anion-exchange chromatography is often used as a polishing step in the purification of monoclonal antibodies (mAbs) and is typically operated in flowthrough (FT) mode (1–3) where the mAb does not bind to the resin while trace impurities (e.g., host cell proteins, DNA, and aggregates) bind to the resin. Based on data collected to date, partitioning of virus and impurities can be predicted based on electrostatic interactions (4). For example, DNA and virus bind more strongly than mAb to the anion exchange chromatography (AEX) resin and are retained on the column in FT mode (5). Higher log reduction values (LRVs) are achieved when there is a significant difference in pI between the mAb and the virus (4). Process performance seems generally to be effective (LRV ⬎4) and robust when operated within a bracketed set of operating conditions for AEX for viral clearance (4). However, there are also examples suggesting that interactions with impurities may make clearance of viruses more complex. Session Overview The common theme among presentations was the ability to reliably achieve effective (⬎4 LRV) retroviral clearance with AEX, further supporting the recommendation of a “generic” viral clearance claim if a constrained operating space is employed (4). Consistent with presentations at the 2009 Symposium, the levels and types of impurities present in the feedstock to the AEX step appear to have an impact on the LRV achieved. This also suggests that the LRV achieved for AEX could be influenced by the position
doi: 10.5731/pdajpst.2014.00963
Vol. 68, No. 1, January-February 2014
of the AEX step in the process. For example, if the AEX step is performed as the second chromatography operation, following the initial affinity (protein A) step, a higher level of host cell DNA (HCDNA) and host cell protein (HCP) would be expected to be present in the feedstock than if AEX is the third or final polishing purification step. Also discussed at the 2011 Symposium was the hypothesis that DNA can compete with virus for binding sites on the surface of the AEX and that the increased levels of DNA would thus correlate with reduced virus binding capacity and reduced LRV. Both DNA length and relative concentration to other impurities (e.g., HCP) and virus could therefore affect the degree of LRV achieved. Although DNA levels are typically reported as a concentration (e.g., mg/L, ppm) and determined via PicoGreen or quantitative polymerase chain reaction (Q-PCR), also discussed was how the length in addition to concentration of DNA could affect its relative diffusivity and thus AEX binding capacity. Specifically, the size of DNA present can affect the relative rejection of the DNA in the AEX step. DNA length could also impact the partitioning of the DNA to the surface versus pores. A literature precedent exists for the impact of pore size and bead size on DNA partitioning for a limiting (single) case of very large plasmid DNA (4000 Å) where capacity is in the range of 0.74 mg/mL resin for a 1000 Å pore size (6). The impact of pore and bead size on virus partitioning is also reported in the literature (7, 8) for Q-Sepharose Fast Flow (QSFF). There is a potential to overcome this challenge via the use of an AEX membrane absorber. For example, capacity for plasmid DNA was increased substantially (10⫻, to ⬃10 mg/mL membrane) when an AEX membrane was employed versus AEX chromatography (9). AEX membrane chromatography is 23
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an emerging unit operation (10 –12) for viral clearance and is explored in more detail later in this issue (see Hubbard, Emerging Unit Operations). In addition to the competitive binding hypothesis, there is also literature precedent (13, 14) that high levels of DNA in the feedstock to the AEX step, especially in the case of low viability bioreactor harvest, could also result in a DNA/mAb complex. This complex could have significantly different partitioning behavior on the AEX column and therefore reduced resolution versus the virus. Recent Insight from Paul-Ehrlich-Institut (J. Blu¨mel, S. A. Baylis, F. Neske, and A. Stu¨hler) At the previous meeting (4), parameters were defined for robust clearance of murine leukemia virus (MuLV) for AEX chromatography (i.e., pH 7.0 – 8.5, conductivity ⬍14 mS/cm, effective loading ⬍100 mg/mL, and resin re-use less than 50 times). These data warranted a review of the impact of buffer/resin combinations on the clearance of small non-enveloped viruses. One recent study, by a manufacturer, has evaluated the design space for effective clearance of the non-enveloped viruses simian virus 40 (SV40) and minute virus of mice (MMV) by non-binding Q-Sepharose Fast Flow chromatography using conditions that encompass those outlined above (15). Following the discussion from the previous Symposium, the viral clearance database from clinical trial applications in Germany was reviewed at the PaulEhrlich-Institut with respect to clearance of non-enveloped viruses (MMV and PPV) and the postulated parameters. The analysis was limited to antibody purification incorporating Q Sepharose FF (AEX chromatography) in the non-binding mode. This specific AEX step seemed to result in most cases in robust clearance of non-enveloped viruses. Effective clearance (defined as an LRV of at least 4 log10) of PPV or MMV, was reported for 51 (82%) out of 62 different antibodies. In 11 (18%) cases, parvovirus clearance was below 4 log10. The individual results and the process parameters studied are summarized in Table I. In some cases, the limited viral clearance may be due to a pH below 7.0 or a high ionic strength, as would be expected with phosphate-buffered saline (PBS). In other cases, however, the limited clearance was not easily explained by deviations from the postulated parameters or else information concern24
ing some or all of these parameters was unavailable for review. Since impurities from the product intermediate might also influence viral clearance, the position of the chromatographic steps in the purification process was also reviewed. In four out of the 11 cases where reduced parvovirus clearance was observed, the non-binding Q-Sepharose step immediately followed an initial protein A affinity chromatography operation, while in six other cases, a Bind and Elute (BE) mode chromatography operation (Sulfopropyl SP) Sepharose (Cation Exchange Chromatography) or ceramic hydroxyapatite chromatography, was placed between the initial protein A step and the non-binding Q Sepharose. In six out of 11 cases with limited parvovirus clearance, effective clearance (ⱖ4 log10) of the model retrovirus was demonstrated; these observations suggest that parvovirus binding is weaker than retrovirus binding. In one case (Table I, no. 4), retroviral clearance was not investigated due to the presence of detergent in the product intermediate. Using High-Throughput Screening (HTS) To Define AEX Design Space for MuLV Clearance (Lisa Connell-Crowley, Amgen) Amgen presented a 96 well HTS method for screening binding of MuLV to Q-Sepharose FF, a resin with established viral clearance capabilities (3). Batch binding HTS has been successfully used to identify operating spaces on chromatography resins for removal of impurities such as product aggregates, host cell protein, and DNA (16). Batch binding HTS is advantageous because it can be used to evaluate multiple conditions simultaneously in a 96 well plate format with minimal mass requirements. Experiments were performed to determine the feasibility of this technique for identifying key operating parameters for virus removal by chromatography resins. The AEX resin Q Sepharose Fast Flow (QSFF) was dispensed into 24 wells of a 96 well filter plate (0.45 m pore size) and equilibrated at pH 5, 6, 7, or 8 with sodium chloride levels ranging from 25 to 400 mM. Each well was then spiked with 1% MuLV in the corresponding pH/salt buffer. After agitation for 60 min, the unbound material was collected by centrifugation and analyzed for MuLV by Q-PCR. Figure 1 shows the impact of pH and salt levels on MuLV binding, quantified as MuLV log10 LRV in the unbound fraction. Consistent with previous observations PDA Journal of Pharmaceutical Science and Technology
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Table I Summary of Paul Erlich Institut Analysis of Antibody Regulatory Dossiers Where no Effective Reduction of Animal Parvoviruses Was Observed by Non-Binding Q-Sepharose Chromatography
No.
mAb pI
pH a
Conductivity or Load Buffer Composition ?
a
Column Load (g mAb/L resin) ?
a
Temp (°C) RT
d
Stepb
Parvovirus LRVf
Retrovirus LRVf ⱖ6.7; ⱖ6.8 X-MuLV
1
8.6–9.0
?
3
3.5; 3.4 PPV
2
6.8–7.4
5.3
10.7 mS/cm
45
?a
3
2.0; 1.9 PPV
6.39; 6.19 X-MuLV
3
5.8–6.9
8.0
25 mM Tris.Cl 250 mM NaCl
20
RTd
2
2.0 PPV
ⱖ 4.39 E-MuLV
4
4.5–6.4
7.0
180 mM NaCl 20 mM Na-phosphate
?a
RTd
1
1.4 MVM
n.d.g
5
9.0
7.5
50 mM Tris 50 mM NaCl 100-225mM Na-acetate
41
15-26
3
3.1; 4.3 MVM
ⱖ4.66 X-MuLV
6
8.7–9.1
8.5
Trish
97
RTd
2
1.0; 0.9 PPV
ⱖ3.6; ⱖ3.9 X-MuLV
7
?a
7.5
5–7 mS/cm 20 mM Tris 17 mM acetic acid 62 mM NaCl
50
16–25
2
0.9 MVM
2.3 X-MuLV
8
7.6
5.9–6.5
?a
59.8
?a
3
0.0; 0.2 MVM
2.9; 4.0 X-MuLV
9
9.5
8.0
140 mM Na-acetate 20 mM MESe
40
17 ⫾ 2.0
3
ⱕ1.0; ⱕ1.0
ⱖ4.8 X-MuLV
10
7.4–8.3
6.5
15 mM Na-phosphate 140 mM NaCl
5–20
RTd
3
1.9; 1.8 MVM
ⱖ4.9 ; ⬎6.7 X-MuLV
11
8.7–9.2
7.4
PBSc
?a
RTd
2
ⱕ1.0; ⱕ1.0 MVM
2.6; 2.7 E-MuLV
a
Information was not provided in the investigational medicinal product dossier. b Sequential number of chromatographic step at the purification process. c PBS: polyphosphate buffered saline. d RT: room temperature. e MES: 2-(n-morpholino) ethanesulfonic acid. f log10 reduction factors from single runs or double runs and model virus. g n.d.: not determined. h Elution intermediate from protein A chromatography was adjusted for pH with Tris base. (4), MuLV binds to QSFF in a salt-dependent manner, with the most binding and thus highest LRV at lowsalt conditions. Surprisingly, pH appeared to have only a small effect on MuLV binding, and binding was observed even at pH as low as 5.0. In contrast, no virus binding was observed during studies using the Sepharose base matrix, indicating that the ligand is required for virus binding. A small amount of virus retention is observed at pH 5.0 at low-salt conditions with and without the presence of the base matrix, suggesting that the virus may be slightly retained by the 0.45 m filter of the filter plate under those conditions. The conclusion from these studies was that MuLV binding was a stronger function of salt (range from 0 to 400 mM NaCl) than pH (range from pH 5 to 8; Figure 1). Data confirm that the primary mechanism for MuLV is electrostatically mediated, as based on comparison of the impact of Q ligand (4). Figure 2 shows the impact of a mAb load containing various levels of impurities on the binding of MuLV to Vol. 68, No. 1, January-February 2014
the resin. While relatively pure mAb appears to have little effect, MuLV binding is significantly reduced in the presence of increased impurities levels, suggesting that one or more impurities may be competing with MuLV for binding. Previously published data has implicated DNA as one of the potential competitors (4), although others may be involved. A comparison of LRV from batch binding and column runs was performed to evaluate whether the batch binding format was predictive of the column format. It was found that similar LRVs were observed between the batch binding and column formats with respect to the effect of salt, pH, and impurity level. These data demonstrate that batch binding HTS is a feasible tool for screening conditions for virus binding to resin. While batch binding experiments cannot provide information on column-specific parameters such as resin bed height and flow rate, they can provide relevant information on other parameters important for viral clearance including the effect of pH, salt level and type, product and impurity load25
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ing, and resin type for one or more viruses. The ability to rapidly screen and identify key parameters and parameter ranges for virus clearance on chromatography resin can aid the definition of robust viral clearance operating window. Impact of Cell Culture and Harvest Changes on One Agarose-Based Strong Ion Exchange Chromatography Method (John Mattila, Regeneron) Regeneron presented a case study on the impact of cell culture and harvest changes on the performance of an agarose-based strong ion exchange chromatography method (the specific brand of media used in this study was not disclosed).
Figure 1 Amgen study of the impact of pH and salt on XMuLV binding to Q Sepharose Fast Flow and naked Sepharose resin using batch binding HTS. Log reduction value (LRV) was determined by using XMuLV QRTPCR on the load and unbound material.
Process parameters influencing retrovirus removal by FT-AEX have previously been extensively evaluated and reported to support a design space that may provide effective viral clearance within a mAb purification platform. One potential knowledge gap is the role of residual process-related impurities that could be affected by upstream process changes. To illustrate this concern, a case study has reported varied virus removal by one AEX method using two developmental feedstocks for mAb X (Figure 3).
Figure 2 Amgen study of the impact of different MAb loads with variable levels of impurities on XMuLV binding to QSFF. 26
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note that the virus breakthrough at 120 g/L is higher than the proposed bracketed operating range of ⬍100 g/L. While process conditions allowed electrostatic interactions that drive removal of virus by AEX, it is speculated the AEX column was subject to saturation by an unidentified negatively charged impurity. The case study highlights the need for prospective studies to account for upstream changes that may alter feedstock impurity composition. One potential explanation, consistent with literature precedent, is that virus partitioning is more strongly affected as the relative level of process residuals and resin loading is increased. Summary
Figure 3 Regeneron case study of MMV (A) and X-MuLV (B) spike/removal studies with model monoclonal antibody X. The study used identical anion exchange media but different feedstocks (X and X’). “*” denotes viruses reduced below assay limit of detection (LOD).
Each feedstock had been previously purified using a platform affinity capture method, whereas the mAb process intermediate had been produced by two different cell lines and harvest methods. Clearance was evaluated for a model retrovirus (MuLV) and a model parvovirus (MMV). Tests with identical AEX media, contact time, bed height, buffers, and antibody resulted in varied capacity of 120 g/L resin for one feedstock and ⬎150 g/L for another. When virus breakthrough was observed for one feedstock (Figure 3), it corresponded roughly with DNA breakthrough. AEX was performed under conditions generally regarded as robust. Specifically, parameters expected to influence electrostatic interactions (e.g., pH, conductivity, buffer system) were identical during tests of both feedstocks and were within published ranges, providing robust clearance for a variety of model viruses. It is important to Vol. 68, No. 1, January-February 2014
Follow-up experimental data presented at the 2011 Symposium confirm that AEX provides effective viral clearance for a range of viruses when operated under the proposed bracketed operating conditions (4). Progress has been made in addressing the gaps identified at the 2009 Symposium, such as the extension of the bracketed viral clearance operating space across mAbs, process conditions, and virus species. Researchers have also subsequently reported that a quality by design (QbD) approach can be applied to viral clearance of MuLV, SV40, and MMV (15). However, overall capacity for virus or indirectly the LRV can be affected by the DNA levels in the feedstock to the AEX step, especially when AEX is the second chromatography (i.e., first polishing) step. Position in the purification process is an important factor, as competitive adsorption from process residuals such as DNA and HCP, which were shown to affect binding capacity to virus, would be at elevated levels when AEX is the second versus third chromatography (i.e., second polishing) step. One potential approach is to define AEX conditions by capacity for impurities as opposed to effective mAb capacity. Challenges remain in that capacity will vary depending on the nature of the impurities (e.g., DNA length). Impurity load is a de facto critical process parameter (CPP) for this unit operation because it can affect virus LRV via competitive adsorption based on a combination of presentations at the 2009 and 2011 Symposia and literature precedent. AEX viral clearance has been demonstrated to be robust for mAb loadings ⬍100 mg/mL, but decreased 27
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LRVs were observed between 100 and 200 mg/mL mAb loadings, in some cases where process residuals (HCP and DNA) are elevated. This observation was further supported by HTS data indicating a decrease in LRV when HCP and DNA process residuals were significantly increased to 45⫻ and 5⫻, respectively. A 2009 Symposium presentation (P. Mensah, Pfizer) suggested a correlation between reduced LRV and increasing DNA levels bound to the resin. Specifically, the LRV for MuLV (Figure 22 in reference 4) dropped below 4 log10 when DNA exceeded ⬃80 ng/mL loading on the resin. These data suggest that there may be a threshold level of process residuals bound to the resin where virus LRV is subsequently reduced. Below this threshold, robust and effective viral clearance is achieved. Further investigation into the impact of high process residuals levels combined with high mAb loadings on virus LRV for AEX could provide further insights into the outliers discussed at the 2011 Symposium. Additional experiments (e.g., potential DoE-based) to decouple the impact of HCP versus DNA concentrations, combined with HTS methodologies and additional characterization of DNA (e.g., influence of length), could provide further insight into competitive adsorption mechanisms. DNA breakthrough experiments performed using a DoE approach as a function of operating conditions in the presence of virus spike would be particularly useful. Literature precedent and data reviewed at the Symposium suggest that characterization of the impurities in an AEX feedstock is important for ensuring robust process performance, and is particularly critical where outliers, such as those observed in the analysis by Paul-Ehrlich-Institut (J. Blu¨ mel), are observed. References 1. Follman, D. K.; Fahrner, R. L. Factorial screening of antibody purification processes using three chromatography steps without protein A. J. Chromatogr., A 2004, 1024 (1), 79 – 85. 2. Ishihara, T.; Kadoya, T. Accelerated purification process development of monoclonal antibodies for shortening time to clinic. Design and case study of chromatography processes. J. Chromatogr., A 2007, 1176 (1), 149 –156. 28
3. Norling, L.; Lute, S.; Emery, R.; Khuu, W.; Voisard, M.; Xu, Y.; Chen, Q.; Blank, G.; Brorson, K. Impact of multiple re-use of anion-exchange chromatography media on virus removal. J. Chromatogr., A 2005, 1069 (1), 79 – 89. 4. Miesegaes, G.; Bailey, M.; Willkommen, H.; Chen, Q.; Roush, D.; Blu¨mel, J.; Brorson, K. Proceedings of the 2009 Viral Clearance Symposium. Dev. Biol. (Basel) 2010, 133, 3–101. 5. Knudsen, H. L.; Fahrner, R. L.; Xu, Y.; Norling, L. A.; Blank, G. S. Membrane ion-exchange chromatography for process-scale antibody purification. J. Chromatogr., A 2001, 907 (1), 145–154. 6. Sagar, S. L.; Chau, T. G.; Watson, M. P.; Lee, A. L. Chapter 8. Case Study: Capacity Challenges in Chromotography-Based Purification of Plasmid DNA. In Scale-Up and Optimization in Preparative Chromatography: Principles and Biopharmaceutical Applications, Rathore, A., Velayudhan, A., Eds.; Marcel Dekker, Inc.: New York, 2003. 7. Ferreira, G. N.; Cabral, J. M.; Prazeres, D. M. Studies on the batch adsorption of plasmid DNA onto anion-exchange chromatographic supports. Biotechnol. Prog. 2000, 16 (3), 416 – 424. 8. Iyer, G.; Ramaswamy, S.; Asher, D.; Mehta, U.; Leahy, A.; Chung, F.; Cheng, K. S. Reduced surface area chromatography for flow-through purification of viruses and virus like particles. J. Chromatogr., A 2011, 1218 (26), 3973–3981. 9. Teeters, M. A.; Conrardy, S. E.; Thomas, B. L.; Root, T. W.; Lightfoot, E. N. Adsorptive membrane chromatography for purification of plasmid DNA. J. Chromatogr., A 2003, 989 (1), 165–173. 10. Phillips, M.; Cormier, J.; Ferrence, J.; Dowd, C.; Kiss, R.; Lutz, H.; Carter, J. Performance of a membrane adsorber for trace impurity removal in biotechnology manufacturing. J. Chromatogr., A 2005, 1078 (1), 74 – 82. 11. Zhou, J. X.; Tressel, T. Basic concepts in Q membrane chromatography for large-scale antibody production. Biotechnol. Prog. 2006, 22 (2), 341–349. 12. Zhou, J. X.; Tressel, T.; Gottschalk, U.; Solamo, F.; Pastor, A.; Dermawan, S.; Hong, T.; Reif, O.; PDA Journal of Pharmaceutical Science and Technology
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Mora, J.; Hutchison, F.; Murphy, M. New Q membrane scale-down model for process-scale antibody purification. J. Chromatogr., A 2006, 1134 (1), 66 –73. 13. Gagnon, P.; Hensel, F.; Lee, S.; Zaidi, S. Chromatographic behavior of IgM:DNA complexes. J. Chromatogr., A 2011, 1218 (1), 2405–2412. 14. Luhrs, K. A.; Harris, D. A.; Summers, S.; Parseghian, M. H. Evicting hitchhiker antigens from purified antibodies. J. Chromatogr., B 2009, 877 (14), 1543–1552.
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15. Strauss, D. M.; Cano, T.; Cai, N.; Delucchi, H.; Plancarte, M.; Coleman, D.; Blank, G. S.; Chen, Q.; Yang, B. Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production. Biotechnol. Prog. 2010, 26 (3), 750 –755. 16. Kelley, B. D.; Switzer, M.; Bastek, P.; Kramarczyk, J. F.; Molnar, K.; Yu, T.; Coffman, J. Highthroughput screening of chromatographic separations: IV. Ion-exchange. Biotechnol. Bioeng. 2008, 100 (5), 950 –963.
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Viral Clearance Using Traditional, Well-Understood Unit Operations (Session I): Anion Exchange Chromatography (AEX) David Roush
PDA J Pharm Sci and Tech 2014, 68 23-29 Access the most recent version at doi:10.5731/pdajpst.2014.00963
Downloaded from journal.pda.org on November 20, 2015
CONFERENCE PROCEEDING – ARTICLE
Viral Clearance Using Traditional, Well-Understood Unit Operations (Session I): Anion Exchange Chromatography (AEX) DAVID ROUSH, Ph.D. Merck, Sharp, and Dohme, BioProcess Development, 2015 Galloping Hill Road, Mailstop K15-2-H206, Kenilworth, New Jersey 07033 ©PDA, Inc. 2014 Background Anion-exchange chromatography is often used as a polishing step in the purification of monoclonal antibodies (mAbs) and is typically operated in flowthrough (FT) mode (1–3) where the mAb does not bind to the resin while trace impurities (e.g., host cell proteins, DNA, and aggregates) bind to the resin. Based on data collected to date, partitioning of virus and impurities can be predicted based on electrostatic interactions (4). For example, DNA and virus bind more strongly than mAb to the anion exchange chromatography (AEX) resin and are retained on the column in FT mode (5). Higher log reduction values (LRVs) are achieved when there is a significant difference in pI between the mAb and the virus (4). Process performance seems generally to be effective (LRV ⬎4) and robust when operated within a bracketed set of operating conditions for AEX for viral clearance (4). However, there are also examples suggesting that interactions with impurities may make clearance of viruses more complex. Session Overview The common theme among presentations was the ability to reliably achieve effective (⬎4 LRV) retroviral clearance with AEX, further supporting the recommendation of a “generic” viral clearance claim if a constrained operating space is employed (4). Consistent with presentations at the 2009 Symposium, the levels and types of impurities present in the feedstock to the AEX step appear to have an impact on the LRV achieved. This also suggests that the LRV achieved for AEX could be influenced by the position
doi: 10.5731/pdajpst.2014.00963
Vol. 68, No. 1, January-February 2014
of the AEX step in the process. For example, if the AEX step is performed as the second chromatography operation, following the initial affinity (protein A) step, a higher level of host cell DNA (HCDNA) and host cell protein (HCP) would be expected to be present in the feedstock than if AEX is the third or final polishing purification step. Also discussed at the 2011 Symposium was the hypothesis that DNA can compete with virus for binding sites on the surface of the AEX and that the increased levels of DNA would thus correlate with reduced virus binding capacity and reduced LRV. Both DNA length and relative concentration to other impurities (e.g., HCP) and virus could therefore affect the degree of LRV achieved. Although DNA levels are typically reported as a concentration (e.g., mg/L, ppm) and determined via PicoGreen or quantitative polymerase chain reaction (Q-PCR), also discussed was how the length in addition to concentration of DNA could affect its relative diffusivity and thus AEX binding capacity. Specifically, the size of DNA present can affect the relative rejection of the DNA in the AEX step. DNA length could also impact the partitioning of the DNA to the surface versus pores. A literature precedent exists for the impact of pore size and bead size on DNA partitioning for a limiting (single) case of very large plasmid DNA (4000 Å) where capacity is in the range of 0.74 mg/mL resin for a 1000 Å pore size (6). The impact of pore and bead size on virus partitioning is also reported in the literature (7, 8) for Q-Sepharose Fast Flow (QSFF). There is a potential to overcome this challenge via the use of an AEX membrane absorber. For example, capacity for plasmid DNA was increased substantially (10⫻, to ⬃10 mg/mL membrane) when an AEX membrane was employed versus AEX chromatography (9). AEX membrane chromatography is 23
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an emerging unit operation (10 –12) for viral clearance and is explored in more detail later in this issue (see Hubbard, Emerging Unit Operations). In addition to the competitive binding hypothesis, there is also literature precedent (13, 14) that high levels of DNA in the feedstock to the AEX step, especially in the case of low viability bioreactor harvest, could also result in a DNA/mAb complex. This complex could have significantly different partitioning behavior on the AEX column and therefore reduced resolution versus the virus. Recent Insight from Paul-Ehrlich-Institut (J. Blu¨mel, S. A. Baylis, F. Neske, and A. Stu¨hler) At the previous meeting (4), parameters were defined for robust clearance of murine leukemia virus (MuLV) for AEX chromatography (i.e., pH 7.0 – 8.5, conductivity ⬍14 mS/cm, effective loading ⬍100 mg/mL, and resin re-use less than 50 times). These data warranted a review of the impact of buffer/resin combinations on the clearance of small non-enveloped viruses. One recent study, by a manufacturer, has evaluated the design space for effective clearance of the non-enveloped viruses simian virus 40 (SV40) and minute virus of mice (MMV) by non-binding Q-Sepharose Fast Flow chromatography using conditions that encompass those outlined above (15). Following the discussion from the previous Symposium, the viral clearance database from clinical trial applications in Germany was reviewed at the PaulEhrlich-Institut with respect to clearance of non-enveloped viruses (MMV and PPV) and the postulated parameters. The analysis was limited to antibody purification incorporating Q Sepharose FF (AEX chromatography) in the non-binding mode. This specific AEX step seemed to result in most cases in robust clearance of non-enveloped viruses. Effective clearance (defined as an LRV of at least 4 log10) of PPV or MMV, was reported for 51 (82%) out of 62 different antibodies. In 11 (18%) cases, parvovirus clearance was below 4 log10. The individual results and the process parameters studied are summarized in Table I. In some cases, the limited viral clearance may be due to a pH below 7.0 or a high ionic strength, as would be expected with phosphate-buffered saline (PBS). In other cases, however, the limited clearance was not easily explained by deviations from the postulated parameters or else information concern24
ing some or all of these parameters was unavailable for review. Since impurities from the product intermediate might also influence viral clearance, the position of the chromatographic steps in the purification process was also reviewed. In four out of the 11 cases where reduced parvovirus clearance was observed, the non-binding Q-Sepharose step immediately followed an initial protein A affinity chromatography operation, while in six other cases, a Bind and Elute (BE) mode chromatography operation (Sulfopropyl SP) Sepharose (Cation Exchange Chromatography) or ceramic hydroxyapatite chromatography, was placed between the initial protein A step and the non-binding Q Sepharose. In six out of 11 cases with limited parvovirus clearance, effective clearance (ⱖ4 log10) of the model retrovirus was demonstrated; these observations suggest that parvovirus binding is weaker than retrovirus binding. In one case (Table I, no. 4), retroviral clearance was not investigated due to the presence of detergent in the product intermediate. Using High-Throughput Screening (HTS) To Define AEX Design Space for MuLV Clearance (Lisa Connell-Crowley, Amgen) Amgen presented a 96 well HTS method for screening binding of MuLV to Q-Sepharose FF, a resin with established viral clearance capabilities (3). Batch binding HTS has been successfully used to identify operating spaces on chromatography resins for removal of impurities such as product aggregates, host cell protein, and DNA (16). Batch binding HTS is advantageous because it can be used to evaluate multiple conditions simultaneously in a 96 well plate format with minimal mass requirements. Experiments were performed to determine the feasibility of this technique for identifying key operating parameters for virus removal by chromatography resins. The AEX resin Q Sepharose Fast Flow (QSFF) was dispensed into 24 wells of a 96 well filter plate (0.45 m pore size) and equilibrated at pH 5, 6, 7, or 8 with sodium chloride levels ranging from 25 to 400 mM. Each well was then spiked with 1% MuLV in the corresponding pH/salt buffer. After agitation for 60 min, the unbound material was collected by centrifugation and analyzed for MuLV by Q-PCR. Figure 1 shows the impact of pH and salt levels on MuLV binding, quantified as MuLV log10 LRV in the unbound fraction. Consistent with previous observations PDA Journal of Pharmaceutical Science and Technology
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Table I Summary of Paul Erlich Institut Analysis of Antibody Regulatory Dossiers Where no Effective Reduction of Animal Parvoviruses Was Observed by Non-Binding Q-Sepharose Chromatography
No.
mAb pI
pH a
Conductivity or Load Buffer Composition ?
a
Column Load (g mAb/L resin) ?
a
Temp (°C) RT
d
Stepb
Parvovirus LRVf
Retrovirus LRVf ⱖ6.7; ⱖ6.8 X-MuLV
1
8.6–9.0
?
3
3.5; 3.4 PPV
2
6.8–7.4
5.3
10.7 mS/cm
45
?a
3
2.0; 1.9 PPV
6.39; 6.19 X-MuLV
3
5.8–6.9
8.0
25 mM Tris.Cl 250 mM NaCl
20
RTd
2
2.0 PPV
ⱖ 4.39 E-MuLV
4
4.5–6.4
7.0
180 mM NaCl 20 mM Na-phosphate
?a
RTd
1
1.4 MVM
n.d.g
5
9.0
7.5
50 mM Tris 50 mM NaCl 100-225mM Na-acetate
41
15-26
3
3.1; 4.3 MVM
ⱖ4.66 X-MuLV
6
8.7–9.1
8.5
Trish
97
RTd
2
1.0; 0.9 PPV
ⱖ3.6; ⱖ3.9 X-MuLV
7
?a
7.5
5–7 mS/cm 20 mM Tris 17 mM acetic acid 62 mM NaCl
50
16–25
2
0.9 MVM
2.3 X-MuLV
8
7.6
5.9–6.5
?a
59.8
?a
3
0.0; 0.2 MVM
2.9; 4.0 X-MuLV
9
9.5
8.0
140 mM Na-acetate 20 mM MESe
40
17 ⫾ 2.0
3
ⱕ1.0; ⱕ1.0
ⱖ4.8 X-MuLV
10
7.4–8.3
6.5
15 mM Na-phosphate 140 mM NaCl
5–20
RTd
3
1.9; 1.8 MVM
ⱖ4.9 ; ⬎6.7 X-MuLV
11
8.7–9.2
7.4
PBSc
?a
RTd
2
ⱕ1.0; ⱕ1.0 MVM
2.6; 2.7 E-MuLV
a
Information was not provided in the investigational medicinal product dossier. b Sequential number of chromatographic step at the purification process. c PBS: polyphosphate buffered saline. d RT: room temperature. e MES: 2-(n-morpholino) ethanesulfonic acid. f log10 reduction factors from single runs or double runs and model virus. g n.d.: not determined. h Elution intermediate from protein A chromatography was adjusted for pH with Tris base. (4), MuLV binds to QSFF in a salt-dependent manner, with the most binding and thus highest LRV at lowsalt conditions. Surprisingly, pH appeared to have only a small effect on MuLV binding, and binding was observed even at pH as low as 5.0. In contrast, no virus binding was observed during studies using the Sepharose base matrix, indicating that the ligand is required for virus binding. A small amount of virus retention is observed at pH 5.0 at low-salt conditions with and without the presence of the base matrix, suggesting that the virus may be slightly retained by the 0.45 m filter of the filter plate under those conditions. The conclusion from these studies was that MuLV binding was a stronger function of salt (range from 0 to 400 mM NaCl) than pH (range from pH 5 to 8; Figure 1). Data confirm that the primary mechanism for MuLV is electrostatically mediated, as based on comparison of the impact of Q ligand (4). Figure 2 shows the impact of a mAb load containing various levels of impurities on the binding of MuLV to Vol. 68, No. 1, January-February 2014
the resin. While relatively pure mAb appears to have little effect, MuLV binding is significantly reduced in the presence of increased impurities levels, suggesting that one or more impurities may be competing with MuLV for binding. Previously published data has implicated DNA as one of the potential competitors (4), although others may be involved. A comparison of LRV from batch binding and column runs was performed to evaluate whether the batch binding format was predictive of the column format. It was found that similar LRVs were observed between the batch binding and column formats with respect to the effect of salt, pH, and impurity level. These data demonstrate that batch binding HTS is a feasible tool for screening conditions for virus binding to resin. While batch binding experiments cannot provide information on column-specific parameters such as resin bed height and flow rate, they can provide relevant information on other parameters important for viral clearance including the effect of pH, salt level and type, product and impurity load25
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ing, and resin type for one or more viruses. The ability to rapidly screen and identify key parameters and parameter ranges for virus clearance on chromatography resin can aid the definition of robust viral clearance operating window. Impact of Cell Culture and Harvest Changes on One Agarose-Based Strong Ion Exchange Chromatography Method (John Mattila, Regeneron) Regeneron presented a case study on the impact of cell culture and harvest changes on the performance of an agarose-based strong ion exchange chromatography method (the specific brand of media used in this study was not disclosed).
Figure 1 Amgen study of the impact of pH and salt on XMuLV binding to Q Sepharose Fast Flow and naked Sepharose resin using batch binding HTS. Log reduction value (LRV) was determined by using XMuLV QRTPCR on the load and unbound material.
Process parameters influencing retrovirus removal by FT-AEX have previously been extensively evaluated and reported to support a design space that may provide effective viral clearance within a mAb purification platform. One potential knowledge gap is the role of residual process-related impurities that could be affected by upstream process changes. To illustrate this concern, a case study has reported varied virus removal by one AEX method using two developmental feedstocks for mAb X (Figure 3).
Figure 2 Amgen study of the impact of different MAb loads with variable levels of impurities on XMuLV binding to QSFF. 26
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note that the virus breakthrough at 120 g/L is higher than the proposed bracketed operating range of ⬍100 g/L. While process conditions allowed electrostatic interactions that drive removal of virus by AEX, it is speculated the AEX column was subject to saturation by an unidentified negatively charged impurity. The case study highlights the need for prospective studies to account for upstream changes that may alter feedstock impurity composition. One potential explanation, consistent with literature precedent, is that virus partitioning is more strongly affected as the relative level of process residuals and resin loading is increased. Summary
Figure 3 Regeneron case study of MMV (A) and X-MuLV (B) spike/removal studies with model monoclonal antibody X. The study used identical anion exchange media but different feedstocks (X and X’). “*” denotes viruses reduced below assay limit of detection (LOD).
Each feedstock had been previously purified using a platform affinity capture method, whereas the mAb process intermediate had been produced by two different cell lines and harvest methods. Clearance was evaluated for a model retrovirus (MuLV) and a model parvovirus (MMV). Tests with identical AEX media, contact time, bed height, buffers, and antibody resulted in varied capacity of 120 g/L resin for one feedstock and ⬎150 g/L for another. When virus breakthrough was observed for one feedstock (Figure 3), it corresponded roughly with DNA breakthrough. AEX was performed under conditions generally regarded as robust. Specifically, parameters expected to influence electrostatic interactions (e.g., pH, conductivity, buffer system) were identical during tests of both feedstocks and were within published ranges, providing robust clearance for a variety of model viruses. It is important to Vol. 68, No. 1, January-February 2014
Follow-up experimental data presented at the 2011 Symposium confirm that AEX provides effective viral clearance for a range of viruses when operated under the proposed bracketed operating conditions (4). Progress has been made in addressing the gaps identified at the 2009 Symposium, such as the extension of the bracketed viral clearance operating space across mAbs, process conditions, and virus species. Researchers have also subsequently reported that a quality by design (QbD) approach can be applied to viral clearance of MuLV, SV40, and MMV (15). However, overall capacity for virus or indirectly the LRV can be affected by the DNA levels in the feedstock to the AEX step, especially when AEX is the second chromatography (i.e., first polishing) step. Position in the purification process is an important factor, as competitive adsorption from process residuals such as DNA and HCP, which were shown to affect binding capacity to virus, would be at elevated levels when AEX is the second versus third chromatography (i.e., second polishing) step. One potential approach is to define AEX conditions by capacity for impurities as opposed to effective mAb capacity. Challenges remain in that capacity will vary depending on the nature of the impurities (e.g., DNA length). Impurity load is a de facto critical process parameter (CPP) for this unit operation because it can affect virus LRV via competitive adsorption based on a combination of presentations at the 2009 and 2011 Symposia and literature precedent. AEX viral clearance has been demonstrated to be robust for mAb loadings ⬍100 mg/mL, but decreased 27
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LRVs were observed between 100 and 200 mg/mL mAb loadings, in some cases where process residuals (HCP and DNA) are elevated. This observation was further supported by HTS data indicating a decrease in LRV when HCP and DNA process residuals were significantly increased to 45⫻ and 5⫻, respectively. A 2009 Symposium presentation (P. Mensah, Pfizer) suggested a correlation between reduced LRV and increasing DNA levels bound to the resin. Specifically, the LRV for MuLV (Figure 22 in reference 4) dropped below 4 log10 when DNA exceeded ⬃80 ng/mL loading on the resin. These data suggest that there may be a threshold level of process residuals bound to the resin where virus LRV is subsequently reduced. Below this threshold, robust and effective viral clearance is achieved. Further investigation into the impact of high process residuals levels combined with high mAb loadings on virus LRV for AEX could provide further insights into the outliers discussed at the 2011 Symposium. Additional experiments (e.g., potential DoE-based) to decouple the impact of HCP versus DNA concentrations, combined with HTS methodologies and additional characterization of DNA (e.g., influence of length), could provide further insight into competitive adsorption mechanisms. DNA breakthrough experiments performed using a DoE approach as a function of operating conditions in the presence of virus spike would be particularly useful. Literature precedent and data reviewed at the Symposium suggest that characterization of the impurities in an AEX feedstock is important for ensuring robust process performance, and is particularly critical where outliers, such as those observed in the analysis by Paul-Ehrlich-Institut (J. Blu¨ mel), are observed. References 1. Follman, D. K.; Fahrner, R. L. Factorial screening of antibody purification processes using three chromatography steps without protein A. J. Chromatogr., A 2004, 1024 (1), 79 – 85. 2. Ishihara, T.; Kadoya, T. Accelerated purification process development of monoclonal antibodies for shortening time to clinic. Design and case study of chromatography processes. J. Chromatogr., A 2007, 1176 (1), 149 –156. 28
3. Norling, L.; Lute, S.; Emery, R.; Khuu, W.; Voisard, M.; Xu, Y.; Chen, Q.; Blank, G.; Brorson, K. Impact of multiple re-use of anion-exchange chromatography media on virus removal. J. Chromatogr., A 2005, 1069 (1), 79 – 89. 4. Miesegaes, G.; Bailey, M.; Willkommen, H.; Chen, Q.; Roush, D.; Blu¨mel, J.; Brorson, K. Proceedings of the 2009 Viral Clearance Symposium. Dev. Biol. (Basel) 2010, 133, 3–101. 5. Knudsen, H. L.; Fahrner, R. L.; Xu, Y.; Norling, L. A.; Blank, G. S. Membrane ion-exchange chromatography for process-scale antibody purification. J. Chromatogr., A 2001, 907 (1), 145–154. 6. Sagar, S. L.; Chau, T. G.; Watson, M. P.; Lee, A. L. Chapter 8. Case Study: Capacity Challenges in Chromotography-Based Purification of Plasmid DNA. In Scale-Up and Optimization in Preparative Chromatography: Principles and Biopharmaceutical Applications, Rathore, A., Velayudhan, A., Eds.; Marcel Dekker, Inc.: New York, 2003. 7. Ferreira, G. N.; Cabral, J. M.; Prazeres, D. M. Studies on the batch adsorption of plasmid DNA onto anion-exchange chromatographic supports. Biotechnol. Prog. 2000, 16 (3), 416 – 424. 8. Iyer, G.; Ramaswamy, S.; Asher, D.; Mehta, U.; Leahy, A.; Chung, F.; Cheng, K. S. Reduced surface area chromatography for flow-through purification of viruses and virus like particles. J. Chromatogr., A 2011, 1218 (26), 3973–3981. 9. Teeters, M. A.; Conrardy, S. E.; Thomas, B. L.; Root, T. W.; Lightfoot, E. N. Adsorptive membrane chromatography for purification of plasmid DNA. J. Chromatogr., A 2003, 989 (1), 165–173. 10. Phillips, M.; Cormier, J.; Ferrence, J.; Dowd, C.; Kiss, R.; Lutz, H.; Carter, J. Performance of a membrane adsorber for trace impurity removal in biotechnology manufacturing. J. Chromatogr., A 2005, 1078 (1), 74 – 82. 11. Zhou, J. X.; Tressel, T. Basic concepts in Q membrane chromatography for large-scale antibody production. Biotechnol. Prog. 2006, 22 (2), 341–349. 12. Zhou, J. X.; Tressel, T.; Gottschalk, U.; Solamo, F.; Pastor, A.; Dermawan, S.; Hong, T.; Reif, O.; PDA Journal of Pharmaceutical Science and Technology
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Mora, J.; Hutchison, F.; Murphy, M. New Q membrane scale-down model for process-scale antibody purification. J. Chromatogr., A 2006, 1134 (1), 66 –73. 13. Gagnon, P.; Hensel, F.; Lee, S.; Zaidi, S. Chromatographic behavior of IgM:DNA complexes. J. Chromatogr., A 2011, 1218 (1), 2405–2412. 14. Luhrs, K. A.; Harris, D. A.; Summers, S.; Parseghian, M. H. Evicting hitchhiker antigens from purified antibodies. J. Chromatogr., B 2009, 877 (14), 1543–1552.
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15. Strauss, D. M.; Cano, T.; Cai, N.; Delucchi, H.; Plancarte, M.; Coleman, D.; Blank, G. S.; Chen, Q.; Yang, B. Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production. Biotechnol. Prog. 2010, 26 (3), 750 –755. 16. Kelley, B. D.; Switzer, M.; Bastek, P.; Kramarczyk, J. F.; Molnar, K.; Yu, T.; Coffman, J. Highthroughput screening of chromatographic separations: IV. Ion-exchange. Biotechnol. Bioeng. 2008, 100 (5), 950 –963.
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