Downloaded from journal.pda.org on June 20, 2015
Overview of 2009 Indianapolis Conference White Paper: The Goal of an Integrated Viral Clearance Strategy Kurt Brorson
PDA J Pharm Sci and Tech 2014, 68 2-5 Access the most recent version at doi:10.5731/pdajpst.2014.00958
Downloaded from journal.pda.org on June 20, 2015
CONFERENCE PROCEEDING – INTRODUCTION
Overview of 2009 Indianapolis Conference White Paper: The Goal of an Integrated Viral Clearance Strategy Indianapolis meeting. In 2009, the 1st Viral Clearance Symposium was held in Indianapolis, IN to interactively discuss methods for virus removal and inactivation during biopharmaceutical manufacture. This conference series was initiated with the goal of gathering the world’s top experts on this issue in a close, informal, collaborative setting to discuss data and strategies to advance this product safety– critical field. A white paper summarizing the 2009 meeting was published in Developments in Biologicals (1). The white paper opened with a statement summarizing the group consensus on the purpose of the meeting: “Worldwide regulatory and industry recognition that challenges, gaps, and opportunities for improvement of viral clearance technology exist, if formally addressed could benefit the field as a whole.” Attendance was restricted to individuals directly responsible for viral clearance studies. They were invited from 17 firms worldwide and two regulatory authorities from the U.S. and Europe. Most gave short presentations of their firm’s experience with viral clearance by particular unit operations. The conference was broken down into sessions devoted to five major unit operations: affinity chromatography (mostly protein A), acid pH inactivation, ion exchange chromatography, detergent-based inactivation, and virus retentive filtration. Each session had 5–12 presentations with three to five slides and ended with an interactive group discussion with the goal of capturing “lessons learned” and “next steps” on flip charts. This activity translated into the final work product of the conference: the white paper that contains a list of conclusions from the sessions (i.e., “What we learned”) and recommendations for further actions (i.e., “What are the next steps”). Integrated viral clearance strategy. One of the stretch goals of the Viral Clearance Symposium series is to define an integrated viral clearance strategy. While a
doi: 10.5731/pdajpst.2014.00958
2
complicated venture, it can be summarized as “If you do process A you can expect viral clearance B”. However, at least for monoclonal antibodies (mAbs), the universe of “process A” and “clearance B” can be defined by platform processes that can be applied across products. In addition, many mAb products have been developed over the past 20 years, generating a multitude of data to meet this goal. Thus, based on available data, a goal of the conference series is to achieve a reliable clearance expectation that can apply to a mAb purification process. It should be scalable, simple (ease of operation), robust, and not subject to product-dependent factors. The clearance strategy should be robust with respect to small/controlled changes in critical process parameters (CPPs) and supported by a defined mechanism of action (MoA), industrial experience, and lab studies. Manufacturing of biopharmaceuticals consists of a sequence of unit operations, each of which may or may not clear viruses. In an overall viral clearance strategy, one must address the individual elements as well as their integration. The individual elements are the viral clearance unit operations where the mechanism of clearance is understood and where CPPs for viral clearance are identified. In an effective strategy, the input for each element would be well-defined, with robust ranges for CPPs and other parameters. The output would be expected overall clearance targets for one to three viruses of different types/classifications (e.g., DNA- and RNA genome, enveloped and nonenveloped, etc.). Unit operation integration requires establishment of orthogonality and the MoA of clearance; the individual elements must work together to achieve additive clearance. The scope of the 2011 version of the Viral Clearance Symposium was expanded to cover integration as well as viral clearance by individual unit operations. A natural by-product for the symposium would be a status check for the “robustness and reliable clearance expectation”, and possibly establishing a control range for at least some unit operations. There are many sources of potential information leading to control strategy: the 2009 Symposium white paper (1), the overall scientific literature, a regulatory submission PDA Journal of Pharmaceutical Science and Technology
Downloaded from journal.pda.org on June 20, 2015
database compiled by CDER (2), and recent advances reported at the 2011 symposium. Based on all available information, unit operations seem to fall into one of three categories: (A) the goal (of a “robustness and reliable clearance expectation”) seems to be in place (low pH, large virus filters), (B) the goal is close (AEX, detergent-based inactivation), and (C) there are significant “gaps, and opportunities for improvement” (protein A, cation exchange chromatography [CEX], small virus filters) and emerging technologies (e.g. membrane adsorbers, mixed mode resins, UV-C inactivation). For category C, the unit operations are likely to be “process-dependent” for at least the near future. This status is evidenced by issues raised in both the 2009 meeting (e.g., feedstock effects on viral clearance by protein A) and the 2011 meeting (e.g., small virus breakthrough related to pressure changes during parvovirus retentive filtration). Category A unit operation: Low pH inactivation. The 2009 symposium white paper contained a multitude of data from low pH inactivation studies and outlined a control strategy. Based on all of the available information at the time, it was clear that the CPPs are pH, time, and temperature. The information reported at the meeting supported a proposed control range of pH ⱕ 3.6, time ⱖ 30 min., temperature ⫽ room temperature. The proposed control range assumes that other factors (protein concentration, buffer types, etc.) will be controlled in “a range compatible with routine manufacturing”. Under these conditions, the log10 reduction value (LRV) expectation for retroviruses is not less than 5 log10. This may be applicable to other, but probably not all, enveloped viruses. The CDER regulatory database analysis supports this proposed control range; most low pH studies reported complete inactivation and the low LRV outliers were mostly pH ⬎ 3.6. Similar observations were reported at the 2009 conference from a regulatory database from the German Paul Ehrlich Institut. The existing published literature, while not extensive, also supports the above ranges and outcomes. An early paper arguing for a control range bounded by a pH of 3.8 (3) has since been updated to pH 3.6. However, its initial claim that the MoA is surface protein denaturation has held up over time. Category A unit operation: Large virus retentive filters. The 2009 symposium also recognized large virus retentive filtration as a mature technology where a control strategy can be proposed for removal of large Vol. 68, No. 1, January-February 2014
viruses. The available sources of information included CDER’s regulatory database with 69 murine leukemia virus (MuLV) removal records, almost all of which were reported as “complete”. An exhaustive literature search reported in the Encyclopedia of Industrial Biotechnology (4) found broad clearance of many viruses with ⱖ3.2 log10 as the lowest reported LRV. Probably most convincing is a clearance study performed in 2004 as part of the PDA virus filter nomenclature standardization effort (5, 6). This study challenged three brands of large virus retentive filters with 9 log10/mL of a medium-sized bacteriophage: 63– 82 nm PR772 (7, 8), a model smaller than the intended target for this filter class, that is, retroviruses (80 – 110 nm) (9). Twenty-one filters were tested; complete clearance was obtained in all but one filter, which nevertheless had still cleared 7.8 log 10 of PR772. Because filtration relies on sized-based sieving and of their reported reliability, normal operation for large virus retentive filters is considered robust. The only CPPs would be pore size relative to target virus (i.e., they won’t clear parvo- or other small viruses) and membrane integrity (i.e., they won’t work if defective, but this is testable). Thus, the proposed control strategy is the following: (I) Follow vendor recommendations for transmembrane pressure (TMP), flux, etc.; (II) Maintain protein load typical of manufacturing; (III) Use buffers compatible with the filter; and (IV) The vendor-recommended integrity test must pass. Based on available data, an expected target LRV would be in excess of 5– 6 log 10 of a large virus such as retrovirus. This target LRV and filtration control strategy coincides with the PDA nomenclature test method published in Technical Report 41 (10), and as based on the above-noted PR772 clearance study. Category B unit operations. The category two unit operations are those that are generally regarded as reliable and robust but still have issues pending for resolution. Category B unit operation: flow through (FT) mode anion exchange chromatography (FT-AEX). The attendee consensus was that FT-AEX falls into this category. Based on the published literature, the CPPs for FT-AEX are clearly process fluid (buffer and feedstock) conductivity and pH. Other factors of frequent concern are probably not CPPs, including contact time (11, 12) and resin re-use cycles (13). However, data 3
Downloaded from journal.pda.org on June 20, 2015
presented at the 2009 conference, and reiterated at the 2011 conference, argues that load composition is also important (i.e., impurities such as host cell proteins [HCP], DNA, and other impurities). This situation seems to have added more complexity to the context of the CDER database analysis. While most records reported high LRV, there were a few difficult to explain low LRV “outliers”. Although these were mostly older products from smaller firms, no mechanistic explanation for these outliers was evident from the database information provided. Additional and possibly relevant features such as feedstock quality is rarely described in regulatory submission study reports, and therefore could not be investigated in this context. The 2009 white paper nevertheless proposed a control strategy for FT-AEX based on the cumulative data— CDER database analysis, presentations at the meeting, and literature precedent: pH 7.0 – 8.5; conductivity ⬍14 mS/cm; effective mAb loading ⬍100 mg/mL; and ⬍50 resin re-uses. However, because of the above noted observations, there is a footnote stating that effective loading can be influenced by host cell impurity load (e.g., host cell DNA). Category B unit operation: Detergent-based inactivation. The second category two unit operation identified at the conference is detergent-based inactivation. Classical solvent/detergent is widely regarded as affording rapid, usually complete inactivation of enveloped viruses. While this perception is probably true, measuring clearance is limited by buffer cytotoxicity issues during the execution of infectivity studies that measure the kinetics of inactivation or the virus present in input and output material. Because these test articles need to be diluted extensively for analysis, the effective LRV window that can be measured in viral clearance studies is modest. In fact, the CDER database analysis found that low pH seems to more consistently achieve higher LRVs (average “incomplete” values of 5.0 vs. 3.2 log10 and “complete” values of ⱖ 5.6 vs. ⱖ4.0 log10 (1, 2). However, this is most probably not because low pH is more effective, but because of the buffer cytotoxicity limitation associated with solvent/ detergent (S/D) studies. The landscape of detergentonly inactivation is more scattered. The CDER database not only found reasonable inactivation in most cases, but also a significant subset of low LRV outliers. This may reflect insufficient concentrations or type(s) of detergent used. Regarding the latter and as discussed in the 2011 conference (see Session II sum4
mary in this issue), the number of usable detergents has recently increased. Also worth considering is the viral clearance impact on detergent when employed with or without the presence of solvent, a discussion topic of the 2009 symposium that remains unresolved. Category C unit operations. These are the least understood unit operations from the standpoint of having substantial knowledge gaps and opportunities for improvement. Based on the 2009 symposium white paper and the scientific literature, protein A chromatography is clearly in this category. While the viral clearance MoA is virus flow-through during loading, and it usually achieves a 2–3 log10 LRV (as measured by quantitative polymerase chain reaction), the CPPs are not entirely clear. Feedstock effects seem important, but it is not known what aspects of a feedstock influence viral clearance. Small virus filters are also in this category. It is widely presumed that they have a sieving-based MoA. As reported in the 2009 symposium white paper and the wider scientific literature, the clearance and breakthrough pattern is filter type– dependent. A key paper from 2007 reported that virus overloading can lead to breakthrough in several filter brands (14). Now in the 2011 conference, more than one firm reported that pressure spikes may also lead to breakthrough. CEX is another operation grouped into category three because little information has been published on its viral clearance MoA and associated critical process parameters. This is slowly changing over time; the CDER database found for example that in general, MuLV clearance by CEX is modest (as protein A) while MMV clearance is poor. Data reported in the 2011 symposium found that CEX can clear viruses when run in a very narrow process space (see session II description in this issue). As mAbs can vary in pI, this process space probably varies between products. CEX is clearly an opportunity for further study. References 1. 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. 2. Miesegaes, G.; Lute, S.; Brorson, K. Analysis of viral clearance unit operations from monoclonal antibody regulatory aubmissions. Biotechnol. Bioeng. 2010, 106, 238 –246. PDA Journal of Pharmaceutical Science and Technology
Downloaded from journal.pda.org on June 20, 2015
3. Brorson, K.; Krejci, S.; Lee, K.; Hamilton, E.; Stein, K.; Xu, Y. Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins. Biotechnol. Bioeng. 2003, 82 (3), 321–329. 4. Miesegaes, G.; Lute, S.; Aranha, H.; Brorson, K. Virus Retentive Filtration. In Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology; Flickinger, M., Ed.; Wiley Interscience: Hoboken, NJ, 2010. 5. Brorson, K.; Sofer, G.; Aranha, H. Nomenclature standardization for “large pore size” virus-retentive filters. BioProcess Int. 2005, 3 (10), 341–345. 6. Brorson, K.; Sofer, G.; Robertson, G.; Lute, S.; Martin, J.; Aranha, H.; Haque, M.; Satoh, S.; Yoshinari, K.; Moroe, I.; Morgan, M.; Yamaguchi, F.; Carter, J.; Krishnan, M.; Stefanyk, J.; Etzel, M.; Riorden, W.; Korneyeva, M.; Sundaram, S.; Wilkommen, H.; Wojciechowski, P. “Large pore size” virus filter test method recommended by the PDA Virus Filter Task Force. PDA J. Pharm. Sci. Technol. 2005, 59 (3), 177–186.
testing. Appl. Environ. Microbiol. 2004, 70 (8), 4864 – 4871. 9. Doane, F. W.; Anderson, N. Electron Microscopy in Diagnostic Virology: A Practical Guide and Atlas; Cambridge University Press, New York, 1987. 10. PDA Technical Report 41: Virus Retentive Filtration. Parenteral Drug Association: Bethesda, MD, 2008. 11. Curtis, S.; Lee, K.; Blank, G. S.; Brorson, K.; Xu, Y. Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flowthrough mode. Biotechnol. Bioeng. 2003, 84 (2), 179 –186. 12. Strauss, D. M.; Gorrell, J.; Plancarte, M.; Blank, G. S.; Chen, Q.; Yang, B. Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009, 102 (1), 168 –175. 13. 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.
7. Bamford, D. H.; Ackermann, H.-W. Virus Taxonomy, Classification and Nomenclature of Viruses: 7th Report of the International Committee on Taxonomy of Viruses. In Van Regenmortel, M., Fauquet, C., Bishop, D., Carstens, E., Estes, M., Lemon, S., Maniloff, M., Mayo, D., Eds.; Academic Press: San Diego, CA, 2000.
14. Lute, S.; Bailey, M.; Combs, J.; Sukumar, M.; Brorson, K. Phage passage after extended processing in small-virus-retentive filters. Biotechnol. Appl. Biochem. 2007, 47 (3), 141–151.
8. Lute, S.; Aranha, H.; Tremblay, D.; Liang, D.; Ackermann, H. W.; Chu, B.; Moineau, S.; Brorson, K. Characterization of coliphage PR772 and evaluation of its use for virus filter performance
Kurt Brorson, Ph.D. Center for Drug Evaluation and Research (CDER) U.S. Food and Drug Administration (FDA) Silver Spring, MD
Vol. 68, No. 1, January-February 2014
5
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Overview of 2009 Indianapolis Conference White Paper: The Goal of an Integrated Viral Clearance Strategy Kurt Brorson
PDA J Pharm Sci and Tech 2014, 68 2-5 Access the most recent version at doi:10.5731/pdajpst.2014.00958
Downloaded from journal.pda.org on June 20, 2015
CONFERENCE PROCEEDING – INTRODUCTION
Overview of 2009 Indianapolis Conference White Paper: The Goal of an Integrated Viral Clearance Strategy Indianapolis meeting. In 2009, the 1st Viral Clearance Symposium was held in Indianapolis, IN to interactively discuss methods for virus removal and inactivation during biopharmaceutical manufacture. This conference series was initiated with the goal of gathering the world’s top experts on this issue in a close, informal, collaborative setting to discuss data and strategies to advance this product safety– critical field. A white paper summarizing the 2009 meeting was published in Developments in Biologicals (1). The white paper opened with a statement summarizing the group consensus on the purpose of the meeting: “Worldwide regulatory and industry recognition that challenges, gaps, and opportunities for improvement of viral clearance technology exist, if formally addressed could benefit the field as a whole.” Attendance was restricted to individuals directly responsible for viral clearance studies. They were invited from 17 firms worldwide and two regulatory authorities from the U.S. and Europe. Most gave short presentations of their firm’s experience with viral clearance by particular unit operations. The conference was broken down into sessions devoted to five major unit operations: affinity chromatography (mostly protein A), acid pH inactivation, ion exchange chromatography, detergent-based inactivation, and virus retentive filtration. Each session had 5–12 presentations with three to five slides and ended with an interactive group discussion with the goal of capturing “lessons learned” and “next steps” on flip charts. This activity translated into the final work product of the conference: the white paper that contains a list of conclusions from the sessions (i.e., “What we learned”) and recommendations for further actions (i.e., “What are the next steps”). Integrated viral clearance strategy. One of the stretch goals of the Viral Clearance Symposium series is to define an integrated viral clearance strategy. While a
doi: 10.5731/pdajpst.2014.00958
2
complicated venture, it can be summarized as “If you do process A you can expect viral clearance B”. However, at least for monoclonal antibodies (mAbs), the universe of “process A” and “clearance B” can be defined by platform processes that can be applied across products. In addition, many mAb products have been developed over the past 20 years, generating a multitude of data to meet this goal. Thus, based on available data, a goal of the conference series is to achieve a reliable clearance expectation that can apply to a mAb purification process. It should be scalable, simple (ease of operation), robust, and not subject to product-dependent factors. The clearance strategy should be robust with respect to small/controlled changes in critical process parameters (CPPs) and supported by a defined mechanism of action (MoA), industrial experience, and lab studies. Manufacturing of biopharmaceuticals consists of a sequence of unit operations, each of which may or may not clear viruses. In an overall viral clearance strategy, one must address the individual elements as well as their integration. The individual elements are the viral clearance unit operations where the mechanism of clearance is understood and where CPPs for viral clearance are identified. In an effective strategy, the input for each element would be well-defined, with robust ranges for CPPs and other parameters. The output would be expected overall clearance targets for one to three viruses of different types/classifications (e.g., DNA- and RNA genome, enveloped and nonenveloped, etc.). Unit operation integration requires establishment of orthogonality and the MoA of clearance; the individual elements must work together to achieve additive clearance. The scope of the 2011 version of the Viral Clearance Symposium was expanded to cover integration as well as viral clearance by individual unit operations. A natural by-product for the symposium would be a status check for the “robustness and reliable clearance expectation”, and possibly establishing a control range for at least some unit operations. There are many sources of potential information leading to control strategy: the 2009 Symposium white paper (1), the overall scientific literature, a regulatory submission PDA Journal of Pharmaceutical Science and Technology
Downloaded from journal.pda.org on June 20, 2015
database compiled by CDER (2), and recent advances reported at the 2011 symposium. Based on all available information, unit operations seem to fall into one of three categories: (A) the goal (of a “robustness and reliable clearance expectation”) seems to be in place (low pH, large virus filters), (B) the goal is close (AEX, detergent-based inactivation), and (C) there are significant “gaps, and opportunities for improvement” (protein A, cation exchange chromatography [CEX], small virus filters) and emerging technologies (e.g. membrane adsorbers, mixed mode resins, UV-C inactivation). For category C, the unit operations are likely to be “process-dependent” for at least the near future. This status is evidenced by issues raised in both the 2009 meeting (e.g., feedstock effects on viral clearance by protein A) and the 2011 meeting (e.g., small virus breakthrough related to pressure changes during parvovirus retentive filtration). Category A unit operation: Low pH inactivation. The 2009 symposium white paper contained a multitude of data from low pH inactivation studies and outlined a control strategy. Based on all of the available information at the time, it was clear that the CPPs are pH, time, and temperature. The information reported at the meeting supported a proposed control range of pH ⱕ 3.6, time ⱖ 30 min., temperature ⫽ room temperature. The proposed control range assumes that other factors (protein concentration, buffer types, etc.) will be controlled in “a range compatible with routine manufacturing”. Under these conditions, the log10 reduction value (LRV) expectation for retroviruses is not less than 5 log10. This may be applicable to other, but probably not all, enveloped viruses. The CDER regulatory database analysis supports this proposed control range; most low pH studies reported complete inactivation and the low LRV outliers were mostly pH ⬎ 3.6. Similar observations were reported at the 2009 conference from a regulatory database from the German Paul Ehrlich Institut. The existing published literature, while not extensive, also supports the above ranges and outcomes. An early paper arguing for a control range bounded by a pH of 3.8 (3) has since been updated to pH 3.6. However, its initial claim that the MoA is surface protein denaturation has held up over time. Category A unit operation: Large virus retentive filters. The 2009 symposium also recognized large virus retentive filtration as a mature technology where a control strategy can be proposed for removal of large Vol. 68, No. 1, January-February 2014
viruses. The available sources of information included CDER’s regulatory database with 69 murine leukemia virus (MuLV) removal records, almost all of which were reported as “complete”. An exhaustive literature search reported in the Encyclopedia of Industrial Biotechnology (4) found broad clearance of many viruses with ⱖ3.2 log10 as the lowest reported LRV. Probably most convincing is a clearance study performed in 2004 as part of the PDA virus filter nomenclature standardization effort (5, 6). This study challenged three brands of large virus retentive filters with 9 log10/mL of a medium-sized bacteriophage: 63– 82 nm PR772 (7, 8), a model smaller than the intended target for this filter class, that is, retroviruses (80 – 110 nm) (9). Twenty-one filters were tested; complete clearance was obtained in all but one filter, which nevertheless had still cleared 7.8 log 10 of PR772. Because filtration relies on sized-based sieving and of their reported reliability, normal operation for large virus retentive filters is considered robust. The only CPPs would be pore size relative to target virus (i.e., they won’t clear parvo- or other small viruses) and membrane integrity (i.e., they won’t work if defective, but this is testable). Thus, the proposed control strategy is the following: (I) Follow vendor recommendations for transmembrane pressure (TMP), flux, etc.; (II) Maintain protein load typical of manufacturing; (III) Use buffers compatible with the filter; and (IV) The vendor-recommended integrity test must pass. Based on available data, an expected target LRV would be in excess of 5– 6 log 10 of a large virus such as retrovirus. This target LRV and filtration control strategy coincides with the PDA nomenclature test method published in Technical Report 41 (10), and as based on the above-noted PR772 clearance study. Category B unit operations. The category two unit operations are those that are generally regarded as reliable and robust but still have issues pending for resolution. Category B unit operation: flow through (FT) mode anion exchange chromatography (FT-AEX). The attendee consensus was that FT-AEX falls into this category. Based on the published literature, the CPPs for FT-AEX are clearly process fluid (buffer and feedstock) conductivity and pH. Other factors of frequent concern are probably not CPPs, including contact time (11, 12) and resin re-use cycles (13). However, data 3
Downloaded from journal.pda.org on June 20, 2015
presented at the 2009 conference, and reiterated at the 2011 conference, argues that load composition is also important (i.e., impurities such as host cell proteins [HCP], DNA, and other impurities). This situation seems to have added more complexity to the context of the CDER database analysis. While most records reported high LRV, there were a few difficult to explain low LRV “outliers”. Although these were mostly older products from smaller firms, no mechanistic explanation for these outliers was evident from the database information provided. Additional and possibly relevant features such as feedstock quality is rarely described in regulatory submission study reports, and therefore could not be investigated in this context. The 2009 white paper nevertheless proposed a control strategy for FT-AEX based on the cumulative data— CDER database analysis, presentations at the meeting, and literature precedent: pH 7.0 – 8.5; conductivity ⬍14 mS/cm; effective mAb loading ⬍100 mg/mL; and ⬍50 resin re-uses. However, because of the above noted observations, there is a footnote stating that effective loading can be influenced by host cell impurity load (e.g., host cell DNA). Category B unit operation: Detergent-based inactivation. The second category two unit operation identified at the conference is detergent-based inactivation. Classical solvent/detergent is widely regarded as affording rapid, usually complete inactivation of enveloped viruses. While this perception is probably true, measuring clearance is limited by buffer cytotoxicity issues during the execution of infectivity studies that measure the kinetics of inactivation or the virus present in input and output material. Because these test articles need to be diluted extensively for analysis, the effective LRV window that can be measured in viral clearance studies is modest. In fact, the CDER database analysis found that low pH seems to more consistently achieve higher LRVs (average “incomplete” values of 5.0 vs. 3.2 log10 and “complete” values of ⱖ 5.6 vs. ⱖ4.0 log10 (1, 2). However, this is most probably not because low pH is more effective, but because of the buffer cytotoxicity limitation associated with solvent/ detergent (S/D) studies. The landscape of detergentonly inactivation is more scattered. The CDER database not only found reasonable inactivation in most cases, but also a significant subset of low LRV outliers. This may reflect insufficient concentrations or type(s) of detergent used. Regarding the latter and as discussed in the 2011 conference (see Session II sum4
mary in this issue), the number of usable detergents has recently increased. Also worth considering is the viral clearance impact on detergent when employed with or without the presence of solvent, a discussion topic of the 2009 symposium that remains unresolved. Category C unit operations. These are the least understood unit operations from the standpoint of having substantial knowledge gaps and opportunities for improvement. Based on the 2009 symposium white paper and the scientific literature, protein A chromatography is clearly in this category. While the viral clearance MoA is virus flow-through during loading, and it usually achieves a 2–3 log10 LRV (as measured by quantitative polymerase chain reaction), the CPPs are not entirely clear. Feedstock effects seem important, but it is not known what aspects of a feedstock influence viral clearance. Small virus filters are also in this category. It is widely presumed that they have a sieving-based MoA. As reported in the 2009 symposium white paper and the wider scientific literature, the clearance and breakthrough pattern is filter type– dependent. A key paper from 2007 reported that virus overloading can lead to breakthrough in several filter brands (14). Now in the 2011 conference, more than one firm reported that pressure spikes may also lead to breakthrough. CEX is another operation grouped into category three because little information has been published on its viral clearance MoA and associated critical process parameters. This is slowly changing over time; the CDER database found for example that in general, MuLV clearance by CEX is modest (as protein A) while MMV clearance is poor. Data reported in the 2011 symposium found that CEX can clear viruses when run in a very narrow process space (see session II description in this issue). As mAbs can vary in pI, this process space probably varies between products. CEX is clearly an opportunity for further study. References 1. 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. 2. Miesegaes, G.; Lute, S.; Brorson, K. Analysis of viral clearance unit operations from monoclonal antibody regulatory aubmissions. Biotechnol. Bioeng. 2010, 106, 238 –246. PDA Journal of Pharmaceutical Science and Technology
Downloaded from journal.pda.org on June 20, 2015
3. Brorson, K.; Krejci, S.; Lee, K.; Hamilton, E.; Stein, K.; Xu, Y. Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins. Biotechnol. Bioeng. 2003, 82 (3), 321–329. 4. Miesegaes, G.; Lute, S.; Aranha, H.; Brorson, K. Virus Retentive Filtration. In Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology; Flickinger, M., Ed.; Wiley Interscience: Hoboken, NJ, 2010. 5. Brorson, K.; Sofer, G.; Aranha, H. Nomenclature standardization for “large pore size” virus-retentive filters. BioProcess Int. 2005, 3 (10), 341–345. 6. Brorson, K.; Sofer, G.; Robertson, G.; Lute, S.; Martin, J.; Aranha, H.; Haque, M.; Satoh, S.; Yoshinari, K.; Moroe, I.; Morgan, M.; Yamaguchi, F.; Carter, J.; Krishnan, M.; Stefanyk, J.; Etzel, M.; Riorden, W.; Korneyeva, M.; Sundaram, S.; Wilkommen, H.; Wojciechowski, P. “Large pore size” virus filter test method recommended by the PDA Virus Filter Task Force. PDA J. Pharm. Sci. Technol. 2005, 59 (3), 177–186.
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14. Lute, S.; Bailey, M.; Combs, J.; Sukumar, M.; Brorson, K. Phage passage after extended processing in small-virus-retentive filters. Biotechnol. Appl. Biochem. 2007, 47 (3), 141–151.
8. Lute, S.; Aranha, H.; Tremblay, D.; Liang, D.; Ackermann, H. W.; Chu, B.; Moineau, S.; Brorson, K. Characterization of coliphage PR772 and evaluation of its use for virus filter performance
Kurt Brorson, Ph.D. Center for Drug Evaluation and Research (CDER) U.S. Food and Drug Administration (FDA) Silver Spring, MD
Vol. 68, No. 1, January-February 2014
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