Viral Clearance by Protein A, Anion Exchange and Cation Exchange Chromatography Steps

• Bioreliance by SAFC

A key component of the viral safety strategy for a biopharmaceutical is an evaluation of the viral clearance potential of the manufacturing process. Certain steps of the process can provide inactivation or removal of a potential viral contaminant, and some steps are wholly dedicated to viral inactivation or removal. Low pH incubation is included in a manufacturing process to provide inactivation of enveloped viruses, and the virus reduction filtration step serves to provide removal of viruses larger than the nominal filter pore size. The purpose of most steps in the manufacturing process, however, is to purify the protein product. The backbone of most purification processes is chromatography, and various types of chromatographic resins are used to remove various process and product impurities. Manufacturers have found that many chromatography steps can also provide virus clearance, the level of clearance dependent on the chromatography operating parameters, such as the composition of the buffer and the way in which the column is run (e.g., bind and elute versus flowthrough mode).

As a contract research organization, we have helped many clients evaluate the viral clearance potential of their chromatography steps. We have evaluated the xenotropic murine leukemia virus (XMuLV) and murine minute virus (MMV) log reduction values (LRVs) for three types of chromatography resins, Protein A, anion exchange (quaternary amine chemistry) and cation exchange. These data are provided to demonstrate the potential viral clearance that has been achieved by these types of chromatography. While some clients provide detailed buffer and process step information, others do not, and in those cases we know little more than the resin type and the log reduction values. Consequently we have not been able to make any correlations between LRV and potentially relevant parameters such as buffer pH, salt concentration or protein pI. It must be understood that the viral clearance potential by any given chromatography resin is dependent on the nature of the virion itself (e.g., pI, etc.), and the conditions under which the chromatography will be performed.

Protein A Chromatography

Protein A chromatography is typically used as an initial capture step for monoclonal antibodies (mAb) and Fc fusion proteins. This step separates the mAb from cell culture harvest through a specific interaction between the Protein A molecule and the Fc region of the immunoglobulin^1,2. The bulk of virus will flow through the column with other non-binding impurities, while immunoglobulins bind to the resin. When evaluating viral clearance across Protein A columns, most of the virus flows through the column³. Usually, however, some virus will bind to the resin or to the bound mAb through non-specific interactions and co-elute with the antibody product. Viral clearance by Protein A chromatography has been evaluated by many manufacturers, and while levels of clearance vary between different mAbs, typically only modest levels of clearance are achieved; complete virus removal is not often observed. Variations in process parameters do not appear to have a significant effect on viral clearance across this resin^4,5.

We have reviewed LRVs for 49 XMuLV-spiked runs and 176 MMVspiked runs. The data were from runs using Protein A resins from various vendors. The range in virus reduction was from no reduction to 5.27 logs for XMuLV and from no reduction to 5.26 logs for MMV (Table 1). The mean and median LRVs were very similar, 2.98 logs and 2.96 logs, respectively for XMuLV and 2.32 logs and 2.23 logs, respectively for MMV.

Table 1. Descriptive statistics for log reduction values across XMuLV- and MMV-spiked Protein A, anion exchange and cation exchange chromatography runs.

Complete clearance of XMuLV was achieved in only 2 runs; some level of MMV was recovered in the product fraction of all MMV-spiked runs (Table 1). The data in Figure 1 show the number of XMuLV or MMVspiked runs achieving various levels of reduction. Reductions are presented in one log increments up to five or more logs. Most XMuLV runs achieved 2-4 logs of reduction, while most MMV-spiked runs achieved 1-3 logs of reduction. The data suggest that XMuLV reduction by Protein A chromatography is somewhat higher than MMV reduction. The distribution of both XMuLV and MMV LRVs appears normal, which might be expected if the process parameters had no impact on virus reduction. This is consistent with the observations of Miesegaes, Lute and Brorson as well as Zhang et al 4,5.This step is usually optimized for immunoglobulin purification and not for virus removal, and therefore virus reduction across this step is serendipitous.

 Figure 1. Log reduction value distributions for XMuLV and MMV spiked Protein A chromatography runs.

Anion Exchange Chromatography

Anion exchange chromatography is often used in flow through mode as a polishing step. The protein product flows through the column, but impurities, such as host cell DNA and any potentially contaminating virus, bind. The isoelectric point of many biotherapeutic proteins is basic and the isoelectric point of most viruses, including the model viruses used in viral clearance studies is acidic. When the chromatography is run under conditions where the viruses are negatively charged, virus removal has been shown to be due to electrostatic interactions with the positively charged resin⁶. Understanding the mechanism of virus removal by anion exchange chromatography has allowed many manufacturers to adjust buffer pH and conductivity for optimal virus removal ^7-9. In fact, some manufacturers have developed operating spaces that achieve good virus reduction ^8-9.These operating spaces are often the basis for anion exchange chromatography as part of a manufacturing platform.

We evaluated the virus reduction from 166 MMV-spiked and 220 XMuLVspiked anion exchange chromatography runs. The runs included both column and membrane anion exchange chromatography based on quaternary amine chemistry and run in flow through mode. The range in virus reduction was from no reduction to 7.26 logs for XMuLV and from no reduction to 8.10 for MMV (Table 1). The mean and median LRVs were 4.22 logs and 4.55 logs, respectively for XMuLV. For MMV, the mean was 3.25 logs and the median, 3.16 logs.

Complete clearance was achieved in over half of the XMuLV-spiked runs and in about one third of the MMV-spiked runs (Table 1). The data in Figure 2 show the number of XMuLV or MMV-spiked runs achieving various levels of reduction. Reductions are presented in one log increments up to five or more logs. For the XMuLV-spiked runs, over 60% achieved four or more logs of reduction and over 90% achieved two or more logs of reduction. For the MMV-spiked runs, 42% of the runs achieved four or more logs of reduction and 65% of the runs achieved two or more logs of reduction. The anion exchange chromatography data appear skewed and not normally distributed, reflecting the fact that this step can be optimized to achieve higher levels of virus reduction.

 Figure 2. Log reduction value distributions for XMuLV and MMV spiked anion exchange chromatography runs.

Cation Exchange Chromatography

Cation exchange chromatography is often used in the purification process for a monoclonal antibody or recombinant protein as a polishing step and occasionally has been explored as an initial capture step. The step is typically run in bind and elute mode and the bound protein is usually eluted with a linear or step gradient of increasing salt concentration. While the viral reduction for this step is often lower than for other types of chromatography⁴, when operated at pH 5.0, it has been shown to provide effective removal of XMuLV, pseudorabies virus (PRV) and reovirus type 3 (Reo 3)^10-11. When the column is operated at pH 5.5 or higher, XMuLV reduction decreases significantly. The virus appears to bind to the resin by a tight electrostatic interaction and remains bound during elution of the product10. Inactivation of XMuLV at pH 5.0 does not appear to be the mechanism of clearance. In contrast to XMuLV, cation exchange chromatography does not achieve good parvovirus clearance; at most, MMV clearance levels were similar to those achieved by Protein A chromatography ^11-12.

We evaluated the LRVs from 24 XMuLV-spiked and 22 MMV-spiked cation exchange chromatography runs. The runs included several cation exchange resins, including SP Sepharose, SP Sepharose HP, Poros HS and Poros XS resins; all runs were bind and elute mode. The range in virus reduction was from no reduction to 4.80 logs for XMuLV and from no reduction to 2.51 logs for MMV (Table 1). For XMuLVspiked runs, the mean clearance was 2.51 logs and the median was 2.07 logs. For MMV-spiked runs mean clearance was 0.92 logs median clearance was 1.07 logs.

Complete clearance was achieved in only 5 of the XMuLV-spiked runs and in none of the MMV-spiked runs (Table 1). The data in Figure 3 show the number of XMuLV or MMV-spiked runs achieving various levels of reduction. Again, reductions are presented in one log increments up to five or more logs. Most of the XMuLV-spiked runs achieved 1-3 logs of reduction, while most of the MMV-spiked runs achieved up to 2 logs of reduction. These data indicate that while effective XMuLV reduction can be achieved, typically only 1-3 logs of this virus is removed. The step does not provide effective MMV reduction.

 Figure 3. Log reduction value distributions for XMuLV and MMV spiked cation exchange chromatography runs.

Conclusions

Evaluation of LRVs from Protein A, anion exchange and cation exchange chromatography steps has provided information about the potential of these steps to provide effective virus reduction. In the absence of process specific information, such as buffer compositions, pH, conductivity, column load composition or biotherapeutic pI, correlations between LRV and run conditions cannot be made. However, general trends were observed. Protein A chromatography typically achieved 2-4 logs of XMuLV reduction and 1-3 logs of MMV reduction. Given the distribution of the LRVs, process parameters do not appear to impact reduction. Anion exchange chromatography has the potential to achieve the most virus reduction, and can even provide complete clearance. A careful evaluation of process parameters can be used to optimize viral clearance. Cation exchange chromatography does not provide significant MMV removal, and in general achieves better reduction of XMuLV. Data suggest that the step can be optimized to achieve effective XMuLV reduction.

The primary purpose of a chromatography step in a manufacturing process is to purify the protein product, but many times the step can be optimized to provide effective removal of potential viral contaminants. Evaluating this potential is an important component of a good viral safety strategy. These data demonstrate that viral clearance for a given chromatography step can vary greatly and suggest that careful evaluation of processing parameters, such as in design space experiments is required to fully explore the clearance potential of a chromatography step.

References

1. Jungbauer A, Hahn R. Engineering protein A affinity chromatography. Curr Opin Drug Discov Devel. 2004; 7(2):248-256.
2. Huse K, Bohme H-J, Scholz GH. Purification of antibodies by affinity chromatography. J Biochem Bioph Methods. 2002;51(3):217-231.
3. Brorson K, Brown J, Hamilton E, Stein KE. Identification of Protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use. J Chromatogr A. 2003;989(1):155-163.
4. Miesegaes G, Lute S, Brorson K. Analysis of viral clearance operations for monoclonal antibodies. Biotechnol Bioeng. 2010;106(2):238-246.
5. Zhang M, Miesegaes GR, Lee M, Coleman D, Yang B, Trexler-Schmidt M, Norling L, Lester P, Brorson KA, Chen Q. Quality by design approach for viral clearance by Protein A chromatography. Biotechnol Bioeng. 2014;111(1):95-103.
6. Strauss DM, Lute S, Tebaykina Z, Frey DD, Ho C, Blank GS, Brorson K, Yang B. Understanding the mechanism of virus removal by Q Sepharose Fast Flow chromatography during the purification of CHO-cell derived biotherapuetics. Biotechnol Bioeng. 2009;104(2):371-380.
7. Curtis S, Lee K, Blank GS, Brorson K, Xu Y. Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flow-through mode. Biotechnol Bioeng. 2003;84(2):179-186.
8. Strauss DM, Cano T, Cai N, Delucchi H, Plancarte M, Coleman D, Blank GS, Chen Q, Yang B. Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production. Biotechnol Progr. 2010;26(3):750-755.
9. Strauss DM, Gorrell J, Plancarte M, Blank GS, 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.
10. Miesegaes G. Viral clearance by traditional operations with significant knowledge gaps (Session II): cation exchange chromatography (CEX) and detergent inactivation. PDA J Pharm Sci Technol. 2014;68(1):30-37.
11. Connell-Crowley L, Nguyen T, Bach J, Chinniah S, Bashiri H, Gillespie R, Moscariello J, Hinckley P, Dehghani H, Vunnum S, Vedanthan G. Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes. Biotechnol Bioeng. 2012;109(1):157-165.
12. Miesegaes GR, Lute S, Strauss DM, Read EK, Venkiteshwaran A, Kreuzman A, Shah R, Shamlou P, Chen D, Brorson, K. Monoclonal antibody capture and viral clearance by cation exchange chromatography. Biotechnol Bioeng. 2012;109(8):2048-2058.