Chromatography: A Two-Column Process To Purify Antibodies Without Protein A - - BioPharm International

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Chromatography: A Two-Column Process To Purify Antibodies Without Protein A


BioPharm International
Volume 20, Issue 5


Table 2. Comparison of in-process contaminant clearance using the cation exchange (CEX) resin for capture and a hydrophobic charge induction (HCI) resin (MEP HyperCel) for polish as the process is scaled-up. The overall yield for HuMAb-6 and HuMAb-7 for large-scale process was about 80% for both antibodies.
Table 2 summarizes the scalability of the proposed process for two other human monoclonal antibodies. The largest variability was observed with the DNA measurements. All remaining parameters were very comparable. Final purified material retained the quality obtained at bench-scale. Recovery values tended to be better with scale-up.

Viral clearance

Regulatory agencies require at least two separate steps for viral inactivation and clearance during the production of therapeutic proteins. These steps must be based on different modes of action, typically, a low pH hold and a viral filtration step.3 Viral inactivation was conveniently positioned between the chromatography steps, but this step can also be positioned at the end of both sequences. Similarly, an additional specific step, such as viral clearance filtration, is performed after the second column.


Table 3. A-MuLV clearance capacity of the purification process using a cation exchange resin for capture and hydrophobic charge induction resin for polish. Results were estimated from bench-scale runs.
Table 3 shows the viral clearance of HuMAb-6 and HuMAb-7 following scheme A. Quantification of virus-like particles in the cell culture supernatant yielded up to 10 logs of infectious and non-infectious viral particles. The minimum safety factor for clearance of A-MuLV for the process ranged between 7 and 10 logs, for HuMAb-6 and HuMAb-7, respectively. Therefore, the final antibody product (antibody and residual process contaminants) had sufficient viral clearance and safety viral factor, allowing its use as a therapeutic.

CONCLUSION

With increasing titers in antibody manufacturing, purification processes must increase process efficiency and productivity. By minimizing the number of purification process steps, the number of buffers to be prepared and process components are also reduced. The two-column purification technology design described here can be used with various types of proteins with only minor process adjustments. The scheme is a starting point for subsequent optimization and iteration steps because chromatography parameters often can be optimized, for example, for higher binding capacity ranges. One additional advantage of using this scheme is the flexibility of choosing the direction of the process steps that is most convenient for a specific product or production facility.

Gisela M. Ferreira, PhD, is a process engineer, and Jill Dembecki and Krina Patel are associate scientists. Alahari Arunakumari, PhD, is the director of process development, all at Medarex, Inc., 908.479.2451,

REFERENCES

1. Rathin D and Morrow, KJ Jr. Progress in antibody therapeutics. Am Biotechnol Lab. 2006; 24(8):8–10.

2. Thompson R. Antibody therapeutics: product development, market trends, and strategic issues. 2006 Oct. Available from: http://www.zangani.com/blog/antibody-therapeutics-product-development-market-trends-and-strategic-issues/

3. Zoon KC. Points to consider in the manufacturing and testing of monoclonal antibody products for human use. US FDA, CBER. Rockville, MD; 1997 Feb 28.


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