PEG Precipitation: A Powerful Tool for Monoclonal Antibody Purification - This alternative purification method to chromatography is readily scalable and fits a fully disposable downstream process. - B

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PEG Precipitation: A Powerful Tool for Monoclonal Antibody Purification
This alternative purification method to chromatography is readily scalable and fits a fully disposable downstream process.


BioPharm International Supplements


Cation Exchange Chromatography


Table 4. Yield and impurity removal for the PEG precipitation operation using microfiltration to recover the product at pilot scale
High capacity cation exchange (CEX) chromatography was evaluated with feeds pretreated with or without PEG precipitation. The precipitation step did not have any significant impact on the step yield or the percentage reduction of HCPs, but the eluate resulting from the precipitated load material had seven-fold less HCP (Table 5).

Conclusions


Table 5. Yield and HCP removal for GigaCap S-650 loaded with precipitated (+PEG) and nonprecipitated (–PEG) antibody
Precipitation has long been used in the plasma protein industry to purify proteins at large scales. The technique has been adapted here to the initial MAb purification from clarified fed-batch and XD media in a scalable manner. Two single-use filtration steps have been developed to capture and wash the precipitated product, eliminating the need for centrifugation. It was shown that the precipitation operation did not negatively affect the yield of the CEX capture step, and it reduced the HCP content of the eluate by a factor of seven.

The ability to reduce the impurity burden so far upstream in the purification train is key to truncating the downstream process or replacing traditional chromatography with other single-use technologies. Lower impurity burdens can improve the loading capacity of flow-through membrane adsorbers and possibly virus filters, which are generally very expensive items in a process.

An added benefit is the ability to redissolve the antibody in a buffer that facilitates the subsequent unit operation. For example, cell culture media typically requires extensive titration and dilution or a UF–DF step to prepare for capture chromatography with a cation exchanger. Here, the precipitated antibody can be dissolved in equilibration buffer at high concentration, thus shortening the processing time. This can be important for products that do not tolerate long exposure to low pH/conductivity conditions. In the case of this particular antibody, the clarified media requires a more than two-fold dilution to be loaded onto a CEX column, whereas the redissolved MAb could be loaded directly at nearly two-fold the concentration of the unadjusted media. This is at least a four-fold reduction in the load volume, which can result in substantial time savings for modern, high-capacity CEX resins.

Acknowledgments

The authors would like to thank the Percivia protein and analytical sciences for assay support in this work and the Percivia upstream process development group for supplying the cell culture media.

Trademark Note

PER.C6 is a registered trademark of Crucell Holland BV Corporation; XD is a registered trademark of DSM NV; and ECS is a registered trademark of DSM NV. All other brand names are trademarks of their respective owners.

MICHAEL KUCZEWSKI is scientist I, EMILY SCHIRMER, PhD, is scientist II, BLANCA LAIN, PhD, is a senior scientist, and GREGORY ZARBIS-PAPASTOITSIS, PhD, is a senior director, all in downstream process development, Percivia LLC, Cambridge, MA, 617. 301.8821,


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