Recovery and purification process development for monoclonal antibody production

Abstract

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.

Figure 1

A typical monoclonal antibody recovery process.

Figure 2

Impact of nonviable cell density at harvest on centrate quality for different disc-stack centrifugation conditions.

Figure 3

Depth Filter experiments showing different modes of failure. Plot on the left shows a tight depth filter protecting a 0.22 um membrane and resistance builds up on this first layer. Plot on the right shows the case using a more open depth layer and particle breakthrough to membrane layer.

Figure 4

High load capacity runs of monoclonal antibody. Under chromatographic conditions at selected optimal Kp (0.8–7), 3 logs of host cell protein reduction were achieved while the load capacity is higher than 250 mg/mL of resin.

Figure 5

Dynamic binding capacity of a monoclonal antibody as a function of pH and conductivity displays two regions. In the first domain (positively sloped capacity trend), the capacity increases with increasing conductivity. In the second region (negatively sloped capacity trend), the capacity decreases with increasing conductivity. (A) SP Sepharose XL, pH 4 (◆), 5 (■) and 6 (▲), (B) SP Sepharose FF, pH 4 (◊), 5 (□) and 6 (△).

Figure 6

Elution condition development for a monoclonal antibody. (A and B) a pH linear gradient elution program and a salt linear gradient elution program applied on a weak cation exchange column (Fractogel COO⁻ resin), respectively. (C and D) the same linear pH and salt elution programs applied on a strong anion exchange column (Fractogel SO- resin), respectively.

Figure 7

High performance size exclusion chromatography analysis does not show a reduction of HMWS across Mustang S/VPro filtrates. The lack of any significant change suggests the levels of HMWS (as parvovirus filter foulants) in the feedstream are low. However, the enrichment of HMWS is presented in the Mustang S elution pool.

Figure 8

Viresolve Pro filter throughput improvement through coupling Mustang S membrane as a pre-filter.

Figure 9

Filtration throughput variation of a mAb in-process purification pool on Viresolve Pro filter. Viresolve Pro filter resistance (psi/LMH) increases faster with the Lot 3 in-process pool. It results a lower filtration throughput with the Lot 3 in-process pool.