Cationic polymer precipitation for enhanced impurity removal in downstream processing

• Published in: Biotechnology and bioengineering Vol. 120; no. 7; pp. 1902 - 1913
• Main Authors: Li, Zhao, Chen, Justin, Martinez‐Fonts, Kirby, Rauscher, Michael, Rivera, Shannon, Welsh, John, Kandula, Sunitha
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.07.2023
• Subjects: Buffers; Cationic polymerization; Deoxyribonucleic acid; DNA; downstream purification; Electrostatic properties; Excipients; Filtrate; Filtration; Immunoglobulin G; Impurities; Intermediates; Ions; lipase removal; Mass spectrometry; Mass spectroscopy; Microfiltration; Monoclonal antibodies; Particle size distribution; Polyallylamine; Polymers; Precipitates; precipitation; Protein A; Proteins; PS‐80 stability; Purification; Purity; Reduction; Scientific imaging; Shelf life; Size distribution; Stability; downstream purification; precipitation; mass spectrometry; lipase removal; shelf life; PS-80 stability
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.28416

Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation–depth filtration and the current platform process for the purification of biologics. Highlights The use of polyallylamine (PAA) precipitation was examined by Li and coworkers to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. The study provides the first quantitative data for the HCP removal as a function of pI distribution for three mAbs with different properties, and illustrates the impact of the electrostatic interactions on the effectiveness of HCP precipitation. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation.