Host cell protein adsorption characteristics during protein a chromatography

Abstract

Protein A chromatography is a critical and 'gold-standard' step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI-TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back-bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D-PAGE was then used to determine individual components associated with resin back-bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs.

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

“…However, the similarity between results for mAb1 P1 vs P2 is encouraging in this regard. Protein A eluate pool Protein A affinity chromatography is the single most effective purification step in platform mAb processes, 23,24 and this was reflected in the large reduction in both numbers and total levels of HCPs after this step ( Table 2). Compared with mAb1, mAb2 has an improved PrA step with MabSelect SuRe resin and a chaotropic salt wash, which could help to more effectively reduce HCP levels.…”

Heterologous recombinant expression of non-originator NISTmAb

“…The purified proteins were shown to be virtually clean of any nucleic acid contamination (Peter Vallone, NIST, personal communication). Protein-A affinity chromatography is the industry standard for purification of mAbs, and has been shown to decrease host cell protein levels in excess of 98% 13 . Specific evaluation of such process impurities was not the focus of this initial report; therefore, additional purification was not conducted.…”

Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review

“…The yield of the desired product is consistently higher than 95%. Process related impurities such as host DNA, HCPs, virus particles and medium components are removed during Protein-A purification step (Shukla and Hinckley, 2008; Liu et al, 2010; Tarrant et al, 2012). …”

“…The major drawback of the Protein-A resin is leachable and non-specific binding host DNA and HCP unwanted impurities, which reduces resin’s DBC and required to be removed in consecutive purification steps (Ghose et al, 2006; Liu et al, 2010; Tarrant et al, 2012). Recently, single use column chromatography is also developed and being used for Protein-A chromatography.…”