Chromatographic clarification overcomes chromatin‐mediated hitch‐hiking interactions on Protein A capture column

Abstract

Protein A capture chromatography is a critical unit operation in the clearance of host cell protein (HCP) impurities in monoclonal antibody (mAb) purification processes. Though one of the most effective purification steps, variable levels of protein impurities are often observed in the eluate. Coelution of HCP impurities is suggested to be strongly affected by the presence of chromatin complexes (Gagnon et al., 2014; Koehler et al., 2019). We investigated the effect of removal of DNA complex and HCP reduction pre-Protein A on the HCP clearance performance of the Protein A capture step itself. We found that only reduction of DNA in the Protein A load consistently lowered HCP in the Protein A eluate. Reduction of HCP in the Protein A load stream did not produce a significant increase in the chromatography HCP clearance performance. These results are consistent across three different biosimilar therapeutic mAbs expressed by the same Chinese hamster ovary (CHO) cell line (i.e., CHO BC® of Polpharma Biologics). This result demonstrates that optimization of the mAb purification process utilizing Protein A as the primary capture step depends primarily on being able to effectively clear DNA and associated complexes early in the process, rather than trying to incorporate HCP reduction at the harvest cell culture fluid.

Single‐use centrifugal separator enables intensification of the clarification process in biomanufacturing of recombinant proteins

“…Figure 2(F) (see colored curves) shows the particle size distribution measured by DLS of the light phase obtained from the centrifuge for all nine operating conditions. The particle size distribution ‘generally’ overlaps for all the conditions and consists of a target protein peak of ~10 nm and a broad peak of ~200 nm indicative of various soluble and insoluble contaminants such as cell debris and rDNA (residual DNA); 32,36‐38 the presence of these contaminants ≤1 μm in size necessitates subsequent secondary‐stage clarification as discussed in the later sections. Further inspection of data shown in Fig.…”

“…We selected 3M Emphaze as a secondary clarifier due to its high‐capacity quaternary ammonium functionality that allows the capture of both negatively charged insoluble and soluble contaminants, including cell debris, rDNA, and HCPs 32,33,36,37,43 . 3M Emphaze typically is operated as a secondary‐stage clarifier after depth‐filtration‐based primary‐stage clarification and has previously been shown to provide ≥95% recovery of the target molecule with ~5‐log reduction in rDNA and ~5‐fold reduction in HCPs 32,33,36,37,43 .…”

“…We selected 3M Emphaze as a secondary clarifier due to its high-capacity quaternary ammonium functionality that allows the capture of both negatively charged insoluble and soluble contaminants, including cell debris, rDNA, and HCPs. 32,33,36,37,43 3M Emphaze typically is operated as a secondary-stage clarifier after depth-filtration-based primary-stage clarification and has previously been shown to provide ≥95% recovery of the target molecule with ∼5-log reduction in rDNA and ∼5-fold reduction in HCPs. 32,33,36,37,43 To understand whether 3M Emphaze can be used as a secondary stage clarifier after primary-stage clarification through the Alfa Laval CultureOne™ centrifuge, we performed filterability tests on four different light phases (turbidity ranging from 85 to 135 NTU) originating from the centrifuge corresponding to the centrifuge operating conditions of experiments 1, 3, 6 and 8 (see Fig.…”

A multisensor approach for improved protein A load phase monitoring by conductivity‐based background subtraction of UV spectra

“…It can be seen, that while the conductivity is stable after this point, the absorption at 280 nm is still increasing due to the displacement of impurities. It has been shown, that DNA and certain HCP species interact with the mAb bound to the Protein A resin (Aboulaich et al, 2014; Nogal et al, 2012; Sisodiya et al, 2012; Van de Velde et al, 2020). This interaction can lead to a retention effect of the interacting impurities in comparison to noninteracting impurities, which could lead to a delayed breakthrough of the interacting impurities.…”

Analytical characterization of host‐cell‐protein‐rich aggregates in monoclonal antibody solutions

“…[35][36][37][38][39][40] The observation of HCPs in mAb aggregates nonetheless warrants deeper investigation, as improvements in aggregate clearance have appeared to reduce HCP levels generally. 29,[41][42][43] HCP persistence in protein A chromatography was also recently correlated with the self-association propensity of mAbs and was attributed to the transient formation of positively-charged mAb clusters that attract HCPs. 44 It seems reasonable that HCPs may likewise associate with more permanent mAb aggregates and their survival through protein A chromatography could be construed as an extension of the product association mechanism.…”