Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography

Abstract

Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.

CHO cell cultures in shake flasks and bioreactors present different host cell protein profiles in the supernatant

“…It should be noted that significant differences in the measured HCP concentration have previously been reported when using different ELISA assay kits. Specifically, when we measured the concentration of HCP in bioreactor supernatant samples using the Cygnus kit, the values were approximately 2.6 times lower than those determined with the Lonza GS-HCP kit [31]. Similar discrepancies were observed by Yuk et al when comparing their results obtained with a proprietary Genentech HCP ELISA kit with those obtained by Tait et al using the Cygnus kit [32,33] The specificity and accuracy thus appear to be different for each kit, hence, only the results obtained with the Lonza GS-HCP kit are presented here for consistency.…”

The N-Glycosylation Processing Potential of the Mammalian Golgi Apparatus

“…The SSA incorporates the inherent noise that is present in biological systems and that becomes increasingly relevant when the number of reactants (and enzymes) are small, as is the case for glycosylation in the Golgi. In contrast to models of glycosylation based on ordinary differential equations (ODEs) (Hossler et al, 2007; Krambeck et al, 2009, 2017; Goey et al, 2018), several factors can be included in each parameter, reducing the need for excessive parameterization in our model. As such, we define parameters for each enzyme by using the term “effective enzymatic rate” to encompass the enzyme’s protein level, availability of its nucleotide-monosaccharide substrates and the chemical enzymatic rate.…”

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

“…It can give an appropriate impression of the impurity level in the supernatant. However, to understand the dynamics and the complexity of HCPs, a qualitative analysis must be further applied …”

“…However, to understand the dynamics and the complexity of HCPs, a qualitative analysis must be further applied. [15,19,36]…”

“…Enzyme-linked immunosorbent assay (ELISA) is a common method used to quantitatively determine the HCP content of a given sample though it lacks coverage of the complete spectrum of HCPs and is likely to miss weak or nonimmunogenic species. [18,19] Moreover, this procedure is costly and can result in a huge work, particularly when many samples need to be analyzed. The application of ELISA for the routine tracking of HCP generation during a bioprocess to uncover cell lysis can be regarded as not economically feasible.…”