Introduction
Chinese hamster ovary (CHO) cells are the most common host organisms for production of recombinant therapeutic proteins, including monoclonal antibodies (mAbs). Fed-batch (FB) cell culture processes using CHO cells have been established as a platform for most mAbs in the biopharmaceutical industry (Zhu, 2012). As cell culture platforms continue to evolve, perfusion process stands out as an attractive next generation alternative to fed-batch process. High demand, competitive landscape and fast-paced biopharmaceutical markets require higher productivity, manufacturing flexibility and reduction in development time (Rodrigues et al., 2010). In order to accept the current market need, continuous downstream manufacturing combined with perfusion upstream process offers great advantage on the intensification of the volumetric productivity with limited investment. In the perfusion process, media is fed on a regular basis and the product containing media is harvested continuously. The continuous product removal and short residence time reduce the variability in product modification and lead higher product heterogeneity. Short residence time adds another advantage for unstable molecules such as enzymes or bispecific antibodies (Karst et al., 2018).
Perfusion cell culture have diverged to two mode of operations, steady state (SS) and non-steady state (NSS) (Bielser et al., 2018). SS perfusion mode maintains constant cell density over a lengthier culture duration. The harvest stream is used to remove excess cells to keep a steady concentration. NSS perfusion mode focuses on a higher intensified cell density, which varies during the culture duration. Therefore, perfusion rate must be adjusted dynamically to meet the cellular demand and allow cells to grow at the maximum rate (Karst et al., 2017). Changing the upstream processes can impact the host cell protein (HCP) profile and the relative abundance of particular proteins in harvest cell culture fluid (HCCF), as well throughout the downstream purification (Hogwood et al., 2013; Park et al., 2017). Despite great research interest in HCPs, there are no published investigations to compare perfusion and fed-batch processes for differences in HCP production and profiles, as well as the two modes of perfusion cell culture process.
In this study, we collected HCCF from various bioreactor cultures (FB, SS perfusion, NSS perfusion) at different growth phases of a mAb1-producing CHO DG44 cell line. A complete analysis of HCPs will establish the early proof of concept to understand the differences in HCP production based on differences in cell culture processes. We utilized liquid chromatography-mass spectrometry (LC-MS) based proteomics throughout the study, which have recently shown great promise for HCP characterization (Bracewell et al., 2015; Walker et al., 2017). Discovery-based proteomics by data-dependent acquisition (DDA) is a powerful technique for unbiased identification of proteins in a sample. However, this method is not ideal for the consistent detection of low abundance proteins. HCPs co-exist with highly concentrated mAb product (dynamic range of concentrations ≥ 5 orders) in the HCCF and purification intermediates, which puts a great challenge for detection. Here, to meet our needs, we developed a parallel reaction monitoring (PRM) approach for the target proteins of interest. PRM uses targeted tandem MS to simultaneously monitor product ions of a targeted peptide with high mass resolution and accuracy (Kreimer et al., 2017).
In our HCP profiling studies, we focused on the comparison of one particular group of HCPs that are known to affect product stability. Particle formation in the polysorbate (PS)-formulated biotherapeutics that has become a major quality concern and potential risk factor in the industry. Recent published data and our own investigation have provided clues for the leading cause of PS degradation by HCPs remaining in drug product. Low levels of residual HCPs that have lipase activity, such as lipoprotein lipase (LPL) (Chiu et al., 2017), hydrolyze polysorbates cleaving them into fatty acids. The accumulation of free fatty acids ultimately precipitates to form particles upon long or in some cases short-term storage.
Particle formation in drug substances was observed in mAb1 and this molecule was used in this study. Source materials were generated by three different modes of cell culture processes, subsequently the enzymes that potentially can hydrolyze polysorbates were identified, quantified and compared. The aim of this body of work was to establish proof of concept and methodology to compare FB and perfusion cell culture processes. Perfusion process is shown to be a viable alternative to utilize on the problematic molecules, such as mAb1 that have product quality issues when cultured in traditional FB mode.