Characterization of Host Cell Proteins (HCPs) in CHO Cell Bioprocesses.

Host cell protein content during bioprocessing of biotherapeutic proteins generated from cultured Chinese hamster ovary (CHO) cells is typically measured using immunological and gel-based methods. Estimation of HCP concentration is usually undertaken using Enzyme-Linked ImmunoSorbent Assays (ELISA), while estimation of HCP clearance/presence can be achieved by comparing 2D-PAGE images of samples and by undertaking western blotting of 2D-PAGE analyzed samples. Here, we describe the analyses of HCP content using these methodologies.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

An ultra scale-down approach identifies host cell protein differences across a panel of mAb producing CHO cell line variants.

During the manufacture of biopharmaceutical products, the final product must lie within strict pre-set specifications, for example the host cell protein (HCP) content. A number of specific HCPs have been identified in particular products and the interactions between product/HCPs have also been recently investigated; however, a comparison of the HCP dynamics between related cell lines and their response to early downstream processing to aid process development and cell line selection has not been published. We have utilised a proteomic approach coupled with an ultra scale-down study to determine the HCP profile dynamics, at harvest and during early downstream processing, across a panel of recombinant GS-CHOK1SV antibody producing cell lines. The results reveal that cell culture viability upon harvest has the greatest impact upon shear sensitivity and HCP concentration. Whilst the general HCP population/profile was broadly similar across the cell lines, the actual amounts of some specific HCPs in the supernatant differed and a number of cell line specific differences in the response to early downstream processing were observed. We anticipate that such knowledge can now be applied to cell line selection and downstream processing development to target reduction/removal of general and specific problematic HCPs before and during downstream processing.

Measurement and control of host cell proteins (HCPs) in CHO cell bioprocesses.

Chinese hamster ovary (CHO) cells are widely used for the production of biotherapeutic recombinant proteins for a range of molecules including monoclonal antibodies and Fc-fusion proteins. Regulatory requirements for the final product include the removal of host cell proteins (HCPs) to acceptable amounts (<100ppm). Recent research has begun to unravel the extent to which upstream process conditions and subsequent product recovery and purification processes impact upon the HCP profile. A number of upstream parameters, including the selection of the cell line, the culturing process (e.g. feeding regime, culture temperature), cell viability at time of harvest/culture duration and cell shear sensitivity can all influence the resulting HCP profile. Further, the molecule itself plays an important role in determining those HCPs that are retained throughout a bioprocess and HCPs can co-elute with the target product during purification. Measurement and monitoring of HCPs is usually undertaken using ELISA technology, however alternative approaches are also now emerging that complement ELISA and allow the detection, identification and monitoring of specific HCPs. Here we discuss our understanding of how the process itself influences those HCPs present throughout the production process and the challenges in their monitoring, measurement and removal.

The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process.

Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D-PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post-protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D-PAGE can be used for monitoring and identification of HCPs post-protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell-engineering approaches can be applied to reduced, or eliminate, such HCPs.