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.

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.

Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.

Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates.

Mammalian cell lines are currently employed as one of the main cellular factories for the expression of recombinant protein-based drugs. The establishment of high-producing cell lines typically begins with a heterogeneous starter population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOK1 cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post-translational mechanisms affecting both protein turnover and protein folding become severely limiting. The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed.