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.
HighlightsAn in investigation in to the use of membrane chromatography for the purification of a γ-retrovirus was undertaken.The first report of a capacity for γ-retrovirus binding to a membrane chromatography device is presented.A process that produces a large increase in concentration and purity of the studied γ-retrovirus was identified.Proteomic techniques were used to identify the protein impurities removed and co-purified with the virus containing eluate.
We describe a design of experiments (DoE) response surface modeling strategy to optimize the concentration of basal variables underpinning polyethylenimine (PEI) mediated transfection of different CHO-K1 derived parental cell populations in a chemically defined medium, specifically the relative concentration of linear 25 kD PEI, host CHO cells and plasmid DNA. Using recombinant secreted alkaline phosphatase (SEAP) reporter activity as the modeled response, a discrete simple maximum was predicted for each CHO host cell population. Differences between the modeled optima derived from host cell specific differences in PEI cytotoxicity, such that the PEI:cell interaction effectively limited PEI-DNA polyplex load at a relatively constant PEI:DNA ratio. However, across the three CHO host cell populations, SEAP reporter production was not proportional to plasmid DNA input at the host cell specific predicted basal variable optima. A 10-fold variation in SEAP reporter output per mass of plasmid DNA delivered was observed. To determine the cellular basis of this difference in transient productivity, host CHO cells were transfected with fluorescently labeled polyplexes followed by flow cytometric analysis. Each CHO host cell population exhibited a distinct functional phenotype, varying in the extent of PEI-DNA polyplex binding to the cell surface and degree of polyplex internalization. SEAP production was directly proportional to the level of polyplex internalization and heparan sulfate proteoglycan level. Taken together, these data show that choice of host CHO cell line is a critical parameter, which should rationally precede cell line specific transient production platform design using DoE methodology.
Physical characteristics critical to chromatography including geometric porosity and tortuosity within the packed column were analysed based upon three dimensional reconstructions of bed structure in-situ. Image acquisition was performed using two X-ray computed tomography systems, with optimisation of column imaging performed for each sample in order to produce three dimensional representations of packed beds at 3μm resolution. Two bead materials, cellulose and ceramic, were studied using the same optimisation strategy but resulted in differing parameters required for X-ray computed tomography image generation. After image reconstruction and processing into a digital three dimensional format, physical characteristics of each packed bed were analysed, including geometric porosity, tortuosity, surface area to volume ratio as well as inter-bead void diameters. Average porosities of 34.0% and 36.1% were found for ceramic and cellulose samples and average tortuosity readings at 1.40 and 1.79 respectively, with greater porosity and reduced tortuosity overall values at the centre compared to the column edges found in each case. X-ray computed tomography is demonstrated to be a viable method for three dimensional imaging of packed bed chromatography systems, enabling geometry based analysis of column axial and radial heterogeneity that is not feasible using traditional techniques for packing quality which provide an ensemble measure.
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