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.

Cell line specific control of polyethylenimine-mediated transient transfection optimized with "Design of experiments" methodology.

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.

Large-scale ion-exchange column chromatography of proteins. Comparison of different formats.

This review describes the performance of various column designs available to process-scale users of low-pressure chromatography for protein purification. By carrying out a range of ion-exchange separations using Whatman microgranular ion-exchange celluloses we are able to compare and contrast the practical performance issues associated with several designs of axial and radial flow columns.

Direct enzyme adsorption from an unclarified microbial feedstock using suspended bed chromatography.

Suspended bed chromatography (SBC) is a new hybrid technique concomitantly benefiting from batch adsorption, the process advantages of an enclosed system, and its compatibility with established commercial chromatographic contactors and adsorbents. SBC was evaluated in the anion-exchange capture and chromatographic fractionation of native glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from the complex mixture of molecular and particulate moieties that constitute wet-milled bakers' yeast. Method scouting established operating conditions exploiting Whatman Express-Ion Exchanger Q at pH 7.5 and a disrupted biomass concentration equivalent to 3.5% (wet mass/v original intact cells). Partially purified G3PDH was recovered directly from the yeast disruptate in a scaled-down process developed at 1/756 process scale. This was used to establish operating parameters to facilitate process scale-up to exploit a 44 cm I.D. Millipore IsoPak column, 18 kg (swollen mass) of Express-Ion Q anion-exchange cellulose and 275 1 of 3.5% (wet w/v) bakers' yeast disruptate. The generic utility of SBC was demonstrated for direct product adsorption from feedstocks characterised by a modest content of bioparticulates (equivalent to < 4% (wet w/v) disrupted cells). Analyses illustrated an enrichment of G3PDH in respect of enzyme concentration and significant reduction in product turbidity and Pico-Green reactivity (correlated with double stranded (ds) DNA content). The application niche for this new approach to primary protein recovery is discussed with particular reference to the downstream processing of coarsely clarified whole broths, cell disruptates and biological extracts.