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.
Defining what Chinese hamster ovary (CHO) host cell proteins (HCPs) are present, and at what abundance, during upstream and downstream bioprocessing of biotherapeutic recombinant proteins can aid in bioprocess development to minimize the amounts of critical host cell proteins. The authors 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, using 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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The performance of Magarose
®, an agarose-based bead containing a paramagnetic component has been evaluated. The anion exchanger DEAE-Magarose is effective at binding DNA from a crude cell lysate. ...The plasmid pBluescript was isolated from 1.5 ml
Escherichia coli JM109 cell culture, following alkaline lysis yielding 8.2 μg high-quality DNA. Under similar binding conditions 21 μg of salmon sperm DNA bound to the ion exchangers. The affinity medium oligo-dT Magarose was demonstrated to bind 75 μmol of an oligo-dA probe/g of medium by hybridization. Under similar conditions mRNA could be isolated from a preparation of baby hamster cell total RNA. The magnetic susceptibility of Magarose is very high, facilitating the use of this separation technique for rapid batch chromatographic processes.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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 l 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.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The performance of two new designs of pump-packed axial flow process chromatography columns have been evaluated for the preparative anion-exchange chromatography of hen egg-white proteins using ...Whatman Express-Ion Exchanger Q. A 16 l Side-Pack column and a 24 l IsoPak column containing Express-Ion Q were used in this study. In each case ca. 20 l feedstock containing 5–7 g protein/l, was applied per litre packed bed at flow-rates of ca. 150 and 300 cm/h. In each case the ovalbumin binding capacity was ca. 70 g/l packed bed with ca. 100% (w/w) recovery of applied protein. A clean-in-place procedure involving storage in 0.5
M NaOH was effective in maintaining chromatographic performance in all cases. These data were consistent with our previous work using the more traditionally configured slurry-packed axial flow columns. Each of these column designs were easy to use facilitating rapid packing with this adsorbent and in the case of IsoPak rapid pump unpacking. The introduction of these column designs significantly improves the task of column packing, hitherto a labour intensive, physically demanding and potentially unreproducible process.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The chromatography of the murine hybridoma cell C595/102 culture supernatant expressing the therapeutic monoclonal antibody C595, on the cation-exchange cellulose Whatman Express-Ion Exchanger S has ...been investigated. Initial method scouting studies using purified C595 in 1-ml mini columns demonstrated that binding capacity and binding efficiency were dependent not only on decreasing pH but also on the buffer salts used to prepare the mobile phase. Under optimised conditions of 0.1
M sodium acetate buffer, pH 5.0, we were able to separate purified C595 from BSA, the major contaminant in tissue culture fluid. Under these conditions immunoreactive C595 could be isolated directly from tissue culture supernatant. A scale-down study was carried out using a 25-ml column operated at a flow-rate of 150 cm/h which also yielded purified immunoreactive antibody. This procedure should now be suitable for scale-up.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
In some applications, the purification and recovery of biomolecules is performed via a cascade of batch adsorption and desorption stages using agitated contactors and related filtration devices. ...Suspended bed chromatography is a recent process-scale innovation that is applicable to these separations. This hybrid technique exploits the benefits of combining batch adsorption in an agitated contactor with elution in an enclosed column system. To some extent, the process is similar to batch contactor chromatography but can be fully contained and significantly quicker. The process has two steps; first the fluid containing the sample is mixed with the adsorbent in a stirred tank. Second, the slurry suspension is transferred directly into a specialized column, such as an IsoPak column. The media with the adsorbed product is formed as a packed bed, whilst the suspension liquid is passed out of the column. The product is then eluted from the packed bed utilizing standard column-chromatography techniques. The performance of the suspended bed and the agitated contactor operations are demonstrated both by full-scale experimental results and process simulations. The purification of ovalbumin from a hen-egg white feedstock by anion-exchange chromatography was used as a case study in order to prove the concept. With the availability of both pump-packed systems and shear-resistant media, suspended bed chromatography is a better alternative for a range of applications than the traditional batch separations using agitated contactors.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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