First, mAbs could be engineered to minimize sticky patches resulting from unique variable region sequences and presumably responsible for much product-specific HCP binding
First, mAbs could be engineered to minimize sticky patches resulting from unique variable region sequences and presumably responsible for much product-specific HCP binding. HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell tradition fluid. These observations hint that minimizing cell lysis during cell tradition/harvest may be useful in minimizing Biotin-HPDP downstream HCP content material. Clusterin and actin are abundant in the protein A eluate swimming pools of most mAbs analyzed. HCP profiling by this strategy can provide useful info to process developers and Biotin-HPDP lead to the Biotin-HPDP refinement of existing purification platforms. of the three analyses were included (n = 74), and these are outlined in Table S4. By excess weight, these HCPs comprised the bulk (av. 6000 ppm) of the measured HCP content of this sample, with individual HCPs ranging from 1000 ppm to 3 ppm. Number?4 shows the correlation of HCP identifications among the 3 runs; 65 (88%) were independently recognized in all three runs, 7 (9%) were recognized in 2 runs and two (3%) were only recognized once. All HCPs that quantified above an average of 22 ppm (n = 43) were independently recognized in every replicate run. Conversely, as HCP levels approached the LOQ of ~13 ppm,7 self-employed identifications became less probable. There was good quantitative agreement for each HCP among the three runs, with an average CV of 27% (Table S4). Scattergrams showing two-way comparisons are offered in Number?5; an average linear correlation coefficient (r2) of 0.91 was obtained between the quantification results from any two analyses. These data are representative of the present study and demonstrate that good results can be obtained even from a single analysis. Open in a separate window Physique?4. Venn diagram of overlap of identifications among 3 analyses of mAb1 P2 PrA pool. All identifications with a PLGS score of 1000 in any run were included in the analysis (n = 74). Open in a separate window Physique?5. Quantitative correlations (in ppm) among three replicate analyses of mAb1 P2 PrA pool, showing each HCP plotted as an individual point. The most abundant HCP, clusterin, was plotted at its nominal value, even though this likely represents an under-quantification (text). FVIP and further downstream Although not designed for this purpose, HCP levels were further reduced following low pH viral inactivation and depth filtration of the PrA column eluate (Table 2). The total HCP reduction by this step was significant (nearly 10-fold) for mAb1. Notably, with mAb2, where the PrA pool HCP content was already much lower, the subsequent viral inactivation/depth filtration step resulted in only a ~2-fold reduction. This difference may be due to differences in depth filters used or due to the difference in composition, differences in the interactions with the antibody, or concentration of the HCPs in the ProA pool sample. As a result, although the mAb1 and mAb2 PrA pool HCP levels differed ~6-fold, the FVIP HCP levels were similar. The process reproducibility of these results was not RICTOR investigated. The second chromatography step for both P1 and P2 of mAb1 consisted of cation-exchange chromatography in the bind/elute mode, although different resins were used. MSE analysis of the CEX pools yielded no confident identifications, demonstrating both the effectiveness of this purification step and the practical sensitivity limitation of our current methodology. The second chromatography step of the mAb2 process was mixed-mode CaptoAdhere chromatography conducted in the flow-through mode. In the CaptoAdhere flow-through pool, six HCPs, totaling 195 ppm, were confidently identified. However, a subsequent ion-exchange chromatography step further reduced HCPs to non-detectable levels. HCP step clearance Quantitative tracking of individual HCPs through purification is usually of interest to process developers because it allows clearance factors at various actions to be calculated and correlated with physiochemical properties of individual HCPs. This, in turn, should allow informed, as opposed to empirical, purification improvements that target HCPs deemed problematic. Because the highly effective PrA affinity purification step reduced the number of identified HCPs to manageable levels, we tracked all HCPs detected in the PrA pools of mAb1 Pr1 and mAb2 throughout all actions of their respective processes. Results for mAb1 P1 are shown in Table 3 (partial data) and Table S5 (complete data). There appear to be considerable variations in the reduction factor for individual HCPs across PrA chromatography, even though accurately estimating these for many of the most abundant HCPs in CCCF is usually difficult. Reduction to at least 23% of the level.