Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

Abstract

The clearance of host cell proteins (HCPs) is of crucial importance in biomanufacturing, given their diversity in composition, structure, abundance, and occasional structural homology with the product. The current approach to HCP clearance in the manufacturing of monoclonal antibodies (mAbs) relies on product capture with Protein A followed by removal of residual HCPs in flow‐through mode using ion exchange or mixed‐mode chromatography. Recent studies have highlighted the presence of “problematic HCP” species, which are either difficult to remove (Group I), can degrade the mAb product (Group II), or trigger immunogenic reactions (Group III). To improve the clearance of these species, we developed a family of synthetic peptides that target HCPs and exhibit low binding to IgG product. In this study, these peptides were conjugated onto chromatographic resins and evaluated in terms of HCP clearance and mAb yield, using an industrial mAb‐producing CHO harvest as model supernatant. To gather detailed knowledge on the binding of individual HCPs, the unbound fractions were subjected to shotgun proteomic analysis by mass spectrometry. It was found that these peptide ligands exhibit superior HCP binding capability compared to those of the benchmark commercial resins commonly used in mAb purification. In addition, some peptide‐based resins resulted in much lower losses of product yield compared to these commercial supports. The proteomic analysis showed effective capture of many “problematic HCPs” by the peptide ligands, especially some that are weakly bound by commercial media. Collectively, these results indicate that these peptides show great promise toward the development of next‐generation adsorbents for safer and cost‐effective manufacturing of biologics.

Purification of polyclonal immunoglobulin G from human serum using peptide‐based adsorbents

“…The resulting profiles of Y pIgG and Q PP vs. loading volume are reported in Figure 9C,E; the corresponding profiles of C pIgG /C pIgG * are reported in Figure S6. The loading of 20‐fold diluted plasma (pIgG ~0.6 mg/ml; non‐Ig plasma proteins ~3.9 mg/ml) resulted in efficient flow‐through purification: at the cut‐off loading (10 CVs), the cumulative pIgG purity in the effluent reached 98.1% (Figure 10A); the global yield, on the other hand, reached 25.4% only: the low protein concentration in the feedstock is unlikely to match the high binding capacity of the second‐generation LigaGuard™ and prevent undesired capture of IgG via weak partitioning mechanism, as described in prior work 26,27 . Accordingly, the loading of 10‐fold diluted plasma resulted in a significant increase in pIgG yield with high purity; specifically, at the loading volume of 4.0 ml (8 CVs), Y pIgG and Q PP , respectively, reached approximately 71% and 27 mg per ml resin, corresponding to a cumulative product purity of approximatley 80%; beyond this point, however, a significant amount of non‐Ig proteins flow through the column, lowering product purity to approximatley 70% at the loading volume of 5.5 ml (Figure 10B).…”

“…In prior work, we demonstrated the use of LigaGuard™ resin as a scrubber of process‐related impurities prior to the affinity capture step in a process purification for monoclonal antibodies 26,27 . In analogy to that study, we attempted the purification of pIgG from plasma using a two‐column process comprising a LigaGuard™ adsorbent that captures the non‐Ig plasma proteins in flow‐through followed by a LigaTrap™ adsorbent that operates in bind‐and‐elute mode to isolate and concentrate pIgG.…”

Purification of polyclonal Immunoglobulin G from human serum using LigaGuardTM and LigaTrapTM adsorbents

“…Following on these results, we proceeded with the flow-through purification of pIgG from cryo-rich plasma using the second-generation LigaGuard adsorbent. This was imputed to the protein concentration in the feedstock being inadequate to match the higher binding capacity of the second-generation LigaGuard adsorbent: specifically, the titer of non-Ig plasma proteins was insufficient to prevent undesired capture of IgG or displace the bound pIgG molecules via the weak partitioning mechanism described in prior work [ 26,27 ]. We therefore adjusted loading conditions by reducing the dilution of the feedstock to 10-fold and 5-fold, which increased the titer of pIgG to˜1.0 and 2.0 mg/mL, and non-Ig plasma proteins to˜7.2 and 14.5 mg/mL, respectively.…”

“…In prior work, we have demonstrated the use of LigaGuard TM resin as a scrubber of process-related impurities prior to the Protein A-based affinity capture step in a process of antibody purification from CHO cell culture fluids [ 26,27 ]. In analogy to that study, we attempted the purification of pIgG from plasma using a twocolumn process comprising a LigaGuard adsorbent that captures the non-Ig plasma proteins in flow-through followed by a LigaTrap TM adsorbent that operates in bind-and-elute mode to isolate and concentrate pIgG.…”

“…Our group has developed the concept of "flow-through affinity chromatography", wherein all the nontarget species in a complex feedstock are captured by peptide ligands immobilized on a chromatographic substrate (LigaGuard TM ), allowing the target protein product to flow through unbound [ 25 ]. A LigaGuard TM adsorbent tailored for the purification of therapeutic mAbs from Chinese Hamster Ovary (CHO) cell culture harvests has been demonstrated in prior work [ 26,27 ]. In this study, a LigaGuard TM adsorbent tailored for IVIg purification from human plasma has been developed, whose peptide ligands bind all non-Ig plasma proteins.…”