The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.

Infectious arthritis is difficult to treat in both human and veterinary clinical practice. Recent literature reports Staphylococcus aureus as well as other gram‐positive and gram‐negative isolates forming free‐floating biofilms in both human and equine synovial fluid that are tolerant to traditional antimicrobial therapy. Using an in vitro equine model, we investigated the ability of platelet‐rich plasma (PRP) formulations to combat synovial fluid biofilm aggregates. Synovial fluid was infected, and biofilm aggregates allowed to form over a 2‐hour period. PRP was collected and processed into different formulations by platelet concentration, leukocyte presence, and activation or lysis. Infected synovial fluid was treated with different PRP formulations with or without aminoglycoside cotreatment. Bacterial load (colony‐forming unit/mL) was determined by serial dilutions and plate counting at 8 hours posttreatment. All PRP formulations displayed antimicrobial properties; however, formulations containing higher concentrations of platelets without leukocytes had increased antimicrobial activity. Lysis of PRP and pooling of the PRP lysate (PRP‐L) from multiple horses as compared to individual horses further increased antimicrobial activity. This activity was lost with the removal of the plasma component or inhibition of the proteolytic activity within the plasma. Fractionation of pooled PRP‐L identified the bioactive components to be cationic and low‐molecular weight (<10 kDa). Overall, PRP‐L exhibited synergism with amikacin against aminoglycoside tolerant biofilm aggregates with greater activity against gram‐positive bacteria. In conclusion, the use of PRP‐L has the potential to augment current antimicrobial treatment regimens which could lead to a decrease in morbidity and mortality associated with infectious arthritis.

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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.