7/28/2023 0 Comments Buufers for capto q![]() A number of “problematic” HCPs have been identified in the context of mAb polishing, including (i) product-bound species (e.g., nidogen-1, secreted protein acidic and cysteine-rich (SPARC) protein, clusterin) (ii) species that co-elute with mAb from protein A (e.g., histone) (iii) proteins that affect product stability (e.g., lipoprotein lipase) and (iv) species that stimulate an immunogenic response (e.g., phospholipase B-like protein). In flow-through mode, the adsorbent features a broad specificity to allow for the capture of not only the HCP species, but also other impurities and contaminants such as DNA, leached Protein A, and media components. Process and product impurities not removed in the capture step are typically cleared in subsequent polishing steps, operated either in bind-and-elute or flow-through modes. This step is performed in bind-and-elute mode, where the mAb product is retained and non-binding impurities flow through, thus achieving both impurity removal and concentration of the target product. ![]() While general guidelines point to a maximum allowable HCP content in the final formulation of a biotherapeutic at 90%) is removed at the product capture step, which currently relies on either affinity chromatography, mainly Protein A, or alternatively ion exchange or multimodal chromatography. The effective removal of host cell proteins (HCPs) from mammalian cell culture supernatants is a crucial issue in the manufacturing of biopharmaceuticals for human therapy. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography. 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. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. ![]() 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. 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. 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. ![]() 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. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. 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. 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.
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