Abstract
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.
Highlights
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
Concerns regarding HCP impurities stem from a variety of risk factors, including (i) an immunogenic response from repeated exposure to low concentration HCPs present in drugs administered over time [2], (ii) reduced therapeutic efficacy caused by product-bound impurities [3], and (iii) low shelf-life of the therapeutic product caused by low-concentration proteolytic enzymes present in the final formulation [4]
Multipolar and hydrophobic positive peptides show promise as next-generation mixed-mode ligands for Chinese hamster ovary (CHO) HCP capture in IgG purification applications
Summary
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. In the manufacturing of therapeutic monoclonal antibodies (mAbs), the majority of HCP impurities (>90%) [9] is removed at the product capture step, which currently relies on either affinity chromatography, mainly Protein A [10,11], or alternatively ion exchange [12] or multimodal chromatography [13]. This step is performed in bind-and-elute mode, where the mAb product is retained and non-binding impurities flow through, achieving both impurity removal and concentration of the target product. While the threshold for bead selection for the HCP fluorescence in this instance may appear substantially lower than observed with the manual screening, differences were expected given that a different Alexa Fluor dye was required for this system (Alexa Fluor 546 [34], which has a lower reported initial brightness compared to Alexa Fluor 594 [35]), in addition to the differences in imaging exposure and intensity required to visualize the beads
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