Evaluating Immunogenicity Risk Due to Host Cell Protein Impurities in Antibody-Based Biotherapeutics

Abstract

A potential risk factor for immunogenicity of a biotherapeutic is the low levels of host cell protein (HCP) impurities that remain in the product following the purification process. During process development, significant attention has been devoted to removing HCPs due to their potential safety risk. Samples from different purification steps of several monoclonal antibodies (mAbs) purified by one type of platform were evaluated for their residual Chinese Hamster Ovary (CHO) cell-derived HCP content. HCPs in both in-process (high levels of HCP) and highly purified (low levels of HCP) samples were identified and quantitated by proteomic analysis via mass spectrometry. The responses to HCPs were evaluated in an in vitro assay using PBMC from a population of healthy and disease state individuals. Results indicated that samples with up to 4000 ppm HCP content (levels 200 times greater than the drug substance) did not pose a higher immunogenicity risk than highly purified mAb samples. As an orthogonal method to predict immunogenicity risk, in silico algorithms that probe amino acid sequence for foreign epitope content were used to evaluate over 20 common HCPs (identified in the different mAb samples). Only a few HCPs were identified as high risk by the algorithms; however, the in vitro assay results indicated that the concentration of these HCPs from in-process biotherapeutic mAb samples was not sufficient to stimulate an immune response. This suggests that high levels of HCP in mAb biotherapeutics purified by this type of platform do not increase the potential risk of immunogenicity of these molecules. Insights from these studies can be applied to HCP control and risk assessment strategies.

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

“…Calculated p ‐values for ANOVA comparisons of each peptide resin compared to both benchmark resins (Capto Q and Capto Adhere) are provided. Species identified by shotgun proteomics in this study identified as “problematic” based on prior art (Aboulaich et al, ; Bailey‐Kellogg et al, ; Chiu et al, ; Fischer et al, ; Gagnon et al, ; Goey et al, ; Jawa et al, ; Levy et al, ; Mechetner et al, ; Zhang et al, ). * p < .05.…”

“…This study focuses on problematic HCPs in three primary categories based on risk factors described above: (a) HCPs co‐eluting with mAbs in the capture step, herein referred to as Group I (Aboulaich et al, ; Gagnon et al, ; Levy et al, ; Mechetner et al, ; Zhang et al, ), including IgG‐associated and Protein A‐binding HCPs, (b) HCPs that cause product degradation, or Group II (Aboulaich et al, ; Bee et al, ; Chiu et al, ; Goey et al, ; Zhang et al, ), and (c) HCPs with high immunogenicity risk, or Group III (Bailey‐Kellogg et al, ; Fischer et al, ; Goey et al, ; Jawa et al, ). In typical mAb platform processes, the large majority of HCPs are removed at the product capture step with Protein A‐based adsorbents; thus, the design of the subsequent intermediate and final polishing steps is highly dependent on their ability to remove the species that co‐elute with IgG from Protein A.…”

Transcriptomic analysis of IgG4 Fc‐fusion protein degradation in a panel of clonally‐derived CHO cell lines using RNASeq

“…DNA, RNA, proteins, lipids, and virus) present in the cell factory and cell culture medium (Bracewell, Francis, & Smales, ). The removal of these “process” impurities from the harvest fluid particularly host cell proteins (HCPs), which even at relatively low levels have the potential to elicit an immunogenic response in patients, is considered to be a critical quality attribute during the process development (Jawa et al, ). The second class of impurity encountered during the manufacture arise from molecular variants of the therapeutic protein itself.…”

Mass Spectrometry Approaches for Identification and Quantitation of Therapeutic Monoclonal Antibodies in the Clinical Laboratory

“…At the same time, novel fragmentation methods were being developed that allowed extensive characterization of MAbs in the gas phase (40–42). Currently, MS plays a central role in the routine quality control of therapeutic-MAb production (43–45), and new MS-based methodologies for monitoring are continuously appearing in the literature. The remainder of this review will focus on translating MS-based methods for quantifying therapeutic MAbs into the clinical laboratory with a focus on selecting methods that result in the highest level of analytical sensitivity and specificity with respect to each patient's endogenous Ig repertoire.…”