Application of ER Stress Biomarkers to Predict Formulated Monoclonal Antibody Stability

Abstract

For a therapeutic monoclonal antibody (mAb) to reach the clinic, the molecule must be produced at an appropriate yield and quality, then formulated to maintain efficacy and stability. The formation of subvisible particles (SVPs) can impact product stability and is monitored during formulation development; however, the potential of a mAb to form such species can be influenced throughout the whole bioprocess. The levels of intracellular endoplasmic reticulum (ER) stress perceived by Chinese hamster ovary (CHO) cell lines, the day of mAb harvest, and the relationship with subsequent product stability of two mAbs (denoted A and B), as determined by the SVP content after accelerated stability studies, are reported here. Here, it is shown that the propensity of mAb A to form SVPs can be predicted by transcript expression of biomarkers of cellular ER stress, heavy/light-chain transcript and polypeptide amounts, and harvest day. Further, mAb A material harvested on day 9 of culture was more stable, in terms of SVP formation, than material harvested on day 13. These data suggest that ER stress perceived by CHO cells can reflect the stability of a mAb, and that biomarkers of such stress could help define culture harvest time as a tool to control SVP formation in formulated mAbs.

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

“…3 and Supplemental Figures S6 c, d). We found the mRNA ChaC glutathione specific gamma-glutamylcyclotransferase 1 (CHAC1) displayed a similar expression profile to that of ATF4, CHOP, and Homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 ( HERPUD1) in both cell lines , while the mRNAs for Catalase (CAT), Peroxiredoxin-1 (PRDX1), and Superoxide dismutase 2 (SOD2) displayed transient increases on day 3 in the IgG 1 producer, consistent with other CHO cell studies 4 – 8 , 18 , 20 , 24 , 50 – 57 . Collectively, these mRNAs which are upregulated on day 3 are involved in glycoprotein folding, ERAD, and oxidative stress and are transcribed through the individual or combined action of ATF6α and XBP1s 25 , 28 , 58 – 61 .…”

Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

“…Thus, there is interest in limiting excessive high mannose glycan formation in IgGs during commercial manufacturing ( Mastrangeli et al, 2020 ). Current trends in biopharmaceutical manufacturing such as process intensification, development of fully humanized mAbs, growth in biosimilar products, development of high concentration mAb formulations, and the emergence of next-generation biopharmaceuticals such as anti-drug conjugates and bispecific antibodies, necessitates the control of high mannose content as a critical aspect in the process development ( Berkel et al, 2009 ; Shi and Goudar, 2014 ; Feinberg et al, 2017 ; Sumit et al, 2019 ; Talbot et al, 2019 ; Mastrangeli et al, 2020 ).…”

Temperature Down‐Shift Modifies Expression of UPR‐/ERAD‐Related Genes and Enhances Production of a Chimeric Fusion Protein in CHO Cells

“…[ 38,39 ] Elevated amounts of grp78 mRNA was associated with the extent of intracellular stress conditions, and therefore, it is no surprise to find high grp78 expression at late culture stages. [ 40 ] Antibody‐producing plasma cells are characterized by high expression of grp78 and grp94 , linked to immunoglobulin biosynthesis and folding. [ 41 ] ER chaperones expression is controlled by several UPR‐specific transcription factors, such as ATF6, ATF4, and XBP1s, that generate a series of feed‐back loop regulation.…”