title: Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.
abstract: In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions.
authors.affiliation: Sterile Product & Analytical Development, Bioprocess Development, Merck Research Laboratories, Kenilworth, New Jersey, 07033.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Rizzo
authors.forename: Shuai
authors.initials: S
authors.lastname: Shi
authors.forename: Yunsong
authors.initials: Y
authors.lastname: Li
authors.forename: Andrew
authors.initials: A
authors.lastname: Semple
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: Esposito
authors.forename: Shenjiang
authors.initials: S
authors.lastname: Yu
authors.forename: Daisy
authors.initials: D
authors.lastname: Richardson
authors.forename: Valentyn
authors.initials: V
authors.lastname: Antochshuk
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shameem
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D002626:Chemistry, Pharmaceutical; D002942:Circular Dichroism / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D066329:Protein Aggregates; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D066329:Protein Aggregates; D011994:Recombinant Proteins
keywords: calorimetry (DSC); chemical denaturation; circular dichroism; formulation; guanidine-HCl; intrinsic fluorescence; protein aggregation; protein folding/refolding; robotic automation; urea