Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles

Abstract

In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

“…The stability of protein structure can be a sensitive probe for the overall higher order structural integrity of proteins, and it has been suggested such an approach is an important part of protein comparability studies and biosimilarity analyses. [27][28][29] In this work, the resulting EPDs were similar overall for the 5 CRM 197 molecules, indicating a similar structural integrity and conformational stability. Not surprisingly, all the CRM 197 molecules showed a notable decrease in the overall conformational stability profile as the solution pH was lowered from 6.8 to 5.8, a result which is consistent with the known conformational change of diphtheria toxin at pH 5.0 related to insertion of the toxin into the endosomal membrane.…”

Correlating the Impact of Well-Defined Oligosaccharide Structures on Physical Stability Profiles of IgG1-Fc Glycoforms

“…45 In addition, site occupancy and amino acid changes at the Asn297 site within IgG1-Fc molecules were shown to affect their physical stability profile by the same experimental approach. [45][46][47][48] Similarly, Yageta et al 49 have recently used EPDs to compare the conformational and colloidal stability of isolated constant domains of human IgGs (including aglycosylated C H 2 domains) under acidic conditions. One key goal of these types of studies is to assess the utility of physical stability profiles in protein comparability assessments.…”

Hydrogen-Deuterium Exchange Mass Spectrometry as an Emerging Analytical Tool for Stabilization and Formulation Development of Therapeutic Monoclonal Antibodies

“…Hence, robust formulation strategies are needed to impart maximal stability to these proteins and to minimize degradation during long‐term storage (e.g., across the shelf life). From a pharmaceutical development perspective, it is also a major challenge to predict the effects of product or process changes on the higher order structure and long‐term stability of mAb drug products as part of comparability assessments . One recent trend in the formulation development field is to explore the interrelationships between antibody dynamics, conformational stability, and pharmaceutical stability .…”

“…From a pharmaceutical development perspective, it is also a major challenge to predict the effects of product or process changes on the higher order structure and long-term stability of mAb drug products as part of comparability assessments. 8 One recent trend in the formulation development field is to explore the interrelationships between antibody dynamics, conformational stability, and pharmaceutical stability. 7,[9][10][11][12][13] If direct relationships can be established between these variables, these relationships can lead to a better understanding of the implications of process and product changes on protein stability.…”