High‐Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation. Issue 11 (24th September 2013)

• Record Type: Journal Article
• Authors: Alsenaidy, Mohammad A.; Kim, Jae Hyun; Majumdar, Ranajoy; Weis, David D.; Joshi, Sangeeta B.; Tolbert, Thomas J.; Middaugh, C. Russell; Volkin, David B.
• Is Part Of: Journal of pharmaceutical sciences. Volume 102:Issue 11(2013:Nov.)
• Page Start: 3942
• Page End: 3956
• Publication Date: 2013-09-24
• Subjects: Pharmacy -- Periodicals; 615.1
• DOI: 10.1002/jps.23730
• Languages: English
• ISSNs: 0022-3549

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial or complete enzymatic removal of the N‐linked Fc glycan, were compared with the untreated mAb over a wide range of temperature (10°C–90°C) and solution pH (3–8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams. Subtle‐to‐larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0–6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in the physical stability of a mAb glycoform. On the basis of these results, a two‐step methodology was used in which conformational stability of a mAb glycoform is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques, and data visualization methods. With this approach, a high‐throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3942–3956, 2013</p> </abstract>