Circular Dichroism Spectroscopy as a Tool for Monitoring Aggregation in Monoclonal Antibody Therapeutics

• Published in: Analytical chemistry (Washington) Vol. 86; no. 23; pp. 11606 - 11613
• Main Authors: Joshi, Varsha, Shivach, Tarun, Yadav, Nitin, Rathore, Anurag S.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02-12-2014
• Subjects: Agglomeration; Analytical chemistry; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - chemistry; Buffers; Chromatography; Circular Dichroism; Dichroism; Formulations; Monoclonal antibodies; Protein Aggregates; Proteins; Scanning; Spectrum analysis; Stability; Temperature
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac503140j

Aggregation continues to be a critical quality attribute for a monoclonal antibody therapeutic product due to its perceived significant impact on immunogenicity. This paper aims to establish the versatility of circular dichorism (CD) spectroscopy toward understanding aggregation of monoclonal antibody (mAb) therapeutics. The first application involves the use of far-UV CD as a complementary analytical technique to size exclusion chromatography (SEC) for understanding protein aggregation. The second application uses thermal scanning CD as a high throughput screening tool for examining stability of a mAb therapeutic in various formulation and downstream buffers. For establishing far-UV CD as an orthogonal technique, a mAb was incubated in different downstream processing buffers and another mAb in formulation buffers, and they were analyzed by SEC and far-UV CD for aggregate content and conformational stability, respectively. To examine thermal scanning as a high throughput screening tool, ellipticity as a function of the temperature was measured at 218 nm from 20 to 90 °C. Far-UV CD was found to display high sensitivity toward early detection of conformational changes in mAb. CD measurements were also able to elucidate the different aggregation mechanisms. Furthermore, thermal stability scan allowed us to estimate T onset which has been found to correlate with aggregation induced by salt, low pH, and buffer species. T onset temperature from thermal scanning at 218 nm using CD was correlated successfully to aggregate content measured by SEC. Results from both the studies demonstrate the usefulness of CD for assessing stability of therapeutic proteins during process development, formulation development, and product characterization.