Chemical Stability

Chemical instability involves formation or destruction of covalent bonds within a peptide or protein, such as deamidation of asparagine and/or glutamine residues, oxidation of methionine and other amino acids, reduction of disulfide bonds and disulfide interchange and hydrolysis of peptide bonds. The samples are generally analyzed by orthogonal techniques to ensure the best understanding of the effects of a stressor on the protein of interest. Many of these techniques quantitate degraded products while others are used to determine their identity.

The following high-throughput instruments are available at MVSC to measure chemical stability of macromolecules and vaccine antigens.

Chromatography

  • Shimadzu HPLCs (reverse phase, ion exchange, and size exclusion)
  • Amersham Pharmacia Biotech AKTA FPLC system
  • Beckman-Coulter ProteomeLab PF 2D System

Charge/Size

  • Convergent Biosciences iCE 280 IEF analyzers (2)
  • SDS-PAGE with Alphar Imager Gel imaging system
  • Western blot for ID

Mass Spectrometers

  • Thermo Fisher Scientific LTQ XL mass spectrometer
  • Waters Micromass MALDI-LR system

Forced Degradation Studies: Studies of the chemical stability of a protein, with an emphasis on Met oxidation, Asn deamidation and Asp isomerization processes as well as proteolytic degradation, are conducted using a combination of higher pH to induce deamidation/isomerization, various oxidizing agents (e.g., H2O2, metals) to produce oxidation, and a combination of pH, temperature and other stresses to enhance proteolytic degradation. Stability-indicating (forced degradation) assays are developed using multiple chromatographic and electrophoretic techniques such as RP-HPLC, cIEF (see below), ion exchange, and SDS-PAGE.

Charge Heterogeneity: Changes in charge heterogeneity of a protein are generally monitored by capillary isoelectric focusing studies with a Convergent Bioscience iCE280 analyzer.  This instrumentation employs capillary electrophoresis to measure pI values on small samples (0.1 mg/ml) in the presence of urea and/or under native conditions.  Thus, Asn deamidation, Asp isomerization, differential glycosylation, and in some cases, oxidized species of a protein can be simply detected.

Peptide Mapping: We employ a combination of RP-HPLC in conjunction with mass spectrometry and peptide mapping to identify the specific amino acid residue sites of chemical degradation in the protein. For peptide mapping, the protein is first digested with appropriate proteases (e.g., trypsin) and the resultant peptides are separated by a RP-HPLC system, quantified by UV spectroscopy, and then analyzed for identity by mass spectrometry. 

Contact Information

Head, David B. Volkin PhD

2030 Becker Drive
Lawrence, KS 66047
volkin@ku.edu
785-864-6262

Director, Sangeeta B. Joshi PhD

2030 Becker Drive
Lawrence, KS 66047
joshi@ku.edu
785-864-3356

Scientific Advisor, C. Russell Middaugh PhD

2030 Becker Drive
Lawrence, KS 66047
middaugh@ku.edu
785-864-5813