Utilization of Zwitterion-based solutions to dissect the relative effects of solution pH and ionic strength on the aggregation behavior and conformational stability of a fusion protein.

Solution pH and ionic strength (I) have complex effects on protein stability. We developed an experimental approach based on exploitation of the zwitterionic characteristic of amino acid molecules to probe the relative contribution from each. A variety of types of amino acid solutions were adopted to investigate the effects of pH and I in a manner that allows independent evaluation of each factor. The same effect could not be achieved using conventional buffer solutions. Size-exclusion chromatography, capillary differential scanning calorimetry, and fluorescence spectroscopy were utilized to probe the protein aggregation and conformation. The results suggested that, in addition to pH, solution ionic strength as a function of ionization state of the amino acid molecules and the ions introduced by pH adjustment played an important role in the aggregation and conformation of the protein studied. This experimental approach offers a useful tool to aid fundamental understanding of the relative effects of solution pH and ionic strength on protein stability.

[1]  Werner Kunz,et al.  Specific ion effects in colloidal and biological systems , 2010 .

[2]  Tim J Kamerzell,et al.  The complex inter-relationships between protein flexibility and stability. , 2008, Journal of pharmaceutical sciences.

[3]  M. Morbidelli,et al.  Aggregation Mechanism of an IgG2 and two IgG1 Monoclonal Antibodies at low pH: From Oligomers to Larger Aggregates , 2012, Pharmaceutical Research.

[4]  Christopher J Roberts,et al.  Nonnative aggregation of an IgG1 antibody in acidic conditions: part 1. Unfolding, colloidal interactions, and formation of high-molecular-weight aggregates. , 2011, Journal of pharmaceutical sciences.

[5]  M. Record,et al.  Thermodynamic origin of hofmeister ion effects. , 2008, The journal of physical chemistry. B.

[6]  M. Manning,et al.  Aggregation of recombinant human interferon gamma: kinetics and structural transitions. , 1998, Journal of pharmaceutical sciences.

[7]  K. Dill.  Dominant forces in protein folding. , 1990, Biochemistry.

[8]  D. Brems,et al.  Self-buffering antibody formulations. , 2008, Journal of pharmaceutical sciences.

[9]  Henry Eyring,et al.  Conformation Changes of Proteins , 1954 .

[10]  C Russell Middaugh,et al.  Effect of ionic strength and pH on the physical and chemical stability of a monoclonal antibody antigen-binding fragment. , 2013, Journal of pharmaceutical sciences.

[11]  John F. Carpenter,et al.  Physical Stability of Proteins in Aqueous Solution: Mechanism and Driving Forces in Nonnative Protein Aggregation , 2003, Pharmaceutical Research.

[12]  J. M. Sanchez-Ruiz,et al.  Theoretical analysis of Lumry-Eyring models in differential scanning calorimetry. , 1992, Biophysical journal.

[13]  A. Mcauley,et al.  Excipients for Protein Drugs , 2006 .

[14]  Dexter S. Moore,et al.  Amino acid and peptide net charges: A simple calculational procedure , 1985 .

[15]  Brian M. Murphy,et al.  Stability of Protein Pharmaceuticals: An Update , 2010, Pharmaceutical Research.

[16]  Charles L Brooks,et al.  The effects of ionic strength on protein stability: the cold shock protein family. , 2002, Journal of molecular biology.

[17]  Massimo Morbidelli,et al.  On the role of salt type and concentration on the stability behavior of a monoclonal antibody solution. , 2012, Biophysical chemistry.

[18]  C. Henry,et al.  Effect of buffer species on the thermally induced aggregation of interferon-tau. , 2006, Journal of Pharmacy and Science.

[19]  S. Shire,et al.  Stability and characterization of recombinant human relaxin. , 1996, Pharmaceutical biotechnology.

[20]  Theodore W Randolph,et al.  Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition. , 2002, Biochemistry.

[21]  T. Siahaan,et al.  Evaluation of the physical stability of the EC5 domain of E-cadherin: effects of pH, temperature, ionic strength, and disulfide bonds. , 2009, Journal of pharmaceutical sciences.

[22]  J. Lecomte,et al.  Temperature dependence of histidine ionization constants in myoglobin. , 1997, Biophysical journal.

[23]  Yatin R. Gokarn,et al.  Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions , 2011, Protein science : a publication of the Protein Society.

[24]  V. Nashine,et al.  Orthogonal High-Throughput Thermal Scanning Method for Rank Ordering Protein Formulations , 2013, AAPS PharmSciTech.

[25]  A. Rosenberg,et al.  Effects of protein aggregates: An immunologic perspective , 2006, The AAPS Journal.

[26]  D. W. Ball,et al.  Quantitative measurement of the solvent accessibility of histidine imidazole groups in proteins. , 2012, Biochemistry.

[27]  Christopher J Roberts,et al.  Aggregation of anti-streptavidin immunoglobulin gamma-1 involves Fab unfolding and competing growth pathways mediated by pH and salt concentration. , 2013, Biophysical chemistry.

[28]  Theodore W Randolph,et al.  Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony‐stimulating factor , 2003, Protein science : a publication of the Protein Society.