Biomedical subjects

Michael R Stoner

Publications and source records attributed to Michael R Stoner.

4 recordsLinked to original sources

PubMed2006

Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged in the biopharmaceutical industry as a technique to detect small quantities of protein aggregates. However, the limits of detection and quantitation of these aggregates are not yet well understood. Although diverse factors (molecule, instrument, technique, and software dependent) preclude an all-encompassing measurement of these limits for the complete system, it is possible to use simulated data to determine the quantitation limits of the data analysis software aspect. The current study examines the performance of the SEDFIT/c(s) data analysis tool with simulated antibody monomer/dimer and monomer/aggregate systems. Under completely ideal conditions (zero noise, known meniscus, and shape factor homogeneity), the software limit of quantitation was 0.01% for the monomer/aggregate system and 0.03% for the less well-resolved monomer/dimer system. Under more realistic conditions (0.005 OD root mean square [RMS] noise, shape factor variability, and long solution column), the software limits of quantitation were 0.2 and 0.6% (0.002 and 0.006 OD) for the monomer/aggregate and monomer/dimer systems, respectively. Interestingly, diminished quantitation accuracy at very low levels of oligomer was not accompanied by deterioration of fit quality (as measured by root mean square deviation [RMSD] and residuals bitmap images).

Animals↗

PubMed2004

Protein-solute interactions affect the outcome of ultrafiltration/diafiltration operations.

Protein production operations often involve a final diafiltration of the protein into formulation buffer. For several Amgen product proteins, post-diafiltration assays revealed a significant difference in molar excipient concentrations on the retentate and the permeate side of the membrane. For example, post-diafiltration assays of formulated 200 mg/mL human interleukin-1 receptor antagonist showed molar chloride concentrations up to 30% lower than those of the diafiltration buffer. Deviations from expected results were also observed in cases where a fusion conjugate protein (AMG-719) was formulated by dialysis in 10 mM acetate and where PEGylated soluble tumor necrosis factor receptor (PEG-sTNF-RI) was formulated in 270 mM glycine and 10 mM histidine. Classical thermodynamic theory describing intermolecular interactions predicts that the partitioning of small solutes during dialysis will be dependent on the protein concentration, charge, and surface area. This study illustrates methods to approximate these effects using readily available protein data (theoretical titration curves based on protein sequence, density information, etc.). Additionally, guidelines are provided to determine when intermolecular interactions are likely to significantly impact the outcome of dialysis/diafiltration operations.

Chemistry, Pharmaceutical↗

PubMedDate not supplied

Surfactant-induced unfolding of cellulase: kinetic studies.

Surfactant-induced unfolding is a significant degradation pathway for detergent enzymes. This study examines the kinetics of surfactant-induced unfolding for endoglucanase III, a detergent cellulase, under conditions of varying pH, temperature, ionic strength, surfactant type, and surfactant concentration. Interactions between protein and surfactant monomer are shown to play a key role in determining the kinetics of the unfolding process. We demonstrate that the unfolding rate can be slowed by (1) modifying protein charge and/or pH conditions to create electrostatic repulsion of ionic surfactants and (2) reducing the amount of monomeric ionic surfactant available for interaction with the enzyme (i.e., by lowering the critical micelle concentration). Additionally, our results illustrate that there is a poor correlation between thermodynamic stability in buffer (DeltaG(unfolding)) and resistance to surfactant-induced unfolding.

Bacteria↗

PubMedDate not supplied

Ca2+-surfactant interactions affect enzyme stability in detergent solutions.

Detergent proteases and amylases generally bind Ca(2+) ions. These bound ions enhance enzyme stability, reducing the rates of degradative reactions such as unfolding and proteolysis. Thus, surfactant aggregates, such as micelles, affect protease and amylase stability indirectly, by competing with the enzymes for Ca(2+) ions. Dissociation constants for Ca(2+) interactions with anionic surfactant micelles are in the 10(-3) to 10(-2) M range. These interactions are weak relative to enzyme-Ca(2+) interactions (K(d) of order 10(-6) M). However, surfactant is typically present at much higher concentration than enzyme, and it is the Ca(2+)-micelle equilibrium that largely determines the amount of free Ca(2+) available for binding to enzymes. The problem of surfactant-mediated Ca(2+) removal from enzymes can be avoided by adding calcium to a detergent formulation in an amount such that the concentration of free Ca(2+) is around 10(-5)M.

Bacillus↗