Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

A critical evaluation of self-interaction chromatography as a predictive tool for the assessment of protein-protein interactions in protein formulation development: a case study of a therapeutic monoclonal antibody.

The aim of this study was to establish and evaluate a screening method for the physical characterization of protein-protein interactions of therapeutic proteins based on the determination of the osmotic second virial coefficient (B(22)). B(22) of an IgG1 was measured by self-interaction chromatography (SIC) and was compared to data obtained from static light scattering (SLS). As assessed by Fourier transform infrared spectroscopy (FTIR), the protein coupling to chromatography particles had no relevant influence on the three-dimensional native structure of the IgG1. B(22) variations could be measured for physiological relevant excipient concentrations. Significant positive B(22) values were observed for the following solution conditions of the investigated antibody: (i) acidic pH conditions, (ii) low buffer concentrations, (iii) low salt concentrations and (iv) high amino acid concentrations. B(22) was compared to IgG1 stability data derived from a study conducted for 12weeks at 40 degrees C. A concentration of 5mM histidine, which was the most promising buffer candidate according to B(22), showed a slightly better physical stability (as assessed by turbidity and size exclusion chromatography) compared to the other tested formulations. This is confirmed in a stress study investigating the colloidal stability. Thus, measuring protein-protein interactions with SIC appeared as a promising screening tool for physical characterization of protein formulations for cases in which the protein stability is governed by interparticle interactions.

Correlation of protein-protein interactions as assessed by affinity chromatography with colloidal protein stability: a case study with lysozyme.

Lysozyme-lysozyme interactions were assessed in the native state at 25 degrees C as well in the denatured state at 80 degrees C by affinity chromatography in order to measure the osmotic second virial coefficient (B). This parameter allows us to better understand protein aggregation pathways and colloidal protein stability. Repulsive interactions (B > 0) were weakened for both protein states by increasing salt concentration and by increasing the pH value toward lysozyme pI. This decrease was more pronounced in the denatured state, most likely caused by changes in electrostatic interactions and the formation of hydrophobic clusters. The lysozyme formulations presenting the more repulsive conditions (B > 0), as derived from the osmotic second virial coefficient, showed better colloidal stability under mechanical and thermal stresses. As expected, B values are much more negative for the interactions in the denatured state compared to the data obtained for the native state, reflecting a strong tendency of denatured lysozyme to aggregate. Thus, measurement of protein interactions by affinity chromatography allows us to gain information on protein interactions in both native and denatured states as well as to predict solution conditions prone for improving protein colloidal stability.

Lysozyme-lysozyme self-interactions as assessed by the osmotic second virial coefficient: impact for physical protein stabilization.

The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein-protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B(22)), a parameter of solution non-ideality, which is obtained using self-interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B(22) was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B(22) is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B(22) decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein-protein interactions. The B(22) parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting-in/salting-out effect. It is also shown that B(22) correlates with protein solubility as well as physical protein stability.