Tangir Ahamed

Multi‐dimensional fractionation and characterization of crude protein mixtures: Toward establishment of a database of protein purification process development parameters

A multi‐dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion‐exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size‐exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt‐gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision‐making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge‐based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation. Biotechnol. Bioeng. 2012; 109: 3070–3083.

The use of self-interaction chromatography in stable formulation and crystallization of proteins

This paper reviews the basic principles of the recently developed self‐interaction chromatography (SIC) technique with regard to protein solution stability and protein crystallization. It gives experimental protocols for both normal‐scale and micro‐scale SIC experiments and reviews recent developments and current applications of this novel technique in the biopharmaceutical area. This paper aims to be a benchmark in the further proliferation of this highly effective and fast technology for the rational design of stable aqueous formulations of therapeutic proteins and the determination of solution conditions favoring protein crystallization.