Systematic purification of salt‐intolerant proteins by ion‐exchange chromatography: The example of human α‐galactosidase A

Abstract

Chromatography is an essential tool for purifying biopharmaceutical products. Many processes are still developed based on traditional routines and empirical procedures. Product losses are mostly due to insufficient optimization of purification setups and product sensitivity to process conditions. In order to eliminate these shortcomings, a systematic strategy for the setup of ion‐exchange chromatography is presented, which considers both product stability as well as operational conditions. The stages—a hybrid approach combining high‐throughput screening and analytical small‐scale chromatography—are as follows: (1) pH stability (short‐term); (2) pH stability (long‐term), followed by a screening of additives to enhance protein stability, if required; (3) analytical pH gradient chromatography for evaluation of the operational pH window; and (4) salt stability (long‐term) in the operational pH window determined. The efficiency and straightforwardness of the strategy were shown in a case study on capturing the human α‐galactosidase A enzyme. Following the above procedure, the enzyme was found to be salt‐unstable; a purification factor of 13.2, a concentration factor of 4, and an overall yield of 84.3% were achieved. The applied strategy allowed for a quick establishment of a dedicated capture step at low salt concentrations under stable conditions by well‐chosen prior screening experiments.

Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches

“…In a study by Baumann et al. , pH gradients served as a systematic screening tool for IEX process development and lysate characterization including salt‐intolerant proteins as demonstrated for human α‐Galactosidase A from a complex Pichia pastoris supernatant. For HIC, such a characterization based on pH gradients is hard to accomplish.…”

“…Kröner et al [32] performed a multi-dimensional analysis for the isolation of Nucleolin from Sf9 feedstocks starting a pre-fractionation using pH gradients. In a study by Baumann et al [7], pH gradients served as a systematic screening tool for IEX process development and lysate characterization including salt-intolerant proteins as demonstrated for human α-Galactosidase A from a complex Pichia pastoris supernatant. For HIC, such a characterization based on pH gradients is hard to accomplish.…”

“…Inversely, the experimental approach is used for characterizing processes and generating new platforms and databases. When defined HT experiments are used for lysate characterization of unknown compositions and a systematic prediction of key parameters for new tailored processes, this can be referred to as a deterministic approach .…”