Choosing the right protein A affinity chromatography media can remove aggregates efficiently
| Autor: | Noriko Yoshimoto, Tomokazu Yada, Koichi Nonaka, Shuichi Yamamoto, Masayuki Yabuta |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Arginine Protein aggregation Antibodies Monoclonal Humanized 01 natural sciences Applied Microbiology and Biotechnology Chromatography Affinity 03 medical and health sciences chemistry.chemical_compound Affinity chromatography Ph gradient Staphylococcal Protein A Chromatography biology Elution 010401 analytical chemistry food and beverages Antibodies Monoclonal General Medicine Hydrogen-Ion Concentration humanities 0104 chemical sciences 030104 developmental biology Monomer chemistry Yield (chemistry) biology.protein Molecular Medicine Electrophoresis Polyacrylamide Gel Protein A |
| Zdroj: | Biotechnology journal. 12(1) |
| ISSN: | 1860-7314 |
| Popis: | Protein A chromatography (PAC) is commonly used as an efficient capture step in monoclonal antibody (mAb) separation processes. Usually dynamic binding capacity is used for choosing the right PAC. However, if aggregates can be efficiently removed during elution, it can make the following polishing steps easier. In this study a method for choosing the right PAC media in terms of mAb aggregate removal is proposed. Linear pH gradient elution experiments of two different mAbs on various PAC columns are carried out, where the elution behavior of aggregates as well as the monomer is measured. Aggregates of one mAb are more strongly retained compared with the mAb monomer. Another mAb showed different elution behavior, where the aggregates are eluted as both the weakly and strongly retained peaks. In order to remove the two types of aggregates by stepwise elution two protocols are tested. The first protocol A consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. No detectable peaks are observed during the wash stage of protocol A whereas significant peaks are monitored during the arginine wash of protocol B. One of the PAC columns showed a smaller peak during the arginine wash. In addition, both aggregate removal and monomer yield are higher with protocol B compared with the other PAC columns. This method is found to be useful for choosing the right PAC column. |
| Databáze: | OpenAIRE |
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