RESUMO
Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure, thereby allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we independently evaluated cation exchangers to facilitate media selection and investigated the relationship between surface modification and chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained high capacities even with high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions with minimal residence/contact time. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity, and could contribute to cost reduction. In this work, we evaluated the dynamic binding capacities of various new ion exchange resins at different binding conductivities for different residence times, and observed that.
Assuntos
Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica/métodos , Humanos , Imunoglobulina G/isolamento & purificação , Modelos Químicos , Muramidase/isolamento & purificação , PressãoRESUMO
We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of these mixed mode resins. Optimal conditions for antibody purification and host cell proteins (HCPs) elimination were determined and then directly up-scaled to laboratory columns. Then we used mass spectrometry to identify the major HCPs potentially coeluted with the antibody. Differences between the four resins in terms of amount, complexity and identity of the HCPs present in the elution fractions were investigated.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida , Modelos Químicos , Proteínas/classificação , Proteínas Recombinantes/isolamento & purificação , Animais , Anticorpos Monoclonais/química , Células CHO , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Cricetinae , Cricetulus , Condutividade Elétrica , Ensaios de Triagem em Larga Escala , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Reagentes de Laboratório , Espectrometria de Massas , Proteínas/química , Proteínas Recombinantes/química , Reprodutibilidade dos TestesRESUMO
Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure and allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we suggested an independent evaluation of major anion exchangers to facilitate media selection, and investigated the relationship between (i) surface modification and (ii) chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained mainly high capacities even with extremely high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity and could contribute to cost reduction.
Assuntos
Cromatografia por Troca Iônica/instrumentação , Cromatografia por Troca Iônica/métodos , Resinas Sintéticas/química , Animais , Bovinos , Cromatografia por Troca Iônica/economia , Conalbumina/química , Conalbumina/isolamento & purificação , Ligação Proteica , Resinas Sintéticas/economia , Albumina Sérica/química , Albumina Sérica/isolamento & purificaçãoRESUMO
Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.
