“…Moreover, the weaker interaction between mAbs and HCX contributed to sufficiently elute proteins avoiding recombination and obstraction. Considering the unique binding properties of HCX, other reported methods to maximize HCP clearance (Ishihara and Hosono 2015 ; Shukla and Hinckley 2008 ) may also be worthy to be investigated with HCX in order to achieve more contaminant removal in a single purification step. Fig.…”
In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings.
“…Moreover, the weaker interaction between mAbs and HCX contributed to sufficiently elute proteins avoiding recombination and obstraction. Considering the unique binding properties of HCX, other reported methods to maximize HCP clearance (Ishihara and Hosono 2015 ; Shukla and Hinckley 2008 ) may also be worthy to be investigated with HCX in order to achieve more contaminant removal in a single purification step. Fig.…”
In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings.
“…For the bind-and-elute mode, the pI differences between HCPs and target proteins can be exploited to remove parts of HCPs prior to protein elution, thus minimizing co-elution of HCPs with the target protein. Additionally, amino acids have been used in various chromatographic separations to enhance the clearance of HCP during mAb purification processes [ 42 , 43 ]. Our own practice showed the use of CEX in combination with additional wash steps and amino acid additives in the elution buffer to achieve effective HCP removal for a recombinant protein with a pI of 5.9 ( Fig.…”
Section: Solutions For Challenging Protein Purificationmentioning
The innovation in recombinant protein technology has brought forth a host of challenges related to the purification of these therapeutic proteins. This article delves into the intricate landscape of developing purification processes for artificially designed therapeutic proteins. The key hurdles include controlling protein reduction, protein capture, ensuring stability, eliminating aggregates, removing host cell proteins (HCPs), and optimizing protein recovery. In this review, we outline the purification strategies in order to obtain products of high purity, highlighting the corresponding solutions to circumvent the unique challenges presented by recombinant therapeutic proteins, and exemplify the practical applications by case studies. Finally, a perspective towards future purification process development is provided.
“…During production and purification of mAbs, impurities are encountered which can result from product itself and the process. Product impurities are aggregates and post-translational modifications [3,4] whereas host cell proteins (HCPs), residual DNA (rDNA) or Protein A are process impurities [4,5]. Protein A chromatography is the initial capture step in manufacturing of mAbs and process impurities like HCPs and rDNA are generally removed during this step [3,6,7].…”
Monoclonal antibodies have been established as a major product class of biotechnology-based drugs. The increasing demand of monoclonal antibodies has led pharmaceutical companies to adopt efficient production processes. Transferring monoclonal antibody production to the industrial manufacturing requires adequate effort in process development. The strategy to reduce development time and cost comprises high-throughput process development which is especially central for the rapid optimization of the purification process. Chromatography process is the backbone of the purification process that can deliver high purity but it requires significant resources. Combined with high-throughput process development approach, the chromatography process is easy to develop and scale-up from laboratory to manufacturing scale. Design of experiments helps high-throughput process development workflow to provide decision-support techniques. This approach ensures significantly decreased time and material needs while improving the chromatography process. Protein A affinity chromatography is one of the most important chromatographic steps because of its great performance and capabilities. Most of the working parameters can be predefined and are identical for several monoclonal antibodies. However, some parameters like elution pH, loading capacity, resin type need further optimization for each monoclonal antibody. In this study, the loading and elution parameters were screened for Protein A chromatography to identify the best purification conditions using the combination of Design of Experiments and high-throughput process development approach in micro-volume columns. Developed working parameters were used for scale-up and tested under robust process conditions. Specific chromatography conditions were applied in pilot-scale and data comparison was done with micro-volume columns, lab-column scale to validate high-throughput strategy approach
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