Capto s impact3/9/2023 ![]() ( en Inventor 李乐 郭秋菊 蒙国基 邓义熹 张铭生 李雪 于玉根 Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.) Granted Application number CN201610901017.3A Other languages Chinese ( zh) Google Patents Purification method of monoclonal antibodiesĭownload PDF Info Publication number CN106380519A CN106380519A CN201610901017.3A CN201610901017A CN106380519A CN 106380519 A CN106380519 A CN 106380519A CN 201610901017 A CN201610901017 A CN 201610901017A CN 106380519 A CN106380519 A CN 106380519A Authority CN China Prior art keywords antibody filler cation exchange chromatography level pad Prior art date Legal status (The legal status is an assumption and is not a legal conclusion. Google Patents CN106380519A - Purification method of monoclonal antibodies Screening in Predictor plates required less than a tenth of the time in columns, with even larger savings in the amount of sample used.CN106380519A - Purification method of monoclonal antibodies Figure 3 compares time and sample amounts spent on this experiment using the different formats. The same results (both regarding optimal binding conditions and capacity shift with pH) were obtained in column chromatography experiments (Figure 1, BOTTOM). The capacity optimum shifted toward a higher conductivities as pH decreased, which can be related to a change in the protein net charge. The results from the screening study in Predictor plates showed nontraditional binding of conalbumin at all pH values, with the highest capacity obtained at pH 4.75 and 85 mM salt (Figure 2, TOP). ![]() Based on this, a contact time of 60 minutes was chosen for the screening study. RESULTSįor all conditions tested, the chromatography medium was saturated with protein after 45 minutes of incubation (Figure 1). Buffers were identical to those used in the plate-based experiments. Columns used were tricorn™ 5/100 with 2 mL column volume. ![]() The binding capacity ( q) was calculated thusly: q = (C initial – C flowthrough) × V sample/V medium.Ĭolumn Experiments: The dynamic binding capacity at 10% breakthrough (QB10%) at 2 minutes residence time (∼90 minutes loading time) was determined using frontal analysis. Samples were collected in UV-readable collection plates, and the concentration of protein was determined spectrophotometrically using lambert- Beer’s law. Using a suitable plate layout, 200 µL of sample at a concentration of 3.5 mg/mL protein solution was added to each well and incubated for 60 minutes at 1,100 rpm. A prestudy was run to determine the effect of contact time on binding capacity. Plate Experiments: PreDictor Capto S plates filled with 2 µL of chromatography medium per well were used. The factors investigated, associated ranges, and response (buffer was sodium acetate at ∼20 mM) The binding capacity for conalbumin on Capto S was investigated at various ionic strengths (20, 52, 85, 117, and 150 mM) and pH values (4.25, 4.75, and 5.25) using design of experiments (Table 1). Corresponding column experiments were run to verify that data correlated between formats. In this study, Predictor plates were used for screening of loading conditions for conalbumin (a protein showing nontraditional behavior) to the cation exchanger Capto™ S. PreDictor™ 96-well filter plates prefilled with GE Healthcare chromatography media allow parallel screening of conditions for binding, wash, or elution. The introduction of high-throughput techniques for process development partially addresses these challenges. This understanding/knowledge can be obtained if a larger experimental space is considered and/or more detailed studies are performed. In addition, the FDA’s new Quality by design initiative may increase demands on process development work because a higher degree of process understanding must be acquired. Finding optimal conditions for a downstream purification process is critical to achieving high productivity and a robust biopharmaceutical manufacturing process at large scale. ![]()
0 Comments
Leave a Reply.AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |