Effective Polishing of Domain Antibodies (DAbs) Using Capto MMC ImpRes

Extracted from Multimodal Chromatography (PDF), GE Healthcare, 2013

Antibody fragments (e.g., Fab, scFv, and DAbs, Fig 4.57) are becoming an important class of protein-based products. The structure and smaller size give antibody fragments properties to suit a range of applications (e.g., easier tissue penetration), and their effective purification is therefore of great interest for manufacturers of biopharmaceuticals.

Structure of a recombinant DAb

Fig 4.57. Structure of a recombinant DAb.

The performance of Capto MMC ImpRes was evaluated in a study where the medium was used after an initial DAb capture step using Capto L. The recombinant DAb, expressed in E. coli, included the kappa light chain (VL). Binding and elution conditions for Capto MMC ImpRes and Capto SP ImpRes were screened using PreDictor 96-well plates using a HTPD approach. The binding capacity calculated using Assist software revealed information on binding and elution conditions (Fig 4.58), with the red areas on the contour maps showing optimal binding conditions while blue areas show optimal elution conditions.

Figure 4.58 shows the DAb binding capacities for Capto MMC ImpRes and Capto SP ImpRes. Both media showed a large pH range for binding. However, Capto MMC ImpRes had a larger window of operation with respect to NaCl concentration.

Contour maps showing SBC of DAb on (A) Capto MMC ImpRes and (B) Capto SP ImpRes at different pH and NaCl concentrations.

Fig 4.58. Contour maps showing SBC of DAb on (A) Capto MMC ImpRes and (B) Capto SP ImpRes at different pH and NaCl concentrations.

Capto MMC ImpRes is effective in removing E. coli protein (ECP) contaminants in the polishing step of DAb purification processes. To study ECP removal using Capto MMC ImpRes, DAb sample was applied to Capto MMC ImpRes at a load of 20 mg/mL, pH 5.0. As shown in Figure 4.58, the salt tolerance at pH 5.0 is high. Three different wash conditions were investigated—0, 100, and 125 mM NaCl. DAb was eluted with 500 mM NaCl and the ECP content in the elution pool and Dab yield are shown in Figure 4.59. The results showed improved ECP clearance at 125 mM NaCl without major impact on yield.

Purification of a recombinant DAb using Capto MMC ImpRes. (A) ECP contaminants in the elution pool and (B) DAb yield using different NaCl concentrations in the binding and wash buffers

Fig 4.59. Purification of a recombinant DAb using Capto MMC ImpRes. (A) ECP contaminants in the elution pool and (B) DAb yield using different NaCl concentrations in the binding and wash buffers.

For more information on this example, see data file 29-0356-74, “Capto MMC ImpRes.”

Materials

     
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