The most important responses in this study were the E. The studied factors were wash pH, NaCl concentration in the wash, and elution pH. Following screening experiments, a three-factor DoE was performed. The elution pH for this Fab was 3.2 when running a gradient from pH 6 to 2.5. This general approach also supports quality by design (QbD), where the plates give the characterized space and the DoE in columns render both the design and the control space.ĭynamic binding capacity for Capto L in the capture step was 21 mg/mL at 4 minutes residence time. Design of experiments (DoE) was used to find the best conditions for the step. This might be especially important for some molecules that are sensitive to acidic pH values. Third, an elution pH study was performed to find the optimal elution pH. Second, capacity studies were performed in small columns with purified protein (only applicable to binding steps). First, different media were screened in a 96-well format using PreDictor™ plates, and wide wash and elution conditions were evaluated. During the set-up of this three-step process, a general workflow was used with the purpose of minimizing the development time. The theoretical pI of the Fab molecule was 8.5, and the molecular weight was 48 kDa. Here we describe an example of using Capto L in a purification process of a kappa subclass Fab originating from an Escherichia coli supernatant. With its recombinant protein L ligand, Capto L is a BioProcess™ chromatography medium with a broad range affinity for antibody fragments of different sizes containing kappa light chains.Ī Three-Step Fab Purification Process Using Capto L
With the introduction of Capto L, the first industrial platform for the purification of antibody fragments, is now emerging (Figure 1). Antibody fragments, however, have previously lacked such a platform solution. The high purification factor and generic conditions associated with this approach have proven particularly attractive to biopharmaceutical manufacturers. The industry standard for purifying MAbs is a platform approach using affinity chromatography with protein A as the capture step. One of the advantages is that due to their structure and smaller size, antibody fragments possess unique properties (e.g., easier tissue penetration) that suit a range of diagnostic and therapeutic applications. Antibody fragments (e.g., Fab, scFv, DAb, etc.) are set to become the next important class of protein-based biotherapeutics after monoclonal antibodies (MAbs).