Purification processes for antibodies have improved progressively over the last 20 years so that a limited number of unit operations can now handle a more than 50-fold increase in cell culture productivity. Most current processes for therapeutic antibodies use Protein A for capture chromatography with one or more subsequent polishing steps. These affinity processes must be modified to handle increasing antibody titers. In particular, the precipitation of cell line–derived contaminants through the addition of chemicals such as caprylic acid can generate cleaner feed streams that require fewer downstream process steps.1, 2Processes that do not incorporate affinity columns for either the capture or polishing steps have also been developed successfully. These processes generally include concentration and diafiltration steps for primary recovery so that the feed stream is conditioned for efficient capture by ion exchange chromatography.3 A TFF step used for this purpose also achieves the partial clearance of process-derived DNA. There are several alternatives to replace the primary recovery TFF step, such as expanded bed chromatography4 or precipitation of the antibody from the cell culture broth.5 Selective precipitation of antibodies can be achieved by adding polyethylene glycol (PEG) for research-scale preparations, but more recently, PEG precipitation followed by two ion exchange polishing steps was recommended for commercial-scale antibody purification processes.5 The antibody precipitation step must be scalable to ensure consistent feed quality and efficient contaminant clearance in the rest of the process.
Here we describe the successful integration of a precipitation step into non-affinity purification processes by optimizing differential binding and elution conditions for cation exchange (CEX) chromatography resins, an approach that is referred to here as the "HCP exclusion strategy."6,7 The comparative performance of non-affinity processes initiated with either filtration or precipitation is discussed in the context of antibody production from CHO cells at a titer of 5 g/L.
Process 1: Ion exchange process scheme with primary recovery TFF
Simple non-affinity purification processes incorporating concentration and diafiltration steps for primary recovery have been developed for many HuMAbs, and have been scaled up to work with ~100-L columns that process up to 5,000 L of cell culture broth.8 These processes include one or two ion exchange or mixed mode chromatography steps for polishing. The process begins with buffer-exchanged clarified cell culture supernatant from the TFF step, which is conditioned to allow antibody capture on the CEX resin. The resin can also clear most of the HCP and DNA by applying the HCP exclusion conditions. In addition, the high binding capacity of CEX resins (50–120 mg/mL of resin) can reduce the number of capture cycles. In the case of two-step non-affinity processes, an anion exchange (AEX) membrane is the only necessary polishing step. This step clears residual DNA, endotoxins, and HCP, and provides an orthogonal viral removal mechanism. The loading capacity of an AEX membrane is influenced by the purity of the process intermediate from the capture step, and is limited to a few g/mL of membrane because of the need for HCP removal (Table 1). However, in some of the three-step non-affinity processes, the Q membrane load can be increased to 20 g/mL of membrane without compromising its ability to remove adventitious viruses.
Process 1 can handle a large-scale 5 g/L cell culture harvest because of the efficient capture step, the low number of capture cycles, and the rapid processing time made possible by disposable membrane chromatography. However, the productivity of this purification process can be improved further by precipitating contaminants or the antibody as the initial step before capture chromatography.