Process 2: Ion exchange process scheme with selective contaminant precipitation
Protein precipitation is often applied in the food, blood-product, and enzyme manufacturing industries9 and is accomplished by the addition of salts or organic solvents, or by pH titration. Antibody manufacturers are becoming
more interested in these practices because they are suitable for processing bulk products at high throughput. Precipitation
technologies have the potential to reduce or eliminate the bottlenecks that will inevitably be encountered when processing
antibodies at the ton scale.10,11 They may also lead to the development of cost-effective downstream processes that require fewer unit operations for purification.
In the current case study, a small molecule (additive 1) was added to the clarified CHO cell culture supernatant and the pH
was adjusted to precipitate most of the HCPs. The resulting precipitate was removed by depth filtration, leaving behind residual
HCP in the feed. Scaling up the precipitation step is subject to variations in process parameters such as mixing speed, time,
temperature, and pH, the results of which are better controlled when CEX conditions are optimized for differential binding,
washing, and elution of the antibody. Transition from the precipitation step to CEX chromatography by applying the HCP exclusion
strategy enables the effective integration of two-step non-affinity process schemes. Using this protocol, HCP levels can fall
below 10 ng/mg after the first column (Figures 1 and 2). This degree of purity cannot be achieved in a single step when using
Process 1, with diafiltration as the primary recovery step. Product quality even exceeds that achieved with Protein A as the
capture step. Precipitation, combined with low pH conditioning, can prepare the feed stream for CEX chromatography with a
binding capacity of up to 100 mg/mL resin, minimizing the number of process cycles while simultaneously providing virus inactivation.
The material from capture is then processed by AEX membrane chromatography to remove negatively charged contaminants and adventitious
viruses. Because the purity of antibodies eluting from the first column is so high, tens of grams of protein can be loaded
per mL of membrane, making the AEX step faster and more economical for the application of disposable unit operations (Table
Process 3. Ion exchange process scheme with selective antibody precipitation
Antibodies can be precipitated by the addition of salt or polyethylene glycol, and the precipitate can be separated from the
bulk feed by centrifugation or filtration. In the current study, the antibody was precipitated with additive 2 and the precipitate
was dissolved in a resuspension buffer with a pH and conductivity suitable for direct loading onto a CEX column. Based on
the known behavior of HCPs on CEX resin, we were able to design a clearance strategy that reduced the level of HCP to 40 ng/mg
of antibody. When further processed by AEX membrane chromatography, HCP levels fell below the detection limit, making a two-step
non-affinity process feasible (Figure 3). Mixing, incubation time, and the temperature during precipitation must be controlled
to reduce variation in the load material and to achieve consistent recovery and quality. Resuspension of the antibody precipitate
requires approximately 30% of the original clarified bulk volume, therefore significantly reducing the processing time for
multiple CEX cycles. The protein load on the AEX membrane is determined to ensure the robustness of the process at large scale.
Dilution of the protein from the CEX column is sufficient for loading on the AEX membrane chromatography, which can be operated
at very high flow rates.