Precipitation of Process-Derived Impurities in Non-Protein A Purification Schemes for Antibodies - Precipitation prior to capture chromatography offers a simple, robust, and economical method to


Precipitation of Process-Derived Impurities in Non-Protein A Purification Schemes for Antibodies
Precipitation prior to capture chromatography offers a simple, robust, and economical method to remove CHO host cell proteins and DNA.

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Integrating Precipitation into Non-Affinity Purification Schemes

Post caprylic acid precipitation, the filtered bulk is suitable to load onto either an affinity column or a non-affinity column. Here, we describe the integration of precipitation into a non-affinity purification scheme with CEX as the first chromatography step.

Figure 6. Product recovery, CHOP, and DNA levels following precipitation and CEX chromatography
The low pH condition after the caprylic acid precipitation procedure prepares the loading condition for high binding (~100 mg/mL) onto CEX resins. The levels of process-derived impurities have already been significantly reduced after precipitation under optimal conditions (Figure 6), leading to a much cleaner feed stream for the capture resin. As a result, the demand on the purification process is low enough that purification can be achieved by only two orthogonal separation steps. Typical step recoveries for precipitation are in the range of 80–99% for various HuMAb processes.

Figure 7. Comparison of the tangential flow filtration (TFF)-based and precipitation-based process schemes
Impurities are at very low levels after CEX capture (<10 ng/mg HCP), making it possible to complete the purification process by adding just an anion exchange (AEX) membrane chromatography step (Figure 7a). The AEX chromatography in this case is mainly for adventitious virus removal; a 3-log reduction of A-MuLV was still observed even at a conductivity level of 12 mS/cm (Figure 8), thereby accommodating the high conductivity CEX eluate with very low or no dilution.

Figure 8. Effect of salt concentration on viral clearance by Sartobind Q at 20 g/mL load
With the most recent improvement in membrane chromatography, salt-tolerant membrane adsorbers have been developed to accommodate a wide range of salt concentrations in the load while still maintaining contaminant clearance and high flow rate characteristics. Using salt-tolerant interaction chromatography (STIC) disposable membrane modules (Sartorius Stedim Biotech, Goettingen, Germany), the process can be developed without any further dilution after CEX chromatography. STIC membrane chromatography was evaluated here for contaminant clearance to replace the AEX membrane step. The STIC membrane was able to significantly reduce CHOP from CEX eluates in a polishing step without any further dilutions (Figure 9). STIC can replace a Q membrane because the viral clearance capacity was reported to be 4–5 LRVs using the model bacteriophage virus ΦX174, especially at a high salt concentration where Q membranes have negligible clearance.9 It is clear that in a precipitation-based process where CHOP levels are extremely low after CEX, STIC may be able to process loads as high as those that Q membrane (20 g/mL) can when developed for viral removal only. Further studies are needed to confirm the scalability of STIC and its robustness to handle various buffer matrixes used in non-affinity processes.

Figure 9. CHO protein (CHOP) removal profile for a salt-tolerant membrane used in flow-through mode (pH 7.0; conductivity 12 mS/cm).
The residual caprylic acid concentration in the product was monitored for clearance during the purification process. In an experiment with 1% caprylic acid addition after depth filtration, only ~0.1% residual caprylic acid was present, which was further reduced to 0.003% after CEX chromatography alone. Several other fatty acids that are constituents of cell culture medium also were removed in a CEX flow-through fraction. CEX chromatography is a crucial purification step to remove cell cuture additives to significantly low levels in a two step purification scheme.

Table 2. Viral clearance comparison for two non-affinity purification schemes
The precipitation step also significantly contributes to the viral removal strategy of non–Protein A purification schemes by adding ~4 LRV for A-MuLV.10 In our HuMAb case study 1, at pH 4.5 and with a mixing time of 2 h, 1% caprylic acid treatment provided a 4-log reduction in A-MuLV (Table 2). These studies will be extended for a range of model viruses in the future. The process capability of TFF-based and precipitation-based non-affinity purification processes to remove viruses is comparable when the CEX and Q membrane steps are performed under the same conditions for both process schemes (Table 2, Figures 7a and 7b).

Figure 10. Economic analysis of precipitation-based non-affinity purification processes
The economics of two different HuMAb production processes, one using a TFF-based three-step non-affinity purification scheme and the other using a precipitation-based two-step process, were compared (Figures 7a and 7b) by modeling the processes with SuperPro Designer (Intelligen, Scotch Plains, NJ). The two models were compared for raw materials, consumables, and labor costs. After analyzing the processing time, overall process recovery, and cost (2 g/L, 5,000 L compared to 10 g/L, 2,000 L), it was obvious that the precipitation process scheme considerably lowers overall costs as a result of the efficiency of the precipitation step and because fewer processing steps are needed (Figure 10). As the product titer increases, these trends become more obvious, indicating that precipitation is favorable for high titer processes.

Product Characterization and Stability

Figure 11. IEF analysis of stability samples following caprylic acid precipitation, stored at 2–8 C
The quality of the product after caprylic acid precipitation and purification by CEX was compared with a reference standard that was purified by a TFF-based non-affinity process. The results of isoelectric focusing (IEF) gel analysis are shown in Figure 11. Furthermore, SDS–PAGE analysis, monomer purity assessment by SEC–HPLC, and activity by binding ELISA, are similar for the two products. The stability of caprylic acid treated filtrate at 2–8 C was also monitored. The results of a case study showed that the filtrate can be stored at 2–8 C for 34 d without any loss of activity (102% on day 50 of storage) and monomer purity. These stability studies will be continued on the final purified antibody to reveal the effect of caprylic acid precipitation process on long-term storage.

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