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
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 6. Product recovery, CHOP, and DNA levels following precipitation and CEX chromatography
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 7. Comparison of the tangential flow filtration (TFF)-based and precipitation-based process schemes
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 8. Effect of salt concentration on viral clearance by Sartobind Q at 20 g/mL load
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.
Figure 9. CHO protein (CHOP) removal profile for a salt-tolerant membrane used in flow-through mode (pH 7.0; conductivity
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).
Table 2. Viral clearance comparison for two non-affinity purification schemes
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.
Figure 10. Economic analysis of precipitation-based non-affinity purification processes
Product Characterization and Stability
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.
Figure 11. IEF analysis of stability samples following caprylic acid precipitation, stored at 2–8 °C