PEG Precipitation: A Powerful Tool for Monoclonal Antibody Purification - This alternative purification method to chromatography is readily scalable and fits a fully disposable downstream process. - B
Mass balance data are summarized in Table 1. The HCP reduction was lower than in the previous experiment (Figure 1), where
only 5,000 ppm of HCP was in the final pool as compared to 8,300 ppm in this experiment. Some depth filters, however, are
known to have hydrophobic and anion exchange (AEX) adsorptive characteristics.17 The precipitated media was loaded at a relatively high pH (8.5) and low conductivity (<10 mS/cm), which may have induced
binding of acidic proteins on an AEX matrix. Furthermore, in the presence of PEG, the protein binding capacity of ion-exchange
matrices has been shown to increase.18,19 Therefore, it is probable that some of the HCPs that remained in solution after precipitation bound to the depth filter
media and eluted into the product during resolubilization. This hypothesis also is supported by the difference in the HCP
content of the precipitation supernatant and the depth filter flow-through (9,900 versus 240 mg) which indicates HCP removal
from the solution by the depth filter. Following immobilization of the antibody onto the depth filter, it was redissolved
at a significantly lower pH (5.3) resulting in the elution of bound HCP, and reducing the purity of the final product. This
phenomenon could be mitigated by using less adsorptive depth filter media, such as Millistak+HC D0HC/C0HC and ZetaPlus SP
filters like 60SP02A, or by redissolving the productin a low-salt, higher-pH buffer, like 20-mM Tris pH 7.5.
Table 2. Yield and impurity removal for the PEG precipitation operation using depth filtration with various filter media to
recover the product
These strategies were tested by loading precipitated fed-batch media onto D0HC, C0HC, X0HC, and 60SP02A filters. All filters
were washed identically with the PEG/Tris buffer, but the resolubilization was done with 20 mM Tris pH 7.5 plus Tween 20 for
the Millipore filters and 85 mM acetate pH 5.3 plus Tween 20 for the Cuno filter. The Millipore filters also were stripped
with a low pH buffer (85 mM acetate pH 5.3) and high salt (1 M NaCl) after the product was recovered to determine if any bound
HCPs could be eluted. Figure 2 shows that there was substantial fouling of all filters tested. Because the fed-batch media
was not clarified by the ECS method, which has been shown to significantly reduce DNA levels in XD harvests, there may be
more DNA present, which precipitates in high PEG concentrations.20 The higher DNA content in the precipitate may have reduced filter capacities, but this has not been investigated. Table
2 shows that each of the experiments resulted in better HCP reduction (84–88% versus 46%), and even though the starting HCP
burden was higher (49,000 ppm versus 13,000 ppm), the redissolved MAb pools generally had lower HCP contents (6,000–7,800
ppm versus 8,300 ppm). The use of low-adsorptive filters and redissolving in a higher pH buffer appear to solve the problem
of HCPs eluting from the depth filter media. SDS-PAGE of the strip fractions revealed that both HCPs and product were bound
to the filters—including the less adsorptive D0HC and C0HC filters—and confirmed that X0HC media were more adsorptive than
D0HC and C0HC media (Figure 3).
Pellet Capture by Microfiltration
Figure 3
Although the HCP burden of the redissolved MAb was reduced by using a buffer with a higher pH and less adsorptive depth filter
media, the capacities of the depth filters were low for fed-batch media (<400 g-MAb/m2 ). Microfiltration (tangential flow filtration, MF TFF) was tested with the aim of improving capacity with the added benefit
of being able to use any buffer for resolubilization because of the low binding characteristic of the hollow fiber. The hydraulic
performance of the MF TFF concentration and washing of fed-batch precipitate is shown in Figure 4. The permeate flux was about
100 L/m2 /h and the TMP was between 1.0 and 2.5 psid for the entire operation. The mass loading of 475 g-MAb/m2 was better than that achieved in any of the depth filtration operations. The product recovery and HCP reduction were both
around 90%, slightly better than that achieved in depth filtration (Table 3).
Figure 4
The resolubilization was much simpler and faster than for the depth filtration; rather than recirculating buffer through the
device, the buffer was pumped through the inside of the lumens into the retentate vessel with the permeate closed and allowed
to mix for about 60 min. This was sufficient to redissolve the antibody, and no excipients were needed. Because of the difficulty
in resolubilization with depth filtration, the product was redissolved at or near the concentration in the feed media. The
final pool from the MF TFF process was nearly two-fold more concentrated than the starting material.
Table 3. Yield and impurity removal for the PEG precipitation operation using microfiltration to recover the product at bench
scale
Because the retentate and permeate were open to atmospheric pressure, minimal instrumentation was required: a crossflow pump,
one pressure sensor, and a transfer pump for the diafiltration. The combination of high capacity, high recovery and HCP clearance,
and operational simplicity make MF TFF a preferable option for pellet capture as compared with depth filtration. The precipitation
and MF TFF step was scaled-up 10-fold to a 0.12 m2 hollow fiber device, and the performance was comparable to the small scale (Table 4).
