The MAb concentration in media-containing samples was determined by analytical Protein A HPLC (Applied Biosystems, Foster
City, CA). Aggregate levels were measured by size-exclusion chromatography (SEC) using a TSKgel G3000SWXL column from Tosoh
Bioscience (Montgomeryville, PA), with peak detection by UV absorbance at 280 nm. HCP levels were quantified by a PER.C6-specific
ELISA from Cygnus Technologies (Southport, NC). SDS-PAGE was performed with NuPAGE 4–12% Bis-Tris gels and staining was done
with SimplyBlue SafeStain, both from Invitrogen (Carlsbad, CA).
Results and Discussion Precipitation Conditions
For both molecular weights of PEG, 3,350 and 6,000 Da, the PEG concentration was the dominant factor in the recovery and purity
of the redissolved MAb. Precipitation with PEG-3350 resulted in the highest recovery (Figure 1). However, the higher recovery
came at the expense of higher HCP burden in the redissolved MAb. In the case of aggregated MAb, the levels were not significantly
different from the starting material, but it should be noted that the particular MAb used in this work is not prone to forming
aggregates. The improvement of the HCP reduction with the use of PEG-6000 was offset by the reduction in product yield. The
final condition selected was 14% (w/v) PEG-3350 (equivalent to 14.4% w/w) and pH 8.5.
Pellet Capture by Depth Filtration
In the first experiment, ECS-clarified XD media was precipitated and the pellet was captured by depth filtration with X0HC
media. No substantial increase in the transmembrane pressure (TMP) was observed during the loading of 361 g-MAb/m2 (data not shown). After washing, resolubilization of the immobilized pellet was done with 85 mM acetate pH 5.3. Even after
recirculation for 2 h, the antibody had not completely redissolved, so 0.1% v/v Tween 20 was added to the resolubilization
buffer. After an additional 30 min of resolubilization, the MAb was fully dissolved.
Table 1. Yield and impurity removal for the PEG precipitation operation using depth filtration with X0HC to recover the product