MATERIAL AND METHODS
Conditioning cell harvest
Cell culture supernatant containing fully human monoclonal antibodies was concentrated and diafiltered in either 35 or 70
mM sodium phosphate (pH 6.2). For experiments in which MEP HyperCel was used as the capture resin, the load was adjusted to
neutral pH prior to load.
Antibodies were loaded onto CEX resins with a binding capacity ranging from 15 to 45 mg/mL. However, these capacity values
can be further increased to over 100 mg/mL with higher binding resins. The product was step-eluted at pH 6.2 with added salt,
ranging from 40 to 75 mM NaCl. MEP HyperCel (Pall Corp., East Hills, NY) was the HCI resin. Equilibration used 10, 35, or
70 mM sodium phosphate (pH 7.0 to 7.2) and the product was loaded to a capacity of up to 22 mg/mL. The resin was post-load
washed with 10 to 45 mM sodium phosphate (pH 6.2 to 7.0) and then washed before elution with a low conductivity buffer. Elution
took place when buffers with a pH range of 4.5–5.2 were used, using another low conductivity buffer such as 10 mM sodium acetate
or a dual acetate and phosphate buffer. A summary scheme of the proposed method can be seen in Figure 3.
Viral inactivation, filtration, and clearance studies
Low pH hold was performed for one hour. Final viral filtration used Planova 20N (Asahi Kasei Pharma, Westbury, NY). Viral
clearance studies were performed by an independent laboratory (Apptec, St. Paul, MN).
Host cell protein and DNA specific concentration
Chinese hamster ovary-host cell protein (CHO HCP) analysis used Cygnus Technologies ELISA Kit (Catalog# F015). DNA was quantified
by a proprietary qPCR assay.
RESULTS AND DISCUSSION
Both versions A and B of the purification process yielded comparable quality in the final purified material. With similar
materials and the two interchangeable sequences, we achieved a final homogeneous composition containing less than 100 ppm
in CHO HCP, 10 pg DNA/mg antibody, and a monomer percentage >95%, measured by size exlcusion HPLC.
Table 1A summarizes the results obtained for A scheme when CEX chromatography is used for capture. This "direction" was adopted
with four different antibodies. Host cell protein content was reduced 50- to 200-fold after the first column, with concentrations
ranging from about 900 to 6,000 ng/mg, which were reduced to levels less than 100 ng/mg after the HCI column. The CEX resin
cleared DNA very efficiently (as much as 7,000-fold), and remaining traces were further reduced by the HCI resin to less than
10 pg DNA/mg antibody.
Table 1A and 1B. Summary of in-process contaminant clearance. In Table 1A, a cation exchange (CEX) resin is used for capture,
and hydrophobic charge-induced (HCI) resin is used for polishing. In Table 1B the resins were reversed.
The reverse scheme (B) yielded a comparable quality of material (Table 1B). HCP was reduced about 90-fold by the HCI. DNA
was cleared much more efficiently, when compared with the CEX resin as capture, bringing the measurements below the set final
specification, which emphasizes the efficiency of MEP HyperCel in the clearance of nucleic acids.
Final purity (% monomer) and overall recovery for both HuMAb-1 and HuMAb-2 purification schemes, in both directions, were
extremely comparable: over 99% and approximately 70%, respectively.