Development of an Alternative Monoclonal Antibody Polishing Step - The authors describe a mAb polishing step using salt tolerant interaction membrane chromatography. - BioPharm International

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Development of an Alternative Monoclonal Antibody Polishing Step
The authors describe a mAb polishing step using salt tolerant interaction membrane chromatography.


BioPharm International
Volume 25, Issue 5, pp. 34-46

MATERIALS AND METHODS

Purification techniques

The mAbs used for this study were fully human IgG1 produced in recombinant Chinese hamster ovary (CHO) cells grown in a serum-free medium. MabSelect SuRe protein A (GE Healthcare, Piscataway, NJ) was used to purify the antibody present in the harvested cell culture fluid (HCCF) using AKTAexplorer under the control of UNICORN 5.0 (GE Healthcare, Piscataway, NJ). Briefly, the protein A column was loaded to approximately 35 mg mAb/mL-resin. The product was eluted using 50 mM acetate buffer, pH 3.5–3.8, which was mapped out for each protein on 96-well plates. Necessary wash steps were introduced to reduce the host cell protein (HCP) level in the eluate. The protein A elution pool was held at room temperature for one hour after pH was adjusted to 3.5 using 1 M acetic acid for viral inactivation. Following low-pH treatment, the product pool was neutralized to the required pH with 2 M Tris base solution, clarified through a 0.22 m filter (EMD Millipore, Billerica, MA), which served as the feed to STIC experiments.

The STIC equilibration buffer conditions were first screened using 96-well plates (gifts of Sartorius Stedim Biotech, Bohemia, NY) with a full factorial design of experiments. The buffer condition was evaluated at 5 pH levels of 6.5, 7.0, 7.5, 8.0, and 8.5, and 6 NaCl concentrations of 0, 25, 50, 75, 100, and 150 mM. Before loading into each well of STIC plates, the protein A eluate was adjusted to the appropriate pH using 2 M Tris base solution and to the target salt concentration using 5 M NaCl stock solution. The flowthrough/subsequent wash from each well was collected as the product. Response parameters, process yield, HCP, and HMW were determined for each experimental run. The response surfaces were defined in a group of optimization experiments using a 96-well STIC plate through a central composite design with 4 center points. All experimental design and data processing were performed using JMP version 8.0 software (SAS Institute, Cary, NC).

Antibody dynamic loading capacity (DLC) on Sartobind STIC was determined at the optimized buffer pH and NaCl conditions using 1 mL STIC Nano capsule (Sartorius Stedim Biotech, NY) at 10 membrane volume (MV)/min. We collected different flowthrough fractions and determined the HCP level in each fraction. The DLC value was the antibody amount applied to the membrane adsorber when HCP in the flowthrough reached 10 ppm or 20 ppm when applicable. The bound materials in the case of Mab-T were eluted using 50 mM Tris, pH 7.2, 2.0 M NaCl and analyzed for the level of HMW species. The process and product related impurities in the STIC purified products were determined using different analytical techniques.


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