Antibody Purification Using Membrane Adsorbers - Results from a process developed for a commercial antibody. - BioPharm International

ADVERTISEMENT

Antibody Purification Using Membrane Adsorbers
Results from a process developed for a commercial antibody.


BioPharm International
Volume 21, Issue 12

PERFORMANCE EVALUATION

The initial capture and purification step using Protein A affinity chromatography yields a product with >90% purity, but further polishing is required to reduce impurities and viruses to acceptable levels and conform with regulatory guidelines. The dual Sartobind Q/S disposable system tackles both positively and negatively charged impurities in a single step. The Q membrane is able to remove nearly 400 µg of DNA, 1.2 mg of Protein A, and nearly 30,000 EU of endotoxin (Sartobind Q5, surface area 5 cm2 equivalent to a 0.137 mL bed volume). Experiments to test the system for virus removal capacity are in progress.


Table 1. Reproducibility and robustness of the dual Q/S polishing system, as determined by measuring yield and elution conductivity. Yield is expressed as [D]/[PD]%, where D is the amount of antibody dimer recovered after polishing and PD is the amount of antibody dimer submitted for processing. Elution conductivities (MC, mSi/cm) represent values between fraction T collection, and the value in parentheses represents conductivity at the edge of the chromatogram peak.
The reproducibility of these results has been tested by monitoring the yield (calculated as the amount of Teleukin obtained after polishing as a percentage of the input amount) and the elution conductivities of several runs. The results, as presented in Table 1, confirm the robustness of the process. The elution conductivities are highly reproducible both among lots and between runs, and the yield ranges from 82.2 to 99.9%, which shows that the process, when optimized, can recover nearly all of the input product. The Q/S dual adsorber system is therefore suitable as a manufacturing process for this product, and probably all MAbs.

NANOFILTRATION

This final step in the process is a nanofiltration operation to mechanically remove endogenous or adventitious virus particles <20 nm in diameter. A 4-inch NFP Millipore cartridge filter (Millipore, Billerica, MA) is used for this step, and the efficiency of this device has been confirmed for a sample load up of up to 1.6 g of Teleukin in a volume of up to 6.5 L, loaded at a constant pressure (4 bar) using the Akta Pilot chromatographic system. The filtration flow rate is automatically adjusted by the Akta system to keep the pressure value constant. The flow rate usually ranges from 100 to 300 mL/min and sample recovery after filtration is usually very high (>90%).

CONCLUSION

Several comparative evaluation studies have been carried out to determine the relative performance of membrane adsorbers and traditional packed-bed columns in flow-through mode, although direct comparisons can be difficult because different standards are used to measure process capacity and flow rate.12 When such issues are taken into account, the performance of Q adsorbers is encouraging, but until now there has been no similar evaluation of either Q or S adsorbers in retention mode.

The process outlined above demonstrates that a membrane adsorber in retention mode can be used efficiently and robustly in a commercial antibody manufacturing process. The reproducibility analysis shows that the dual adsorber format operates successfully to remove both positively and negatively charged impurities from the feedstream, and because of the in-series linkage between the devices the dual system can be regarded as essentially a single, integrated polishing step. As membrane adsorbers become more widely accepted in antibody purification, it seems that not only flow-through but also retention chromatography steps in antibody polishing could be replaced by disposable membrane devices. Even so, the superior capacity of column chromatography for the capture of antibodies and other recombinant proteins means that resins are unlikely to be replaced for the foreseeable future in the primary capture step, which is the most significant bottleneck of all.13,14 .

Leonardo Giovannoni, PhD, is the head of CMC production and Marco Ventani is the head of downstream processing, both at Philogen S.p.A., Sienna, Italy, +39 0577 588539,
Uwe Gottschalk, PhD, is vice president of purification technologies at Sartorius Stedim Biotech GmbH, Göttingen, Germany. Gottschalk is also a member of BioPharm International's Editorial Advisory Board.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

EMA Warns of Falsified Herceptin Vials
April 16, 2014
Mallinckrodt to Acquire Questcor Pharmaceuticals
April 16, 2014
American CryoStem and Rutgers University File Joint Patent on Stem Cell Platform
April 11, 2014
Center for Biologics Evaluation and Research Relocates
April 11, 2014
PhRMA Report Reveals Growth Trajectories and Policy Factors Affecting Biopharmaceutical Growth
April 11, 2014
Author Guidelines
Source: BioPharm International,
Click here