Five presentations on protein purification at last month’s “Bioprocess International Europe Conference” in Prague provided clear and detailed insights on the latest thinking on how to best handle therapeutic biomolecules.
Günther Jagschies, Ph.D., strategic customer relations leader at GE Healthcare, reviewed the pluses and minuses of affinity chromatography for both monoclonal antibodies and “novel applications.” These include purification of kappa and lambda Fab fragments, coagulation factors VII and VIII, the nonpathogenic adeno-associated virus, and alfa-1 antitrypsin. Competition has heated up for protein A resins, with several small players introducing new mAb capture resins with binding capacities approaching 60 g/L with low residence times and high caustic stability.
Most experts agree, and actual practice validates, that affinity chromatography is the capture step of choice for mAbs due to its high selectivity, volume reduction, suitability for platform separations, and reduction of subsequent purification steps.
“But cost becomes an issue when the capacity and lifetime features of affinity resins are not fully utilized, such as during clinical manufacturing or for products requiring a limited number of batches per year,” said Dr. Jagschies.
Correctly sizing a capture column, particularly for legacy mAbs, is the most straightforward way to minimize cost issues, he added.
Ligand leakage and resin lifetime were considered issues in the past, but are rarely problems with today’s well-designed and -supported resins. Subsequent purification steps almost always clear ligands, which in any case do not lead to significant capacity losses. Lifetime has reached the low hundreds of cycles for hydroxide-stable ligands and the fifties for resins that are not as stable to CIP.
Although costs dominate discussions of affinity resin negatives, according to Dr. Jagschies, interest in protein A and other affinity-based separations has never been higher. This is somewhat of a contradiction since costs dominate, especially at lower scale. “I do not believe that cost will prevent manufacturers from using affinity capture as it is too powerful a method to miss out on,” he explained.
Efficient Workflow for the Production of Unstable IgM Antibodies
Xuemei He, Ph.D., a process chromatography expert at Bio-Rad Laboratories, discussed a new three-column workflow for purifying a monoclonal IgM antibody. Because the protein was unstable below pH 5 and prone to aggregation, cation exchange and hydrophobic interaction chromatography was problematic.
By taking advantage of the novel mixed mode medium’s salt tolerance, Dr. He was able to first remove DNA and host cell proteins by flow through chromatography over AG1-X8 resin followed directly by binding the antibody onto the novel mixed mode medium at high salt and close to neutral pH. The third step, polishing with a ceramic hydroxyapatite (CHT), gave active protein free of aggregates.
Purifying IgMs is challenging due to the molecules’ large size and instability. Several capture modes have been tried, including ion exchange, HIC, size exclusion, and affinity chromatography. IgMs often denature or precipitate under these conditions.
Although size-exclusion chromatography is gentle it does not exhibit production-scale volumetric productivity. Affinity chromatography media for IgM purification are currently available, but the molecules frequently do not survive the harsh elution conditions, resulting in low recovery.
IgMs tend to be more stable in high-salt buffers, which makes them unsuitable for conventional ion-exchange purification. Dilution to reduce feedstream conductivity for IgM binding on an ion-exchange column may lead to target molecule precipitation, while the higher process volumes are problems in their own right.
Dr. He’s approach works around IgM’s large molecular mass and heavy glycosylation, which lead to slow diffusion through conventional media. Her development-stage resin is based on rigid macroporous polymeric base matrices that allow efficient mass transfer. The precise chemistry involved is still under wraps. Dr. He did disclose that it contains “structural elements that interact with target molecules via one or more modes of interaction that include one or more electrostatic or hydrophobic interactions and hydrogen bonding.
“Under optimized conditions, this mixed-mode media offers unique selectivity unmatched by single-mode chromatography.”
Bio-Rad is currently working with collaborators to develop an intuitive approach to method development using this medium.
CHT is the ceramic form of the ages-old, naturally occurring mixed-mode chromatography material, hydroxyapatite or (Ca5(PO4)3OH)2. Electrostatic interactions between calcium and phosphate groups and the surface charges on biomolecules are what make CHT the “polishing workhorse” for removing process- and product-related impurities such as host cell proteins/DNAs, viruses, endotoxins, leached ligand from affinity chromatography, and antibody aggregates/fragments.
“Our method has general applicability to a potentially large variety of proteins, particularly where affinity capture is not a viable option,” explained Dr. He. But depending on the specific process and proteins the sequence of chromatographic steps and purification conditions may need to be adjusted to achieve the best separation.
“The final process based on this medium is simple, with minimal conditioning steps, yet offers products with high purity and biological activity,” said Dr. He.