Designing for PTMs
Anne B. Tolstrup, Ph.D., director for cell culture II process science at Boehringer Ingelheim Pharma, spoke on tailoring product quality through cell-line development and process optimization.
Quality attributes vary depending on the molecule’s desired effector function. For example, anticancer antibodies should exhibit high antibody-dependent cellular cytotoxicity (ADCC), while biosimilars must closely match the physico-chemical profile and activity of originator molecules.
Dr. Tolstrup’s production cell, the proprietary Bi-Hex® (Boehringer Ingelheim high-expression) platform, consists of DG44 CHO cells and other elements enabling selection of cells for desired quality attributes.
Bi-Hex cells include two subclones that differ with respect to the number of galactose residues present in the N-linked sugar on Asn297 in the constant domain of the heavy chain. By manipulating and optimizing these characteristics and others related to glycosylation, Dr. Tolstrup can finetune safety and efficacy-related properties, like ADCC and complement-dependent cytotoxicity for anticancer biologics.
For biosimilars, the goal is to replicate the originator efficacy and safety while preserving physico-chemical properties.
In addition to the Bi-Hex subclones varying in glycosylation pattern, Boehringer Ingelheim has in-licensed GlymaxX®, a glycosylation-optimization technology, from ProBioGen. Glymax enhances ADCC and is suitable for development of biobetters and novel antibodies with enhanced anticancer activity, according to Dr. Tolstrup.
“There’s a significant opportunity to finetune attributes like ADCC through process and media development as well,” she noted. “After clone selection, we typically do a round of media optimization, where we adjust parameters like the concentrations of essential nutrients in the medium and adjust the feeds.
“Then, in a second round of DOE, we fine-tune process parameters like pH, gassing, temperature, and agitation. We’ve shown examples where ADCC can vary from 40 percent to almost 400 percent for the same clone taken through different media or process-development schemes.”
Bi-Hex integrates novel vectors, serum-free transfection, optimized host cells, serum-free flow-based screening, and chemically defined production media. Early assessment of phenotypic stability and product quality attributes is done to generate cells that are stable and highly productive in fed-batch cultures.
Bi-Hex also involves the traditional exercises of optimization of cells, media, and process, but uses a highly integrated and interdiciplinary approach and relies heavily on automation as well as on design-of-experiment approaches, continued Dr. Tolstrup.