Ensuring Biologic API Uniformity

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Biologic drug substances must meet numerous specifications based on critical quality attributes that impact the safety and efficacy of the active ingredient. These important properties must be consistent from batch to batch and within batches if, after final filtration, the API is collected in multiple containers rather than one large vessel. Biologic API uniformity can, however, be influenced by many aspects of the production process, including the quality and variability of the raw materials. Effective harvesting and purification processes play an essential role in ensuring that biopharmaceutical manufacturing processes provide biologic drug substances with uniform and consistent properties.

Importance of consistency
When formulating biopharmaceutical drug products, it is essential that the biologic API have consistent product properties, regardless of the production batch or container in which it was collected following final filtration. Variation in the concentration of product- and/or process-related impurities is of paramount importance as it can affect the safety and/or efficacy of the product, according to Brian G. Turner, senior director of manufacturing sciences and technology with the Specialty Care Operations group within Sanofi. “Product-related impurities such as aggregates can be difficult to control at low levels, and certain process-related impurities can be challenging to reduce to acceptable levels if their physicochemical properties are similar to the desired product,” he notes.

Upstream impact
API consistency begins upstream with cell line selection, according to Tim Hill, director of upstream process development for Fujifilm Diosynth Biotechnologies USA. “If a mammalian cell line is not entirely clonal or is sensitive to small changes in processing controls such as pH, temperature, gassing, and feeds, we typically see wide swings in cell growth, metabolites, and production profiles and impacts on product quality attributes such as aggregates, charged variants, and glycans,” he explains.

The use of undefined media components such as soy or yeast hydrolysates are particularly problematic for API product quality consistency. Lot-to-lot variability of the undefined components impacts cell cultures more than fermentation processes and typically requires raw-material use testing prior to acceptance into manufacturing, according to Hill. Fortunately, he notes that several media vendors have developed very good chemically defined (CD) alternatives to traditional growth media and feeds for common cell culture applications.

Isolating a stable cell line with robust growth and protein expression capability is still the major challenge in cell culture, Hill adds. “Because of the increased interest in the manufacture of biosimilar monoclonal antibodies, cell clone selection with a close eye on product quality is even more important to ensure that undesirable qualities such as aggregates, half-antibodies, and high mannose-5 glycan derivatives are screened out of the process,” he says.

Role of harvesting
While some products may benefit from maintaining tight control of harvesting pH and temperature, Turner finds that the impact of harvesting on product purity is often minimal for secreted proteins, but can be significant for proteins expressed intracellularly, such as insulin expressed as insoluble inclusion bodies in Escherichia coli.

The harvesting process must, however, provide a relatively uniform in-process material to the start of downstream operations with each production batch. To achieve this goal, according to Hill, requires consistency in processing times to avoid over exposure to proteases from lysed cells and unfavorable physiochemical conditions for the protein. Tighter control of the harvest tank environment to minimize degradation of certain products may also have a positive impact on API purity, according to Turner. “When raw materials or process controls vary too much, the consistency of the harvested material can change, which can impact downstream process performance,” he says. “Clearance of potential contaminants including viruses and chemical process impurities during downstream processing can often be impacted by upstream variability, leading to failure in release specifications related to process safety measures,” Hill continues.

Some advances in technology are helping biologic API manufacturers improve the harvesting process with respect to achieving more consistent harvest cell-culture fluids (HCCFs). For instance, Turner notes that the use of flocculants to improve flows (throughput) in centrifugation can improve the efficiency and increase the capacity of post-centrifuge depth filters. In addition, charged depth filters can reduce process-related impurities such as DNA and basic proteins. Other technology advances are leading to improved efficiencies while also ensuring product consistency. “High-capacity depth filters that can process high-density cultures without centrifugation provide increased efficiencies, while process intensification (e.g., concentrated fed-batch culture) using alternating tangential flow (ATF) filtration allows for the continuous harvest of clarified supernatant,” Turner notes.

For Hill, single-use tubular bowl centrifuges represent a key technology development for harvesting. These centrifuges collect cells in a gentle manner to minimize cell breakage. “Because this type of centrifuge enables harvesting at high-cell viability, it allows for lower protease, host-cell protein (HCP), and DNA content in the clarified harvest, and thus better capture column performance downstream,” he observes.

With respect to future developments in harvesting technology, Turner would welcome advances in depth filter construction that would enable the effective processing of high-density cultures. The key remaining challenge, according to Hill, relates to the need to reduce impurity loads associated with in-process streams; lower impurity concentrations would enable the reduction in size of purification columns. Fujifilm Diosynth Biotechnologies is, in fact, working with 3M to evaluate the capability of 3M’s new Emphase AEX Hybrid Purifier to decrease DNA and HCP concentrations in cell-culture harvests. “This additional filtration technology appears to significantly reduce DNA loads onto Protein A capture columns, thus enabling the capture columns to more effectively separate host-cell proteins from biologic APIs,” comments Hill.

Downstream influence
“A well-designed purification process should deliver a consistent biologic API,” states Michael Murray, director of downstream process development with Fujifilm Diosynth Biotechnologies. Each purification step in the downstream processing of biologic APIs serves a particular purpose, according to Turner. For monoclonal antibodies, he notes that Protein A chromatography effectively separates the product from most process-related impurities. Anion-exchange chromatography, meanwhile, is an excellent method for reducing DNA levels. For some products, hydrophobic-interaction chromatography can reduce aggregates to acceptable levels. Nanofiltration is typically included in modern purification processes as a dedicated viral clearance step.

“If a process is challenged to obtain multiple logs of clearance of an impurity, then multiple removal steps are often required not only to meet the needed quality attributes but also to ensure process robustness, which leads to product consistency,” Murray observes. If the demand for clearance is lower, he notes that a single step can often be designed to ensure that quality attributes are met, but process robustness must be built into the step, either through thoughtful design or characterization of the design space.

Advances in chromatography technology have been crucial to the improved performance of downstream purification operations. “Chromatography manufacturers continue to increase their offerings of different functional groups and bead structures from which a process development scientist can choose. For instance, resins are now available with increased capacity, improved selectivity, and tolerance to a wider range of conditions (i.e., salt tolerance),” Murray notes. A wider range of membrane chromatography options are also available that are enabling more optimized purification processes.

There remains, however, growing pressure to increase the throughput of downstream processes to match upstream improvements in productivity and cell-culture titers. “The need is particularly true for monoclonal antibodies that are approaching commercial manufacturing. The challenge for development, engineering, and manufacturing will be to create unit operations that consistently achieve the desired quality attributes for a given step, but at a higher output,” says Murray.

Further advances in downstream purification technologies could have positive operational and financial impacts and also influence biologic drug substance quality and consistency, according to Turner. These advances include low-cost alternatives to Protein A chromatography (e.g., synthetic affinity ligands), high-capacity membrane adsorbers to replace column resins, spray drying or bulk lyophilization of APIs, and room-temperature storage of APIs for increased shelf life. Murray adds that the combination of multiple unit operations into a continuous mode shows promise for improving throughput while still maintaining quality.

Article DetailsPharmaceutical Technology
Vol. 40, No. 5
Pages: 44–47

Citation: 
When referring to this article, please cite as C. Challener, Upstream and Downstream Operations Can Impact Biologic API Uniformity," Pharmaceutical Technology 40 (5) 2016.