In 2012, BioPlan Associates estimated the global market for biopharmaceuticals at $140 billion, of which $100 billion was for recombinant proteins. The monoclonal antibody (mAb) market, a subclass of recombinant proteins, was estimated at $40 billion.
The development of biopharmaceuticals, from initial lab work through production and final regulatory approval, is a complex process. Downstream processing steps can represent up to 60% of the production costs for a protein, illustrating the need to develop efficient processes that can cut costs and improve productivity at commercial scale.
Advances in downstream processing were one of the many technology topics at the recent IBC conference “Biopharmaceutical Development and Production”.
“Multimodal chromatography is a powerful tool for difficult separation challenges, including mAb-aggregate removal,” explained Peter Hagwall, product manager, bioprocess media, GE Healthcare Life Sciences.
“Aggregates are tricky; they are composed of the same mAb molecule so a lot of the properties are similar to the monomer. Size differences are difficult to exploit and general techniques like ion exchange and hydrophobic-interaction chromatography often give unsatisfactory results. Poor separation usually means that yield of pure product is compromised.”
MAbs, although all the same class of molecules, have individual properties. The tendency to aggregate can vary, and cannot always be genetically engineered-in or predicted.
A trend over the past five years has been to apply multimodal media to the difficult polishing challenges downstream of the Protein A capture step. This allows optimization of fewer steps and further reduction of aggregate levels.
“A rapidly growing part of the biological pipeline is antibody fragments (Fabs),” said Hagwall. “In Fab purification, Capto adhere is already established as a workhorse for aggregate removal. Additionally, Capto MMC does a good job separating domain antibodies (dAbs), the smallest functional entity of antibodies, and can be used as part of a platform with Capto L, which purifies conventional Fabs as well as dAbs.
“Our next-generation multimodal ligands, Capto adhere ImpRes and Capto MMC ImpRes, display improved capability to reduce aggregate content while maintaining high yields and, thereby, good process economy,” he continued. “The high yields are achieved through the combination of fast mass transport of the Capto ImpRes base matrix and high selectivity of multimodal ligands, resulting in high-resolution separations. The Capto ImpRes base matrix has a smaller bead size than the Capto base matrix without compromising industrial demands, such as pressure/flow properties.
“Although future polishing challenges will be similar to those of today, new challenges will emerge related to the removal of undesirable product variants such as charge variants, glycosylation-, folding-, and truncated mAb-variants.
“As biomanufacturing continues to grow along with cost and time pressures, process development issues will need to be resolved quickly, and we are already seeing the use of high-throughput approaches to enable more rapid screening and optimization. An improved coupling between upstream and downstream processing will, in the long run, further improve process economy.”
Next-Generation CEX Resins
“Ion-exchange chromatography has been established within mAb platform purification processes for many years; however research shows that this technology can still be significantly optimized,” discussed Lars H. Peeck, Ph.D., head of polymer sorbents, chromatography R&D, EMD Millipore.
“Since new ion-exchange resin development can be viewed as the refinement of a very mature technology, the risk of implementing new resins into a purification scheme can be considered comparatively low with respect to the value the technology implementation can add.”
Productivity gains, resulting in increased mAbs titers in cell culture production processes, make the separation of product-related impurities, such as mAb dimers or higher molecular weight species, very challenging.
To identify cation-exchange (CEX) resin properties that impact mAb monomer/aggregate separation efficiency, EMD Millipore synthesized several hundred CEX resin prototypes, varying in pore and base-bead size, as well as in type of surface modification and ligand density. Out of this prototype series, about two dozen samples were systematically selected and tested in the purification of post Protein A mAb feedstreams.
The impurity-removal abilities of the prototypes were analyzed and related to the resin characteristics, with the main focus on aggregate removal. The study revealed clear trends between resin parameters and aggregate removal efficiency.
These results led to the development of Eshmuno® CPX, a combination of a new 50 µm base-bead technology with an optimized tentacle-type CEX surface. Eshmuno CPX removes mAb dimers and higher molecular-weight species, as well as impurities such as host cell proteins (HCP) and leached Protein A, allowing purification of post Protein A mAb pools at elevated protein loadings without the loss of separation selectivity.
“Resin performance is determined not only by the chromatographic selectivity but also by the protein-binding capacity and process flow rate. Using resins that perform poorly in one, or more, of these parameters often leads to limited yield, or purity, of the therapeutic protein and, consequently, results in poor process economics.
“With EMD Millipore’s Eshmuno CPX resin, efficient removal of process-related impurities is combined with high protein loadings and elevated process flow rates, tackling existing bottlenecks in mAb downstream processing and enabling high mAb yields and purity,” concluded Dr. Peeck.