April 15, 2013 (Vol. 33, No. 8)
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.
High Capacity, Salt-Tolerant Resins
Life Technologies’ POROS® cation and anion exchange chromatography resins are rigid and incompressible polymeric beads, comprised of polystyrene-divinylbenzene, with a high degree of physical and chemical stability and robustness, according to Christine Gebski, head, POROS business unit and global applications.
The beads have unique, large pore structures, which offer higher binding capacity, efficient chromatography, salt tolerance, and the ability to purify both small and large biomolecules, she noted. The average bead size is 50 µm, which provides high-resolution capability, allowing for effective protein separations.
“POROS resins enable high protein-binding capacities, which reduce column sizes and therefore reduce water and buffer consumption. High-resolution capability enables efficient protein separation, the ability to clearly separate product-of-interest from product- and process-related impurities, resulting in better recovery, and higher yield of the protein of interest.
“Enhanced separation allows for simpler product collection and the elimination of the need for peak fractionation, off-line analytical analysis, and the design of product-pooling schemes. These improvements drive efficiencies in manufacturing and quality operations, and enable a better cost of goods in the production of biotherapeutics,” commented Gebski.
POROS XS is a high-performance CEX resin enabling protein binding capacities of greater than 100 mg/mL and the effective separation of product-related impurities like aggregate, clipped species, charge variants, and process-related impurities, such as HCP and DNA. The resin enables effective polish chromatography and the use of CEX for direct load capture chromatography.
“Many blockbusters already include HPLC in their purification process: recombinant insulins, interferons, human growth factors, erythropoietin, etc.,” remarked Margit Holzer, Ph.D., R&D director, Novasep. “This demonstrates that HPLC and biopharmaceuticals downstream processing are compatible, not only technically but also in terms of regulatory compliance and profitability. We have columns in operation with diameters up to 1,600 mm and working pressure up to 70 bars.”
Biomolecules are larger than synthetic pharmaceutical products; HPLC can deal with biomolecules up to 50—60,000 Daltons. This value is the result of the stationary phase performances, specifically in terms of pore size (diffusivity).
Due to the diversity of biomolecules, HPLC is a choice technology in regard to its separating power, and allows more flexibility when dealing with impurities in a post-fermentation or post-culture broth. A good illustration can be found in the recombinant insulin purification process, where HPLC is the only cost-effective solution to allow separation of several isoforms of the protein to reach the required purity.
“Quality requirements for biopharmaceuticals are constantly increasing. It is essential that processing technologies catch up. For example, the use of water-organic eluents mixtures in HPLC avoids microbial contamination.
“HPLC is well established in purification processes, and its reliability and reproducibility have been demonstrated. It is not adapted to every product, but Novasep believes the technology allows an indispensable creativity and flexibility for process design and optimization. We have developed user-friendly tools, software, and services to support the equipment.
“Several improvements are still to come; one example is stationary-phase performances in terms of diffusivity limitation,” concluded Dr. Holzer.
Virus Filtration Optimization
“The pore size of a virus filter must allow the mAb, or other target molecules, to pass through, which limits the minimum pore size possible for viral removal filters,” said Anika Meyer, product manager, viral clearance, Sartorius Stedim Biotech.
Virus filters and Protein A resins are the most costly components in a bill of materials for a mAb production batch. Filters must be optimized to achieve the highest throughput without compromising the flow rate, and the desired log retention value for virus removal.
The virus filter, or inactivation method, must get adjusted to the hydrophilic or hydrophobic properties of the antibody, the isoelectric point, the tendency to aggregate, as well as to the viscosity under the conditions the filtration or inactivation will be performed. There is not one filter or method that does it all.
In practice the most robust solution, not necessarily the most economical solution, gets established. A robust solution allows the establishment of a platform process, which has its own economic impacts.
New generation virus filters have improved performance and robustness, however testing and validation can become more costly as larger volumes of more highly purified virus spike material are needed. Overall, the most important aspect is the positive impact the filters have on the production process cost of goods.
“High titer processes are posing new challenges for final virus filters as product concentration and viscosity increases. The identification of viruses smaller than 20 nm also will challenge the currently used viral clearance strategies.
“Additional economic challenges are related to the discussion of extending viral clearance strategies upstream to raw materials and cell culture media. In this scenario, a filter or inactivation method does not need to balance between virus removal efficiency and target protein transmission. New, cost-efficient virus filters specifically designed for these applications will help to overcome economic constraints.”