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.”