Ion-exchange chromatography is often a crucial component of the downstream processing of biopharmaceuticals. Innovative new exchangers with new ligand chemistries are making it possible to increase selectivity and capacity. Some also are tolerant of high salt conditions, enabling the elimination of some preparative steps. Jim Powell, business development manager with Asahi Kasei Bioprocess, Estelle Zelter, global product manager of ion-exchange chromatography at EMD Millipore, and Sylvio Bengio, head of scientific communications for the global chromatography group at Pall Life Sciences, spoke with Cynthia Challener, editor of the Pharmaceutical Sciences, Manufacturing & Marketplace Report, about recent developments in ion-exchange chromatography technology that are benefiting their pharmaceutical customers.
Capacity and selectivity are key
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What are the key attributes of high-performing ion-exchange chromatography media intended for use in the biopharmaceutical/pharmaceutical industry and why are they important?
Zelter (EMD Millipore): Binding capacity, selectivity, resolution, scalability, predictable pressure/flow behavior, and ease of use are certainly the most important key attributes of a high-performing ion-exchange chromatography media for the biopharmaceutical industry. They allow the end user to best purify the molecule of interest without compromising on yield and productivity.
Powell (Asahi Kasei Bioprocess): Each application requires a mix of key attributes that best solve the application's problem. In preparative biochromatography, and assuming good product recoveries, these factors include primarily capacity and selectivity under the conditions needed for that separation. Toward the end of the purification scheme, smaller bead-size resins might be used to provide higher resolution separations to remove trace contaminants if a liquid chromatography (LC) chemical with the needed selectivity can't be found.
Bengio (Pall Life Sciences): Most importantly, high selectivity is needed either for the protein that is to be captured or the main impurity that is to be removed. Achieving such a level of selectivity cannot be accomplished by guesswork; it requires resin screening, case by case, to determine which sorbent performs best for a specific separation. When ion-exchange chromatography is used at the capture step, a high binding capacity at high flow rates (or short residence times below four minutes) is necessary in order to have high productivity (in kg/protein purified/day or per cycle). Resins also need to be easy to pack/unpack in process columns and provide long service life due to easy and efficient cleaning in place (with 1 M NaOH).
Matching the media with the application
Pharmaceutical Sciences, Manufacturing and Marketplace Report What are the most commonly used types of ion-exchange chromatography media and for what specific applications?
Powell (Asahi Kasei Bioprocess): Strong ion-exchangers (quaternary amines and sulfate ligands) on natural carbohydrate-based resins (agarose and cellulose) are the most popular; however, new polymerics (polystyrene-divinylbenzene or polymethacrylate) are also popular. The resin that solves the separation problem best is the one chosen as long as the vendor is stable and reputable.
Bengio (Pall Life Sciences):The type of media (anionic or cationic) depends on the relative isoelectric point of the target protein that needs to be purified or the impurities that need to be removed. Ion-exchange chromatography can be used at all downstream processing steps for all types of biomolecules: monoclonal antibodies (using cation exchangers for capture as a potential lower-cost alternative to protein A in some cases), plasma derivatives, various recombinant proteins, etc.
Zelter (EMD Millipore): Cation and anion exchanger chromatography media are typically used in downstream processing of monoclonal antibodies, or for intermediate and polishing purification to remove process- and product-related contaminants. Cation and anion exchanger chromatography media, as well as multi-modal resins, can also be used in capture and later purification steps for recombinant protein processes. Depending on the molecule and the process conditions, two or three chromatography steps may be required.
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What is driving development of new technologies?
Bengio (Pall Life Sciences): There is a continual need for higher capacity, greater efficiency, and higher recoveries. While most modern ion exchangers now offer good capacities, depending on the feedstock conductivity, the crude feedstock often should be diluted or first subjected to ultrafiltration/ diafiltration in order to achieve the highest possible capacities and throughput. For polishing steps, membrane adsorbers offer a very attractive approach because they perform at flow rates that are orders of magnitude higher than conventional beads and are also disposable.
Zelter (EMD Millipore): The biopharmaceutical industry is continuously evolving and trying to increase titers on the one hand while also striving for higher molecule purity (separation of low molecular weight impurities and other contaminants) and molecular activity to lower manufacturing costs. In order to adapt to these new drivers, our chromatography media offer higher binding capacities, shorter processing times, reduced buffer volumes, and improved plant utilization.
Powell (Asahi Kasei Bioprocess): The need for greater selectivity seems to be the biggest driver of new ion-exchange chromatography media development in the general market today. Vendors are working hard to develop useful mixed-mode resins, such as those bearing new ligands with both ion-exchange and hydrophobic interaction properties, in order to provide selectivities that may solve real-world problems. As a result, there are many new ligand technologies being developed.
New salt-tolerant sorbents
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What recent advances have been introduced in response to these market needs?
Powell (Asahi Kasei Bioprocess): Smaller bead LC chemicals with higher rigidity provide better process economy and faster high-resolution separations. New selectivities allow high capacity at higher conductivities than ever before. Providing a ligand that allows good binding capacity in high salt conditions may increase recovery or reduce the need or amount of diafiltration required, making the process faster and more economical.
Bengio (Pall Life Sciences): New ‘salt-tolerant’ chromatography sorbents are indeed one leading example. Pall has introduced an anion exchanger based on a primary amine ligand that allows the capture of protein targets or impurities in a broad (2-15 mS/cm) conductivity range, which enables the direct processing of crude feed without dilution or tangential flow filtration.At the process scale, elimination of this step translates into process economic benefits, including buffer and tankage savings and reduced labor time and cost.
Zelter (EMD Millipore): Our newly developed base bead material, which is made of a rigid hydrophilic polyvinyl ether polymer, allows us to produce smaller rigid particles combined with improved mass transfer, delivering superior dynamic binding capacity, high-resolution purification, and outstanding pressure-flow behavior, while also enabling large-scale column packing. These key attributes respond perfectly to the evolving needs of the biopharmaceutical industry.
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What limitations remain with respect to the use and performance of ion-exchange chromatography media in pharmaceutical applications, and what work is being done to address these issues?
Zelter (EMD Millipore): The need for large buffer quantities is about to be solved with the development of salt-tolerant resins, allowing the loading of the protein without prior dilution. In addition, the development of disposable chromatography will also eliminate the limitations associated with reusable systems for certain applications and scales.
Powell (Asahi Kasei Bioprocess): There is a column diameter upper limit of 2 meters i.d. (inner diameter). This limit for packable bed heights is due to a range of reasons, including bead stability above a certain bed height and pressure/flow curves. Separately, there is a large effort in our industry to develop continuous chromatography-based separations, which require resins that can be quickly and efficiently regenerated and cleaned for rapid re-use. In addition, economical, disposable column technologies to reduce cleaning validation costs are of interest; these columns also require rapid and easy packing of the resins.
Bengio (Pall Life Sciences): For separation of impurities closely related to the target, screening of the "right" ion exchanger selectivity is key, but can be time consuming. Selectivity depends not only on the type of ion exchanger functional ligand (e.g., quaternary amine or sulfate, etc.), but also on the chemical nature of the chromatography matrix itself (agarose, polymer, cellulose, other). Therefore, high-throughput screening using 96-well plates with automated operations (robotics and high-throughput analytics) are being used to address this critical aspect of ion-exchange chromatography process development and help speed up the selection of the best ion exchanger candidate.
Suppliers working to meet industry needs
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What specific efforts are ongoing within your company?
Bengio (Pall Life Sciences): Pall has been a pioneer in ion-exchange chromatography since the development of its ‘gel in a shell’ technology in the 1990's, which was a precursor of modern sorbents. More recently, the company has developed innovative ion exchangers with specific differentiated selectivities based on other industry-scalable matrices, and most recently a new ion exchanger allowing the direct processing of crude feeds. Our range of membrane chromatography adsorbers are broadly used for both the capture of large molecular species and plasmids, as well as in polishing steps for removal of contaminant DNA, host cell proteins, and virus clearance at the end of the purification process.
Powell (Asahi Kasei Bioprocess): Asahi Kasei Bioprocess has moved forward with offering carbohydrate-based ion-exchange chromatography resins due to their very high base stability (for effective clean-in-place processes), as well as their renowned high recoveries due to their hydrophilic nature. We now provide small extra-rigid agarose bead ion-exchange chromatography hemistries for intermediate and polishing steps (including anion exchangers that provide high capacity at 40-100 mM NaCl), as well as other chemistries for capture with extremely high capacities.
Zelter (EMD Millipore): We are working on different areas to meet future customer needs.