Sponsored by Purolite
In 2010, life-sciences company Repligen began to develop its own agarose-based protein A affinity resin. However, work on that resin halted when two critical elements became scarce: high-performance agarose beads and capacity for good manufacturing practice (GMP) production. In 2015, Purolite, an Ecolab company, took on a similar project. Purolite’s challenge was limited access to protein-ligand technology and commercial-scale, GMP-ready ligand manufacturing. Sensing potential for collaboration, Repligen and Purolite codeveloped three affinity resins.
A 2024 Ask the Experts webinar with four representatives from those companies explored the benefits of such technology partnerships. Steve Tingley (vice president of sales) and Jamie Peyser (vice president of fluid management) represented Repligen. Patrick Gilbert (director of research, development, and engineering) and Andrew Masters (head of business development and strategy) represented Purolite.
The presenters discussed evolving needs in the biomanufacturing industry and how technical collaborations can meet such demands. New processing models, purification requirements, and raw-material supply chains in particular are driving the industrialization of monoclonal antibodies (mAbs) for affinity chromatography. Collaborations between biomanufacturing companies can produce novel products to fulfill unmet demands as well as establish viable, alternative sources.
The Purolite–Repligen Partnership: Successful technology collaborations are rare because of four common obstacles: mutual profitability concerns, commercial control issues, access to partner intellectual properties (IPs) and research and development (R&D), and customer-adoption risks. Purolite and Repligen addressed those concerns early in their partnership. They pledged to codevelop a cost-effective affinity resin and created clear strategic focus across all affinity modalities. The companies also agreed to share core technologies and IPs so that their combined R&D teams could enhance product performance.
To determine the best use of their combined team’s resources, Purolite and Repligen broke down customer engagement into four categories. The first category centered on working with customers to secure long-term agreements with renewals and thus secure future technology access to products for customers. The second factor was the integration of supply chains for ligands, beads, resins, and prepacked columns. The third factor described collaborative customer support to maximize application expertise. And the final factor described “voice of customer” research to create a proactive identification of market needs to drive future innovation.
The speakers noted that the current model for vendor–customer interaction presumes one-to-one relationships between single vendors and customers. However, that model can entail supply-chain risks and discourage technology development. For example, one supplier historically has controlled resin stock. Such a monopoly can lead to supply chain risks and halt future technology development. Thus, vendors must adapt to a new model in which each customer engages with multiple partnered vendors to address its needs.
Such collaborations create technical benefits. The Purolite–Repligen partnership has resulted in novel affinity ligands derived from Purolite’s agarose-jetting technologies and Repligen’s affinity-ligand libraries. Technologies resulting from that partnership include the Praesto Jetted A50 resin with the NGL-Impact A ligand, the Praesto Jetted A50 HipH resin with the NGL HipH ligand, and the Praesto 70 CH1 resin with the AVIPure CH1 ligand.
Past and Future: Repligen has a deep history with protein A. In the 1980s, it was the first biomanufacturer to clone the protein, and since then, it has manufactured >90% of the
protein A ligands used in commercial mAb-purification processes. Repligen has grown its technology portfolio significantly in the past years. Other product areas include prepacked columns, technologies for upstream intensification, and process analytics.
The Repligen–Purolite partnership began nearly a decade ago. At that time, Purolite focused on agarose-bead capabilities. With Repligen’s history in protein A manufacturing and supply, collaboration made sense. As the partnership has matured, the companies’ work has turned to other market needs.
Resins: Praesto Jetted A50 HipH resin combines Purolite’s proprietary jetting technology and an NGL HipH protein A ligand from Repligen. The resin increases recovery yields for pH-sensitive molecules, particularly for bispecific antibodies (bsAbs), fragment-crystallizable (Fc) fusions, and other nonstandard antibodies that are prone to precipitation and aggregation. The Praesto Jetted A50 HipH resin can be a direct replacement for standard
protein A resins.
The Purolite–Repligen team screened multiple ligands to develop the Praesto Jetted A50 HipH resin. Having compared elution conditions for an Fc-fusion protein in standard protein A resins, the team observed that the protein eluted at pH 3.4–3.6. Thus, the companies sought to develop a resin that could extend the pH conditions. Of all tested candidates, the NGL HipH ligand was chosen because it is not influenced by VH3 binding, enabling its use across many types of antibodies without unwanted binding. That provides users with a high-capacity resin that can provide a 60-g/L binding capacity at relatively short residence times. The bead-jetting technology also gives the resin good alkaline stability and favorable mass transport effects. In the end, the Purolite–Repligen team developed the first resin on the market that can elute an antibody up to pH 5.
Bispecific Purification: At the 2023 BioProcess International Conference and Exhibition in Boston, MA, Kayla Barry from MabPlex International presented a case study comparing the Jetted A50 HipH resin with a standard protein resin for purification of three bsAbs. MabPlex’s interest in the resin arose from challenges that it had experienced with bispecific purity when using standard protein A resins. MabPlex scientists observed that some bsAbs became unstable during protein elution conditions at pH 3.5–3.7, leading to aggregation and fragmentation. One such molecule under investigation was an antibody that the company called BsAb Z. Attempts to purify it would yield only 60% of the monomer remaining after protein A steps using typical elution conditions. The MabPlex team tried the Praesto Jetted A50 HipH resin in the hopes that more acidic bsAbs would benefit from the ability to elute under broader pH conditions.
MabPlex first performed purification of BsAb Z over a small-scale column. The goals were to prevent aggregation and achieve better separation between target proteins and contaminants (e.g., species of high molecular weight) in standard conditions. The team compared a standard protein A resin and the Praesto Jetted A50 HipH resin. The former binds to molecules contained in the Fc and VH3 regions of immunoglobulin G (IgG), whereas the latter binds mainly to the Fc region with negligible interaction with the VH3 region. MabPlex packed a 3.42-mm column and carried out a high-salt wash after loading, then eluted at pH 3.5–5. The resin resulted in ~96% cumulative monomer retention. The team also confirmed an initial finding that eluting at lower pH caused aggregation issues for standard protein A resins. From those data, MabPlex tried to develop and optimize a high-yield isocratic elution of BsAb Z using the Praesto Jetted A50 HipH resin. However, that wasn’t possible because of a lack of additional feed material for BsAb Z. Therefore, MabPlex ran that analysis with a different molecule. The two candidates were BsAb X and
BsAb Y: BsAb X was mostly stable with some aggregation and fragmentation, whereas BsAb Y experienced much more aggregation and fragmentation.
Despite those results, the Praesto Jetted A50 HipH resin demonstrated clear purification benefits for the evaluated bsAbs. The MabPlex team found that low-pH conditions on a standard protein resin can lead to aggregation and coalition of monomer with undesirable species. Using the Praesto resin improved separation and recovery of the monomer species.
How must the industry operate differently to take advantage of technology collaborations? The industry is used to working in a one-to-one model, in which one party is responsible for developing and commercializing a product and another party is responsible for using it. We suggest a one-to-many model as we move toward combination products. For example, collaborating parties must share information and documentation and determine ownership of such data. We’ve observed the greatest success in collaborations with responsibilities assigned swiftly, clearly, and early in the partnership.
What flow rates do you recommend for the Praesto Jetted A50 HipH resin? We recommend a 20-cm bed height to run the column at 200–250 cm/h as a maximum flow rate. For faster operations, we recommend a shorter bed height, which can increase productivity while reducing buffer consumption.
Did MabPlex consider using that resin for its purification platform? When we spoke with Kayla Barry, MabPlex had been considering that possibility already because of issues with aggregation and fragmentation using standard resins. MabPlex wanted to expedite purification; its staff already have been able to apply general conditions to that effect.
What should users of collaboration-driven products expect in terms of quality, technical support, and availability? Quality, technical support, and availability expectations should remain the same for both standard and codeveloped products. Partnering companies must define a precise contact to support end users.
At what scales are the Praesto Jetted A50 and HipH resins packed? Both resins use the same beads. We pack the HipH resins on 1.6-m columns with 17-cm bed heights, 1-m columns with 20-cm bed heights, and 0.6-m columns with 12-cm bed heights. That resin’s high capacity enables shorter residence times to support process intensification.
What elution volumes and pH values should be used for the HipH resin? It depends on the antibody you are using. During our evaluations of the HipH resin, elution volumes increased proportionately with pH levels. We recommend running pH gradients to find the highest pH level to obtain the elution volume needed.
Find the full webinar online at www.bioprocessintl.com/category/webinars.
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