bioProcessUK insight: new approaches to biopharmaceutical purification
In this guest blog, Ian Harwood, PhD, Senior Global Product Manager at Process Chromatography, Bio-Rad Laboratories, Inc., talks of the growing demand for innovative therapies and how it is driving the expansion of biopharmaceutical development.
In response to the global rise in demand for innovative therapies, the development and production of novel biopharmaceuticals continue to expand. The pharmaceutical market, projected to grow at a compound annual growth rate (CAGR) of 4.71%, is expected to reach a value of USD 1,454 billion by 2029 (1). This growth, alongside the increasing complexity of biologics, gene therapies, and vaccines, presents new challenges in large-scale bioprocessing.
Chromatography remains a cornerstone of downstream bioprocessing, utilized across the biopharmaceutical industry for the selective isolation and purification of biomolecules. Depending on the physicochemical properties of the target molecules, different modes of chromatography, such as affinity, ion exchange (IEX), size exclusion (SEC), and hydrophobic interaction (HIC), are employed. Mixed-mode chromatography (MMC), also known as multimodal chromatography, integrates multiple interaction modes on a single resin and continues to grow as an increasingly essential approach: it can be a highly selective and efficient purification technique. This approach allows for the removal of a wide array of impurities in fewer purification steps and can offer significant time and cost savings (2).
Principles of Mixed-Mode Chromatography
MMC combines two or more interaction principles between the stationary phase and solutes using a single chromatographic media or resin. The resultant interactions enable a unique selectivity that conventional single-mode chromatography cannot provide, facilitating the separation of closely related proteins and contaminants. This versatility is particularly advantageous in purifying complex biomolecules encountered in biopharmaceutical production.
The selectivity of MMC during binding, washing, and elution can be controlled through the modulation of buffer composition, conductivity, and pH. For instance, the charge on the typically weak IEX groups can be adjusted by altering the buffer pH, converting amino-based ligands from neutral to positive or carboxyl-based ligands from neutral to negative. The protein’s charge can be similarly altered to enhance binding or elution based on the complementary or similar charges of the ligand and protein. When the ligand and protein possess opposite charges, binding is strengthened; when their charges are alike, elution is promoted. Furthermore, in cases where both ligand and protein binding sites are neutral, hydrophobic interactions dominate.
The dual effects of salt concentration on ionic and hydrophobic interactions are particularly important in MMC. While increasing salt concentration typically weakens ionic interactions, it enhances hydrophobic interactions. This duality helps maintain the relatively stable binding capacity of MMC resins across a wide range of conductivities. Additionally, mobile-phase modifiers can influence the interaction between resins and biomolecules, offering further flexibility in process optimization.
Applications in Biopharmaceutical Manufacturing
Recent applications of MMC have demonstrated its potential for improving bioprocessing efficiency. For instance, combining HIC and IEX within a single resin has been shown to optimize recombinant adenovirus purification (3). Similarly, hydroxyapatite chromatography (HAC), a MMC media, can efficiently bind a broad spectrum of viral particles, separating them from impurities through calcium affinity and cation exchange (4).
As biologics, viral vectors, and vaccines gain traction in clinical applications, the demand for scalable and rapid bioprocessing solutions continues to rise. The complexities associated with purifying these biotherapeutics present a significant challenge, driving the need for innovative, cost-effective purification strategies. MMC offers substantial advantages in process design flexibility, improving end-product purity and homogeneity across a diverse range of biopharmaceutical products.
- “Pharmaceuticals - Worldwide.” Statista, https://www.statista.com/outlook/hmo/pharmaceuticals/worldwide. Accessed 2 Oct. 2024.
- He, Xuemei et al. “Purification of Antibodies with Nuvia aPrime 4A Hydrophobic Anion Exchange Resin.” Bio-Rad, Bulletin 7193. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_7193.pdf. Accessed 2 Oct. 2024.
- Chapman, Philip and Schaefer, Katie. Labcompare, https://www.labcompare.com/10-Featured-Articles/596048-Overcoming-Challenges-and-Improving-Efficiency-in-Protein-Purification/. Accessed 2 Oct. 2024.
- Snyder, Mark and Kronbetter, Laura. “Optimising Viral Vector Purification Strategies with Multimodal Chromatography.” Labmate Online, https://www.labmate-online.com/article/chromatography/1/bio-raddigilab/optimising-viral-vector-purification-strategies-with-multimodal-chromatography/3206. Accessed 2 Oct. 2024.