Heidi Jones, market development manager, Chromatography, Bio-Rad Laboratories discusses streamlining biopharmaceutical purification with continuous chromatography.
Bio-Rad Laboratories
Figure 1
Innovations in the discovery and development of modern biopharmaceutical therapeutics, such as monoclonal antibodies (mAbs), offer new and effective treatment approaches for a multitude of diseases, ranging from cancers and infectious diseases to autoimmune and inflammatory conditions. With rising biotherapeutic demand comes an increased need for efficient downstream purification processes. Column chromatography remains the gold standard for protein purification, offering a widely applicable solution for use in drug development. However, the process is typically limited by column capacity, creating downstream production bottlenecks. This creates a significant opportunity for innovative technologies to accelerate and streamline purification.
Batch vs continuous chromatography
Batch chromatography typically involves the use of a single large column, with each step (i.e., capture, intermediate, and polishing steps) run sequentially before the column can be cleaned and set up for the next run. This standard approach is suitable for most purification processes, and allows for parameter adjustments between runs. However, this creates a disjointed system that requires considerable manual input, large volumes of buffer and resin/media – much of which is not utilised to its full capacity – and longer processing times due to downtime between runs. Additionally, each purification is constrained by the capacity of the column in use, thereby limiting the rate of protein production.
In continuous chromatography, many smaller columns (up to 16) can be used in parallel over a larger number of cycles, allowing simultaneous operation of columns. Running columns in series enables continuous loading, washing, and elution cycles, minimising downtime and increasing throughput, while reduced buffer and consumable use minimises waste output and associated costs, amongst other advantages (Figure 1). While both batch and continuous chromatography utilise the same resins and buffers, it can be difficult to transition between the two, often requiring dedicated multi-column instruments and large up front investment.
Multi-column chromatography (MCC) is a popular format of continuous chromatography which consists of a series of small columns operated in a coordinated cycle, enabling simultaneous purification runs. The most established type of MCC is periodic counter current chromatography (PCC), which continuously switches between a minimum of two columns in a loop fashion. Other variants include simulated moving bed (SMB), which is suited to sugar refining and mimics the movement of the solid phase through sequential switching of column inlet and outlet positions, and twin column MCC systems, which facilitate continuous loading, washing, elution, and regeneration.
Customisable operating platform to optimise multi-column chromatography
By improving resin capacity and eliminating single column constraints, continuous chromatography offers improved yields, greater efficiency, and faster purification processes, providing a cost-efficient approach to protein purification. This approach is particularly well-suited for affinity and capture stages, allowing researchers to streamline workflows and optimize material use.
For example, during mAb purification, multiple smaller columns operate in a coordinated cycle to enable continuous processes. This step leverages the binding affinity of a protein-based ligand (Protein A) with the Fc region of antibodies. A two column MCC approach controlled by a customisable platform can offer an effective continuous chromatography technique for mAb purification, achieving an increased purity for Protein A affinity purification with a decreased run time in comparison to batch chromatography. In this study, the platform was configured to perform parallel purifications using standard column hardware, which opens opportunities for wider applications of continuous chromatography.
A case study demonstrated that such a customisable chromatography instrument can also be modified to run a three column purification including protein A, buffer exchange, and size exclusion (SEC) steps, referred to as the Triple Tandem System. The automated, continuous process enabled tandem SEC purification while minimising contamination of the SEC column, and is capable of purifying six samples simultaneously, reducing manual input and increasing product yield. The reduction in column fouling and contamination also increases the lifespan of columns, further minimising associated costs.
Membrane adsorbers to further enhance productivity
Alongside the use of customisable platforms to operate chromatography workflows, integration of new separation technologies can further accelerate continuous chromatography. One example involves the use of convective diffusive protein A membrane adsorbers (MAs) in a semi-continuous process. Chromatography often relies on protein A resins for the capture step, but this is limited by the diffusion-controlled mass transport which requires a low flow rate. MAs offer a promising alternative to protein A resins by using a novel stationary phase with a higher surface area. The MAs can be connected in series for either rapid cycling parallel MCC (RC-PMCC) or rapid cycling simulated moving bed (RC-BioSMB), utilizing a smaller buffer volume in comparison to batch processes due to the higher loaded mass per cycle.
A study by Schmitz et al. 2024, aimed to investigate the process performance of MAs operated in batch and continuous MCC modes (RC-PMCC and RC-BioSMB) for mAb capture. All processes were compared regarding membrane utilisation, buffer consumption, and productivity whilst considering varying loading flow rates (s1.25–10 MV min⁻¹) and mAb loading concentrations (0.5–5.2 g L⁻¹). Membrane utilisation was up to 86 % higher in RC-BioSMB, while also achieving the lowest buffer consumption (1.1 L g⁻¹). Batch and RC-PMCC achieved peak productivities of 176 g L⁻¹ h⁻¹ at high mAb concentrations and low flow rates, whereas RC-BioSMB excelled at high concentrations and high flow, attaining 217 g L⁻¹ h⁻¹. These results underscore the importance of tailoring loading parameters and considering each system’s optimal operating conditions to maximize productivity and minimise costs.
Continuous chromatography to meet purification demands
With the ever-increasing demand for mAbs, focus is shifting to enhancing protein purification processes; increasing the effectiveness and efficiency of chromatography. Implementation of continuous chromatography into manufacturing workflows increases production rate without compromising on purity. The continuous bioprocessing market is projected to reach over $599 million by 2028, driven by the rising demand for pharmaceuticals and growing adoption of continuous chromatography by manufacturers, representing a significant opportunity for new integrative platform technologies.
Alongside the reduction in cost and time, continuous chromatography has a lesser environmental impact than batch chromatography, reducing both the consumption of reagents and wasting of materials, supporting manufacturers to meet sustainability targets. Ongoing innovations in downstream processing technologies are continuing to advance chromatography, increasing productivity and facilitating the manufacture of safe, effective antibody-based therapeutics.