Setting New Standards in Bioprocess Chromatography
Chromatography is essential for achieving high purity and yield in downstream bioprocessing. Accelerate scale-up and improve product quality with systems, pre-packed columns, resins, and advanced process analytics solutions backed by Repligen’s proven history of innovation in bioproduction.
A cornerstone of downstream bioprocessing, chromatography enables the precise separation and purification of biomolecules to achieve high quality and yield. Its value lies in delivering consistent purity, maximizing recovery, and ensuring process efficiency: critical priorities for bioprocessing professionals working to meet regulatory requirements and accelerate time to market.
What is chromatography?
Chromatography is a separation technique used to isolate and purify molecules based on differences in their physical or chemical properties, such as size, charge, or hydrophobicity. In bioprocessing, chromatography plays a critical role in downstream purification, ensuring that therapeutic proteins, viral vectors, and other complex biologics meet stringent purity targets and (thereby) quality standards.
In bioprocessing, chromatography offers flexibility to address diverse purification challenges, from initial capture to intermediate steps and final polishing. Modern chromatographic media offer high resolution, high capacity, scalability, and reproducibility, which make them indispensable for achieving optimal yield and process economics across scales, from process development to GMP production.
How does chromatography work?
At its core, chromatography has two key phases: the mobile phase and the stationary phase. Chromatography works by first dissolving a mixture of interest into a mobile phase, which then carries the mixture through a stationary phase (such as a resin-packed column). The different components of the mixture travel through the stationary phase at different rates depending on differences in interactions between the phases. In bioproduction, these interactions can be based on molecule size, electrostatic forces, hydrophobic properties, or specific affinity of ligands for the product or other components.
If optimized well, chromatography allows for precise separation and high recovery of the target product from the starting material. This performance and level of control are essential for biopharmaceutical manufacturing, where consistency, scalability, and regulatory compliance are key requirements.
Types of chromatography
Affinity chromatography
Affinity chromatography relies on specific binding affinity between the target (e.g., an antibody) and a ligand immobilized on the stationary phase (e.g., resin backbone). First, the mixture is loaded into the resin-packed column, membrane, or monolith, and binding occurs under conditions that favor the interaction between the target molecule and the ligand. Various wash steps can be applied between load and elution to remove the weakly bound species and thereby improve the purity of the eluate (or improve the impurity clearance). Elution occurs by introducing an elution buffer to disrupt the interaction and release the target molecule from the resin.
Ion exchange chromatography
Ion exchange chromatography separates molecules based on differences in their net surface charge. The stationary phase contains anion or cation exchange ligands, which bind the oppositely charged components from the mobile phase. Elution is promoted by changing the pH and/or increasing the conductivity of the mobile phase. Components with lower charge density are bind weakly and are eluted earlier than components with higher charge density. In bioprocessing, ion exchange chromatography is often used to capture plasmid DNA, purify and polish proteins, and separate empty and full AAV capsids.
Hydrophobic interaction chromatography
Hydrophobic interaction chromatography (HIC) separates molecules based on their hydrophobic properties. It relies on the interaction between the hydrophobic regions of proteins and the hydrophobic ligands immobilized on the stationary phase. HIC often uses a high concentration of a chaotropic salt to promote hydrophobic interactions and binding. Target molecules are then eluted by decreasing the conductivity of the mobile phase.
Size exclusion chromatography
Size exclusion chromatography separates molecules based on their size (more specifically, their hydrodynamic radius) by filtration through a soft, porous resin (often referred to as a gel). Small molecules can enter the pores, while larger molecules are excluded. Unlike other chromatography methods, size exclusion chromatography does not rely on molecules binding to the resin.
Examples of chromatography applications in bioprocessing
Chromatography is essential for capture and polishing steps for multiple modalities in bioprocessing, including:
Antibodies and proteins
Affinity chromatography, most commonly protein A, is the industry standard for selectively isolating antibodies from harvested and clarified cell culture. High selectivity, capacity, reproducibility, and scalability are crucial.
Viral vectors
Chromatography is increasingly important in gene therapy manufacturing. Affinity chromatography is an industry standard for the purification of adeno-associated virus (AAV) and lentivirus. Anion exchange chromatography is also frequently used to separate empty and full viral capsids, one of the critical quality attributes in adeno-associated virus (AAV) purification. Maintaining high capacity and high selectivity for capture, capsid integrity, and high resolution for polishing are essential.
mRNA
Oligo-dT affinity chromatography is often used for mRNA capture. Additionally, removing immunogenic double-stranded (dsRNA) byproducts is a crucial step to ensure the safety of mRNA therapeutics. Affinity chromatography has been shown to remove unwanted dsRNA and eliminate immunogenicity, even when using optimized in vitro transcription processes.1
Plasmid DNA
For pDNA processes, where scalability and shear sensitivity are needed, chromatography equipment must be designed for low-shear performance while maximizing product purity and recovery.
Optimizing performance in bioprocess chromatography
While chromatography is indispensable for achieving high purity and yield across therapeutic modalities, bioprocessing professionals must navigate several technical and operational challenges.
Manufacturers must consider capacity, selectivity, and throughput of the chromatographic media, as well as the accuracy, repeatability, and hold-up volume of the chromatographic system.
A higher loading ratio can improve process economics, but may reduce separation efficiency or increase the risk of breakthrough. Precise gradient control and minimized system hold-up volume help preserve resolution while enabling higher loading, allowing manufacturers to better utilize binding capacity.
Higher flow rates shorten cycle times and improve process efficiency, but reduce binding capacity of traditional resins due to mass transfer limitations. Optimizing flow rates or residence times ensures a balance between productivity and column size. The latter is a crucial consideration for any affinity capture step.
Scaling up from process development to large-scale manufacturing requires maintaining the same bed height, product load, and linear velocities or residence times to obtain comparable capacity, separation efficiency, and pressure-flow characteristics at significantly larger column diameters. Even small variations in packing quality can impact resolution and thereby the purity and/or yield during scale-up. Automated process control and recipe management capabilities can support consistent performance across scales, helping to minimize variability during tech transfer and scale-up.
Column packing is often described as both art and science. It requires specialized training, extensive hands-on expertise, and rigorous validation. Packing expertise is a critical yet often limiting factor for manufacturers. Insufficient column packing experience may result in delays or inconsistencies between batches.
Modern biomanufacturing requires comprehensive process monitoring for effective and data-driven method development and process control. Advanced software capabilities support seamless system integration, as well as data collection and data integrity requirements throughout the product lifecycle.
A history of innovation in chromatography
For decades, Repligen has been trusted by leading biopharma manufacturers to deliver reliable, high-performance chromatography solutions. Our commitment to innovation is reflected in fully integrated KRM® chromatography systems with single-use flow paths, market-leading OPUS® pre-packed columns, and high-efficiency AVIPure® and HiPer™ resins. Backed by integrated process analytics, Repligen solutions empower you to improve process economics, accelerate timelines, and confidently meet the demands of modern bioprocessing.
KRM chromatography systems maximize recovery of high-value biomolecules while reducing operational complexity across process development, validation, and commercial manufacturing. Achieve consistent, high-quality separations with a precision-engineered platform that minimizes product loss through innovative flow-kit design and unparalleled gradient control.
This precision translates to operational efficiency. KRM systems save time and reduce risk through streamlined automation and intelligent process control. Our innovative design minimizes hold-up volume while maintaining robust and comparable performance across scales. Additionally, KlariFi™ software enables seamless SCADA integration, real-time process monitoring, and adaptable recipe management—empowering faster method development, simplified tech transfer, and data-driven decision making and process control.
OPUS pre-packed chromatography columns provide linear scale-up from process development to clinical and commercial GMP manufacturing. Select from the broadest portfolio of pre-packed column sizes, which are easily configurable for bed height, resin type, and application. Repligen packs over 300 commercially available and custom chromatography resins.
OPUS pre-packed columns offer significant time and cost savings compared to conventional column technology. Additionally, OPUS 36R cm, 45R cm, 60R cm, and 80R cm columns are the first pre-packed chromatography columns designed to process high-volume feed streams from large single-use bioreactors (1000L and 2000L). Let us handle the column packing, so you can focus on achieving your process goals.
Repligen combines trusted AVIPure affinity ligands with macroporous base bead technology to deliver high-performance chromatography resins. Innovative HiPer resins are characterized by uniquely designed spherical beads with 50 µm diameter and 1.6 µm average pore size. This architecture maximizes surface area for high dynamic binding capacity while supporting convective mass transfer and rapid flow rates. HiPer resins are ideal for the purification of large-molecule biologics, such as viral vectors, viruses, virus-like particles (VLP), and nucleic acids.
Built on revolutionary variable pathlength technology (VPT), the PATsmart™ FlowVPX® spectrophotometer enables in-line concentration measurement for advanced process control and is designed to support process development and scale-up all the way to commercial manufacturing in GMP.
Installed directly in-line, the FlowVPX System provides continuous concentration measurements by varying the pathlength instead of the concentration, removing the need for dilution, manual sampling, or human intervention that may risk contamination or error. With robust, dedicated software that manages concentration with custom setpoints and alerts, you can precisely control your process like never before and adapt in real time to any variations in your sample.
KRM™ Chromatography Systems
CHROMATOGRAPHY REDESIGNED
The KRM System embodies decades of transformative technology innovation, resulting in a rapidly scalable single-use platform for unprecedented recovery of complex biomolecules and new modalities.
OPUS® Pre-packed Chromatography Columns
Accelerate process development and scale-up with consistent performance, reduced operational complexity, and faster deployment from lab to commercial production.
AVIPure® Albumin Affinity Resin
Discover a high performance affinity chromatography resin for the primary capture of human albumin from complex feedstocks.
Ion Exchange Resins
Designed for the purification of large-molecule biologics, our ion exchange resins use innovative HiPer™ macroporous bead technology to deliver high binding capacity and rapid processing.
PATsmart™ FlowVPX® System
Continuous in-line concentration measurement for real-time process control
Designed for GMP environments, the PATsmart™ FlowVPX® spectrophotometer delivers continuous, in-line concentration measurement during downstream processing, providing real-time data to actively control the process as it runs. This level of control is made possible by Variable Pathlength Technology (VPT), which enables accurate, dilution-free concentration measurement across development, scale-up, and GMP manufacturing.
Fluid Management
Repligen offers innovative, designed-for-purpose storage and transfer solutions for fluid management within and between unit operations. These include flow paths, tubing, valves and gaskets, bottles and containers, totes, carts and cleanroom process equipment.
Reference
- Clark NE, Kozarski M, Asci SD, et al. Removal of dsRNA byproducts using affinity chromatography. Molecular Therapy Nucleic Acids. 2025; 36(2), 102549.
