Bioprocessing Chromatography Techniques

Chromatography techniques in bioprocessing

Downstream purification is an integral stage in bioprocessing that isolates the target biomolecule from impurities through multiple separation techniques. Chromatographic separations rely on physicochemical interactions between the stationary phase and the molecules in solution. Although each purification step can improve product quality, additional steps can also decrease productivity and yield.

Advances in chromatography resins with higher binding capacity, improved selectivity, and faster flow rates help improve recovery and purity, shorten processing time, and lower overall production costs. Designing an optimal process requires balancing purity, recovery, and cost to achieve robust, scalable, transferable purification strategies in biopharmaceutical manufacturing.

Chromatography resin chemistries

Chromatographic resins are matrices that separate biomolecules based on chemical interactions, such as charge, hydrophobicity, or affinity-based binding.

Affinity chromatography

Affinity chromatography is commonly used as the capture step in downstream purification. The target biomolecule, produced in a complex mixture during upstream processing, binds to a ligand on the affinity resin. Ideally, the target binds to the resin while many impurities flow through. Elution is achieved using a buffer that disrupts the binding interaction, releasing the purified target from the column. This technique enables high recovery and purity in one step, making it an efficient capture method in bioprocessing.

Ion exchange chromatography

Ion exchange chromatography (IEX) separates biomolecules based on differences in surface charge using resins functionalized with positively or negatively charged groups. The target molecule binds to the resin through electrostatic interactions, and changes in ionic strength are used to elute bound molecules. IEX can serve as both a capture and polishing step. This scalable technique improves purity by reducing product-related impurities such as aggregates and process-related impurities like host cell proteins.

Hydrophobic interaction chromatography


Hydrophobic interaction chromatography (HIC) separates biomolecules based on hydrophobicity. Resins are functionalized with a range of hydrophobic ligands and can be operated in bind/elute or flow-through mode as a polishing step. In general, a protein binds to the resin in high-salt conditions, which promotes hydrophobic interactions. By gradually lowering the salt concentration, elution occurs as hydrophobic interactions decrease. HIC can contribute to the reduction of aggregates and impurities in bioprocessing.

Mixed-mode chromatography


Mixed-mode chromatography (MMC), also known as multimodal chromatography, combines multiple interaction types, such as ionic and hydrophobic, within a single stationary phase, offering distinct, orthogonal selectivity. By engaging multiple physicochemical mechanisms, MMC may help address challenging impurity profiles when conventional single-mode resins are insufficient, and could reduce the need for additional polishing steps. Its versatility makes MMC an additional, valuable tool for purifying complex bioprocess feedstreams.

Developing chromatography steps


Designing an effective purification process requires balancing selectivity, yield, and scalability. Scientists evaluate each step using analytical tools, such as HPLC, mass spectrometry, and SDS-PAGE, to assess yield, purity, and process consistency. Nucleic acid analytical techniques enable rapid detection and quantification of impurities, improving process efficiency and allowing adherence to regulatory guidelines. Insights from these analyses guide resin selection, buffer conditions, and column parameters to build a robust, reproducible downstream workflow

Modes of operation in chromatography

Chromatography performance depends not just on resin chemistry but also on how each step is operated. Variables, such as buffering scheme and loading concentration, can be selected to operate the resin in either bind and elute or flow-through mode.

Bind and elute mode

In bind and elute mode or positive mode the target binds to the resin while impurities flow through. The bound molecule is then released by changing pH or conductivity to recover the purified product.

 

Flow-through mode

Flow-through or negative mode allows impurities to bind while the target molecule passes through the column. Another type of negative mode is overloaded, or frontal mode.

 

Batch binding

Batch binding or static binding uses loose resin mixed with the sample rather than a packed column. It is often used for screening or early process development because it yields quick results but is not typically representative of scale-up.

Other modalities and applications supported

ADCs

Antibody-Drug Conjugates (ADCs) workflows rely on chromatography steps to separate complex conjugates and reduce impurities. These approaches support consistent production of ADCs for therapeutic research and biomanufacturing.
 

Cell Therapy

Cell therapies utilize living cells as treatments, necessitating strict control over inputs and processing steps. Chromatography is applied to prepare ancillary materials, such as cytokines or viral vectors, that are used during cell manufacturing.
 

Gene Therapy

Gene therapy often relies on viral vectors, such as adeno-associated virus (AAV) or lentivirus, to facilitate the transfer of genetic material. Chromatography separates drug product from product- and process-related impurities in both capture and polishing steps.
 

mAbs

Monoclonal antibody purification typically starts with Protein A chromatography to capture the antibody from cell culture fluid. Additional steps, such as ion exchange, are used to help reduce aggregates, variants, or process-related impurities.

mRNA

Chromatography in mRNA production separates full-length transcripts from double-stranded RNA and truncated products. The purified RNA can then be used in formulation or downstream steps, such as lipid nanoparticle assembly.

Vaccines

Chromatography is often used in vaccine manufacturing for both the capture and polishing of the vaccine drug product. The choice of method depends on the platform, for example, protein subunits, viral vectors, or nucleic acid vaccines.

Frequently asked questions

Chromatography in downstream processing combines several modes, each serving a defined role. Affinity chromatography is typically used to capture the target molecule with high specificity and selectivity. Ion exchange and hydrophobic interaction steps are used to separate charge- or hydrophobic-variant proteins. In some workflows, mixed-mode resins serve as another tool, offering different selectivity through multiple interaction mechanisms in a single immobilized ligand.

Chromatography methods are used throughout downstream bioprocessing to purify biomolecules produced from upstream cell culture or fermentation. Each technique, such as affinity, ion exchange, hydrophobic interaction, mixed-mode, and size exclusion chromatography, targets specific molecular properties to reduce impurities. When combined into a purification process, these methods offer reproducibility and consistency suitable for the manufacturing of biologics.

Selecting a chromatography method depends on the molecule’s properties and overall process goals, often focusing on the minimization of process-related impurities. One of the first steps is to characterize the target molecule and impurities based on key factors, such as charge profile, isoelectric point (pI), hydrophobicity, and buffer stability. This information offers insight into which chromatography resin and separation techniques may be effective. Once a resin type is identified, resin screening can be performed using high-throughput formats, such as 96-well screening plates, to evaluate different buffer conditions to determine binding, wash, and elution parameters. Thermo Fisher Scientific has a team of technical specialists that can help with technique selection and process optimization. Please contact us for more information.

Traditional chromatography methods continue to be applied to emerging therapeutic formats, including mRNA, viral vectors, and ADCs. The same matrices enable selective separation of biomolecules based on chemical interactions, such as charge, hydrophobicity, or affinity-based binding. Innovations in resin chemistry, buffer systems, and operating modes have further advanced the purification and manufacturability of these molecules.

In purification optimization, it involves evaluating factors, such as buffer composition, pH, salt concentration, flow rate, resin selection, and loading conditions, to maximize yield and purity.

Proper scale-up enables process performance, purity, and recovery to remain consistent from laboratory to pilot and production scale. When scaling a purification step, several critical column parameters must be evaluated to maintain consistent product quality and performance. Key factors include column geometry, bed height, flow rate, residence time, load density, buffer composition, and resin packing. Additional considerations, such as hold-up volume, equipment compatibility, and automation control, also influence scale-up success.

Explore other chromatography products

From resins and columns to complete purification solutions, these chromatography products enable efficient operation across capture, intermediate, and polishing steps.

CaptureSelect and POROS chromatography

CaptureSelect and POROS resins support the purification of biologics, such as antibodies, viral vectors, and recombinant proteins. CaptureSelect resins offer selectivity and gentle elution, while POROS resins offer speed and scalability for large-scale chromatography performance.

Chromatography columns

Pre-packed chromatography columns simplify process operations and reduce preparation time. Available in multiple sizes, these columns support capture, intermediate, and polishing steps, enabling reliable data for process development and scale-up.
 

Chromatography resins

Batch binding—or static binding—uses loose resin mixed with the sample rather than a packed column. It is often used for screening or early process development because it yields quick results but is not typically representative of scale-up.
 

Chromatography resources

Access application notes, protocols, and videos that support resin evaluation and process development. Topics include resin selection, column operation, method transfer, and troubleshooting for antibodies, viral vectors, and mRNA. These materials offer guidance for downstream workflows, spanning from early research to manufacturing.
 

Intensify downstream processing with chromatographic techniques

Intensify downstream processing

For Research Use or Further Manufacturing. Not for diagnostic use or direct administration into humans or animals.