Affinity Chromatography Hub
Affinity chromatography is a bind-and-elute method designed to isolate a specific biomolecule or protein rather than separating different peaks in a mixture, making it distinct from other chromatography methods. In affinity chromatography, a ligand with a specific biological interaction to the protein of interest (POI) links or becomes immobilized to a particle or matrix.
The most common ligand, Protein A, binds to a sequence in the Fc region of antibodies. This robust interaction is widely used in therapeutic antibody manufacturing to determine titer — a critical quality attribute (CQA) in Process Analytical Technology (PAT) labs that indicates antibody concentration in a sample. Titer measurement helps identify the most productive clones for clone selection and determine optimal conditions to maximize product quality and yield at cell harvest.
While extremely common, there are still challenges to measuring titer using analytical Protein A.
Key Challenges
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Low-Concentration Sensitivity
Protein A cannot detect antibodies at low concentrations, which typically reach measurable levels 3-5 days after inoculation. Lowering quantitation limits, as seen in the application note, enables earlier detection, saving time and resources by identifying viable clones sooner. Increased sensitivity also reduces the amount of sample used while maintaining accurate titer results.
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Complexity of Modern Therapeutics
As antibody therapeutics are becoming more complex and requiring more analyses, timelines for new therapeutics are shrinking. Innovative workflows that combine Protein A titer with other techniques like SEC aggregate analysis or mass spectrometry is the key to getting these insights in less time than ever before.
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Reproducibility and Column Longevity
Titer results need to be consistent across the board, including run-to-run, column-to-column, and batch-to-batch. If the column experiences drift or failure during a bioprocessing campaign, recalibration with a new column is required, which results in a significant and unexpected time loss. New Protein A columns need to last longer than before, and Clean-in-Place (CIP) procedures help maintain performance without frequent column replacement.
Explore this content hub and resources to discover how Protein A is advancing to overcome today’s analytical challenges and enable faster, more reliable workflows.
Waters analytical workflow solutions deliver confidence, efficiency, and scalability across your biotherapeutic workflows, including BioResolve Protein A Affinity Columns designed for robust, high-performance antibody titer analysis.
On Demand Webcast
Intact and Subunit LC-MS Characterization of mAb and msAb using Advanced Protein A Columns
Webcast overview
Obtaining MS-based intact and subunit characterization of monoclonal antibodies (mAbs) and multispecifics (msAbs) is a crucial step in the development workflow. However, older analytical methods often fall short, lacking the depth of information required to support regulatory approval for today’s advanced therapeutics. This webinar introduces an innovative Protein A-MS pH-elution gradient method for achieving effective separations of protein variants with modulated binding affinities prior to direct intact high-resolution MS analysis. We will discuss how advanced ProA-MS methods can overcome traditional limitations and deliver the characterization needed to meet the evolving demands of advancing protein therapeutics.
Topics include
- How to achieve in-depth characterization of mAbs and msAbs without prior sample preparation
- How new Protein A Affinity columns can provide valuable information beyond just titer
- How Protein A Affinity columns can be used in methods to offer additional characterization insights
- How to implement unique LC-MS analytical methods for both mAbs and msAbs
Speakers
Dr. Stephan Koza (Consulting Scientist, Waters Corporation)
Arnaud Delobel (Director, R&D and Innovation, Quality Assistance).
Application Note
Lowering Quantitation Limits for mAb Titer Measurements Using Small Volume 3.5 µm Particle-Size Protein-A Affinity Columns
Protein A (ProA) affinity chromatography (PAAC) is often used for the measurement of monoclonal antibody (mAb) titer in cell culture conditioned media (CM) samples. Here we introduce high-pressure capable and low volume (70 µL) ProA columns that show 5-fold to 7-fold lower LLQ values versus a ProA column constructed with 20 µm porous particles packed into 2.1 x 30 mm PEEK hardware while also delivering comparable performance at higher sample loads. A reduced LLQ can be advantageous when mAb concentrations are low and sample volumes are extremely limited, as when determining titer for milliliter scale micro-bioreactor samples
Application Note
An Easy-to-Execute Direct-Connect 2D Protein A–SEC Method for the Analysis of Monoclonal Antibody Titer and Size Variants in Cell Culture
Analytical Protein A affinity columns have been routinely used to monitor the titer of mAb and other Fc containing constructs such as fusion proteins and bi-specific antibodies in cell culture. In addition, ProA columns have been deployed for online 2D purification in front of SEC to enable the direct size variant analysis of mAbs in cell culture by either connecting the two columns via switching valves or in series without switching valves. Here, we present a direct-connect 2D ProA-SEC method that makes use of a recently developed high efficiency, low volume ProA column.
Application Note
Extended Gradients with Efficient Protein A Columns for the ProA-MS Analysis of mAb and msAb Variants
Analytical methods that enable the rapid structural evaluation of monoclonal antibodies mAb, msAb, and fusion proteins are sought after throughout the multiple stages of biopharmaceutical development. One such approach is ProA-MS for the direct analysis of CCCF samples. It has also been demonstrated that the differential dissociation of ProA bound mAb variants can be used analytically when using pH gradient elution. In this study, the impact of using high efficiency affinity columns is evaluated along with extended gradient elution methods for ProA-MS analyses.
Application Note
Maximizing Protein A Column Performance, Lifetime, and Reliability Through Clean-in-Place
The useful lifetime of protein A analytical columns in bioprocessing applications can vary quite drastically depending on method conditions and sample composition. A common failure mode for such columns is fouling caused by the build-up of cell culture media components. This application note demonstrates how employing best practices can help mitigate these potential problems to realize the longest possible column lifetimes.
Infographic
BioResolve Protein A Sensitive and Efficient Antibody Titer Anaylsis
Learn about new tools for achieving faster decision-making during cell harvest and better process control.
