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Best Practices for Chromatography Method Development for Monoclonal Antibody Analysis

Applications and benefits of size exclusion chromatography

Lauren Everett
headshot Helen Whitby
Helen Whitby, PhD

Managing editor Lauren Everett speaks with Helen Whitby, an HPLC expert, on how size exclusion chromatography (SEC) can be used within various applications and the intricacies and challenges of chromatography method development for monoclonal antibody analysis.


Q: Can you explain size exclusion chromatography (SEC) and the applications it is best suited for? 

A: Size exclusion chromatography is a technique which separates molecules based on their size, specifically their hydrodynamic radius in solution. It is known as an effective and efficient method commonly used to analyze size variants in protein therapeutics including monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs). 

Inside the chromatography column, an aqueous or organic solution containing molecules of different sizes, referred to as the mobile phase, is passed through a chromatography column filled with porous packing material, referred to as the stationary phase. Larger molecules which cannot permeate all the pores in the chromatographic media are partially excluded and thus elute from the column earlier, wherein smaller molecules which can permeate more pores, elute later. It is a non-adsorptive, efficiency-driven separation. 

One of the areas where SEC is particularly valuable is in quality control workflows, especially in an application known as aggregate quantitation analysis, which can monitor how biotherapeutic proteins react to the drug development and storage process and whether certain critical quality attributes (CQAs) have remained within desired ranges. Because the presence of protein aggregates has been known to illicit immunogenic responses and differences in potency, proper characterization is critical throughout the drug development process. 

Size exclusion chromatography can also be used in fragment identification and other liquid chromatography mass spectrometry (LCMS) based analytical solutions such as drug-to-antibody ratio (DAR) analysis of ADCs and intact mass analysis for oligonucleotides when coupled to a high-resolution mass spectrometer (HRMS).

Q: What are some of the key factors that need to be considered when developing a robust and reproducible chromatography method for monoclonal antibody analysis?

A: When building robustness into your method, it is critical during column evaluation that you test your method with multiple columns from at least three different batches and ideally on numerous instruments with different end-users. This helps to ensure variability in the operator and column batch does not lead to variability in your results. With the global nature and connectivity of organizations today, size exclusion methods need to consider potential use on different system types in different labs around the world. This underlies the need to test multiple particle sizes and column lengths during development a variety of systems and set-ups that may introduce extra column effects such as dwell volume or even slight fluctuations in pH

When developing a method for aggregate analysis of mAbs by SEC, it is critical to optimize mobile phase conditions to prevent non-specific secondary interactions. Phosphate is a commonly used buffer for SEC and supports stabilization of the protein. To prevent adsorption of the protein to the stationary phase a moderate amount of salt should also be added, however the total amount will very much depend on the phase and nature of the protein as too much may result in salting out of the protein resulting in hydrophobic retention on the surface of the particle. When there is a need for a platform method, a good starting point for SEC is 50mM monopotassium phosphate 250mM potassium chloride, pH 6.8. Additionally, testing the effect of pH on your method should also form part of the development process, as subtle differences in pH can alter selectivity.

It is equally important when developing a method for aggregate quantitation that good recovery is achieved to allow low level quantitation. Adsorption can occur from the hardware of both the column and instrument and also the silica packing material itself. Through tailoring the mobile phase conditions, adsorption to the silica can be minimized by using salt. However, for maximum recovery, bio-inert HPLC systems and hardware are recommended. 

Q: What benefits does SEC offer compared to other methods? 

A: Size exclusion chromatography creates well-defined separations in a relatively short amount of time, with no sample loss, less solvent, good sensitivity, and an adjustable flow rate. Further, SEC is a relatively easy to implement technique, which offers good robustness and transferability between liquid chromatography (LC) platforms, even between ultra-high performance liquid chromatography (UHPLC) and high-performance liquid chromatography (HPLC) techniques. This is especially useful as UHPLC instruments are increasingly being used in quality control labs, along with conventional HPLC instruments. As you run in native conditions for proteins, size exclusion chromatography is a non-denaturing technique that allows for sample analysis in a native form. With size exclusion you can quantify aggregate and fragment peaks relative to the protein monomer. 

Q: What challenges need to be overcome to improve SEC method development?

A: Size exclusion columns are historically packed with inherently weak silica particles. These SEC particles are typically overly porous, which leads to poor mechanical strength. Additionally, a loosely or poorly packed SEC column will typically show poor column lifetime in a routine environment. Column lifetime can overall be improved by using a low pore volume silica column and ensuring appropriate guard columns are placed inline.

Additionally, some moderate to highly hydrophobic analytes like ADCs present challenges in SEC due to their propensity to interact with the stationary phase of the SEC column. In order to mitigate these unwanted secondary interactions, the incorporation of isopropanol into your mobile phase is an option to test out during development. We have found that the use of 10% isopropanol has been beneficial in many cases where we needed separation between aggregate and an ADC.

Q: Are there any emerging trends or advancements you are observing in the industry that may prove beneficial for labs in this space?

A: Inside the lab, there are several exciting developments. Especially when available levels of sample are incredibly low and valuable, bio-inert column hardware, column fittings and instrumentation offer the potential for low protein adsorption and an improvement in the levels of potential quantitation. Stronger, inert silica SEC particles with a more consistent pore structure such as Biozen dSEC-2 provide more robust methods and greatly minimize secondary interactions, while also providing significant improvements in column lifetime. These new SEC particles are available in both HPLC (3 μm) and UHPLC (Sub-2 μm) formats, allowing for the use of shorter columns packed with sub-2 μm media, which offer the opportunity to improve separation efficiency and method throughput.


Helen Whitby, PhD, is the senior product manager for biopharma at Phenomenex. Whitby has had several roles in the company, including as a biopharma specialist in the technical team supporting customers across all biopharma products globally before moving to product management in 2022. Whitby holds a PhD from the University of Manchester where her research focused on analogues of Shikimic acid to treat malaria. Whitby was a post-doctoral researcher at the University of Illinois in Chicago working on electron-deficient divalent reactive intermediates specifically nitrenium ions for the development of antiviral agents to treat Ebola.