Guest Author – Namrata Saxena, Technical Specialist – Phenomenex Asia/Pacific
Monoclonal Antibodies are large and complex biomolecules which falls in the prominent section of biotherapeutic drugs market. The complex structure and composition make it a necessity to fully characterize the critical quality attributes of these biomolecules before they can be approved as drugs for human use. Aggregation is one such critical attribute which can materialize during any of the steps of biotherapeutic drug manufacturing process. Which is why I will be breaking down how size exclusion chromatography (SEC) is the best tool for antibody aggregate analysis.
Proteins are typically only marginally stable in their folded state and thus, conversely in their unfolded or partially folded state they are extremely prone to aggregation because it provides stability to the protein structure. Aggregation is a post translational modification (PTM) that can alter the proteins biological activity and render it immunogenic characteristics. It is thus, absolutely essential that robust analytical methods are developed to qualify and quantify the aggregate content of a biotherapeutic drug.
Aggregate quantitation requires that the protein has to be in its native state, and therefore separation modes require that denaturing conditions in the method cannot be employed. Gel Filtration Chromatography (GFC) or Aqueous Size Exclusion Chromatography (SEC) is the most widely used chromatographic technique to characterize and monitor the aggregates of mAbs. GFC/Aqueous-SEC is a very simple technique, however it is equally prone to errors because of the lack of understanding of the basic principles of this chromatography.
Principle: SEC is essentially a non-adsorptive technique which separates analytes based upon their size or more correctly on the basis of their hydrodynamic radius or volume which is also known as “Stokes radius”. Technically, with GFC it is separation by the hydrodynamic volume of the molecule i.e., how much space a particular biomolecule takes up when it is in solution, and not according to its mass. Hydrodynamic volume, however of course, has a direct correlation to the molecular weight of the protein. So, if a protein unfolds while in solution, parts of the molecule will be able to go into and out of the pores of the stationary phase and achieve some type of separation. If that same protein happens to stay compact and globular, the entire molecule may be too large to fit into any of the pores and is effectively excluded. Therefore, even though the molecular weight of the protein is still the same but running under different mobile phase conditions can change its hydrodynamic volume and thus affects its separation.
Standard Calibration Curve: This is the very reason why SEC standard calibration curves are made specifically for the mobile phase running conditions, or else the extrapolation can be wrong if something can alter the molecules hydrodynamic volume.
In ideal SEC method running conditions the larger the molecule, the less stationary phase pores it has access to and more often it is excluded and as a result elutes earlier, the smaller the analyte, the more pores it has access to and thus, longer the elution.
Therefore, in GFC separation occurs because analytes diffuse in and out of the pores of the stationary phase. Pore size will determine whether an analyte will be included or excluded from the media, and ultimately will determine its elution volume or its retention time.
Non-Adsorptive Technique: Analytes will elute according to their MW only if the separation is under ideal SEC conditions, which is more often not the case. Therefore, the most important requirement is that analytes should NOT interact with the stationary phase.
Consider this entropy equation, it’s well known that most chromatographic separations can be explained by this equation for equilibrium. However, in SEC which is a non-adsorptive chromatography, the separation should be driven by ENTROPY- in which case, delta H or enthalpy, is zero. This is very critical since we need to minimize the protein adsorption on stationary phase, and this is done through optimization of mobile phase.
Van Deemter Equation: Now with the Van Deemter equation, because the SEC separation is entropy driven, diffusion plays a significant role in chromatographic separation specifically in the “C-term.” The larger the analyte, the more pronounced will be the “drop off”, of efficiency especially at higher linear velocities. One way to mitigate this effect is to minimize the “A-term” so smaller particle sizes can increase efficiency.
Another thing to point out is, that at very low linear velocities, efficiency is much higher in SEC which is especially true for larger analytes which have a higher diffusion coefficient.
Stationary Phase: Fully porous silica based stationary phases are most commonly used which can be of various pore sizes suitable for different MW range of proteins. Although the modern, highly pure silica-based phases are made extremely inert but silica is inherently acidic in nature and the free silanol groups can lead to some degree of chemical interaction between the proteins and stationary phase. For this reason, sometimes polymeric stationary phases are used, or the better way is to optimize the mobile phase conditions to reduce these secondary interactions.
Effect of hardware: Another challenge is the adsorption of proteins on the column hardware which inadvertently leads to irreproducible results. The age-old solution to this was considerable priming of the columns, however Phenomenex has an easy solution to this issue – the BioTi™ hardware. Extremely inert titanium lined stainless steel hardware called as BioTi™ significantly reduces the priming time for SEC columns and will provide reproducible results almost from the 1st injection.
Particle Size: A source of band broadening in SEC is because of the effect of MW on HETP factor. This means that larger biomolecules, because of slower mass transfer will experience more band broadening at higher linear velocities. One way to mitigate this band broadening is to use smaller sub-2 µm particles. Using a smaller particle allows to run higher linear velocities to improve analysis and shorten the run times.
Mobile Phase: Mobile phase in SEC fulfils some very important goals, the significant ones being:
- Keep the proteins in solution.
- Keep the proteins in native state.
- Deactivate the residual active sites on stationary phase.
Like in any other chromatography one of the major roles of SEC mobile phase is to keep the analytes soluble, however the buffers that are typically used in GFC are often selected in order to the keep proteins in their native state if possible. Proteins in their native forms are generally very dense structures due to strong intramolecular interactions, which results in protein folding.
Some of the common denaturing agents and chaotropic salts such as guanidine (GnHCl) and sodium di-lauryl sulphate (SDS) are typically used in the mobile phase to unfold proteins. These chaotropic salts can break the hydrogen bonds between the proteins and unfold them, this can be a reversible process and often improve the accuracy of the SEC results.
Mobile phase buffers not just denature the proteins, but also help to neutralize the active sites present on either the analyte and/or stationary phase. These buffers help to minimize the secondary interactions that may occur between the basic protein functionalities and the acidic surface silanols present on the stationary phase. However, low pH buffering is often avoided as it can result in the complete breakdown of proteins.
Buffers & Salts:
SEC mobile phase buffers can be selected on the basis of Hoffmeister series- this series is typically used in the context of Hydrophobic Interaction Chromatography (HIC) but it can also be used for GFC buffer selection. The fig. shows the mobile phase additives commonly used in Size Exclusion Chromatography methods i.e. K+, Na+ cations and phosphate and sulfate anions which are “Kosmotropic” in nature and encourages hydrophobic interactions.
Phosphate buffer is the most common buffer for GFC, typically used at physiological pH of 7.4, phosphate also tends to stabilize the proteins which is an important requirement with aggregate analysis. However, when using Phosphate buffer, it’s “Kosmotropic effect” i.e. the disposition to stabilize and “salt out” the protein should always be considered.
Furthermore, the cation used in the cosolvent (e.g. Na+ vs K+) should also be given perceptible consideration. As mentioned in the fig. above and as hypothesized by “Goyon et al at the University of Geneva”, K+ is more effective at intercepting secondary silanol interactions in comparison to more commonly used Na+ ion in the salt form.
Flow Rate: Flow rate can be very effectively utilized as a method optimization parameter in SEC to improve the resolution and over all chromatography. Lower flow rate increases resolution, this can be considered counterintuitive as we have recommended to use smaller particle size to improve the “H Term” in Van Deemter Equation. However, recall in SEC the separation is driven by entropy and diffusion in an isocratic, non-adsorptive method, is very pronounced, therefore the slower flow-rates allow proteins to ingress and egress in and out of the pores more effectively and thus improves the resolution.
Organic Solvents: Organic Solvents like ACN, Methanol and IPA can be added in mobile phase as organic modifiers to improve the recovery and peak shape of biomolecules in GFC. Typically, 10~15% concentration of an organic modifier is enough to improve the chromatography and recovery values, increasing the organic concentration more than 20% can change the native state of the protein and render it biologically inactive. In case of Antibody Drug Conjugates (ADCs) protic solvent like IPA has proven to improve the recoveries and peak shapes of analytes, by forming hydrogen bonding with proteins and simultaneously solvating the hydrophobic regions of the proteins. Compellingly, Methanol does not show this effect due to its less solvation and elution power than IPA. Thus, IPA can be effectively used as an organic modifier to improve the recoveries and peak shapes of ADCs.
Typical values:
Below I have listed some typical ranges for mobile phase parameters, these parameters have a significant effect on the selectivity, peak shape, recovery and resolution of the analytes and should be optimized in the given method.
Conclusion: The important point in size exclusion chromatography separation of antibody aggregate analysis is there is no general rule that can be applied to a category of biomolecules. Antibodies and their aggregates are extremely complex and convoluted analytes and although they may have a very little variation in sequence, even then these small variations have capabilities to cause significant effect on protein folding and thus on the protein structure and its hydrodynamic volume in solution. Therefore, each of these biomolecules should have a method that is customized to give optimized chromatographic results.
Therefore, having an absolute understanding of the factual principles of Size Exclusion Chromatography (SEC) and customizing the stationary phase and mobile phase parameters subject to the complexity and requirements of the antibody aggregate analysis will assist in developing an extremely robust and reproducible method.
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