In our blog series we have looked at the idea of orthogonality in various aspects of structural characterization (read more here). We’ve already seen how this approach is a powerful tool for strengthening structural conclusions drawn from individual techniques, as well as being a requirement from the regulatory authorities. One area that we haven’t discussed yet, and that I would like to discuss in this blog, is the assessment of aggregation.
Aggregates are noted as being either product or process related in the ICH Q6B structural characterization guidelines (1) and as such are expected to be assessed. Aggregation is also specifically mentioned in the EMA and FDA guidelines on biosimilar structural characterization along with the idea, in each case, of orthogonal assessment of this structural feature (2,3). In addition, the assessment of degradation profiles (e.g. in Forced Degradation studies) of the product is also detailed and includes the use of aggregation assessments as part of this investigation (2, 3).
So, what is the best way to examine aggregation in a manner that draws on the expectation of orthogonality in this context?
There are several ways that aggregation can be studied, each drawing on a different mechanism of analysis and thus providing orthogonality for one another. What we need to consider though, is the nature of what we are attempting to examine in these types of study. Aggregates (and we are considering only subvisible components here) are delicate non-covalent assemblies of individual protein or glycoprotein units such as dimers, trimers, tetramers or larger. These are not usually the expected assemblies of the product that give activity (Insulin hexamers being a noted exception) but are most frequently impurities within the product.
These aggregates, being non-covalent, have less inherent stability than would be found for covalently linked units. Consequently, their stability may be disrupted to different degrees by the locality they find themselves in i.e. the experimental environment in which they exist (this has echoes of Werner Heisenberg’s quote that “The very act of observing disturbs the system” or what is known in physics as “The Observer Effect”). So, we need to remember that any approach we take will give an answer only within the parameters of the system. Bearing this in mind, at BioPharmaSpec we use the techniques of size exclusion chromatography with multiangle laserlight scattering (SEC-MALS) together with sedimentation velocity-analytical ultracentrifugation (SV-AUC) as our orthogonal methods for aggregation analysis.
SEC-MALS relies on chromatographic separation of the variously sized aggregates with photon scattering detection used as a measurement of aggregate mass and UV and refractive index to measure sample response (and so provide relative amounts of each component). This technique provides aggregate information but it must be borne in mind that shear forces within the column, along with significant sample dilution during passage of the sample can cause disruption to aggregates and lead to lower aggregation values for the sample.
As an orthogonal procedure to SEC-MALS we use SV-AUC, whereby samples are subject to separation via high-speed centrifugation. The generated intense gravitational field causes sequential partitioning and separation of the different sized aggregates. Regular UV measurements of the sample tubes in the rotor are taken and processed through computer algorithms to determine the aggregation profile and provide relative quantitation of the components. The nature of the AUC processing software has evolved over time with new techniques being incorporated to interrogate the data more deeply.
AUC, being a column-free technique, does not subject the sample to the same dispersive forces that SEC-MALS does. Thus AUC analyses tend to result in higher relative percentages of aggregates detected compared to SEC-MALS. The technique of AUC is well considered by regulatory agencies.
The use of SEC-MALS and SV-AUC, with their opposing theories of separation and thus of system disturbance, result in relative abundances of protein monomer and smaller oligomer states that will give upper and lower ranges for the degree of aggregation seen in the sample. There are other techniques available for the analysis of aggregates, such as asymmetric flow field flow fractionation (AF4). Again, these will provide data relative to the system being used and the effect it causes on aggregation. In the case of AF4, there will be forces associated with the channel flow and membrane interactions as well. In our experience, the use of SEC-MALS in conjunction with SV-AUC provides meaningful data for aggregation studies that fulfil the need for orthogonal analysis in this area of structural characterization.
