As part of the requirements for full structural characterization of a biopharmaceutical, we are required to investigate the higher order structure (HOS) of the product to get information on the 3-dimensional shape of the molecule. This therefore begs the question: what is the best way to do this and how can we interrogate structure to obtain as full a knowledge as possible?
What constitutes Higher Order Structure?
First of all, it’s important to recognize that the higher order structures of molecules can be composed of several different types of spatial features. These form as a direct result of the primary amino acid sequence and how the constituent amino acid side-chains interact with each other in the solution environment to produce the final 3-dimensional shape.
At the secondary structural level, ordered structural features such as alpha helices or beta sheet structures can be produced, depending on the amino acid sequence, but regions of more random-type structure can also exist. How these secondary structural units interact with one another and assemble in three dimensions gives rise to the tertiary level of molecular structure. Our investigations are therefore not based on the assessment of a single higher order structural feature, but rather on the presence of multiple features that will be present in varying relative levels depending on the nature of the primary amino acid sequence, as well as how the molecule folds to achieve the precise energy state it ultimately adopts. We will only be considering secondary and tertiary structure in this blog. The orthogonal assessment of multimeric assemblies and aggregates has been considered in this previous blog.
What tools do we have to investigate HOS?
How do we investigate a system where a range of HOS features are present and abundances can vary? Not surprisingly the approach that should be taken is one of orthogonality, where different analytical techniques are applied and data brought together in a comparative sense (when considering biosimilarity, for example). The concept of building orthogonality into structural characterization has been covered in a separate blog (Part 1 here and Part 2 here) but it is worth expanding on how this can be specifically applied to HOS analysis.
The idea and application of orthogonality in structural characterization is very highly regarded and encouraged by the regulatory agencies. Thus, it naturally follows that orthogonality in HOS investigations is important for the same reasons. The application of an orthogonal approach to HOS analysis means that data will be derived from various techniques, each of which has its own relative strengths for assessing specific structural features. So, in order to get the best and most meaningful data, different structural techniques need to be employed, thus playing to the strengths of each technique.
At BioPharmaSpec, we employ the techniques of Circular Dichroism (CD), Fourier Transform Infra-Red spectroscopy (FT-IR), fluorescence analysis (both intrinsic and extrinsic) and Nuclear Magnetic Resonance (NMR, 1D and 2D) for HOS analysis at both the secondary and tertiary levels. This allows us the use of techniques with particular sensitivities for the secondary level structures of alpha helices (i.e. CD) or beta sheet (i.e. FT-IR) as well as the ability to assess, in fine detail, the spatial distributions and chemical environments of amino acids within the structure as a whole, through the use of NMR. Intrinsic and extrinsic fluorescence are also often used to investigate surface profiles of proteins through inherent fluorescence and fluorophore interactions.