The higher order secondary- and tertiary-structure of a protein is critical for its pharmacological activity. When carrying out biosimilar comparability studies, knowledge of this structure is required and must be ascertained through careful evaluation. Several techniques exist to determine higher order protein structure. In a 2011 European conference [2], it was strongly recommended that a minimum of one method be used to assess the integrity of the secondary structure and another to assess tertiary structure.

The authors of the recent GaBIJ paper have investigated all possible methods to determine higher order protein structure via a thorough literature review and a summary is outlined below.

Techniques to determine higher order structure
Classical X-ray diffraction methods can only be used with solid state materials. However, for pharmaceutical products that are commonly produced in solutions, small-angle X-rays scattering (SAXS) can be performed but will only give information on the overall shape of proteins in a solution.

The derivative fast-transform Fourier infrared (FTIR) method is commonly used to determine secondary structure. It can calculate the relative percentage of α-helix, β-sheets in a secondary structure.

Circular dichroism (CD) is commonly used to determine secondary and tertiary structure.

Second derivative UV spectroscopy assesses the integrity of a tertiary structure and determine if alterations have occurred. The method focusses on modifications to the UV spectrum region (from 250 nm to 300 nm), where aromatic amino acids strongly absorb. This is commonly used.

Fluorescence spectroscopy is commonly used to determine tertiary structure.

Raman or nuclear magnetic resonance (NMR) spectroscopy can be used to determine tertiary structure.

A global evaluation of the free energy of the protein can assess its global thermodynamic properties. Generally, proteins exist in their native state under a thermodynamically favoured folded state (lowest free internal energy). Micro differential scanning calorimetry (DSC) is used to find differences in energy of folded and unfolded states that occur during denaturation of the protein. This denaturation process can be followed with the melting point of transition (Tm) determined through DSC, or by the change of the emission fluorescence of the protein if it is in aqueous solution.

Chromatographic methods based on global charge and hydrophobicity of a protein, can be used to detect change in the higher order structure. These include ionic chromatography (IC) and hydrophobic interaction chromatography (HIC). However, the GaBIJ paper authors [1] found no published paper only uses these methods to assess conformational changes for stability studies or to compare a biosimilar to its originator. Thus, it appears that chromatographic methods are only used to complement spectroscopic methods when assessing protein higher order structure. In addition, chromatographic bioassays are used to test the pharmacological activity of a protein. However, their sensitivity will not unambiguously demonstrate the absence of modification of the tertiary structure.

Conclusions
Following extensive literature review, the study concluded that spectroscopic methods remain most used to assess protein’s secondary and tertiary structures. These include: X-ray crystallography, NMR, absorption, fluorescence, CD, DSC. However, IR spectroscopy, CD and fluorescent spectroscopy are the most widespread and convenient for proteins in solution. This is in line with the 2011 recommendations [2] that second derivative FTIR and UV should be used, together with a global evaluation that should be performed through a thermodynamic stability study or methods such as Raman or nuclear magnetic resonance (NMR) spectroscopy. In addition, the authors of this GaBIJ paper stated that chromatographic methods (such as HIC and bioassays) cannot characterize higher order structure for biosimilar comparison exercises and stability studies and should be only used as confirmatory techniques.

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References
1. Astier A. Importance of the determination of the higher order structure in the in-use stability studies of biopharmaceuticals. Generics and Biosimilars Initiative Journal (GaBI Journal). 2020;9(2):49-51. doi:10.5639/gabij.2020.0902.009
2. Bardin C, Astier A, Vulto A, Sewell G, Vigeron J, Trittler R, et al. Guidelines for the practical stability studies of anticancer drugs: a European consensus conference. Ann Pharm Fr. 2011;69(4):221-31.

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