Submitted: 7 February 2020; Revised: 7 March 2020; Accepted: 12 March 2020; Published online first: 25 March 2020
The higher order secondary-and tertiary-structure of a protein is critical for its pharmacological activity. Knowledge of this structure is required when carrying out biosimilar comparability studies, when ascertaining the integrity of a protein’s activity after stress, and when determining the extent of possible chemical degradation such as deamidation and extensive evaluation of aggregation. For such studies to be well designed, they must include a careful evaluation of the higher order structure which can be performed by several techniques. Currently, structural analysis is mainly based on spectroscopic methods. Classical X-ray diffraction methods can only be used with solid state materials. However, pharmaceutical products are commonly produced in solutions. Although Small-Angle X-Rays Scattering (SAXS) can be performed on solutions, this method only gives information on the overall shape of proteins in a solution.
In practice, the most commonly used methods to determine higher order protein structure are derivative Fast-transform Fourier Infrared (FTIR) for secondary structure, circular dichroism (CD) for secondary and tertiary structure, derivative UV spectroscopy and fluorescence for tertiary structure. These methods have been frequently applied to assess higher order structure modification in numerous conditions known to destabilize a protein [1–7]. The 2011 ‘Guidelines for the practical stability studies of anticancer drugs: a European consensus conference’ strongly recommends using at the minimum one method to assess the integrity of the secondary structure and another to assess tertiary structure . Specifically, second derivative FTIR and UV are recommended, together with a global evaluation that should be performed through a thermodynamic stability study. However, alternative methods are also accepted, such as Raman or nuclear magnetic resonance (NMR) spectroscopy.
For example, the derivative FTIR method can calculate the relative percentage of α-helix, β-sheets in a secondary structure as has been demonstrated in several published papers such as Sonveaux et al. . Here, the authors studied the secondary structure of P-glycoprotein in the absence and presence of substrate and were able to show ±2% of secondary sub-structures formed after binding. These included α-helix, β-sheets, U-turn or were random.
Second derivative UV spectroscopy is also useful to assess the integrity of a tertiary structure. The method focusses on modifications to the UV spectrum region (from 250 nm to 300 nm), where aromatic amino acids (tyrosine, phenylalanine and tryptophan) strongly absorb . These amino acids are highly hydrophobic and contribute to the formation of internal pockets by hydrophobic interactions. Destabilization of the tertiary structure may destroy these interactions and thus change the environment of the corresponding amino acids, inducing modifications in the absorption of the protein in this spectral region (red shifts). Second derivatives of the spectra permit determination of potential alterations .
A global evaluation of the free energy of the protein is also necessary to assess its global thermodynamic properties. In general, proteins exist in their native state under a folded conformation which is thermodynamically favoured (lowest free internal energy). Following stress, stabilizing internal bonds that maintain the 3D-structure (hydrophobic, hydrogen, ionic bonds) can be broken, inducing a series of unfolded states of higher energies, see Figure 1. This transition can be reversible (equilibrium) or irreversible. The energy required to totally unfold a protein (transition folded/unfolded or also called denaturation) is the activation energy Ea or ΔG ‡.. The energy difference between the native to the unfolded state is ΔG ‡, the variation of Gibbs free energy, expressed in Kcal/mole, see Figure 2. This value can be directly obtained by micro differential scanning calorimetry (DSC), which also permits determination of the melting point of transition Tm which corresponds to a ratio unfolded/folded of 0.5 . Higher Tm indicates a more stable protein. Thus, the determination of Tm before and after a stress, enables visualization of protein destabilization as its Tm will be decreased compared to the initial state. It is easier to plot the ratio unfolded/folded as a function of the temperature (thermal denaturation curve) or the concentration of a denaturing chemical, such as guanidine hydrochloride or urea (isothermal chemical denaturation curve) , see Figure 2. The denaturation process can be easily followed by the change of the emission fluorescence of the protein which shifts from the maximum of 335 nm (0% unfolded) to 352 nm (100% unfolded), see Figure 3. In aqueous solution, hydrophobic amino acids such as tryptophan are hidden inside the protein structure forming hydrophobic pockets with a conformation presenting the hydrophilic domains in the outside shell, directly in contact with the water and ions. If the high order structure is modified by a stress, i.e. destabilized, the hydrophobic environment of the tryptophan will be modified, exposing it to the protein outside. The native fluorescence of tryptophan being very dependent on its hydrophobic environment, permits determination of the degree of structural perturbation of the protein and calculation of the corresponding Tm, see Figure 3. However, the comparison at a single temperature of the fluorescence spectra before and after a stress leads only to verification of whether the tertiary structure has been altered or not.
The global charge and hydrophobicity of a protein will depend on its higher order structure. Thus, some authors consider that chromatographic methods based on these properties, such as ionic chromatography (IC) or hydrophobic interaction chromatography (HIC), can be used to detect change in the higher order structure. Indeed, modification of tertiary structure, such as denaturation that may expose hidden hydrophobic residues, could be theoretically detected by this method. However, no published paper uses only these methods to assess conformational changes for stability studies or to compare a biosimilar to its originator. Moreover, IC profiles can be changed by chemical modifications of a protein such as deamidation (it is the mean goal to use this method in comparability exercises to assess the distribution of ionic variants of a protein and for stability studies), but without clear conformational modification and vice versa. Finally, a few older published papers have proposed using chromatographic methods to study higher order structure . Moreover, in the last decade there have been dramatic improvements of methods such as microfluidic modulation spectroscopy, and of the mathematical treatment of analytical data by powerful informatics, that permits very accurate detection of subtle structural changes [15–17]. Thus, it appears that chromatographic methods are only complementary to the spectroscopic methods to assess higher order structure of a protein and must prove their stability indicating capacities in this domain. Moreover, in all recent papers devoted to compare biosimilars to originators, only spectroscopic methods and DSC were used to study higher order structure [18–25]. Bioassays that test the pharmacological activity of a protein cannot unambiguously demonstrate the absence of modification of the tertiary structure even if it is obvious that the biological activity is directly linked to the tertiary structure. However, the intrinsic variability of any biological method cannot assess a small variability of a tertiary structure. Indeed, the ‘˜Yellow book’ published by UK’s National Health Service (NHS) about the in-use stability of biopharmaceuticals noted, ‘Due to inherent variability in cellular responses a pragmatic view has to be taken on reproducibility. Nevertheless, the cell-based assay selected should allow for an acceptable level of reproducibility’ . Moreover, if an alteration of the tertiary structure happens in a protein domain which is not implicated in the pharmacological activity of a protein, the determination of this activity will not detect it.
As an example, using the proliferation inhibition on BT-474 cells overexpressing human epidermal growth factor receptor 2 (HER2) antigen to demonstrate that a biosimilar of trastuzumab remained unaltered after stress conditions is not relevant . This test only determines the direct cytotoxicity of biosimilar trastuzumab on cells expressing HER2 but does not explore the antibody-dependent cell-mediated cytotoxicity (ADCC) which is also a part of the total anticancer action of this monoclonal antibody (mAb) . Indeed, if a modification of the tertiary structure implies solely the fragment crystallizable region (Fc) domain of the mAb, it is unlikely that a corresponding alteration can be detected by the direct cytotoxicity test which implicates only the fixation of the antigen binding fragment (Fab) on its target. Consequently, this test is not enough to claim that the tertiary structure was unaltered. This kind of test would have only been acceptable as complementary proof if another orthogonal spectroscopic method (fluorescence, derivative UV and CD) showed no sign of modification.
In conclusion, spectroscopic methods remain commonly used to assess protein’s secondary and tertiary structures. Numerous techniques are available, such as X-ray crystallography, NMR, absorption, fluorescence, CD, DSC. However, IR spectroscopy, CD and fluorescent spectroscopy are the most widespread techniques and the most convenient for proteins in solution. 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.
Competing interests: None.
Provenance and peer review: Commissioned; externally peer reviewed.
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Author: Professor Alain Astier, PharmD, PhD, Honorary Head of the Pharmacy Department, Henri Mondor University Hospital, Créteil, France; President, Biotopic Pharmaceuticals, Paris, France
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