NMR spectroscopic Higher-Order Structure Analysis of Biosimilars

This article discusses the power of NMR spectroscopy in comparative higher-order structure analysis (HOS) of biosimilars. As noted in the previous post on this subject, biologics are much more complex than small-molecule drugs. They are produced in living cells and may undergo uncontrolled post-translational modifications (PTMs) and glycosylation, which can affect the drug’s three-dimensional structure and modify its biological potency. Other factors, like changes in pH, ionic strength, buffers, or the presence of various additives in the formulation solution, may also affect the structure. All these factors must be considered when developing biosimilar biologics, the follow-on versions of a therapeutic monoclonal antibody (once the original drug’s patent protection has expired). The costs of biosimilars, like those of generic drugs, are expected to be lower, making them more affordable in clinics.

Assessing Biosimilar’s Comparability

Comparability assessment of biosimilars must involve physicochemical and functional characterization of the molecules in solution. Additional studies must include animal toxicity, human pharmacokinetics/ pharmacodynamics, immunogenicity, clinical safety, and effectiveness. When considering physicochemical quality attributes, the regulatory requirements list three essential factors: post-translational modifications (PTMs), higher-order structure (i.e., secondary, tertiary, and quaternary structure) comparability, and protein aggregation.

While techniques for detecting, characterizing, and quantifying PTMs and aggregates are well established and widely applied in antibody characterization, higher-order structure analysis to assess differences between the biosimilar and its originator that may arise from PTMs, glycosylation, or other factors remains challenging. X-ray crystallography, cryo-electron microscopy (cryo-EM), and solution NMR spectroscopy are the gold-standard techniques for high-resolution 3D structure determination of proteins. However, opening a well-cited review by Berkowitz et. al., Nat Rev Drug Disc, 2012, we can read that “…the application of these technologies (crystallography and NMR spectroscopy) for higher-order structure studies presents major problems”. Eight years later, in a 2020 paper by A. Asrier in Generic & Biosimilars J, we can still read that “Classical X-ray diffraction can only be used with solid-state materials” and it is “not suitable for pharmaceutical products which are commonly produced in solution”!

3D Structure in Antibody Studies

What about the 10000+ antibody and antibody-antigen complex higher-order structures in the PDB or SabDab database? Let us also imagine how much we would know about antibodies without access to their X-ray structures or where small-molecule drug discovery would be today without NMR spectroscopy or X-ray crystallography! Not surprisingly, NMR is also rejected as a method for comparability studies. This is despite the developments that have shown that NMR fingerprinting techniques based on natural abundance can be used time-efficiently for the analysis of antibody higher-order structure (Poppe et al., 2013; Poppe et al., 2015). In addition, the feasibility of NMR-based mapping of monoclonal antibody structure using a 2D ¹³C NMR methyl fingerprint method was also shown by Arbogast et al., 2014. The authors showed that a single 2D-NMR spectroscopy experiment, acquired in approximately 30 min, can yield a spectral map that provides an atomic-level fingerprint of a protein therapeutic’s higher-order structure: its secondary, tertiary, and quaternary structures. The fingerprints of the reference antibody and its biosimilar higher-order structures are subsequently compared to assess the degree of similarity.

Experimental Techniques for Higher-Order Structure Analysis

What are the standard techniques currently recommended for biosimilar comparative higher-order structure analysis? The authors of the above-cited 2020 review in Generic & Biosimilars Journal refer to the 2011 ‘Guidelines for the practical stability studies of anticancer drugs: A European consensus conference’ in which it is recommended to “use at a minimum one method to assess the integrity of the secondary structure and another to assess tertiary structure”. And “specifically, second derivative FTIR (Fourier-transform infrared) and UV are recommended, together with a global evaluation that should be performed through a thermodynamic stability study”. While FTIR is recommended for assessing secondary structure, UV spectroscopy (specifically, tryptophan fluorescence) is recommended for evaluating tertiary structure integrity.

Without going into the details of these methods and the recent history of the development of solution NMR spectroscopy methods, we jump to a couple of papers, the first one from 2020 with the title “A Comparison Between Emerging and Current Biophysical Methods for the Assessment of Higher-Order Structure of Biopharmaceuticals” published in J Pharm Sc, 2020, by Mats Wikström and colleagues at Amgen, California. The authors used a set of monoclonal antibody samples belonging to the IgG1 and IgG2 subclasses, mixed in different proportions, to assess the methods’ performance in detecting the two IgGs. They note that the amino acid sequence identity between the two samples was 95%, meaning that a mixture containing, e.g., 90% IgG1 and 10% IgG2 would correspond to only minor differences in overall structural properties. This suggests that, if the differences are still detectable in this sample, the setup is appropriate for evaluating the experimental method’s ability to differentiate.

NMR Superiority for Comparative Higher-Order Structure Analysis

The authors compared the commonly recommended higher-order structure comparability analysis methods, FTIR, circular dichroism (CD), UV fluorescence spectroscopy, and differential scanning calorimetry (DSC), to 1D 1H Profile NMR spectroscopy. They show that NMR can distinguish between most sample combinations (93%), DSC can differentiate 61%, and CD spectroscopy can differentiate 52% of the combinations. In contrast, no significant distinction between FTIR and intrinsic fluorescence samples could be detected. 

The results speak for themselves; the ability of NMR structure analysis to distinguish between samples was proved far superior to all other methods, especially the spectroscopic methods, which are essentially useless in this application. Another study (Brinson et al., 2019) involved 26 industrial CROs, government, and academic laboratories. It aimed to establish benchmarks for adopting the 2D NMR technique under controlled sample conditions to enable accurate measurements and comparability assessment of biosimilar higher-order structure. The experimental results demonstrated that the method is reliable and can be harmonized for routine measurements, providing highly accurate higher-order structure analysis of monoclonal antibody therapeutics.

NMR Spectroscopy Services at SARomics Biostructures

SARomics Biostructures offers NMR-based comparative analysis of higher-order structures. The details can be found on our antibody structure and epitope mapping services and protein NMR spectroscopy services pages. We use the 2D ¹³C NMR method described above. The fingerprint spectra of the methyl groups of the originator antibody and its biosimilar are registered and a analysed, revealing any differences directly between the two. A significant advantage is that no extra labeling is required for these experiments, meaning no additional material needs to be produced. Molecules as large as whole antibodies can be analyzed using this approach.

Additionally, NMR higher-order structure analysis can be used for formulation optimization and batch comparison. In such cases, the biomolecules can be studied in the actual formulation buffer. Depending on the biomolecule, around 1-2 mg of protein can be sufficient for HOS analysis.