Epitope Mapping and Binding Assessment by Solid-State NMR Provide a Way for the Development of Biologics under the Quality by Design Paradigm

Abstract

Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.

Solid-state NMR – a complementary technique for protein framework characterization

“…One such method is solid-state NMR (ssNMR), which thanks to on-going experimental advances yields spectra of microcrystalline, sedimented or freeze-dried proteins comparable in quality to solution-state spectra used for structural studies. [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] The sample conditions, in particular the protein hydration state (e.g. rehydration of freeze-dried samples), are crucial for achieving good quality spectra.…”

“…13,14,19 Recently, we showed that solid-state spectral quality is sufficient to ensure resonance assignment, epitope mapping, and the calculation of structural models. 22,27 Here, we present a ssNMR characterization of microcrystalline RSL -sclx 8 precipitates, 11 which form spontaneously at low pH and low salt. Complete solution-state NMR assignments of RSL in the sugar-free (BMRB 25952), D-mannose-(BMRB 25950) or L-fucosebound (BMRB 25951) forms made this protein an ideal candidate.…”

Combining Solid‐State NMR with Structural and Biophysical Techniques to Design Challenging Protein‐Drug Conjugates

“…[14][15][16] The rational design of protein-drug conjugates to maximize effectiveness, pharmacokinetics, and stability in vivo while minimizing their structural complexity is receiving more and more interest, and could benefit from highly accurate structural information from X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. [17][18][19][20][21][22][23][24][25][26] However, proteins that are covalently bound to, or that interact strongly with, relatively large drugs through long linkers can be difficult to crystallize. Furthermore, these systems are too big for NMR spectroscopy in solution, but still neither big nor rigid enough to allow for the use of cryoelectron microscopy.…”

2D NMR Analysis as a Sensitive Tool for Evaluating the Higher-Order Structural Integrity of Monoclonal Antibody against COVID-19

“…The use of nuclear magnetic resonance (NMR) has been suggested as a technology with the potential to more accurately assess the differences between HOS and other well-established methods [ 8 , 9 , 10 , 11 , 12 ].…”