Ranking mAb–excipient interactions in biologics formulations by NMR spectroscopy and computational approaches
ABSTRACT
Excipients are added to biopharmaceutical formulations to enhance protein stability and enable the development of robust formulations with acceptable physicochemical properties, but the mechanism by which they confer stability is not fully understood. Here, we aimed to elucidate the mechanism through direct experimental evidence of the binding affinity of an excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. We ranked a series of excipients with respect to their dissociation constant (KD) and nonspecific binding constants (Ns). In parallel, molecular dynamic and site identification by ligand competitive saturation (SILCS)-Monte Carlo simulations were done to rank the excipient proximity to the proteins, thereby corroborating the ranking by STD NMR. Finally, the excipient ranking by NMR was correlated with mAb conformational and colloidal stability. Our approach can aid excipient selection in biologic formulations by providing insights into mAb–excipient affinities before conventional and time-consuming excipient screening studies are conducted.
Introduction
Therapeutic proteins, if not formulated well, are susceptible to physical (e.g., aggregation) and chemical degradation from small perturbations in their structures that can affect drug potency and lead to immune response in vivo. Citation1,Citation2 Overcoming stability issues and maintaining the stability of therapeutic proteins expected to be commercially viable have been substantial challenges for formulation development.Citation3,Citation4 The addition of excipients to the protein solution is the most commonly used approach to enhance protein stability and stabilize the drug product.Citation5 The most common composition of a biopharmaceutical formulation consists of a buffer (e.g., histidine, phosphate), stabilizer (e.g., carbohydrates, sugars, polyols, amino acids), surfactant (e.g., Polysorbate 20, Polysorbate 80) and antioxidant (e.g., ethylenediaminetetraacetic acid (EDTA); diethylenetriaminepentaacetic acid (DTPA)).Citation6–11 The stability of proteins varies widely, depending on the protein concentration, pH, buffer, buffer concentration, and the type of excipients. Excipient compatibility screening and physicochemical stress studies at accelerated conditions are conducted to finalize the excipient selection and composition.Citation12–15 Lyophilization (freeze-drying) of the protein formulation in selected excipients is also done to further enhance protein stability, especially for formulations with significant stability challenges.Citation16–18
Systematic screening is conducted to choose the right excipients for a given protein, but the mechanisms by which the excipients provide stability to the protein are not fully understood. Understanding why some excipients are better stabilizers of proteins can help with developing robust biopharmaceutical formulations in an accelerated manner. Moreover, having an analytical tool to quantify and rank the factors leading to their stability simplifies the excipient selection process, making it systematic and practical. However, few studies that provide a mechanistic understanding of the stabilizing effect of excipients to maintain protein stability have been published, and no direct evidence for protein–excipient interactions was identified.Citation19–22 Preferential exclusion by carbohydrates is one of the most prevalent mechanisms by which protein can be stabilized, adding beneficial effects on aggregation and the conformational stability of the protein.Citation19–21 Using isothermal titration calorimetry (ITC), Kim et al. identified proteins with a high binding affinity to carbohydrates, probably due to the hydrogen bond formation between the protein-binding sites with the carbohydrate molecules.Citation19 Souillac et al. used Fourier transform infra-red (FTIR) spectroscopic studies to show that, in the presence of carbohydrates, the secondary structure was replenished by hydrogen bonds formed between the polar groups on the surface of the protein and carbohydrate moieties during the lyophilization process.Citation22
The aim of our study is to provide direct experimental evidence of the binding affinity of the excipient to a monoclonal antibody (mAb), using a saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. A series of excipients were ranked with respect to their dissociation constant (KD) and nonspecific binding constants (Ns),Citation23,Citation24 and, in parallel, molecular dynamic (MD) and Monte Carlo (MC) simulations were conducted to rank the excipient proximity to the proteins, thereby corroborating the STD NMR results and ranking. The study also provided insights into potential specific binding sites on proteins and the preferential conformation of the excipient to the protein, especially for sucrose and mannitol. Finally, the excipient ranking by NMR was correlated to mAb stability by comparing both thermal stability (Tm, the mid-point of thermal transition) measurements and colloidal stability (B22, the second osmotic virial coefficient) measurement. Tm represents where the folded and unfolded states of the protein are in equilibrium, and a higher value of Tm indicates a higher conformational stability of the protein, provided by the excipient.Citation25–27 B22 indicates the direction and magnitude of the interaction between two protein molecules in solution in presence of the chosen excipients as measured by static light scattering (SLS), with a positive value indicating net repulsive interactions, and a negative value indicating net attractive interactions. Citation26, Citation27 This approach has aided and accelerated the excipient selection in biologic formulations by providing insights into mAb–excipient affinities before conducting a conventional and time-consuming excipient screening study.
Results
Ranking of excipients by STD NMR
In this study, six excipients (sucrose, trehalose, mannitol, sorbitol, succinic acid, and glycine) were chosen to investigate and compare their binding affinities with a Bristol Myers Squibb mAb (BMSmAb, a type of IgG4). From the basic principles of saturation transfer, STD experiments rely on intermolecular transfer of magnetization from protein (mAb) to its binding ligand molecule (excipient) during a selective saturation time. Ligand (excipient) protons in closer proximity to the protein (mAb) receive higher degrees of saturation, which reflects in greater STD effects and can be used to map the mAb–excipient molecular interaction at atomic resolution and estimate their apparent binding affinities. It can be inferred that the intensity of an STD signal corrected by the excess of ligand (STD amplification factor, STDaf) gives direct information about the concentrations of mAb–excipient complexes in solution. STD NMR results from titration experiments (using a concentration series) can thus be used to derive mAb–excipient binding affinities.24,28 The derived mAb–excipient binding can be specific or nonspecific in nature. For specific interactions, the ligand resides in a specific binding pocket of the protein, and for nonspecific interactions the ligand is most likely distributed on the surface of the protein with no specific conformation.
It is to be noted that the mAb-excipient molecular interactions that are too strong could not be detected by STD. Also, when interactions between excipients and BMSmAb are not strong enough to be maintained in vivo, the addition of excipients is unlikely to affect the biological activity of BMSmAb. The pseudo-2D STD NMR quantitative experiments were used to get specific dissociation constants (KD) and nonspecific binding constants (Ns) of the chosen excipients of the BMSmAb. The STD NMR experiments were acquired at 283 K and 293 K for each BMSmAb–excipient series. The structures with numbering and 1D NMR spectral proton assignments of each excipient are shown in Figure 1. KD and Ns were determined by monitoring the excipient STD NMR signals as a function of the excipient concentration. The concentration titrations of excipients ranged from 0 mM to 500 mM, which is a large excess compared to a typical excipient concentration in a formulation, which is ~250 mM. The STD amplification factor (STDaf),23 which represents the experimentally observed STD effects, was calculated using Eq (1)
where (I0 Isat) represents the ligand signal intensity in the STD NMR spectrum, I0 is the ligand peak intensity in an offresonance NMR spectrum (reference), and Isat is the ligand peak intensity in an on-resonance NMR spectrum; ½L total ½P total is the ligand excess relative to a fixed and constant protein concentration.
All the STD NMR experiments were collected using a recycle time of 12 s (longer than 5 × T1) and the STDaf was corrected for different T1 relaxation times as proposed by Kemper et al. 24 The STDafs belonging to the same proton were combined first. The experimental buildup curves of STDaf of each proton with respect to ligand concentration were first fitted with the modified Michaelis–Menten equation, which assumes single-site binding and includes a nonspecific binding term in Eq (2)
A 5% permissible error threshold was chosen for 1 H STD NMR and T1 measurement experiments, given the uncertainty of peak position and imperfection of baseline correction. For the fitting, the concentration was cut off at 250 mM since a typical excipient concentration in a biologic formulation is at or below 250 mM due to high viscosity effects at higher concentrations. Moreover, excipient–excipient interactions could dominate excipient–protein interactions at higher concentrations. The viscosity measurement results are shown in Supplementary Table S2.
Among all the excipients, only the experimental data for sucrose is well fitted to Eq (2) at both 283 K and 293 K. The fitted plots for all the protons at both temperatures are shown in Figure 2. The values of KD and Ns are shown in Table 1. KD values of proton H5/H9 at 283 K and proton H5/H9/H17/H19 at 293 K were rejected by P test. Ns values of proton H7/H13 at both 283 K and 293 K were rejected by Q test. The average KD values are 67.83 mM and 88.63 mM at 283 K and 293 K, respectively. As for trehalose, mannitol, sorbitol, succinic acid and glycine, the buildup curves are fitted to Eq (3) and the fitted plots are shown in Figure 3. The values of Ns are shown in Table 2. The proton H4/H12/H17/H19 at 283 K and mannitol H9 at 293 K were rejected by Q test. All outliers were excluded for further investigation.
The nonspecific binding constant (Ns) for all six excipients was determined and ranked in Figure 4A. At 283 K, sucrose has the largest binding constant (0.065 mM−1 in average), trehalose ranks second (0.034 mM−1 in average), and the other four excipients have a similar binding constant (range of 0.010–0.020 mM−1 in average). The ranking is the same at 293 K. We observed that the STD effects are stronger at 283 K than at 293 K for all the excipient protons.
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Chunting Zhang, Steven T. Gossert, Jonathan Williams, Michael Little, Marilia Barros, Barton Dear, Bradley Falk, Ankit D. Kanthe, Robert Garmise, Luciano Mueller, Andrew Ilott & Anuji Abraham (2023) Ranking mAb–excipient interactions in biologics formulations by NMR spectroscopy and computational approaches, mAbs, 15:1, 2212416, DOI:10.1080/19420862.2023.2212416, https://doi.org/10.1080/19420862.2023.2212416
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