Quantification of Biopharmaceutically Relevant Nonionic Surfactant Excipients Using Benchtop qNMR.

Nonionic surfactant excipients (NISEs) are commonly added to biologics formulations to mitigate the effects of stress incurred by the active biotherapeutic during manufacturing, transport, and storage. During manufacturing, NISEs are added by dilution of a stock solution directly into a protein formulation, and their accurate addition is critical in maintaining the quality and integrity of the drug product and thus ensuring patient safety. This is especially true for the common NISEs, polysorbates 20 and 80 (PS20 and PS80, respectively) and poloxamer 188 (P188). With the increasing diversity of biologic modalities within modern pharmaceutical pipelines, there is thus a critical need to develop and deploy convenient and user-accessible analytical techniques that can rapidly and reliably quantify these NISEs under biopharmaceutically relevant conditions. We thus pursued 60 MHz benchtop quantitative NMR (qNMR) as a nondestructive and user-friendly analytical technique for the quantification of PS20, PS80, and P188 under such conditions. We demonstrated the ability of benchtop qNMR (1) to quantify simulated PS20, PS80, and P188 stock solutions representative of those used during the drug substance (DS) formulation step in biomanufacturing and (2) to quantify these NISEs at and below their target concentrations (≤0.025% w/v) directly in biologics formulations containing histidine, sucrose, and one of three biotherapeutic modalities (monoclonal antibody, antibody-drug conjugate, and Fc-fusion protein). Our results demonstrate that benchtop qNMR offers a fit-for-purpose, reliable, user-friendly, and green analytical route by which NISE of interest to the biopharmaceutical industry may be readily and reliably quantified. We conclude that benchtop qNMR has the potential to be applied to other excipient formulation components in the presence of various biological modalities as well as the potential for routine integration within analytical and QC laboratories across pharmaceutical development and manufacturing sites.

A Monte Carlo Method for Analyzing Systematic and Random Uncertainty in Quantitative Nuclear Magnetic Resonance Measurements.

Quantitative nuclear magnetic resonance (qNMR) is a powerful analytical technology that is capable of quantifying the concentration of any analyte with exquisite accuracy and precision so long as it contains at least one nonlabile nuclear magnetic resonance (NMR)-active nucleus. Unlike with traditional analytical technologies, the concentrations of analytes do not directly influence the uncertainty in the quantification of NMR signals because an ideal NMR response depends only on the nature and amount of the nucleus being observed. Rather, in the absence of spectral artifacts and under favorable experimental conditions, the measurement uncertainty may be influenced by the following factors: (1) spectroscopic parameters such as the spectral width, number of time domain points, and acquisition time; (2) postacquisition data processing, such as apodization and zero-filling; (3) the signal-to-noise ratios (SNRs) and lineshapes of the two signals being used in a qNMR measurement; and (4) the method of signal quantification employed, such as numerical integration or lineshape fitting (LF). Here, a general Monte Carlo (MC) method that considers these factors is presented, with which the random and systematic contributions to qNMR measurement uncertainty may be calculated. Autocorrelation analysis of synthetic and experimental noise is used in a fingerprint-like approach to demonstrate the validity of the simulations. The MC method allows for a general quantitative assessment of measurement uncertainty without the need to acquire spectral replicates and without reference to the molecular structures and concentrations of analytes. Representative examples of qNMR measurement uncertainty simulations are provided in which the metrological performances of integration and LF are contrasted for signal pairs obtained using various acquisition and processing schemes in the low-SNR regime-an area where application of the proposed MC method may prove to be particularly salient.

Measurement and Characterization of Hydrogen-Deuterium Exchange Chemistry Using Relaxation Dispersion NMR Spectroscopy.

One-dimensional heteronuclear relaxation dispersion NMR spectroscopy at 13C natural abundance successfully characterized the dynamics of the hydrogen-deuterium exchange reaction occurring at the Nε position in l-arginine by monitoring Cδ in varying amounts of D2O. A small equilibrium isotope effect was observed and quantified, corresponding to ΔG = -0.14 kcal mol-1. A bimolecular rate constant of kD = 5.1 × 109 s-1 M-1 was determined from the pH*-dependence of kex (where pH* is the direct electrode reading of pH in 10% D2O and kex is the nuclear spin exchange rate constant), consistent with diffusion-controlled kinetics. The measurement of ΔG serves to bridge the millisecond time scale lifetimes of the detectable positively charged arginine species with the nanosecond time scale lifetime of the nonobservable low-populated neutral arginine intermediate species, thus allowing for characterization of the equilibrium lifetimes of the various arginine species in solution as a function of fractional solvent deuterium content. Despite the system being in fast exchange on the chemical shift time scale, the magnitude of the secondary isotope shift due to the exchange reaction at Nε was accurately measured to be 0.12 ppm directly from curve-fitting D2O-dependent dispersion data collected at a single static field strength. These results indicate that relaxation dispersion NMR spectroscopy is a robust and general method for studying base-catalyzed hydrogen-deuterium exchange chemistry at equilibrium.

Complexity of protein energy landscapes studied by solution NMR relaxation dispersion experiments.

The millisecond time scale motions in ribonuclease A (RNase A) were studied by solution NMR CPMG and off-resonance R1ρ relaxation dispersion experiments over a wide pH and temperature range. These experiments identify three separate protein regions termed Cluster 1, Cluster 2, and R33, whose motions are governed by distinct thermodynamic parameters. Moreover, each of these regions has motions with different pH dependencies. Cluster 1 shows an increase in activation enthalpy and activation entropy as the pH is lowered, whereas Cluster 2 exhibits the opposite behavior. In contrast, the activation enthalpy and entropy of R33 show no pH dependence. Compounding the differences, Δω values for Cluster 2 are characteristic of two-site conformational exchange, yet similar analysis for Cluster 1 indicates that this region of the enzyme exhibits conformational fluctuations between a major conformer and a pH-dependent average of protonated and deprotonated minor conformers.

What's in your buffer? Solute altered millisecond motions detected by solution NMR.

To date, little work has been conducted on the relationship between solute and buffer molecules and conformational exchange motion in enzymes. This study uses solution NMR to examine the effects of phosphate, sulfate, and acetate in comparison to MES- and HEPES-buffered references on the chemical shift perturbation and millisecond, chemical, or conformational exchange motions in the enzyme ribonuclease A (RNase A), triosephosphate isomerase (TIM) and HisF. The results indicate that addition of these solutes has a small effect on (1)H and (15)N chemical shifts for RNase A and TIM but a significant effect for HisF. For RNase A and TIM, Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, however, show significant solute-dependent changes in conformational exchange motions. Some residues show loss of millisecond motions relative to the reference sample upon addition of solute, whereas others experience an enhancement. Comparison of exchange parameters obtained from fits of dispersion data indicates changes in either or both equilibrium populations and chemical shifts between conformations. Furthermore, the exchange kinetics are altered in many cases. The results demonstrate that common solute molecules can alter observed enzyme millisecond motions and play a more active role than what is routinely believed.

Alteration of hydrogen bonding in the vicinity of histidine 48 disrupts millisecond motions in RNase A.

The motion of amino acid residues on the millisecond (ms) time scale is involved in the tight regulation of catalytic function in numerous enzyme systems. Using a combination of mutational, enzymological, and relaxation-compensated (15)N Carr-Purcell-Meiboom-Gill (CPMG) methods, we have previously established the conformational significance of the distant His48 residue and the neighboring loop 1 in RNase A function. These studies suggested that RNase A relies on an intricate network of hydrogen bonding interactions involved in propagating functionally relevant, long-range ms motions to the catalytic site of the enzyme. To further investigate the dynamic importance of this H-bonding network, this study focuses on the individual replacement of Thr17 and Thr82 with alanine, effectively altering the key H-bonding interactions that connect loop 1 and His48 to the rest of the protein. (15)N CPMG dispersion studies, nuclear magnetic resonance (NMR) chemical shift analysis, and NMR line shape analysis of point mutants T17A and T82A demonstrate that the evolutionarily conserved single H-bond linking His48 to Thr82 is essential for propagating ms motions from His48 to the active site of RNase A on the time scale of catalytic turnover, whereas the T17A mutation increases the off rate and conformational exchange motions in loop 1. Accumulating evidence from our mutational studies indicates that residues experiencing conformational exchange in RNase A can be grouped into two separate clusters displaying distinct dynamical features, which appear to be independently affected by mutation. Overall, this study illuminates how tightly controlled and finely tuned ms motions are in RNase A, suggesting that designed modulation of protein motions may be possible.