Ranking mAb-excipient interactions in biologics formulations by NMR spectroscopy and computational approaches.

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.

New Development in Understanding Drug-Polymer Interactions in Pharmaceutical Amorphous Solid Dispersions from Solid-State Nuclear Magnetic Resonance.

Pharmaceutical amorphous solid dispersions (ASDs) represent a widely used technology to increase the bioavailability of active pharmaceutical ingredients (APIs). ASDs are based on an amorphous API dispersed in a polymer, and their stability is driven by the presence of strong intermolecular interactions between these two species (e.g., hydrogen bond, electrostatic interactions, etc.). The understanding of these interactions at the atomic level is therefore crucial, and solid-state nuclear magnetic resonance (NMR) has demonstrated itself as a very powerful technique for probing API-polymer interactions. Other reviews have also reported exciting approaches to study the structures and dynamic properties of ASDs and largely focused on the study of API-polymer miscibility and on the identification of API-polymer interactions. Considering the increased use of NMR in the field, the aim of this Review is to specifically highlight recent experimental strategies used to identify API-polymer interactions and report promising recent examples using one-dimensional (1D) and two-dimensional (2D) experiments by exploiting the following emerging approaches of very-high magnetic field and ultrafast magic angle spinning (MAS). A range of different ASDs spanning APIs and polymers with varied structural motifs is targeted to illustrate new ways to understand the mechanism of stability of ASDs to enable the design of new dispersions.

Drug-Polymer Interactions in Acetaminophen/Hydroxypropylmethylcellulose Acetyl Succinate Amorphous Solid Dispersions Revealed by Multidimensional Multinuclear Solid-State NMR Spectroscopy.

The bioavailability of insoluble crystalline active pharmaceutical ingredients (APIs) can be enhanced by formulation as amorphous solid dispersions (ASDs). One of the key factors of ASD stabilization is the formation of drug-polymer interactions at the molecular level. Here, we used a range of multidimensional and multinuclear nuclear magnetic resonance (NMR) experiments to identify these interactions in amorphous acetaminophen (paracetamol)/hydroxypropylmethylcellulose acetyl succinate (HPMC-AS) ASDs at various drug loadings. At low drug loading (<20 wt %), we showed that 1H-13C through-space heteronuclear correlation experiments identify proximity between aromatic protons in acetaminophen with cellulose backbone protons in HPMC-AS. We also show that 14N-1H heteronuclear multiple quantum coherence (HMQC) experiments are a powerful approach in probing spatial interactions in amorphous materials and establish the presence of hydrogen bonds (H-bond) between the amide nitrogen of acetaminophen with the cellulose ring methyl protons in these ASDs. In contrast, at higher drug loading (40 wt %), no acetaminophen/HPMC-AS spatial proximity was identified and domains of recrystallization of amorphous acetaminophen into its crystalline form I, the most thermodynamically stable polymorph, and form II are identified. These results provide atomic scale understanding of the interactions in the acetaminophen/HPMC-AS ASD occurring via H-bond interactions.

Analysis of biopharmaceutical formulations by Time Domain Nuclear Magnetic Resonance (TD-NMR) spectroscopy: A potential method for detection of counterfeit biologic pharmaceuticals.

1H Time-Domain Nuclear Magnetic Resonance (TD-NMR) is used to characterize solutions of antibodies that simulate biologic pharmaceutical formulations. The results from these measurements are compared with those from solutions in which the concentration or identity of the antibody has been altered. TD-NMR is shown to be very sensitive to differences in the amount of antibody in solution, with the ability to detect variations in as low as 2 mg/mL. It is therefore capable, by comparison with data from known formulations, of determining whether a particular sample is likely to be of an authentic biologic formulation. This method expands on the previous use of HPLC, UV/VIS, Near-IR and High-Resolution NMR to detect adulterated pharmaceutical materials. While the sensitivity of the method is high, it is a fingerprinting methodology, illustrating differences but not elucidating their origin. The extracted relaxation times reflect the combined effect of all solutes (antibody, buffer components, etc.) on the solvent (water).

Effect of coating excipients on chemical stability of active coated tablets.

The objective of this study was to understand the impact of coating excipients on the chemical stability of active pan coated peliglitazar, which was prone to acid as well as base-catalyzed degradation. Four different coating formulations containing either polyvinyl alcohol (PVA) or hydroxypropyl methylcellulose (HPMC) as a coating polymer and triacetin (glycerol triacetate) or polyethylene glycol (PEG) as a plasticizer/detackifier were used for coating of peliglitazar in a perforated pan coater. Tablets of one-milligram strength were manufactured by suspending the drug in the coating suspension and spray coating onto inert core tablets. The active coated tablets were placed on stability (40 °C/75% RH) in high-density polyethylene (HDPE) bottles in closed condition with desiccants or in open condition. Tablet samples were withdrawn and analyzed for degradants using a stability-indicating HPLC method. The overall stability for the film-forming polymer-plasticizer/detackifier combination showed the rank order: HPMC-triacetin > PVA-triacetin > HPMC-PEG > PVA-PEG. Higher stability of triacetin systems over PEG systems was attributed to lower solubility of peliglitazar in triacetin coating systems. For the same plasticizer/detackifier, higher stability of HPMC over PVA-based formulations was attributed to lower solubility and mobility of peliglitazar in HPMC compared with the PVA-based coating.

Fast understanding of phases and phase separation in liquid crystal drug delivery systems using deuterium solid-state NMR.

Characterization of lipid based (SPC/GDO/H2O) liquid crystal (LC) drug delivery system is non-trivial and highly complex, especially when multiple and intermediate phases are present. The phase behavior of such mixtures during hydration or delivery is still poorly understood and therefore, characterizing these systems is crucially important towards controlling their function and enhancing the understanding of their drug release behavior. Current work has established an easy way to identify liquid crystal phases and phase mixtures using deuterium (2H) solid-state nuclear magnetic (NMR) spectroscopy under static conditions without disrupting the three dimensional structure and phases, as magic-angle spinning (MAS) could lead to disruption of the phases. Small angle X-ray scattering (SAXS) technique and optical microscopy were also employed to corroborate the study.

Solid state nuclear magnetic resonance studies of hydroxypropylmethylcellulose acetyl succinate polymer, a useful carrier in pharmaceutical solid dispersions.

Hydroxypropylmethylcellulose (HPMC) acetyl succinate (HPMC-AS) is a key polymer used for the enablement of amorphous solid dispersions (ASDs) in oral solid dosage forms. Choice of the appropriate grade within the material is often made empirically by the manufacturer of small-scale formulations, followed by extensive real time stability. A key factor in understanding and predicting the performance of an ASD is related to the presence of hydrogen (or other) bonds between the polymer and active pharmaceutical ingredient (API), which will increase stability over the parameters captured by miscibility and predicted by the Gordon-Taylor equation. Solid state nuclear magnetic resonance (NMR) is particularly well equipped to probe spatial proximities, for example, between polymer and API; however, in the case of HPMC-AS, these interactions have been sometimes difficult to identity as the carbon-13 NMR spectra assignment is yet to be firmly established. Using feedstock, selectively substituted HPMC polymers, and NMR editing experiments, we propose here a comprehensive understanding of the chemical structure of HPMC-AS and a definitive spectral assignment of the 13 C NMR spectra of this polymer. The NMR data also capture the molar ratios of the acetate and succinate moieties present in HPMC-AS of various grades without the need for post treatment required by chromatography methods commonly use in pharmacopoeia. This knowledge will allow the prediction and measurement of interactions between polymers and APIs and therefore a rational choice of polymer grade to enhance the solid state stability of ASDs.

A nondestructive and noninvasive method to determine water content in lyophilized proteins using low-field time-domain NMR.

Determining the moisture content in lyophilized solids is a fundamental step towards predicting the quality and stability of lyophilized products, but conventional methods are time-consuming, invasive, and destructive. High levels of residual moisture in a lyophilized product can lead to cake collapse, product degradation, and reduced shelf life. The aim of this study was to develop a fast, noninvasive, nondestructive, and inexpensive method for determining the moisture content in a lyophilized monoclonal antibody (mAb) formulation using benchtop low-field time-domain nuclear magnetic resonance spectroscopy.

Correlation of Phosphorus Cross-Linking to Hydration Rates in Sodium Starch Glycolate Tablet Disintegrants Using MRI.

Understanding the behavior of tablet disintegrants is valuable in the development of pharmaceutical solid dosage formulations. In this study, high-resolution magnetic resonance imaging has been used to understand the hydration behavior of a series of commercial sodium starch glycolate (SSG) samples, providing robust estimates of tablet disintegration rate that could be correlated with physicochemical properties of the SSGs, such as the extent of phosphorus (P) cross-linking as obtained from infra-red spectroscopy. Furthermore, elemental analysis together with powder X-ray diffraction has been used to quantify the presence of carboxymethyl groups and salt impurities, which also contribute to the disintegration behavior. The utility of Fast Low Angle SHot magnetic resonance imaging has been demonstrated as an approach to rapidly acquire approximations of the volume of a disintegrating tablet and, together with a robust voxel analysis routine, extract tablet disintegration rates. In this manner, a complete characterization of a series of SSG grades from different sources has been performed, showing the variability in their physicochemical properties and demonstrating a correlation between their disintegration rates and intrinsic characteristics. The insights obtained will be a valuable aid in the choice of disintegrant source as well as in managing SSG variability to ensure robustness of drug products containing SSG.

Understanding API-polymer proximities in amorphous stabilized composite drug products using fluorine-carbon 2D HETCOR solid-state NMR.

A simple and robust method for obtaining fluorine-carbon proximities was established using a (19)F-(13)C heteronuclear correlation (HETCOR) two-dimensional (2D) solid-state nuclear magnetic resonance (ssNMR) experiment under magic-angle spinning (MAS). The method was applied to study a crystalline active pharmaceutical ingredient (API), avagacestat, containing two types of fluorine atoms and its API-polymer composite drug product. These results provide insight into the molecular structure, aid with assigning the carbon resonances, and probe API-polymer proximities in amorphous spray dried dispersions (SDD). This method has an advantage over the commonly used (1)H-(13)C HETCOR because of the large chemical shift dispersion in the fluorine dimension. In the present study, fluorine-carbon distances up to 8 Å were probed, giving insight into the API structure, crystal packing, and assignments. Most importantly, the study demonstrates a method for probing an intimate molecular level contact between an amorphous API and a polymer in an SDD, giving insights into molecular association and understanding of the role of the polymer in API stability (such as recrystallization, degradation, etc.) in such novel composite drug products.

(1)H MAS NMR study of cysteine-coated gold nanoparticles.

(1)H MAS NMR experiments were performed on gold nanoparticles coated with l-cysteine. The experiments show that l-cysteine molecules are zwitterions and support a structural model of cysteine forming two layers. The inner layer is composed of cysteine molecules chemisorbed to the gold surface via the sulfur atom. The outer layer interacts with the chemisorbed layer. The (1)H NMR suggests that the cysteine in the outer layer exhibits large amplitude motion about specific carbon-carbon bonds.

A multi-technique approach to the study of structural stability and desolvation of two unusual channel hydrate solvates of finasteride.

The dehydration/desolvation of two hydrate solvates of the pharmaceutically important compound finasteride (namely, bisfinasteride monohydrate monotetrahydrofuran and bisfinasteride monohydrate mono-1,4-dioxane) has been studied by solid-state nuclear magnetic resonance, powder X-ray diffraction, thermogravimetric analysis (including coupling with mass spectrometry) and dynamic vapour sorption. The structure is unusual in that water holds the host finasteride molecules together by hydrogen bonding to form channels in which the solvent is sited. Whilst the solvent guest molecules are not strongly bound to the host, their presence is essential for structural stability. Desolvation is not found to occur at a well-defined temperature or even to consistently produce the same anhydrous form (form I vs. form II), but is instead highly dependent on the physical environment and, therefore, on the technique used. This behaviour complicates investigations, but the combination of complementary methods does allow the desolvation to be understood. Water and solvent are shown to be lost simultaneously, with no evidence of an intermediate form or increased mobility of the hydrogen-bonded water molecules. The results are consistent with a model in which structural collapse and rearrangement follows the loss of a small fraction of the solvent molecules from the channel structure, with the final form produced being very sensitive to the presence of water vapour during desolvation.

Line-narrowing in proton-detected nitrogen-14 NMR.

In solids spinning at the magic angle, the indirect detection of single-quantum (SQ) and double-quantum (DQ) (14)N spectra (I=1) via spy nuclei S=1/2 such as protons can be achieved in the manner of heteronuclear single- or multiple-quantum correlation (HSQC or HMQC) spectroscopy. The HMQC method relies on the excitation of two-spin coherences of the type T(11)(I)T(11)(S) and T(21)(I)T(11)(S) at the beginning of the evolution interval t(1). The spectra obtained by Fourier transformation from t(1) to omega(1) may be broadened by the homogenous decay of the transverse terms of the spy nuclei S. This broadening is mostly due to homonuclear dipolar S-S' interactions between the proton spy nuclei. In this work we have investigated the possibility of inserting rotor-synchronized symmetry-based C or R sequences and decoupling schemes such as Phase-Modulated Lee-Goldburg (PMLG) sequences in the evolution period. These schemes reduce the homonuclear proton-proton interactions and lead to an enhancement of the resolution of both SQ and DQ proton-detected (14)N HMQC spectra. In addition, we have investigated the combination of HSQC with symmetry-based sequences and PMLG and shown that the highest resolution in the (14)N dimension is achieved by using HSQC in combination with symmetry-based sequences of the R-type. We show improvements in resolution in samples of l-alanine and the tripeptide ala-ala-gly (AAG). In particular, for l-alanine the width of the (14)N SQ peak is reduced from 2 to 1.2 kHz, in agreement with simulations. We report accurate measurements of quadrupolar coupling constants and asymmetry parameters for amide (14)N in AAG peptide bonds.

TRAPDOR double-resonance and high-resolution MAS NMR for structural and template studies in zeolite ZSM-5.

A combination of (27)Al magic-angle spinning (MAS)/multiple-quantum (MQ) MAS, and (27)Al-{(14)N} TRAnsfer of Population in DOuble-Resonance (TRAPDOR) nuclear magnetic resonance (NMR) was used to study aluminium environments in zeolite ZSM-5. (27)Al-{(14)N} TRAPDOR experiments, in combination with (14)N NMR were employed to show that the two tetrahedral peaks observed in the (27)Al MAS/3Q-MAS spectra of as-synthesized ZSM-5 are due to aluminium atoms occupying crystallographically inequivalent T-sites. A (13)C-{(27)Al} TRAPDOR experiment was used to study the template, tetrapropyl ammonium bromide (TPABr), in the three-dimensional pore system of ZSM-5. The inequivalency of the methyl groups of TPA was observed in the (13)C-{(27)Al} TRAPDOR spectra of as-synthesized ZSM-5 and the motion of the methyl end of the propyl chain appeared to be more restricted in the sinusoidal channel than in the straight channel.

Evidence for dynamics on a 100 ns time scale from single- and double-quantum nitrogen-14 NMR in solid peptides.

The indirect detection of 14N spectra via protons in the manner of heteronuclear multiple-quantum correlation (HMQC) allows one to obtain single- (SQ) and double-quantum (DQ) 14N spectra in solids. A comparison of the SQ and DQ line widths as a function of temperature with simulations reveals motions in the tripeptide AAG with rates on the order of 107 s(-1) at 49 degrees C.

Coherence transfer between spy nuclei and nitrogen-14 in solids.

Coherence transfer from 'spy nuclei' such as (1)H or (13)C (S=1/2) was used to excite single- or double-quantum coherences of (14)N nuclei (I=1) while the S spins were aligned along the static field, in the manner of heteronuclear single-quantum correlation (HSQC) spectroscopy. For samples spinning at the magic angle, coherence transfer can be achieved through a combination of scalar couplings J(I,S) and second-order quadrupole-dipole cross-terms, also known as residual dipolar splittings (RDS). The second-order quadrupolar powder patterns in the two-dimensional spectra allow one to determine the quadrupolar parameters of (14)N in amino acids.

Multinuclear solid-state high-resolution and 13C -{27Al} double-resonance magic-angle spinning NMR studies on aluminum alkoxides.

A combination of 27Al magic-angle spinning (MAS)/multiple quantum (MQ)-MAS, 13C-1H CPMAS, and 13C-{27Al} transfer of population in double-resonance (TRAPDOR) nuclear magnetic resonance (NMR) were used for the structural elucidation of the aluminum alkoxides aluminum ethoxide, aluminum isopropoxide, and aluminum tertiarybutoxide. Aluminum alkoxides exist as oligomers with aluminum in different coordinations. High-resolution 27Al MAS NMR experiments with high-spinning speed distinguished the aluminum atoms in different environments. The 27Al MAS NMR spectrum gave well-resolved powder patterns with different coordinations. Z-filter MQ-MAS was performed to obtain the number and types of aluminum environments in the oligomeric structure. 13C-1H CPMAS chemical shifts resolved the different carbon species (-CH3, =CH2, =CH-, and =C=) in the structures. 13C-{27Al} TRAPDOR experiments were employed to obtain relative Al-C dipolar interactions and to distinguish between terminal and bridging alkoxides in the crystallographic structures. The complete characterization of selected aluminum alkoxides using advanced NMR methods has evidenced the tetrameric structure for aluminum isopropoxide and the dimeric structure for aluminum tertiary-butoxide, as reported in the literature, and proposed a polymeric structure for aluminum ethoxide.