John P. Marino

Mapping Monoclonal Antibody Structure by 2D 13C NMR at Natural Abundance

Monoclonal antibodies (mAbs) represent an important and rapidly growing class of biotherapeutics. Correct folding of a mAb is critical for drug efficacy, while misfolding can impact safety by eliciting unwanted immune or other off-target responses. Robust methods are therefore needed for the precise measurement of mAb structure for drug quality assessment and comparability. To date, the perception in the field has been that NMR could not be applied practically to mAbs due to the size (∼150 kDa) and complexity of these molecules, as well as the insensitivity of the method. The feasibility of applying NMR methods to stable isotope-labeled, protease-cleaved, mAb domains (Fab and Fc) has been demonstrated from both E. coli and Chinese hamster ovaries (CHO) cell expression platforms; however, isotopic labeling is not typically available when analyzing drug products. Here, we address the issue of feasibility of NMR-based mapping of mAb structure by demonstrating for the first time the application of a 2D (13)C NMR methyl fingerprint method for structural mapping of an intact mAb at natural isotopic abundance. Further, we show that 2D (13)C NMR spectra of protease-cleaved Fc and Fab fragments can provide accurate reporters on the domain structures that can be mapped directly to the intact mAb. Through combined use of rapid acquisition and nonuniform sampling techniques, we show that these Fab and Fc fingerprint spectra can be rapidly acquired in as short as approximately 30 min.

RNA and RNA-Protein Complexes as Targets for Therapeutic Intervention

Today, the majority of pharmaceuticals developed to treat cancers and viral/bacterial infections target cellular, bacterial or viral proteins known to be associated with a given pathology. Although proteins are the focus of most current drug discovery efforts, exciting new research has recently begun which aims to exploit ribonucleic acid (RNA) and RNP particles as novel targets for pharmaceutical development. These RNA-targeted research efforts have been fueled by an increased appreciation for the central role played by RNA and RNA-protein interactions in many biological processes and diseases, together with a better understanding of RNA structure and an improvement in biophysical/biochemical techniques available to study RNA. As for protein targets, genome sequencing is greatly accelerating the identification of human and microbial RNA transcripts for targeted drug discovery. With this explosion in the number of potential RNA and RNP targets, the effective development of specific small molecule RNA-based drugs requires robust and general approaches for detecting and quantifying RNA-ligand interactions, which can be used as high-throughput screens (HTS) and for obtaining rapid structural information to guide rational drug design. In this review, an overview of the potential for therapeutic intervention based on RNA and RNP targets is presented, together with recent efforts to develop generally useful nuclear magnetic resonance (NMR) and fluorescence binding assays for screening and optimizing drugs aimed at RNA and RNP targets.

Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit NMR ANALYSIS OF GTPγS-BOUND STATES

Solution NMR studies of a 15N-labeled G-protein α-subunit (Gα) chimera (15N-ChiT)-reconstituted heterotrimer have shown previously that G-protein βγ-subunit (Gβγ) association induces a “pre-activated” conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the 15N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5′-O-3-thiotriphosphate (GTPγS)/Mg2+-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPγS/Mg2+-bound ChiT revealed 1HN, 15N chemical shift changes relative to GDP/Mg2+-bound ChiT that were similar, but not identical, to those observed for the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GDP}{\cdot}\mathrm{AlF}_{4}^{-}{/}\mathrm{Mg}^{2+}\) \end{document}-bound state. Line widths observed for R*-generated GTPγS/Mg2+-bound 15N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg2+- and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GDP}{\cdot}\mathrm{AlF}_{4}^{-}{/}\mathrm{Mg}^{2+}\) \end{document}-bound states. The increased dynamics appeared to be correlated with Gβγ and R* interactions because they are not observed for GTPγS/Mg2+-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPγS/Mg2+-exchanged heterotrimer. The HSQC spectrum of 15N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPγS/Mg2+-bound ChiT. Overall, these results demonstrated that R*-induced changes in Gα can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.

Measurement and application of 1H-19F dipolar couplings in the structure determination of 2′-fluorolabeled RNA

Residual dipolar couplings can provide powerful restraints for determination and refinement of the solution structure of macromolecules. The application of these couplings in nucleic acid structure elucidation can have an especially dramatic impact, since they provide long-range restraints, typically absent in NOE and J-coupling measurements. Here we describe sensitive X-filtered-E.COSY-type methods designed to measure both the sign and magnitude of long-range 1H-19F dipolar couplings in selectively fluorine labeled RNA oligonucleotides oriented in solution by a liquid crystalline medium. The techniques for measuring 1H-19F dipolar couplings are demonstrated on a 21-mer RNA hairpin, which has been specifically labeled with fluorine at the 2′-hydroxyl position of three ribose sugars. Experimentally measured 1H-19F dipolar couplings for the 2′-deoxy-2′-fluoro-sugars located in the helical region of the RNA hairpin were found to be in excellent agreement with values predicted using canonical A-form helical geometry, demonstrating that these couplings can provide accurate restraints for the refinement of RNA structures determined by NMR.

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein α-subunit (Gα) chimera (ChiT) associated with G-protein βγ-subunit (Gβγ) and activated receptor (R*) interactions. Here, we show that ChiT can be functionally reconstituted with Gβγ as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that 15N-ChiT can be titrated with Gβγ to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the 15N-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg2+-bound 15N-ChiT in the presence and absence of aluminum fluoride and guanosine 5′-3-O-(thio)triphosphate (GTPγS)/Mg2+-bound 15N-ChiT. As anticipated, Gβγ association with 15N-ChiT results in 1HN, 15N chemical shift changes relative to the GDP/Mg2+-bound state. Strikingly, however, most 1HN, 15N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GDP}{\cdot}\mathrm{Al}\mathrm{F}_{4}^{-}{/}\mathrm{Mg}^{2+}\) \end{document}- and GTPγS/Mg2+-bound states. Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of Gα (“preactivation”) that may facilitate the interaction with R* and subsequent GDP/GTP exchange.

Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands

The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5′(rA)4:(dT)4 tract and the 3′ PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

JE-TROSY: combined J- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules.

With the application of RDCs in high-resolution NMR studies of macromolecules, there has been an interest in the development of accurate, sensitive methods for measuring 15N-1H and 13C-1H one-bond coupling constants. Most methods for determining these couplings are based on the measurement of the displacement between cross-peak components in J-coupled spectra. However, for large macromolecules and macromolecular complexes, these methods are often unreliable since differential relaxation can significantly broaden one of the multiplet components (i.e., the anti-TROSY component) and thereby make accurate determination of its position difficult. To overcome this problem, a J-evolved transverse relaxation optimized (JE-TROSY) method is presented for the determination of one-bond couplings that involves J-evolution of the sharpest cross-peak multiplet component selected in a TROSY experiment. Couplings are measured from the displacement of the TROSY component in the additional J-evolution dimension relative to a zero frequency origin. The JE-TROSY method is demonstrated on uniformly labeled 15N, 13C-labeled RNA and peptide samples, as well as with an RNA-protein complex, in which the protein is uniformly 15N, 13C-labeled. In all cases, resolved, sensitive spectra are obtained from which heteronuclear one-bond J-couplings could be accurately and easily measured.

2D (1)H(N), (15)N Correlated NMR Methods at Natural Abundance for Obtaining Structural Maps and Statistical Comparability of Monoclonal Antibodies.

PurposeHigh-resolution nuclear magnetic resonance spectroscopy (NMR) provides a robust approach for producing unique spectral signatures of protein higher order structure at atomic resolution. Such signatures can be used as a tool to establish consistency of protein folding for the assessment of monoclonal antibody (mAb) drug quality and comparability.MethodsUsing the NIST monoclonal antibody (NISTmAb) and a commercial-sourced polyclonal antibody, both IgG1κ isotype, we apply 2D NMR methods at natural abundance for the acquisition and unbiased statistical analysis of 1HN -15N correlated spectra of intact antibody (Ab) and protease-cleaved Fab and Fc fragments.ResultsThe study demonstrates the feasibility of applying 2D NMR techniques to Abs and the precision with which these methods can be used to map structure and establish comparability between samples at atomic resolution.ConclusionsThe statistical analyses suggests that, within the limit of detection, no significant structural differences are observed between the Fab and Fc domains of each respective intact Ab and its corresponding fragments. Discrimination between dissimilar species, such as between the Fab domains of both Abs or between the glycosylated and deglycosylated Fc domains, was further demonstrated. As such, these methods should find general utility for the assessment of mAb higher order structure.

Precision and robustness of 2D-NMR for structure assessment of filgrastim biosimilars

139 and Supplementary Tables 11 and 12; refs. 9,10). The chemical shift of each nucleus is an absolute frequency position, and perturbations in the higher-order structure, such as altered hydrogen bonding, can influence the local electronic environments of nuclei, leading to changes in chemical shifts. Thus, amide 1HN and 15N chemical shift changes can be assessed individually using RMSD from the average values or in aggregate by CCSD (which involves taking a weighted average of the observed changes in 1HN and 15N shift values9). With either of these methods, the chemical shift assignment of the primary sequence allows atomic-level mapping of spectral signals to a known protein structure, but this is not required for the method to be useful. From RMSD and CCSD analyses, an experimental precision of 8 p.p.b. was determined across the six spectrometers in the four laboratories. Notably, 8 p.p.b. is close to the digital resolution of approximately 5 p.p.b. of an individual spectrum (see Supplementary Methods), which establishes a threshold for the precision of the measurement. Similarly, an intralaboratory precision of approximately 4 p.p.b. was found that falls well within the digital resolution of a spectrum (Supplementary Table 12), and a related study found an experimental precision for the same sample of 2.4 p.p.b.10. These precision limits are well below any chemical shift changes that could be induced by a significant structural change (such as a point mutation or a modification of a residue by oxidation or local conformational change). These approaches also readily identify the temperature deviation of the non-calibrated spectrometers (Fig. 1b), further demonstrating the need for matched experimental conditions if the method is to be used as a structure comparability tool. As expected, the recalibrated spectra were well within the determined experimental precision (Fig. 1c). Although comparison of spectral overlays and chemical shifts provides the most straightforward method for the analysis of two or three samples, it can be cumbersome when large numbers of datasets need to be compared, as in the monitoring of lot-to-lot consistency; in such instances a multivariate statistical analysis approach may be more appropriate. Here, we used PCA because it provides a single readout of variance in all To the Editor: With the advent of biosimilar versions of brand biologics, regulatory authorities in all major jurisdictions throughout the world have developed guidance documents to facilitate their approval1–4. The common theme in the new documents is the stipulation that sponsors must demonstrate biosimilarity between their proposed product and an approved reference product using state-of-the-art analytical technologies. In the US guidance documents, a “totality of evidence” approach is described that can be used to establish the degree of similarity and guide regulatory decision-making3,4. Higher-order structure is an important quality attribute of biosimilars that must be assessed by comparison to the reference licensed drug. To date, higher-order structure has been evaluated with low-resolution techniques, such as circular dichroism, Fourier transform infrared and Raman spectroscopies, and indirectly with several biological and stability assays5. In 2008, a two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy approach was first applied to the high-resolution assessment of the higher-order structure of a native recombinant protein therapeutic6. The technique resolves, in a 2D frequency map, the positions of proton–nitrogen atom pairs from each amide and amino group in a biologic molecule. Each signal correlates to the specific local chemical and structural environment of the atom pair; thus, the entire spectrum provides a comprehensive readout of the drug substance conformation along the polypeptide chain at atomic resolution, providing a potentially useful tool to establish drug substance consistency across manufacturing changes or comparability of the higher-order structure of biosimilars. In this work, an interlaboratory comparative study was performed on filgrastim (methionyl granulocyte colony-stimulating factor; Met-G-CSF) to demonstrate the precision and robustness of the 2D-NMR approach. Filgrastim was selected because of its therapeutic importance, because the drug had been characterized by NMR7 and because a number of filgrastim biosimilars have already been approved in Europe, the United States and other jurisdictions. Here, a USapproved originator product (Neupogen) and three unapproved, non-US-sourced filgrastim products were used in their fully formulated states (Supplementary Table 1) to prepare a set of four samples for analysis using heteronuclear 2D-NMR correlation at 15N natural isotopic abundance. Spectra were acquired on the same samples on six different spectrometers, at four different field strengths ranging from 500 MHz to 900 MHz, in four different laboratories (for instrument specifications and experimental parameters, see Supplementary Tables 2–9). All the NMR data were analyzed and evaluated using the same software packages for visual evaluation, chemical shift analysis and principal-component analysis (PCA) to assess spectral similarity through multiple approaches. The interlaboratory study data show that the resolution of the NMR method allows one to quickly visualize the high degree of similarity of the spectral patterns obtained from the 2D-NMR experiments run on different spectrometers. In this respect, visual comparison of spectral overlays allows an operator to directly assess sample similarity (Fig. 1a, Supplementary Table 10 and Supplementary Figs. 1–4). Differences between two spectral patterns can result from variation in solution conditions (such as pH or ionic strength), variation in the temperature of the sample, and/or differences in the higher-order structure of the protein. As an example, in our study, the overlay of spectra acquired for the same filgrastim sample on two spectrometers revealed a number of peak signal deviations that could be mapped to solvent-exposed residues (Fig. 1a, inset). Four representative residues in loop regions that exhibited these deviations are shown in Supplementary Figure 2b. We found that these differences could be explained by differences in temperature of 2 °C and 4 °C for the HC 700 and HC 600 spectrometers, respectively. The change of temperature does not affect all amide resonances equally7 and has been described by Baxter and Williamson8. Proper instrument calibration removed these discrepancies (Supplementary Fig. 2c). For a more quantitative analysis, chemical shifts of each individual signal in the 2D map were compared using a root mean square deviation (RMSD) analysis or combined chemical shift difference (CCSD) methods (Fig. 1b,c, Supplementary Figs. 5 and 6, Precision and robustness of 2D-NMR for structure assessment of filgrastim biosimilars CORRESPONDENCE

High-Resolution NMR Analysis of the Conformations of Native and Base Analog Substituted Retroviral and LTR-Retrotransposon PPT Primers

A purine-rich region of the plus-strand RNA genome of retroviruses and long terminal repeat (LTR)-containing retrotransposons, known as the polypurine tract (PPT), is resistant to hydrolysis by the RNase H domain of reverse transcriptase (RT) and ultimately serves as a primer for plus-strand DNA synthesis. The mechanisms underlying PPT resistance and selective processing remain largely unknown. Here, two RNA/DNA hybrids derived from the PPTs of HIV-1 and Ty3 were probed using high-resolution NMR for preexisting structural distortions in the absence of RT. The PPTs were selectively modified through base-pair changes or by incorporation of the thymine isostere, 2,4-difluoro-5-methylbenzene (dF), into the DNA strand. Although both wild-type (WT) and mutated hybrids adopted global A-form-like helical geometries, observed structural perturbations in the base-pair and dF-modified hybrids suggested that the PPT hybrids may function as structurally coupled domains.