Robert G. Brinson

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.

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.

Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein

Tissue inhibitor of metalloprotease-2 (TIMP-2) is a secreted 21 kDa multifunctional protein first described as an endogenous inhibitor of matrix metalloproteinases (MMPs) that prevents breakdown of the extracellular matrix often observed in chronic diseases. TIMP-2 diminishes the level of growth factor-mediated cell proliferation in vitro, as well as neoangiogenesis and tumor growth in vivo independent of its MMP inhibitory activity. These physiological properties make TIMP-2 an excellent candidate for further preclinical development as a biologic therapy of cancer. Here we present a straightforward bioprocessing methodology that yields >35 mg/L recombinant human TIMP-2 6XHis-tagged protein (rhTIMP-2) from suspension cultures of HEK-293-F cells. Enhanced rhTIMP-2-6XHis yields were achieved by optimization of both TIMP-2 cDNA codon sequence and cell culture conditions. Using a two-step chromatographic process, we achieved >95% purity with minimal processing losses. Purified rhTIMP-2-6XHis was free of mouse antigen contamination. Circular dichroism spectroscopy indicated a well-folded rhTIMP-2-6XHis that is highly stable and refractory to pH changes. Two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance of full length rhTIMP-2-6XHis also indicated a monodisperse, well-folded protein preparation. Purified rhTIMP-2-6XHis inhibited MMP-2 enzymatic activity in a dose-dependent fashion with an IC50 of ∼1.4 nM. Pretreatment of A549 lung cancer and JygMC(A) triple-negative breast cancer cells with rhTIMP-2-6XHis in low-nanomolar amounts inhibited EGF-induced proliferation to basal (unstimulated) levels. This study therefore not only offers a robust bioprocess methodology for rhTIMP-2 production but also characterizes critical physicochemical and biological attributes that are useful for monitoring quality control of the production process.