Bruker (France)

ABSTRACT The analytical comparability of biologic products and their biosimilars, including higher-order structure (HOS) assessment, ensures product quality and is required for regulatory approval. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to evaluate the HOS of Humira (adalimumab) and its biosimilars under normal and photo-stressed conditions. Under normal conditions, 1D and 2D NMR spectra showed strong structural similarity among all products. However, photo-stressed samples exhibited distinct structural differences, including increased methionine oxidation, and localized conformational changes, most notably in the reference product. These changes correlated with findings from size-exclusion chromatography, capillary isoelectric focusing, and mass spectrometry (MS), which revealed size and charge heterogeneity, as well as site-specific methionine oxidation in the heavy chains. The differences in photostability were found to be influenced by container closure systems (CCSs) and formulations. In contrast, circular dichroism spectral analysis showed minimal variation in secondary structures among stressed and unstressed samples. These results underscore the utility of NMR as a sensitive tool for comparative structural analysis of monoclonal antibodies and their biosimilars, particularly under stress conditions, and highlight the impact of formulation and CCS on product stability.

A new 3.2mm 1H-19F-X magic angle spinning dynamic nuclear polarization NMR (MAS DNP-NMR) probe was developed with a unique coil design with separate radiofrequency channels for 1H excitation and 13C or 19F detection to enable acquisition of 1H-19F cross-polarization (CP) MAS experiments, direct-detected 19F spectra with proton decoupling, and acquisition on 13C with simultaneous double decoupling on the 1H and 19F channels as well as 1H-19F-13C double-CP experiments under low temperature MAS DNP conditions. We use these sequences to study AZD2811, which is an active pharmaceutical ingredient (API), in its pure dry state as well as in its corresponding drug delivery formulation consisting of drug-loaded polymeric nanoparticles (PNPs). Included in this study are also small interfering RNAs (siRNAs) for therapeutic targeting of peptidyl-prolyl cis-trans isomerase B (Ppib) mRNA. We demonstrate that 1H-19F CP MAS experiments performed on the new HFX probe represent a notable advantage over usually acquired direct-detected 19F experiments. The indirect 19F DNP enhancement εon/off(19F)=26 was obtained via 1H-19F CP for the pure API impregnated with DNP solution, with an overall 30-fold sensitivity gain compared to the direct-detected 19F experiment under similar conditions. DNP enhancement value of εon/off(19F)=42 was obtained via 1H-19F CP for the polymeric nanoparticle suspension and εon/off(19F)≈150 were obtained for two different siRNAs in frozen DNP solution.

This contribution engages with the emergence of commented editions of the Bible in the early medieval Latin West and illustrates their spread in the Carolingian Empire on the basis of the extant manuscript evidence. It describes their codicological and exegetical features starting from a comparison with the contemporary genre of the Byzantine frame catenae. Since the completion of a remarkably high number of commented editions can be ascribed to Otfrid of Wissembourg (d. after 871), the chapter discusses whether Otfrid might be regarded as a »cultural broker« of this new tool for biblical studies. To this end, it focuses on Otfrid’s networks, interests and intended audiences, using three short poems by Walahfrid Strabo and Otfrid’s own output, in particular his commented edition on Acts, as evidence.

The front cover artwork front cover artwork is provided by NMRCoRe, the Flemish NMR/X-Ray platform for Convergence Research and was designed by Ir. Ewoud Vaneeckhaute and Dr. Eric Breynaert. The image shows the reciprocity between parahydrogen, deuterated ammonia and iridium allowing for hyperpolarized 2D NMR via long-term availability of longitudinal spin order. Read the full text of the Article at 10.1002/cphc.202100079.

<strong class="journal-contentHeaderColor">Abstract.</strong> <span id="page448"/>Half-integer spin quadrupolar nuclei are the only magnetic isotopes for the majority of the chemical elements. Therefore, the transfer of polarization from protons to these isotopes under magic-angle spinning (MAS) can provide precious insights into the interatomic proximities in hydrogen-containing solids, including organic, hybrid, nanostructured and biological solids. This transfer has recently been combined with dynamic nuclear polarization (DNP) in order to enhance the NMR signal of half-integer quadrupolar isotopes. However, the cross-polarization transfer lacks robustness in the case of quadrupolar nuclei, and we have recently introduced as an alternative technique a <span class="inline-formula"><i>D</i></span>-RINEPT (through-space refocused insensitive nuclei enhancement by polarization transfer) scheme combining a heteronuclear dipolar recoupling built from adiabatic pulses and a continuous-wave decoupling. This technique has been demonstrated at 9.4 T with moderate MAS frequencies, <span class="inline-formula"><i>ν</i>_R≈10</span>–15 kHz, in order to transfer the DNP-enhanced <span class="inline-formula">¹</span>H polarization to quadrupolar nuclei. Nevertheless, polarization transfers from protons to quadrupolar nuclei are also required at higher MAS frequencies in order to improve the <span class="inline-formula">¹</span>H resolution. We investigate here how this transfer can be achieved at <span class="inline-formula"><i>ν</i>_R≈20</span> and 60 kHz. We demonstrate that the <span class="inline-formula"><i>D</i></span>-RINEPT sequence using adiabatic pulses still produces efficient and robust transfers but requires large radio-frequency (rf) fields, which may not be compatible with the specifications of most MAS probes. As an alternative, we introduce robust and efficient variants of the <span class="inline-formula"><i>D</i></span>-RINEPT and PRESTO (phase-shifted recoupling effects a smooth transfer of order) sequences using symmetry-based recoupling schemes built from single and composite <span class="inline-formula"><i>π</i></span> pulses. Their performances are compared using the average Hamiltonian theory and experiments at <span class="inline-formula"><i>B</i>₀=18.8</span> T on <span class="inline-formula"><i>γ</i></span>-alumina and isopropylamine-templated microporous aluminophosphate (AlPO<span class="inline-formula">₄</span>-14), featuring low and significant <span class="inline-formula">¹</span>H–<span class="inline-formula">¹</span>H dipolar interactions, respectively. These experiments demonstrate that the <span class="inline-formula">¹</span>H magnetization can be efficiently transferred to <span class="inline-formula">²⁷</span>Al nuclei using <span class="inline-formula"><i>D</i></span>-RINEPT with <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M17" display="inline" overflow="scroll" dspmath="mathml"><mrow><mi mathvariant="normal">SR</mi><msubsup><mn mathvariant="normal">4</mn><mn mathvariant="normal">1</mn><mn mathvariant="normal">2</mn></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="28pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="bfb1b3a831bd4cb203459286d6469a1c"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-2-447-2021-ie00001.svg" width="28pt" height="16pt" src="mr-2-447-2021-ie00001.png"/></svg:svg></span></span>(270<span class="inline-formula">₀</span>90<span class="inline-formula">₁₈₀</span>) recoupling and using PRESTO with <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M20" display="inline" overflow="scroll" dspmath="mathml"><mrow><mi mathvariant="normal">R</mi><msubsup><mn mathvariant="normal">22</mn><mn mathvariant="normal">2</mn><mn mathvariant="normal">7</mn></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="27pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="1467477ecea38809b6b6f06c82d8832a"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-2-447-2021-ie00002.svg" width="27pt" height="16pt" src="mr-2-447-2021-ie00002.png"/></svg:svg></span></span>(180<span class="inline-formula">₀</span>) or <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M22" display="inline" overflow="scroll" dspmath="mathml"><mrow><mi mathvariant="normal">R</mi><msubsup><mn mathvariant="normal">16</mn><mn mathvariant="normal">7</mn><mn mathvariant="normal">6</mn></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="27pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="77936c715f231774d5e767150eec791c"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-2-447-2021-ie00003.svg" width="27pt" height="16pt" src="mr-2-447-2021-ie00003.png"/></svg:svg></span></span>(270<span class="inline-formula">₀</span>90<span class="inline-formula">₁₈₀</span>) schemes at <span class="inline-formula"><i>ν</i>_R=20</span> or 62.5 kHz, respectively. The <span class="inline-formula"><i>D</i></span>-RINEPT and PRESTO recoupling schemes complement each other since the latter is affected by dipolar truncation, whereas the former is not. We also analyze the losses during these recoupling schemes, and we show how these magnetization transfers can be used at <span class="inline-formula"><i>ν</i>_R=62.5</span> kHz to acquire in 72 min 2D HETCOR (heteronuclear correlation) spectra between <span class="inline-formula">¹</span>H and quadrupolar nuclei, with a non-uniform sampling (NUS).

Targeted proteomics allows the highly sensitive detection of specific peptides and proteins in complex biological samples. Here, we describe a methodology for targeted peptide quantification using a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro). The prm-PASEF method exploits the multiplexing capability provided by the trapped ion mobility separation, allowing more than 200 peptides to be monitored over a 30 min liquid chromatography separation. Compared to conventional parallel reaction monitoring (PRM), precursor ions are accumulated in the trapped ion mobility spectrometry (TIMS) cells and separated according to their shape and charge before eluting into the quadrupole time-of-flight (QTOF) part of the mass spectrometer. The ion mobility trap allows measuring up to six peptides from a single 100 ms ion mobility separation with the current setup. Using these improved mass spectrometric capabilities, we detected and quantified 216 isotope-labeled synthetic peptides (AQUA peptides) spiked in HeLa human cell extract with limits of quantification of 17.2 amol for some peptides. The acquisition method is highly reproducible between injections and enables accurate quantification in biological samples, as demonstrated by quantifying KRas, NRas, and HRas as well as several Ras mutations in lung and colon cancer cell lines on fast 10 min gradient separations.

Dissolution dynamic nuclear polarization (D-DNP) probes are usually designed for one or at most two specific nuclei. Investigation of multiple nuclei usually requires manufacturing a number of costly probes. In addition, changing the probe is a time-consuming process since a system that works at low temperature (usually between 1.2 and 4.2 K) must be warmed up, thus increasing the risks of contamination. Here, an efficient apparatus is described for D-DNP designed not only for microwave-enhanced direct observation of a wide range of nuclei S such as 1 H, 13 C, 2 H, 23 Na, and 17 O, but also for cross-polarization (CP) from I=1 H to such S nuclei. Unlike most conventional designs, the tuning and matching circuits are partly immersed in superfluid helium at temperatures down to 1.2 K. Intense radio-frequency (RF) fields with amplitudes on the order of 50 kHz or better can be applied simultaneously to both nuclei I and S using RF amplifiers with powers on the order of 90 and 80 W, respectively, without significant losses of liquid helium. The system can operate at temperatures over a wide range between 1.2 and 300 K.

Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy. Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). By applying DNP NMR to frozen cells, we overcame limitations both of solution-state in-cell NMR spectroscopy (e.g., size, stability and sensitivity) and of visualization techniques, in which (e.g., fluorescent) tagging of the ASO decreases its activity. The capability to detect an untagged, active drug, interacting in its natural environment, represents a first step towards studying molecular mechanisms in intact cells.