The clinical efficacy and safety of protein-based drugs such as monoclonal antibodies (mAbs) rely on the integrity of the protein higher order structure (HOS) during product development, ...manufacturing, storage, and patient administration. As mAb-based drugs are becoming more prevalent in the treatment of many illnesses, the need to establish metrics for quality attributes of mAb therapeutics through high-resolution techniques is also becoming evident. To this end, here we used a forced degradation method, time-dependent oxidation by hydrogen peroxide, on the model biotherapeutic NISTmAb and evaluated the effects on HOS with orthogonal analytical methods and a functional assay. To monitor the oxidation process, the experimental workflow involved incubation of NISTmAb with hydrogen peroxide in a benchtop nuclear magnetic resonance spectrometer (NMR) that followed the reaction kinetics, in real-time through the water proton transverse relaxation rate
(
H
O). Aliquots taken at defined time points were further analyzed by high-field 2D
H-
C methyl correlation fingerprint spectra in parallel with other analytical techniques, including thermal unfolding, size-exclusion chromatography, and surface plasmon resonance, to assess changes in stability, heterogeneity, and binding affinities. The complementary measurement outputs from the different techniques demonstrate the utility of combining NMR with other analytical tools to monitor oxidation kinetics and extract the resulting structural changes in mAbs that are functionally relevant, allowing rigorous assessment of HOS attributes relevant to the efficacy and safety of mAb-based drug products.
Protein therapeutics have numerous critical quality attributes (CQA) that must be evaluated to ensure safety and efficacy, including the requirement to adopt and retain the correct three-dimensional ...fold without forming unintended aggregates. Therefore, the ability to monitor protein higher order structure (HOS) can be valuable throughout the lifecycle of a protein therapeutic, from development to manufacture. 2D NMR has been introduced as a robust and precise tool to assess the HOS of a protein biotherapeutic. A common use case is to decide whether two groups of spectra are substantially different, as an indicator of difference in HOS. We demonstrate a quantitative use of principal component analysis (PCA) scores to perform this decision-making, and demonstrate the effect of acquisition and processing details on class separation using samples of NISTmAb monoclonal antibody Reference Material subjected to two different oxidative stress protocols. The work introduces an approach to computing similarity from PCA scores based upon the technique of histogram intersection, a method originally developed for retrieval of images from large databases. Results show that class separation can be robust with respect to random noise, reconstruction method, and analysis region selection. By contrast, details such as baseline distortion can have a pronounced effect, and so must be controlled carefully. Since the classification approach can be performed without the need to identify peaks, results suggest that it is possible to use even more efficient measurement strategies that do not produce spectra that can be analyzed visually, but nevertheless allow useful decision-making that is objective and automated.
Abstract
The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied ...extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the genome 3′ untranslated region is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the SARS-CoV-2 s2m element dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular microRNA (miRNA) 1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.
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•2D NMR provides fingerprints of the higher-order-structure of protein therapeutics.•Fingerprint of 1Hn-1Hα correlations acquired with 2D J-correlated NMR experiments.•Selective TOCSY ...(TACSY) enables selective J-correlation and optimal sensitivity.
The higher order structure (HOS) of protein therapeutics is essential for drug safety and efficacy and can be evaluated by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. 1Hn-15N amide correlated and 1H-13C methyl correlated NMR spectroscopies at natural isotopic abundance have been demonstrated as feasible on protein therapeutics as large as monoclonal antibodies and show great promise for use in establishing drug substance structural consistency across manufacturing changes and in comparing a biosimilar to an originator reference product. Spectral fingerprints from 1Hn-1Hα correlations acquired using 2D homonuclear proton-proton J-correlated NMR experiments provide a complementary approach for high-resolution assessment of the HOS of lower molecular weight (<25 kDa) protein therapeutics. Here, we evaluate different pulse sequences (COSY, TOCSY and TACSY) used to generate proton-proton J-correlated NMR spectral fingerprints and appraise the performance of each method for application to protein therapeutic HOS assessment and comparability.
We report the discovery of an ultrahot Jupiter with an extremely short orbital period of 0.67247414 ± 0.00000028 days (∼16 hr). The 1.347 ± 0.047 RJup planet, initially identified by the Transiting ...Exoplanet Survey Satellite (TESS) mission, orbits TOI-2109 (TIC 392476080)—a T(eff) ∼ 6500 K F-type star with a mass of 1.447 ± 0.077 Mꙩ, a radius of 1.698 ± 0.060 Rꙩ, and a rotational velocity of v sin i =81.9 ± 1.7 km/s. The planetary nature of TOI-2109b was confirmed through radial-velocity measurements, which yielded a planet mass of 5.02 ± 0.75 M(Jup). Analysis of the Doppler shadow in spectroscopic transit observations indicates a well-aligned system, with a sky-projected obliquity of λ = 1.°7± 1.°7. From the TESS full-orbit light curve, we measured a secondary eclipse depth of 731 ± 46 ppm, as well as phase-curve variations from the planet's longitudinal brightness modulation and ellipsoidal distortion of the host star. Combining the TESS-band occultation measurement with a K(s)-band secondary eclipse depth (2012 ± 80 ppm) derived from ground-based observations, we find that the dayside emission of TOI-2109b is consistent with a brightness temperature of 3631 ± 69 K, making it the second hottest exoplanet hitherto discovered. By virtue of its extreme irradiation and strong planet–star gravitational interaction, TOI-2109b is an exceptionally promising target for intensive follow-up studies using current and near-future telescope facilities to probe for orbital decay, detect tidally driven atmospheric escape, and assess the impacts of H2 dissociation and recombination on the global heat transport.
Purpose
Antisense oligonucleotide (ASO) therapeutics are an emerging class of biopharmaceuticals to treat and prevent diseases, particularly those involving “undruggable” protein targets. Impurities ...generated throughout the ASO drug manufacturing and formulation pipeline can be detrimental to drug safety and efficacy. Therefore, analytical techniques are needed to rigorously characterize these molecules for quality assurance purposes.
Methods
We demonstrate 1D and 2D nuclear magnetic resonance (NMR) spectroscopy methods that can generate high-resolution structural “fingerprints” of ASOs.
Results and Conclusions
1D
1
H and
31
P measurements are shown to provide rapid initial assessment of the ASO integrity. In particular, a well-resolved pair of
31
P signals arising from the 5´-end of the phosphorodiamidate morpholino oligomer (PMO) are sensitive to complex formation and oligomerization state. 2D
1
H-
1
H,
1
H-
13
C, and
1
H-
15
N experiments, although less sensitive, are further shown to enable resonance assignment, which will allow the tracking of structural changes at high-resolution during the drug development and manufacturing processes. We further anticipate that the described NMR approaches will be broadly applicable to fully formulated ASO therapeutics, including modalities other than PMOs.
One of the central challenges in the development of single-molecule protein sequencing technologies is achieving high-fidelity sequential recognition and detection of specific amino acids that ...comprise the peptide sequence. An approach towards achieving this goal is to leverage naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs). One such protein, the N-end rule pathway adaptor protein ClpS, natively recognizes NAAs on a peptide chain. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes the residues Trp, Tyr, and Leu at the N-terminal position. Here, we employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, we identified two promising variants of the
Agrobacterium tumefaciens
ClpS protein with native residues 34–36 ProArgGlu mutated to ProMetSer and CysProSer. In vitro surface binding assays indicate that the ProMetSer variant has enhanced affinity for Phe at the N-terminus with sevenfold tighter binding relative to wild-type ClpS, and that the CysProSer variant binds selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe. Taken together, this work demonstrates the utility of engineering ClpS to make it more effective for potential use in peptide sequencing applications.
Next-generation approaches for protein sequencing are now emerging that could have the potential to revolutionize the field in proteomics. One such sequencing method involves fluorescence-based ...imaging of immobilized peptides in which the N-terminal amino acid of a polypeptide is readout sequentially by a series of fluorescently labeled biomolecules. When selectively bound to a specific N-terminal amino acid, the NAAB (
N
-terminal amino acid binder) affinity reagent identifies the amino acid through its associated fluorescence tag. A key technical challenge in implementing this fluoro-sequencing approach is the need to develop NAAB affinity reagents with the high affinity and selectivity for specific N-terminal amino acids required for this biotechnology application. One approach to develop such a NAAB affinity reagent is to leverage naturally occurring biomolecules that bind amino acids and/or peptides. Here, we describe several candidate biomolecules that could be considered for this purpose and discuss the potential for developability of each.
Key points
• Next-generation sequencing methods are emerging that could revolutionize proteomics.
• Sequential readout of N-terminal amino acids by fluorescent-tagged affinity reagents.
• Native peptide/amino acid binders can be engineered into affinity reagents.
• Protein size and structure contribute to feasibility of reagent developability.
We studied the effect of allele-level matching at human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 in 1568 single umbilical cord blood (UCB) transplantations for hematologic malignancy. The primary ...end point was nonrelapse mortality (NRM). Only 7% of units were allele matched at HLA-A, -B, -C, and -DRB1; 15% were mismatched at 1, 26% at 2, 30% at 3, 16% at 4, and 5% at 5 alleles. In a subset, allele-level HLA match was assigned using imputation; concordance between HLA-match assignment and outcome correlation was confirmed between the actual and imputed HLA-match groups. Compared with HLA-matched units, neutrophil recovery was lower with mismatches at 3, 4, or 5, but not 1 or 2 alleles. NRM was higher with units mismatched at 1, 2, 3, 4, or 5 alleles compared with HLA-matched units. The observed effects are independent of cell dose and patient age. These data support allele-level HLA matching in the selection of single UCB units.
Key Points
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