Physicochemical stability of PF-05280014 (trastuzumab-qyyp; Trazimera), a trastuzumab biosimilar, under extended in-use conditions.

Introduction: The stability and functional activity of the trastuzumab biosimilar PF-05280014 (trastuzumab-qyyp; Trazimera), was assessed under extended in-use conditions.

Methods: PF-05280014 was diluted in 0.9% sodium chloride to final concentrations of 0.

Biophysical Screens Identify Fragments That Bind to the Viral DNA-Binding Proteins EBNA1 and LANA.

The human gamma-herpesviruses Epstein-Barr virus (EBV) (HHV-4) and Kaposi's sarcoma-associated herpesvirus (KSHV) (HHV-8) are responsible for a number of diseases, including various types of cancer. Epstein-Barr nuclear antigen 1 (EBNA1) from EBV and latency-associated nuclear antigen (LANA) from KSHV are viral-encoded DNA-binding proteins that are essential for the replication and maintenance of their respective viral genomes during latent, oncogenic infection. As such, EBNA1 and LANA are attractive targets for the development of small-molecule inhibitors.

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs.

Fragonomics: the -omics with real impact.

Fragonomics is the process of using small, relatively simple molecules to generate chemical starting points for hit generation. Fragonomics has come of age and is now one of the major concepts in hit generation. What is its future?

Saturation transfer difference NMR for fragment screening.

Fragment screening by saturation transfer difference nuclear magnetic resonance (STD-NMR) is a robust method for identifying small molecule binders and is well suited to a broad set of biological targets. STD-NMR is exquisitely sensitive for detecting weakly binding compounds (a common characteristic of fragments), which is a crucial step in finding promising compounds for a fragment-based drug discovery campaign. This protocol describes the development of a library suitable for STD-NMR fragment screening, as well as preparation of protein samples, optimization of experimental conditions, and procedures for data collection and analysis.

Streptococcus pneumoniae serotype 11D has a bispecific glycosyltransferase and expresses two different capsular polysaccharide repeating units.

Streptococcus pneumoniae (pneumococcus) expresses a capsular polysaccharide (CPS) that protects against host immunity and is synthesized by enzymes in the capsular polysaccharide synthesis (cps) locus. Serogroup 11 has six members (11A to -E) and the CPS structure of all members has been solved, except for serotype 11D. The cps loci of 11A and 11D differ by one codon (N112S) in wcrL, which putatively encodes a glycosyltransferase that adds the fourth sugar of the CPS repeating unit (RU).

The use of 1H-31P GHMBC and covariance NMR to unambiguously determine phosphate ester linkages in complex polysaccharide mixtures.

Poly- and oligo-saccharides are commonly employed as antigens in many vaccines. These antigens contain phosphoester structural elements that are crucial to the antigenicity, and hence the effectiveness of the vaccine. Nuclear Magnetic Resonance (NMR) is a powerful tool for the site-specific identification of phosphoesters in saccharides.

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules.

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol.

Structural elucidation of an α-1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris.

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P.

Identification of 3-O-acetylglycerol, a novel structural element in bacterial polysaccharides.

We have discovered a novel bacterial polysaccharide structural element, 3-O-acetylglycerol, in the Streptococcus pneumoniae ST11A polysaccharide: This moiety was elucidated through a combination of homonuclear and heteronuclear 1D and 2D NMR experiments using (1)H, (13)C, and (31)P in various combinations. The 3-O-acetylglycerol moiety is substoichiometrically O-acetylated in ST11A; yet, key connectivities that unequivocally show O-acetylation at the glycerol are provided by the long-range correlations from the acetate methyl groups to the glycerol in the (1)H-(13)C HMBC spectrum. Additionally, we clarify the (1)H-(31)P assignments previously presented.

Structure of the capsular polysaccharide of pneumococcal serotype 11A reveals a novel acetylglycerol that is the structural basis for 11A subtypes.

We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11Aalpha and 11Abeta. The result of our experiments is a revision to the old structure for S.

Practical aspects of NMR-based fragment discovery.

Fragment-based drug discovery (FBDD) needs a biophysical assay to complement, or even replace, biochemical screening. NMR is the best choice for this because NMR delivers many different types of data that impacts medicinal chemistry decisions. There are a multitude of different NMR methods which can be employed to these ends.

Fragonomics: fragment-based drug discovery.

The use of smaller molecules (fragments) in the drug discovery process has led to success in delivering novel leads for many different targets. This process is a highly integrated process, starting from library design to screening and medicinal chemistry. An overview of this process is presented with particular emphasis placed on the NMR aspect of screening.

Application of residual dipolar couplings in organic compounds.

Residual dipolar couplings (RDCs) induced by anisotropic media are a powerful tool for the structure determination of biomolecules through NMR spectroscopy. Recent advances have proven it to be a valuable tool for determination of the stereochemistry of organic molecules. By simple inspection or order matrix calculations, RDCs provide unambiguous information about the relative configurations or complete stereochemistry of organic compounds.

The absolute sign of J coupling constants determined using the order matrix calculation.

A novel methodology using the order matrix calculation to determine the absolute sign of spin-spin couplings based on the structure of organic compounds is presented. The sign of the residual dipolar coupling (RDC) depends on the sign of corresponding scalar spin-spin coupling constant and the sign of the RDC has a dramatic influence on the order matrix calculation. Therefore, the sign of the spin-spin coupling constant can be obtained by an order matrix calculation through the corresponding RDC.

Complete relative stereochemistry of multiple stereocenters using only residual dipolar couplings.

Residual dipolar couplings (RDCs), in combination with molecular order matrix calculations, were used to unambiguously determine the complete relative stereochemistry of an organic compound with five stereocenters. Three simple one-dimensional experiments were utilized for the measurements of (13)C-(1)H, (13)C-(19)F, (19)F-(1)H, and (1)H-(1)H RDCs. The order matrix calculation was performed on each chiral isomer independently.

RAMPED-UP NMR: multiplexed NMR-based screening for drug discovery.

The crucial step in drug discovery is the identification of a lead compound from a vast chemical library by any number of screening techniques. NMR-based screening has the advantage of directly detecting binding of a compound to the target. The spectra resulting from these screens can also be very complex and difficult to analyze, making this an inefficient process.

The effect of relaxation on the epitope mapping by saturation transfer difference NMR.

The effect of longitudinal relaxation of ligand protons on saturation transfer difference (STD) was investigated by using a known binding system, dihydrofolate reductase and trimethoprim. The results indicate that T1 relaxation of ligand protons has a severe interference on the epitope map derived from a STD measurement. When the T1s of individual ligand protons are distinctly different, STD experiments may not give an accurate epitope map for the ligand-target interactions.

A novel method for the determination of stereochemistry in six-membered chairlike rings using residual dipolar couplings.

A novel method for the determination of the relative stereochemistry of six-membered chairlike ring molecules by residual dipolar couplings is presented. C-H residual dipolar couplings were used to investigate the relative stereochemistry of 4,6-O-ethylidene-d-glucopyranose. For this and similar systems it is not necessary to acquire redundant dipolar couplings and to calculate the orientation order tensor.

Transferred nuclear overhauser effect in nuclear magnetic resonance diffusion measurements of ligand-protein binding.

The drug discovery process relies on characterizing structure-activity relationships, since specific ligand-target interactions often result in important biological functions. Measuring diffusion coefficients by nuclear magnetic resonance spectroscopy is a useful way to study binding, because changes can be detected when a small ligand interacts with a macromolecular target. Diffusion coefficients can be miscalculated, however, due to magnetization transfer between the receptor and ligand.

ID NMR Methods in ligand-receptor interactions.

The drug discovery process often involves the screening of compound libraries to identify drug candidates capable of binding to target macromolecules. New approaches in biological and chemical research are driving a change in the pharmaceutical industry. Recent advances in NMR spectroscopy such as affinity NMR techniques, which detect binding of a small molecule with a "receptor", have been shown to be valuable tools to perform rapid screening of compounds for biological activity.

Epitope mapping of ligand-receptor interactions by diffusion NMR.

A novel method based on diffusion NMR for the epitope mapping of ligand binding is presented. The intermolecular NOE builds up during a long diffusion period and creates a deviation from the linearity. The ligand proton nearest the protein generates the strongest NOE from protein during the diffusion period and has the largest deviation.