The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution ...such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either
Komagataella phaffii
(
Pichia pastoris
) or
Escherichia coli
that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The
E. coli
approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while
K. phaffii
secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10–45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-
1
H-
15
N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of
2
H-
13
C-
15
N-adalimumab-scFab and
2
H-
13
C-
15
N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.
•NMR study of mutants of the catalytic domain of diphtheria toxin.•Mutation of residue 52 destabilizes the conformation of DTA.•The lack of toxicity of CRM197 results from an unstable conformation of ...its catalytic domain.
Cross-reacting-material 197 (CRM197) is a naturally occurring non-toxic mutant of diphtheria toxin (DT) that is one of the few carrier protein used in the manufacture of polysaccharide vaccines targeting bacterial pathogens such as Neisseria meningitidis, Streptococcus pneumaniae and Haemophilus influenzae. A detailed explanation in structural terms for the lack of toxicity has started to emerge with the report of the X-ray structure of CRM197. Here, we present an NMR spectroscopy study of the wild-type catalytic domain of diphtheria toxin and the effects of mutations at residue 52 on its conformation. We utilized a strategy that consisted of gradually inducing steric perturbations by increasing the side chain size of the residue. Results show that the catalytic domain does not tolerate even the smallest perturbation, such as a glycine to alanine substitution, resulting in the destabilization of domain fold leading to protein aggregation. The observed behavior is further exacerbated with the substitution of amino acids with larger side chains. These findings support the concept that the lack of toxicity observed for CRM197 is the result of a highly unstable conformation of its catalytic domain that, upon insertion into the cell, cannot properly refold and perform its catalytic activity responsible for the arrest of all cellular protein synthesis.
Purpose
Filgrastim is the generic name for recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF). It is marketed under the brand name Neupogen® by Amgen. Since this product ...has lost patent protection, many biosimilar versions have been approved or are in the process of filing for market authorization throughout the world. Here we show that NMR spectroscopy can be used to assess the three-dimensional structure of the active ingredient in the formulated approved product Neupogen®.
Methods
Recombinant metHuG-CSF was prepared in
E. coli
and isotopically enriched with
13
C and
15
N isotopes. NMR spectroscopy was used to study the effects of excipients on the conformation.
Results
The effects of pH variation on the amide chemical shifts suggest the presence of cation-pi interactions between His-79 and Trp-118, and His-156-Trp-58-His-52 that stabilizes the conformation at low pH. This may be associated with a small local conformational change. The NMR data showed that polysorbate does not interact significantly with filgrastim thus allowing the collection of spectra in the presence of 20 times the formulation concentration in the sample. However, at higher detergent concentrations a reduction of signal intensity is observed. Conclusions
The NMR fingerprint assay applied to filgrastim (Neupogen® and a CRS from the European Pharmacopeia (EP)) provided residue specific information of the structure of the drug substance. In addition to current methods, the ability to assess the conformation with a high degree of resolution can greatly facilitate comparability exercises.
Unsymmetrically coupled biaryls are synthesized in high yield starting from different aryl bromides and bis(pinacolato)diboron by carrying out the Miyaura borylation reaction followed by the ...Suzuki-Miyaura reaction in the same reaction pot over 1-2 mol % SiliaCat DPP-Pd. The SiliaCat DPP-Pd catalyst is air-stable and the method does not require the use of inert conditions. The use of non-toxic isopropanol or 2-butanol as reaction solvent further adds to the environmental benefits of this new green synthetic methodology.
The three-dimensional structure of PrP110–136, a peptide encompassing the conserved hydrophobic region of the human prion protein, has been determined at high resolution in dodecylphosphocholine ...micelles by NMR. The results support the conclusion that the CtmPrP, a transmembrane form of the prion protein, adopts a different conformation than the reported structures of the normal prion protein determined in solution. Paramagnetic relaxation enhancement studies with gadolinium-diethylenetriaminepentaacetic acid indicated that the conserved hydrophobic region peptide is not inserted symmetrically in the micelle, thus suggesting the presence of a guanidium-phosphate ion pair involving the side chain of the terminal arginine and the detergent headgroup. Titration of dodecylphosphocholine into a solution of PrP110–136 revealed the presence of a surface-bound species. In addition, paramagnetic probes located the surface-bound peptide somewhere below the micelle-water interface when using the inserted helix as a positional reference. This localization of the unknown population would allow a similar ion pair interaction.
Summary
Employment and working conditions having an impact on health and safety are some of the most important concerns of workers. Amongst the various means by which trade unions contribute to ...prevention, the contribution of Worker Safety Representatives (WSR) is well-established and the most studied, including their participation in joint occupational health and safety committees (JOHSC). However, there are surprisingly few studies examining the place of OHS as an issue of workers’ collective action. Conducted with a large Quebec Central Labour Body, this study aims to understand why and how local-level unions concentrate upon these issues, the repertoire of means that they employ and the context that supports or hindus such actions. The conceptual framework is based on previous realistic evaluations of OHS preventive interventions and includes Levesque and Murray’s (2010) trade union power resources and strategic capabilities.
In phase I, eleven semi-structured interviews were conducted with union staff members and elected representatives from different sectors, covering a wide array of activities such as unionization, training, negotiation, OHS prevention and compensation. Results also refer to five case studies (phase 2) of local-level trade unions identified by phase 1 respondents as particularly active in relation to prevention.
The process by which working conditions having a negative impact on OHS are framed (or not) as trade union issues is examined. Levers and barriers are also identified. Factors affecting the presence of resources for trade union autonomous action aimed at prevention (like the integration of WSR to the union core structure, release time for prevention, etc.) are highlighted. A widely diverse repertoire of workplace-level trade union means of action for OHS is also highlighted by the interviews and case studies, not limited just to those provided by the Quebec OHS regime. It includes the recourse to labour relations mechanisms (e.g. negotiation and strike) and is based on an autonomous agenda, including mobilization. The potential of OHS issues for union revitalization is discussed, as well as the barriers that must be overcome.
The new spherical sol–gel hybrid material SiliaCat Pd0 selectively mediates the hydrogenolysis of aromatic alcohols, aldehydes, and ketones by using an ultralow catalytic amount (0.1 mol % Pd) under ...mild reaction conditions. The broad reaction scope as well as the catalyst's superior activity and pronounced stability open the route to green and convenient reductive deoxygenation processes of primary synthetic relevance in chemical research as well as in the fine chemical and petrochemical industries.
Perfectness of sphericity: A new‐generation spherical hybrid catalyst SiliaCat Pd0 selectively mediates the hydrogenolysis of aromatic alcohols, aldehydes, and ketones by using an ultralow amount of palladium under mild reaction conditions. The broad reaction scope as well as the catalyst's superior activity and pronounced stability open the route to green and convenient reductive deoxygenation processes of primary synthetic relevance in chemical research as well as in the fine chemical and petrochemical industries.
We investigate the heterogeneously catalyzed direct synthesis of boronic acid pinacol esters using a wide range of aryl chlorides, bromides, and iodides, and bis(pinacolato)diboron as the borylating ...agent over the sol–gel entrapped SiliaCat diphenylphosphine palladium(II) catalyst. Optimization of the reaction conditions, scale‐up of the optimized process, and analysis of palladium leaching enabled us to establish a new selective route for direct access to a diverse set of boronic acid pinacol esters.
Clean borylation for scale‐up: With the easy access and broad availability of diverse borylated species, the Suzuki–Miyaura reaction has become routine in industry and in research labs. We report a new selective route for direct access to a diverse set of boronic acid pinacol esters over the sol–gel entrapped SiliaCat diphenylphosphine palladium(II) catalyst that can be easily scaled‐up.
Quantification of the antigens hemagglutinin and neuraminidase in influenza vaccines has been reported using an antibody-free liquid chromatography–mass spectrometry (LC–MS) based method known as MSE ...“Hi3”. This approach is based on the average signal intensity of the three most intense tryptic peptides relative to a primary standard. This strategy assumes that the Hi3 signal responses are consistent for all proteins, and therefore comparable to a spiked reference for absolute quantification. This method is much faster than the current standard methods; however, the results can vary significantly which brought the method’s accuracy into question. To address this question we generated synthetic proteins comprising a concatenation of the peptides used to quantify the proteins of interest (QconCAT). Complete tryptic digestion of a QconCAT protein produces equal molar peptide amounts, allowing verification of equal signal response of Hi3 peptides for the proteins of interest. The generation of an intact, stable, QconCAT protein that digest completely is challenging. We have designed and analyzed five QconCAT proteins with unique design elements to address these challenges. We conclude that a suitable QconCAT protein can be produced and that the results obtained reinforce the validity of the Hi3 approach for quantifying proteins in annual influenza vaccine formulations.
The advances in quantitative proteomics have allowed the adaptation and application of these methods to numerous fields. In this paper we have validated a Hi3 approach to augment the antigen quantification for influenza vaccines injected into many millions annually. This methodology allows analysis of multiple antigens simultaneously without the need to generate antibodies. Key circumstances where this is advantageous are for quantitation of very similar antigens, such as the new quadravalent products and when time is critical such as in a flu pandemic.
Display omitted
•Demonstrates the utility of LC–MS for quantifying antigens in influenza vaccines•Uses a QconCAT to validate Hi3 quantification of specific proteins of interest•Identifies challenges in designing and manufacturing a suitable QconCAT protein•Proposes an optimized QconCAT design for reproducible peptide production•Confirms the suitability of Hi3 quantification for influenza vaccine antigens
Regulation, change and the work environment Lippel, Katherine; Johnstone, Richard; Baril-Gingras, Genevieve
Relations industrielles (Québec, Québec),
01/2017, Letnik:
72, Številka:
1
Journal Article
Recenzirano
This special issue of Relations industrielles/Industrial Relations presents articles based on original research on fundamental changes to work and work arrangements that undermine workers’ health and ...safety, exacerbate health inequalities and pose major challenges to those who want to resist a 'race to the bottom' in working conditions and imagine and promote regulatory reforms to better protect workers and their health. The papers in this special issue not only make a contribution to knowledge about work intensification and employment precariousness and their impact on health and safety, but also shed light on further challenges presented by the current globalized work environment: the association of precariousness with international, regional and local employment-related mobility, both in developed and developing countries; the commuting difficulties faced by some workers in precarious employment; the non-standard work schedules resulting from work intensification pressures and the consequential health and work family balance difficulties; and the dilution of responsibility for health and safety and for workers’ compensation in international supply chains. One paper illustrates an old but still pervasive challenge: the production of a 'paradigm of doubt' which uses and even produces scientific uncertainty to obscure the effects of hazards to workers’ health, thus delaying the prevention and compensation of their negative effects on health.
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