Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.

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-1H-15N-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 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-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.

Backbone NMR assignment of the yeast expressed Fab fragment of the NISTmAb reference antibody.

The monoclonal antibody (mAb) protein class has become a primary therapeutic platform for the production of new life saving drug products. MAbs are comprised of two domains: the antigen-binding fragment (Fab) and crystallizable fragment (Fc). Despite the success in the clinic, NMR assignments of the complete Fab domain have been elusive, in part due to problems in production of properly folded, triply-labeled 2H,13C,15N Fab domain. Here, we report the successful recombinant expression of a triply-labeled Fab domain, derived from the standard IgG1κ known as NISTmAb, in yeast. Using the 2H,13C,15N Fab domain, we assigned 94% of the 1H, 13C, and 15N backbone atoms.

Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity.

Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a biosynthetic process that conjugates glycans to proteins, which plays crucial roles in mAb bioactivities including effector functions and pharmacokinetics. These glycans are heterogeneous and have diverse chemical structures whose composition is sensitive to manufacturing conditions, rendering the understanding of how specific glycan structures affect mAb bioactivity challenging. There is a need to delineate the effects of specific glycans on mAb bioactivity to determine whether changes in certain glycosylation profiles (that can occur during manufacturing) will significantly affect product quality. Using enzymatic transglycosylation with chemically-defined N-glycans, we show that galactosylation at a specific location of N-glycans in an afucosylated anti-viral mAb is responsible for FcγRIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We report a facile method to obtain purified asymmetric mono-galactosylated biantennary complex N-glycans, and their influence on bioactivity upon incorporation into an afucosylated mAb. Using ELISA, surface plasmon resonance and flow cytometry, we show that galactosylation of the α6 antenna, but not the α3 antenna, consistently increases FcγRIIIA binding affinity. We confirm its relevance in an anti-viral model of respiratory syncytial virus (RSV) using an adapted ADCC reporter assay. We further correlate this structure-function relationship to the interaction of the galactose residue of the α6 antenna with the protein backbone using 2D-1H-15N-NMR, which showed that galactosylation of at this location exhibited chemical shift perturbations compared to glycoforms lacking this galactose residue. Our results highlight the importance of identifying and quantifying specific glycan isomers to ensure adequate quality control in batch-to-batch and biosimilar comparisons.

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. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

Hydrogenolysis of C-O Chemical Bonds of Broad Scope Mediated by a New Spherical Sol-Gel Catalyst.

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.

NMR study of mutations of glycine-52 of the catalytic domain of diphtheria toxin.

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.

Data generated from three quantitative mass spectral methods for the analysis of trivalent influenza vaccine antigens are compared.

Herein we present the data necessary for generation of alternative means to produce equimolar mixtures of peptides ("Design and Expression of a QconCAT Protein to Validate Hi3 Protein Quantification of Influenza Vaccine Antigens" (D.G.S. Smith, G. Gingras, Y. Aubin, T.D. Cyr, 2016) [1]), such as QConCAT ("Trends in QconCATs for targeted proteomics" (J. Chen, I.V. Turko, 2014) [2] , "Natural flanking sequences for peptides included in a quantification concatamer internal standard" (C.S. Cheung, K.W. Anderson, M. Wang, I.V. Turko, 2015) [3]) and SpikeTides versus the label free Hi3 approach. The experimental design and the interpretation of results are discussed in the original article [1].

Design and expression of a QconCAT protein to validate Hi3 protein quantification of influenza vaccine antigens.

UNLABELLED: 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 MS(E) "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. SIGNIFICANCE: 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.

Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR.

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.

Changes implemented during a workplace psychosocial intervention and their consistency with intervention priorities.

OBJECTIVES: To describe the changes implemented as part of a workplace psychosocial intervention. METHODS: The intervention was conducted in a public organization employing 1630 white-collar workers. The intervention was defined as all changes implemented to reduce adverse psychosocial work factors. A logbook was held to describe the changes implemented in the intervention group and in a comparable control group (N = 1282). RESULTS: Social support and reward were the psychosocial factors most targeted (41% to 83%). In comparison with the control group, the intervention group implemented four times more major changes and implemented changes, improving the employees' workload. CONCLUSIONS: Changes mainly targeted social support and reward. The intervention group implemented four times more major changes than the control group. The intervention group implemented changes targeting the workload, whereas no such changes were implemented in the control group.

Clean and fast cross-coupling of aryl halides in one-pot.

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.

Interactions between the conserved hydrophobic region of the prion protein and dodecylphosphocholine micelles.

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 (Ctm)PrP, 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.

Implementation of an organizational-level intervention on the psychosocial environment of work: comparison of managers' and employees' views.

OBJECTIVE: To compare employees' and managers' perceptions of implemented changes in an organizational-level workplace intervention. METHODS: Nine departments participated in an intervention aimed at reducing adverse psychosocial work environment factors. On the basis of a prior risk evaluation, department managers were responsible for decisions and implementation of the intervention, that is, work changes. These were recorded in logbooks, which were compared to employees' questionnaire ratings of the changes. RESULTS: For half of the changes, proportion of employees reporting that a specific change was implemented did not correspond with the amount of manager-reported changes. CONCLUSIONS: Evaluation of employees' and managers' perceptions of an organizational-level intervention is crucial, since these perceptions do not necessarily fully correspond. Employees perceived changes that were not reported by department managers and they also did not perceive exposure to all changes reported by the managers.

Backbone and side chain 1H, 13C and 15N resonance assignments of the catalytic domain of diphtheria toxin.

Diphtheria is a serious upper respiratory tract disease caused by the diphtheria toxin (DT) secreted from the bacteria Corynebacterium diphtheriae. Vaccination is the best way to protect against this infectious disease. Diphtheria vaccines are prepared by isolating, purifying and chemically deactivating DT. Although toxoids have been used for decades in immunization, there is still little understanding at the molecular level of the process of toxoid preparation, and how chemical treatment enhances their immunogenicity. We have undertaken an NMR study of the catalytic domain as a first step in understanding the molecular details involved in vaccine antigen preparation. Here we report a near complete assignment for the backbone and side chain resonances of the diphtheria toxin catalytic domain.

Intervention study on psychosocial work factors and mental health and musculoskeletal outcomes.

Organizations are facing ever-stiffer competition in the current globalized economy, and employees are consequently being exposed to increasingly adverse psychosocial work factors. Psychosocial work factors, also called psychosocial stressors, refer to all organizational factors and interpersonal relationships in the workplace that may affect workers' health. Two well-defined and internationally recognized theoretical models are used to assess these factors: the Karasek demand-latitude-support model and the Siegrist effort-reward imbalance model. The Karasek and Siegrist models reflect specific components of the work environment for which there is empirical evidence of a deleterious effect on health. Preventive interventions targeting these factors are conducted in workplaces. However, few studies have rigorously documented these interventions and their effectiveness in reducing adverse work factors and improving health outcomes. Most previous intervention studies were limited by (1) a short follow-up that may not have provided sufficient time for effects to appear, (2) small sample sizes (N ≤ 100) that limited the statistical power and the possibility of detecting results and (3) rare assessment of the Siegrist model. The current paper presents the overall design and the main results of an intervention study on psychosocial work factors and related mental health and musculoskeletal outcomes. The study integrated (1) a development phase that aimed at identifying the changes needed to reduce psychosocial factors in the target population and the best ways to bring about these changes, (2) an implementation phase that systematically documented how the intervention was carried out and (3) an effectiveness phase that evaluated whether the intervention was successful in reducing adverse psychosocial work factors and health problems. In addition, the study used repeated measurements of psychosocial work factors and health indicators at baseline and six and 30 months post-intervention to assess short- and medium-term effects of the intervention.

Qualitative and quantitative analyses of virtually all subtypes of influenza A and B viral neuraminidases using antibodies targeting the universally conserved sequences.

Neuraminidase-induced immune responses are correlated with protection of humans and animals from influenza. However, the amounts of neuraminidase in influenza vaccines are yet to be standardized. Thus, a simple method capable of quantifying neuraminidase would be desirable. Here we identified two universally conserved sequences in all influenza A and B neuraminidases, one representing a novel finding of nearly 100% conservation near the enzymatically active site. Antibodies generated against the two highly conserved sequences bound to all nine subtypes of influenza A neuraminidase and demonstrated remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Importantly, employing these antibodies for the analyses of vaccines from eight manufacturers using the same vaccine seeds revealed marked variations of neuraminidase levels in addition to considerable differences between lots from the same producer. The reasons for the absence or low level of neuraminidase in vaccine preparations are complex and could be multi-factorial. The antibody-based assays reported here could be of practical value for better vaccine quality control.

Assessment of the three-dimensional structure of recombinant protein therapeutics by NMR fingerprinting: demonstration on recombinant human granulocyte macrophage-colony stimulation factor.

We describe a simple, powerful, and robust NMR-based method that has the potential to greatly impact the characterization of recombinant protein therapeutics. The method ascertains the bioactive conformational identity of recombinant human granulocyte macrophage-colony stimulation factor (rhGM-CSF) produced in Streptomyces lividans versus Escherichia coli by overlaying their 2D 1H,15N HSQC correlation spectra. An identical match of all resonances implies that rhGM-CSF from both processes share indistinguishable conformations that correlate with in vitro activity. The result of this method is unique among existing methods. It can detect and quantify the active ingredient. Moreover it provides a complete assessment of the conformation with high sensitivity to minor structural changes.

NMR assignment of human granulocyte-macrophage colony-stimulating factor.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF), also known as sargramostim or molgramostin, is a cytokine that functions as a hematopoietic cell growth factor. Here we report a near complete assignment for the backbone and side chain resonances for the mature polypeptide.

NMR assignment of the N-terminal TRAF-like RING zinc finger domain of human FLN29.

Resistance of cancer cells to oncotherapeutics designed to trigger programmed cell death (a.k.a. apoptosis) greatly limits clinical efficacy. The human FLN29 protein may play a role in this process via protein-protein interactions. Here we report the NMR spectral assignment of the N-terminal TRAF2/6-RING-zinc finger-like domain of this protein.

Steady-state kinetics and inhibition of anaerobically purified human homogentisate 1,2-dioxygenase.

HGO (homogentisate 1,2-dioxygenase; EC 1.13.11.5) catalyses the O2-dependent cleavage of HGA (homogentisate) to maleylacetoacetate in the catabolism of tyrosine. Anaerobic purification of heterologously expressed Fe(II)-containing human HGO yielded an enzyme preparation with a specific activity of 28.3+/- 0.6 micromol x min(-1) x mg(-1) (20 mM Mes, 80 mM NaCl, pH 6.2, 25 degrees C), which is almost twice that of the most active preparation described to date. Moreover, the addition of reducing agents or other additives did not increase the specific activity, in contrast with previous reports. The apparent specificity of HGO for HGA was highest at pH 6.2 and the steady-state cleavage of HGA fit a compulsory-order ternary-complex mechanism (K(m) value of 28.6+/-6.2 microM for HGA, K(m) value of 1240+/-160 microM for O2). Free HGO was subject to inactivation in the presence of O2 and during the steady-state cleavage of HGA. Both cases involved the oxidation of the active site Fe(II). 3-Cl HGA, a potential inhibitor of HGO, and its isosteric analogue, 3-Me HGO, were synthesized. At saturating substrate concentrations, HGO cleaved 3-Me and 3-Cl HGA 10 and 100 times slower than HGA respectively. The apparent specificity of HGO for HGA was approx. two orders of magnitude higher than for either 3-Me or 3-Cl HGA. Interestingly, 3-Cl HGA inactivated HGO only twice as rapidly as HGA. This contrasts with what has been observed in mechanistically related dioxygenases, which are rapidly inactivated by chlorinated substrate analogues, such as 3-hydroxyanthranilate dioxygenase by 4-Cl 3-hydroxyanthranilate.