NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

The Rnf Complex Is an Energy-Coupled Transhydrogenase Essential To Reversibly Link Cellular NADH and Ferredoxin Pools in the Acetogen Acetobacterium woodii.

The Rnf complex is a respiratory enzyme that catalyzes the oxidation of reduced ferredoxin to the reduction of NAD+, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H2 plus CO2, nor did it produce acetate or ATP from H2 plus CO2, and ferredoxin:NAD+ oxidoreductase activity and Na+ translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD+, and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. The electron-bifurcating hydrogenase of A. woodii reduces ferredoxin, and indeed, the addition of H2 to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates.IMPORTANCE Ferredoxin and NAD+ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of ferredoxin is lower than that of the NADH/NAD+ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD+ When ferredoxin is greater than NADH, exergonic electron flow from ferredoxin to NAD+ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria.

Regulation of lactate metabolism in the acetogenic bacterium Acetobacterium woodii.

Acetogenic bacteria compete in an energy-limited environment by coupling different metabolic routes to their central metabolism of CO2 fixation. The underlying regulatory mechanisms are often still not understood. In this work, we analysed how lactate metabolism is regulated in the model acetogen Acetobacterium woodii. Construction of a ΔlctCDEF mutant and growth analyses demonstrated that the genes are essential for growth on lactate. Subsequent bridging PCR and quantitative PCR analyses revealed that the lctBCDEF genes form an operon that was expressed only during lactate metabolism. The lctA gene was cloned, expressed in Escherichia coli and purified. LctA bound to the intergenic DNA region between lctA and the lct operon in electromobility shift assays, and binding was revoked in the presence of lactate. Further restriction site protection analyses consolidated the lactate-dependent binding of LctA and identified the binding site within the DNA. Cells grew mixotrophically on lactate and another energy source and showed no diauxic growth. From these data, we conclude that the catabolic lactate metabolism is encoded by the lct operon and its expression is negatively regulated by the DNA-binding repressor LctA.

Endemic Mimosa species from Mexico prefer alphaproteobacterial rhizobial symbionts.

The legume genus Mimosa has > 500 species, with two major centres of diversity, Brazil (c. 350 spp.) and Mexico (c. 100 spp.). In Brazil most species are nodulated by Burkholderia. Here we asked whether this is also true of native and endemic Mexican species. We have tested this apparent affinity for betaproteobacteria by examining the symbionts of native and endemic species of Mimosa in Mexico, especially from the central highlands where Mimosa spp. have diversified. Nodules were tested for betaproteobacteria using in situ immunolocalization. Rhizobia isolated from the nodules were genetically characterized and tested for their ability to nodulate Mimosa spp. Immunological analysis of 25 host taxa suggested that most (including all the highland endemics) were not nodulated by betaproteobacteria. Phylogenetic analyses of 16S rRNA, recA, nodA, nodC and nifH genes from 87 strains isolated from 20 taxa confirmed that the endemic Mexican Mimosa species favoured alphaproteobacteria in the genera Rhizobium and Ensifer: this was confirmed by nodulation tests. Host phylogeny, geographic isolation and coevolution with symbionts derived from very different soils have potentially contributed to the striking difference in the choice of symbiotic partners by Mexican and Brazilian Mimosa species.

Energy Conservation in the Acetogenic Bacterium Clostridium aceticum.

In times of global warming caused by the extensive use of fossil fuels, the need to capture gaseous carbon compounds is growing bigger. Several groups of microorganisms can fix the greenhouse gas CO2. Out of these, acetogenic bacteria are role models in their ability to reduce CO2 with hydrogen to acetate, which makes acetogens prime candidates for genetic modification towards biotechnological production of value-added compounds from CO2, such as biofuels. However, growth of acetogens on gaseous substrates is strongly energy-limited, and successful metabolic engineering requires a detailed knowledge of the bioenergetics. In 1939, Clostridium aceticum was the first acetogen to be described. A recent genomic study revealed that this organism contains cytochromes and therefore may use a proton gradient in its respiratory chain. We have followed up these studies and will present data that C. aceticum does not use a H+ but a Na+ gradient for ATP synthesis, established by a Na+-Rnf. Experimental data and in silico analyses enabled us to propose the biochemistry and bioenergetics of acetogenesis from H2 + CO2 in C. aceticum.

Homologous production, one-step purification, and proof of Na+ transport by the Rnf complex from Acetobacterium woodii, a model for acetogenic conversion of C1 substrates to biofuels.

BACKGROUND: Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood-Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study. RESULTS: We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+-NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis. DISCUSSION: We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme.

The Rnf complex from the acetogenic bacterium Acetobacterium woodii: Purification and characterization of RnfC and RnfB.

rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with reduced ferredoxin as a reductant to vectorial ion (Na+, H+) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from reduced ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe4S] centers. The FeS centers in RnfB were reduced with reduced ferredoxin as reductant. These data are consistent with RnfB as the ferredoxin-binding subunit of the complex.

It does not always take two to tango: "Syntrophy" via hydrogen cycling in one bacterial cell.

Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H2 + CO2 or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO2 reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy."

Revealing formate production from carbon monoxide in wild type and mutants of Rnf- and Ech-containing acetogens, Acetobacterium woodii and Thermoanaerobacter kivui.

Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar-based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2 -dependent CO2 reductase and furthermore belong to different classes of 'Rnf'- and 'Ech-acetogens'. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood-Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g-1  h-1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO-based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.

Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing.

The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the analytical data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called "bottom-up" approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall analysis time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their reference sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced - the "Sequence Validation Percentage." Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up molecular weight determination and middle-down protein sequencing. Three errors in the reference amino acid sequence of natalizumab, causing a cumulative mass shift of only -2 Da in the natalizumab Fd domain, were corrected as a result of this work.