Theoretical charge plots as a tool for targeted and accelerated ion exchange chromatography method development of NANOBODYⓇ molecules.

NANOBODYⓇ molecules are an innovative class of biotherapeutics based on heavy chain only VHH immunoglobulins. Much like canonical antibodies, they are prone to the formation of charge variants and other post-translational modifications, which can potentially impact their critical quality attributes. Therefore, establishing high-resolution product-specific methods, such as IEX chromatography, is essential for evaluating the purity of these molecules. However, due to the lower surface charge of NANOBODYⓇ molecules, their charge-based elution behavior can differ considerably from that of classical antibodies, resulting in a more extensive method development set-up for these smaller molecules. Using an initial pH screening gradient based on theoretical protein charge plots, we investigated the IEX retention behavior of eight NANOBODYⓇ molecules with a wide range of pI values (pI 5.0 to 10.0). Our findings reveal that the charge-based chromatographic behavior of NANOBODYⓇ molecules cannot be solely attributed to the isoelectric point (pI) of the protein. Rather, a molecule-specific charge threshold was identified as a critical parameter for NANOBODYⓇ molecule retention. Furthermore, the protein charge plot also showed that NANOBODYⓇ molecule elution can be characterized by a charge plateau where the net charge of the protein remains constant over a certain pH range (∼ pH 5.5 to pH 8.0), further challenging the paradigm that elution pH and pI are fixed values. The application of this theoretical approach using protein charge plots to define NANOBODYⓇ molecule charge threshold and charge plateau parameters, can reduce overall IEX method development turnaround time by at least 2-fold.

Dual Data-Independent Acquisition Approach Combining Global HCP Profiling and Absolute Quantification of Key Impurities during Bioprocess Development.

Host cell proteins (HCP) are a major class of impurities derived from recombinant protein production processes. While HCP are usually monitored by ELISA, mass spectrometry (MS)-based approaches are emerging as promising orthogonal methods. Here, we developed an original method relying on data-independent acquisition (DIA) coupling global HCP amount estimation (Top 3) and absolute quantification with isotope dilution (ID). The method named Top 3-ID-DIA was benchmarked against ELISA and a gold-standard selected reaction monitoring assay (ID-SRM). Various samples generated at different steps and conditions of the purification process, including different culture durations, harvest procedures, and purification protocols were used to compare the methods. Overall, HCP were quantified over 5 orders of magnitude and down to the sub-ppm level. The Top 3-ID-DIA strategy proved to be equivalent to the gold-standard ID-SRM in terms of sensitivity (1-10 ppm), accuracy, and precision. Moreover, 81% of the Top 3 estimations were accurate within a factor of 2 when compared to ID-SRM. Thus, our approach aggregates global HCP profiling for comprehensive process understanding with absolute quantification of key HCP within a single analysis and provides an improved support for bioprocess development and product purity assessment.

Galactosaminogalactan, a new immunosuppressive polysaccharide of Aspergillus fumigatus.

A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of α1-4 linked galactose and α1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates.

Linear osmoregulated periplasmic glucans are encoded by the opgGH locus of Pseudomonas aeruginosa.

Osmoregulated periplasmic glucans (OPGs) are produced by many proteobacteria and are important for bacterial-host interactions. The opgG and opgH genes involved in the synthesis of OPGs are the most widely distributed genes in proteobacterial genomes. Two other non-homologous genes, both named ndvB, are also involved in OPG biosynthesis in several species. The Pseudomonas aeruginosa genome possesses two ORFs, PA5077 and PA5078, that show similarity to opgH and opgG of Pseudomonas syringae, respectively, and one ORF, PA1163, similar to ndvB of Sinorhizobium meliloti. Here, we report that the opgGH locus of P. aeruginosa PA14 is involved in the synthesis of linear polymers with beta-1,2-linked glucosyl residues branched with a few beta-1,6 glucosyl residues. Succinyl residues also substitute this glucose backbone. Transcription of opgGH is repressed by high osmolarity. Low osmolarity promotes the formation of highly structured biofilms, but biofilm development is slower and the area of biomass is reduced under high osmolarity. Biofilm development of an opgGH mutant grown under low osmolarity presents a similar phenotype to the wild-type biofilm grown under high osmolarity. These results suggest that OPGs are important for biofilm formation under conditions of low osmolarity. A previous study suggested that the P. aeruginosa ndvB gene is involved in the resistance of biofilms to antibiotics. We have shown that ndvB is not involved in the biosynthesis of the OPG described here, and opgGH do not appear to be involved in the resistance of P. aeruginosa PA14 biofilms to antibiotics.

Characterization of the Erwinia chrysanthemi Gan locus, involved in galactan catabolism.

beta-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK(2). Degradation of galactans would be catalyzed by the periplasmic 1,4-beta-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-beta-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny.

Proteomic analysis of a non-virulent mutant of the phytopathogenic bacterium Erwinia chrysanthemi deficient in osmoregulated periplasmic glucans: change in protein expression is not restricted to the envelope, but affects general metabolism.

Osmoregulated periplasmic glucans (OPGs) are general constituents of the envelope of Gram-negative bacteria. They are required for full virulence of bacterial phytopathogens such as Pseudomonas syringae, Xanthomonas campestris and Erwinia chrysanthemi. E. chrysanthemi is a pectinolytic gamma-proteobacterium that causes soft rot disease on a wide range of plant species. In addition to the loss of virulence, opg mutants exhibit a pleiotropic phenotype that affects motility, bile-salt resistance, exoenzyme secretion, exopolysaccharide synthesis and membrane lipid composition. This is believed to be the first proteomic analysis of an OPG-defective mutant of E. chrysanthemi and it revealed that, in addition to the effects described, catabolic enzyme synthesis was enhanced and there was a greater abundance of some proteins catalysing the folding and degradation of proteins needed for various stress responses. Thus, in the opg mutant strain, loss of virulence was the result of a combination of envelope structure changes and cellular metabolism modifications.