Characterization of Therapeutic Antibody Charge Heterogeneity Under Stress Conditions by Microfluidic Capillary Electrophoresis Coupled with Mass Spectrometry.

Therapeutic antibodies are a major class of biopharmaceutics that are applied in disease treatment because of their many advantages, including high specificity and high affinity to molecular targets. Between their production and administration, therapeutic antibodies are exposed to multiple stress conditions. Forced degradation and stress stability studies are conducted to simulate the risk of degradation and the effects of these stresses, thereby enhancing understanding of the drug product to support strategies to mitigate the impact from stressed conditions. These types of studies are also routinely conducted to evaluate product comparability when major process changes are implemented during the production. Charge variant analysis helps understand the changes in the electrostatic environment of biotherapeutics and can uncover underlying molecular level alterations associated with charge variants. Herein, we used ZipChip native capillary electrophoresis-mass spectrometry (nCE-MS) to elucidate the changes in charge variant profiles at the molecular level. In two case studies under thermal stress conditions, we observed that charge variants arose from both post-translational modifications (including deamidation, oxidation, and pyroglutamate formation) and sequence truncations at the hinge regions. Under oxidative stress conditions, oxidation was found to be the major contributor to the changes in the charge variant profiles. Under pH stress conditions, the changes in the charge variant profile were due to increased levels of deamidation, oxidation, and pyroglutamate formation. ZipChip nCE-MS analysis enables identification of charge variant species under various stress conditions, thus supporting process and formulation development of biotherapeutics.

Physicochemical and physiological changes during the ripening of Banana (Musaceae) fruit grown in Colombia.

The physicochemical and physiological attributes of three contrasting commercial varieties of Musaceae, Dominico Harton (plantain), Guineo (cooking banana) and Gros Michel (dessert banana), were evaluated and statistically analysed during post-harvest ripening. Quality attributes differed markedly among varieties, both in fresh fruits and during ripening. Variety (V) had a significant effect (P < 0.001) on all attributes except total soluble solids (TSS), carotenes and total chlorophyll. Storage time (ST) had a significant effect on all attributes except colour parameter b* and total carotenes. Starch levels decreased significantly (P < 0.001) during ripening, with nearly complete hydrolysis in Gros Michel, followed by Guineo and Dominico Harton. Discriminant analysis showed that central diameter, TSS of the pulp, colour parameter a* and total starch had the highest weight in the differentiation among varieties. These results point out which parameters may help improve current methods for monitoring ripening of bananas, in particular the commercially important varieties in this study.

Plant-Based Cellulase Assay Systems as Alternatives for Synthetic Substrates.

Dissociative enzymes such as cellulases are greatly desired for a variety of applications in the food, fuel, and fiber industries. Cellulases and other cell wall-degrading enzymes are currently being engineered with improved traits for application in the breakdown of lignocellulosic biomass. Biochemical assays using these "designer" enzymes have traditionally been carried out using synthetic substrates such as crystalline bacterial microcellulose (BMCC). However, the use of synthetic substrates may not reflect the actual action of these cellulases on real plant biomass. We examined the potential of suspension cell walls from several plant species as possible alternatives for synthetic cellulose substrates. Suspension cells grow synchronously; hence, their cell walls are more uniform than those derived from mature plants. This work will help to establish a new assay system that is more genuine than using synthetic substrates. In addition to this, we have demonstrated that it is feasible to produce cellulases inexpensively and at high concentrations and activities in plants using a recombinant plant virus expression system. Our long-term goals are to use this system to develop tailored cocktails of cellulases that have been engineered to function optimally for specific tasks (i.e., the conversion of biomass into biofuel or the enhancement of nutrients available in livestock feed). The broad impact would be to provide a facile and economic system for generating industrial enzymes that offer green solutions to valorize biomass in industrialized communities and specifically in developing countries.

Aromatic residues surrounding the active site tunnel of TfCel48A influence activity, processivity, and synergistic interactions with other cellulases.

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well-studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes.

Transformations to reduce the effect of particle size in mid-infrared spectra of biomass.

Fourier Transform InfraRed (FTIR) spectroscopy is a very powerful technique for the characterization of the chemical composition of biomass and its modifications occurring during thermochemical and chemical pretreatments. However, method development is necessary to generate reproducible signals that can be used in combination with multivariate techniques (such as principal component analysis, PCA) to extract meaningful information on biomass composition and bond cleavage. Particle size is a great source of spectra variability in FTIR of biomass. The FTIR signal for an array of particle sizes (2-0.075 mm) was evaluated for hardwood and switchgrass, revealing that 0.5 mm renders higher intensity and spectral reproducibility for both the FTIR sampling techniques investigated (ATR and HTS-XT). Furthermore, the suitability of different signal processing approaches to decrease particle size variability of spectral signals was tested (signal normalization, derivation, and their combination). Normalization showed the highest contribution to enhance ATR spectral reproducibility of both biomass, as statistically shown by the 5-fold decrease of the ratio of signal variance with magnitude of spectral features (VM ratio) with respect to the unprocessed signal. Spectral signal analysis in combination with multivariate statistics (PCA) was used to extract information about the chemical differences between hardwood and switchgrass. The agreement of the biomass composition findings from FTIR-PCA and literature wet chemistry results (acid hydrolysis) contributed to corroborating that FTIR combined with PCA is a clean, quick, efficient, and versatile technique with potential to analyze and characterize biomass composition.