Monoclonal antibody charge variant characterization by fully automated four-dimensional liquid chromatography-mass spectrometry.

Fully automated characterization of monoclonal antibody (mAb) charge variants using four-dimensional liquid chromatography-mass spectrometry (4D-LC-MS) is reported and illustrated. Charge variants resolved by cation-exchange chromatography (CEX) using a salt- or pH-gradient are collected in loops installed on a multiple heart-cutting valve and consequently subjected to online desalting, denaturation, reduction and trypsin digestion prior to LC-MS based peptide mapping. This innovation which substantially reduces turnaround time, sample manipulation, loss and artefacts and increases information gathering, is described in great technical detail, and applied to characterize the charge heterogeneity associated with three therapeutic mAbs.

Middle-up characterization of monoclonal antibodies by online reduction liquid chromatography-mass spectrometry.

This study describes the fully automated middle-up characterization of monoclonal antibodies (mAbs) and next-generation variants by online reduction liquid chromatography-mass spectrometry (LC-MS). Proteins were trapped on-column and subjected to online desalting, denaturation and reduction prior to reversed phase elution of the created subunits in the MS. The evaluation of more than 20 different therapeutic proteins including full length mAbs (subclasses IgG1, IgG2 and IgG4), bispecific antibodies, antibody fragments, fusion proteins and antibody-drug conjugates (ADC) revealed that the online reduction method is as powerful as the widely applied offline sample preparation with dithiothreitol (DTT) as reducing agent and guanidine hydrochloride (Gnd.

Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis.

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide variety of acceptor molecules, but its low thermostability is a serious drawback for industrial applications. In this work, the stability of the enzyme from Bifidobacterium adolescentis has been significantly improved by a combination of smart and rational mutagenesis. The former consists of substituting the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, while the latter is based on a careful inspection of the enzyme's crystal structure to promote electrostatic interactions.

Sucrose phosphorylase as cross-linked enzyme aggregate: improved thermal stability for industrial applications.

Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme.

Increasing the thermostability of sucrose phosphorylase by multipoint covalent immobilization.

Sucrose phosphorylase from Bifidobacterium adolescentis was recombinantly expressed in Escherichia coli and purified by use of a His-tag. Kinetic characterization of the enzyme revealed an optimal temperature for phosphorolytic activity of 58°C, which is surprisingly high for an enzyme from a mesophilic source. The temperature optimum could be further increased to 65°C by multipoint covalent immobilization on Sepabeads EC-HFA.

Dextran dextrinase and dextran of Gluconobacter oxydans.

Certain strains of Gluconobacter oxydans have been known since the 1940s to produce the enzyme dextran dextrinase (DDase; EC2.4.1.