Controlling fab terminal sialylation of antibodies through culture conditions

Biologics are used for the treatment of a wide-range of diseases with specificity and minimal side effects. Safety and efficacy of the drugs has been linked to carbohydrate structures found on the antibody, termed N-linked glycans. These glycans are mainly found within the Fc-region of an antibody but 20% of all IgG antibodies also contain Fab glycans1. Glycans are composed of a range of sugars whose presence or absence affects the biological qualities of a drug. Sialic acid is one such sugar; its role is to “cap” the glycan chain, protecting the internal sugars, which when exposed are bound by receptors and cleared to host lysosomes2. The presence of sialic acid is linked to an increase in biologic half-life along with a reduced inflammatory response3. It has been established that bioprocess conditions such as cell culture temperature and pH directly impact glycan composition and site occupancy. A shift in the cell culture pH and its effect on the sialic acid content within the Fab region of an antibody product was examined. The product was produced in three CHO cell clones, each innately producing varying levels of sialic acid. An initial experiment utilized the Ambr 24® to run cultures at pH 7.1, 6.8 and with a shift from 7.1 to 6.8 on day 6. Data from this was used to establish a second experiment, utilizing an Ambr 48® system. This experiment looked at the supplementation of ManNAc, copper as well as the effect of pH and temperature shifts on sialylation. Glycan analysis was undertaken using a novel method for triple-quadrupole MS. A pH shift was found to produce overall more processed glycans. Although cell growth was negatively affected, antibody productivity and specific rate of sialylation were both increased at reduced culture pH (Figure 1). The extent of the effect differed between the clones and was correlated to how early the shift occurred. Due to the negative effects on growth, overall antibody yield was reduced with some clones having less than half that of the respective control. To determine the origin of the effect and the differences between the clones further analysis is being undertaken. The Ambr 48® experiment determined the effect of different supplements on sialylation, as well as the effect of a temperature shift and further understanding of the role of cell culture pH in increasing sialylation. Flux balance analysis and expression analysis of the enzymes involved in terminal glycosylation process is underway. Please click Additional Files below to see the full abstract

A de novo peptide hexamer with a mutable channel

The design of new proteins that expand the repertoire of natural protein structures represents a formidable challenge. Success in this area would increase understanding of protein structure and present new scaffolds that could be exploited in biotechnology and synthetic biology. Here we describe the design, characterization and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, six-helix bundle with a channel running through it. Although lined exclusively by hydrophobic leucine and isoleucine side chains, the 6-Å channel is permeable to water. One layer of leucine residues within the channel is mutable, accepting polar aspartic acid and histidine side chains, which leads to subdivision and organization of solvent within the lumen. Moreover, these mutants can be combined to form a stable and unique (Asp-His)3 heterohexamer. These new structures provide a basis for engineering de novo proteins with new functions