Articles tagged ”Glycation”
A Middle-up Approach using FabALACTICA for Characterization of Bispecific Antibodies
In recent years, bispecifics have gained popularity due to their therapeutic advantages over conventional IgG’s. In particular, the T-cell bi-specifics have received a great deal of attention due to their potential for improved efficacy. However, because of their complex TCB formats, there are multiple challenges associated with manufacturing and analysis of these type of biomolecules. A number of product and process related side products are formed which require close monitoring and identification. Moreover, the existence of various charge variants is common which can be challenging to fully characterize and understand.
Read more »
Monitoring Glycation Levels on Bispecific Biologics using FabRICATOR®
Bispecific monoclonal antibodies (BsAbs) are multi-functioning and complex biologics with the ability to recognize two different epitopes for improved therapeutic properties. Characterizing protein modifications such as glycation on biologics is vital to ensure consistency in stability and function. The structural complexity of BsAbs requires robust analytical methods, where conventional top-down and bottom-up strategies may lack in sensitivity or even introduce further modifications. Middle-level analysis using site-specific proteases such as GingisKHAN®(Kgp) and FabRICATOR®(IdeS) is an intermediate strategy that enables complementary analysis of intact or reduced Fab and Fc fragments.
In a recent article by Gstöttner et al. (2019) from Leiden University together with Roche Pharma Technical Development, the authors analyzed a BsAb for protein modification levels, N- and C-terminal sequencing and modification localization using top-down, middle-level and bottom-up strategies. The BsAb was analyzed for changes in glycation levels over time using middle-up FT-ICR MS on Fc/2, LC and Fd’ fragments obtained by FabRICATOR digestion. The scientists also localized glycation hot spots on the heavy chain backbone of the FabRICATOR-digested BsAb using sequential in source decay (ISD) MALDI fragmentation.
Using FabRICATOR in a novel middle-up MS strategy, the scientists were able to analyze all antibody subunits in a single high-resolution mass spectrum. By implementing the method in a forced-glycation experiment, changes in glycation levels were successfully monitored over time. The authors were also able to localize several glycation hot spots by intact top-down and FabRICATOR-assisted middle-down analyses. The use of middle-level strategies in combination with conventional MS-based methods successfully provided complementary data for monitoring the level of glycation.
Learn more about FabRICATOR and our other proteases.
Antibody Glycation Study using Intact LC-MS
A new study from Janssen by Mo et al, demonstrates the use of intact mass spectrometry to determine the levels of glycation on therapeutic antibodies. To perform the assay, the authors used IgGZERO for rapid removal of the Fc-glycans.
Glycation occurs when reducing sugars such as glucose, galactose or fructose, reacts with protein amino acids through the Maillard reaction, and results in attachment of sugars to the protein. For therapeutic antibodies, glycation not only increases the heterogeneity of the drug but may also affect safety and efficacy.
To study the level of glycation on antibodies, the authors used both intact mass of the reduced antibody and peptide mapping to find the +162 Da mass shift indicating an addition of a hexose sugar. The Fc-glycan of an antibody contain 0, 1 or 2 galactose sugars that also gives a mass shift of 162 Da. To specifically remove the Fc-glycans, the scientist used IgGZERO (EndoS) from Genovis. Using this enzymatic pretreatment, the authors could determine glycation levels using intact mass spectrometry.
The authors found the peptide mapping and the intact LC-MS to give correlating results but conclude: “intact LC- MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing”(Mo et al. 2018).
Find the full text of the paper here:
New references on IgG glycosylation, glycation and ADC characterization using IgGZERO and FabRICATOR
New references are out using Genovis enzymes to study antibody glycation, pairing of high-mannose glycans and ADC characterization using CE-MS.