Articles tagged ”GlycINATOR”

SmartEnzymes™ in a new approach to characterize ADCs

October 25, 2019 | Applications, References |

Antibody drug conjugates (ADCs) consist of monoclonal antibodies chemically linked to a cytotoxic agent. The target specificity of the monoclonal antibody in combination with the potency of the cytotoxic drug make ADCs promising therapeutic agents. However, the molecules are often complex, making evaluation of the quality attributes for the ADC challenging.

 

In order to characterize the ADCs, the predominant analysis of choice is peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry. However, the sample preparation steps in a bottom-up approach are often time-consuming and a comprehensive view of ADCs with different sequence variants and post-translational modifications is lacking.

 

In this recently published article by Chen et al., a middle-down RPLC-MS strategy with electron transfer disscociation (ETD) was developed to analyze lysine and cysteine conjugated ADCs at the subunit level. FabRICATOR® (IdeS) and GingisKHAN® (KGB) were used to generate the subunits. FabRICATOR digests below the hinge, generating F(ab’)2 and Fc/2 fragments, and GingisKHAN digests above the hinge, generating intact Fab and Fc fragments. For the deglycosylation, the IgG-specific endoglycosidase GlycINATOR® (EndoS2) was used.

 

This middle-down approach enabled high-resolution evaluation of several ADC quality attributes at the subunit level, including drug to antibody ratio (DAR), conjugation sites and micro-variants. The approach shows great potential for investigating quality attributes during the development and characterization of novel ADCs.

 

Read more about FabRICATOR, GingisKHAN and GlycINATOR.

 

Chen, B et al., 2019. Middle-Down Multi-Attribute Analysis of Antibody-Drug Conjugates with Electron Transfer Dissociation. Anal. Chem. 91(18). 11661-11669.

Glycan analysis by LC/MS in regulated environments

April 28, 2017 | References |

A team of scientist from Quality Assistance in collaboration with Alain Beck from Pierre Fabre, have published a detailed article highlighting the analytical strategies for both N- and O-linked glycan analysis of biotherapeutics using LC/MS. The workflows for glycan analysis on antibodies includes FabRICATOR digestion and study of the Fc/2 fragment for identification of the Fc glycoforms. The researchers used widepore HILIC-MS to separate the individual glycoforms of the Fc/2 subunit of adalimumab and obtained the glycoprofile with increased chromatography and MS resolution as compared to intact analysis. When applying the same workflow to cetuximab, site specific glycan profiles of both the Fc and Fd glycosylation sites were obtained.

 

In the same paper, a workflow combining GlycINATOR (EndoS2) and FabRICATOR (IdeS) was applied to study the level of core afucosylation. This digestions, that can be carried out simultaneously in 30 min, results in a dramatic reduction of the complexity of the Fc/2 subunit. After this simple sample processing the level of GlcNAc, GlcNAc + Fucose, or aglycosylated Fc/2 fragments can be quantified using LC/MS.

 

FabRICATOR_Logo_Gubbe GlycINATOR_Logo_Gubbe

Find the paper available in open access here:

Largy, E. et al., 2017. Orthogonal liquid chromatography-mass spectrometry methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level. Journal of chromatography. A, 1498, pp.128–146.