Articles tagged ”bispecific”

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 »

FabALACTICA

Using FabALACTICA® to Elucidate Proline Trans-cis Isomerization on a Trispecific Antibody

January 30, 2020 | References |
FabALACTICA digestion

FabALACTICA digestion above hinge of a trispecific anti-HIV antibody (Masiero et al 2020).

Proline isomerization can occur in the antigen binding complementary determining regions (CDRs) of an antibody and impact the interaction with the antibody target. In this paper, scientists at Sanofi in Vitry-sur-Seine, France, found an unusual size exclusion chromatography profile of a trispecific anti-HIV antibody and determined that the heterogeneity originated from a proline isomerization.

 

Peptide bonds are planar due to the partial double bond character of the C-N bond and typically occur in a trans conformation since the cis confirmation is energetically unfavorable. However, proline with its ring structure, has a significantly lower energetic threshold and cis conformers occur more frequently as observed in crystal structures. The proline trans-cis isomerization plays various roles in biology where it can act as a molecular switch in immune function and cell signaling but it also plays a role in pathologies such as cancer and Alzheimer’s disease.

 

Scientists at Sanofi in Vitry-sur-Seine, France, found an unusual size exclusion profile of a trispecific anti-HIV antibody and determined that the heterogeneity originates from a proline isomerization.  In this paper,  Masiero et al., studied a trispecific antibody carrying three variable domains that displayed two non-resolved peaks in a UHPLC-SEC analysis. In combination with mass spectrometry, identical masses for the two peaks were observed. To dissect the origin of the heterogeneity, FabALACTICA was used to digest the antibody above the hinge and generate three fragments; an intact Fc fragment, a Fab fragment binding one antigen and a second Fab fragment with two antigen binding domains. Due to the specificity of FabALACTICA, the fragments could be analyzed using SEC-MS with high accuracy and the origin of the double peak was attributed to the domain with two antigen binding domains.

 

In summary, proline trans-cis isomerization can occur in the CDRs of antibodies and impact the analytical profile of the antibody. The complexity of multispecific antibodies can be reduced using specific enzymatic tools such as FabALACTICA for more detailed analysis.

 

  1. Masiero, A. et al., 2020. The impact of proline isomerization on antigen binding and the analytical profile of a trispecific anti-HIV antibody. mAbs, 12(1), p.1698128.

 

Link to FabALACTICA Product page and Poster below