Articles tagged ”FabALACTICA”
Using FabALACTICA® to Elucidate Proline Trans-cis Isomerization on a Trispecific Antibody
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.
Link to FabALACTICA Product page and Poster below
Free Thiols using FabRICATOR® and FabALACTICA®
In biopharmaceutical product development and manufacturing, free thiol content is one of the product quality attributes of interest as its presence could impact structure, stability and function of the product.
At Biogen, Yi Pu et al have optimized a label-free LC (UV) / MS method for free thiol quantification at a subunit level of IgG1 and IgG4. The new method, which is based on a method developed by Faid et al*, was compared to two conventional approaches, Ellman’s assay and peptide mapping.
It is very challenging to identify free thiol forms by mass spectrometry at the intact antibody level. By combining the highly specific proteolytic enzymes FabALACTICA (IgdE) and FabRICATOR (IdeS) the authors generated the subunits Fab, hinge and Fc/2, suited for confident mass determination. The subunits were subsequently separated on a polyphenyl reversed phase column in order to separate free thiol forms from their corresponding disulphide bond-linked form. A baseline or near baseline separation was obtained making it possible to calculate the free thiol content on each subunit.
The result of the quantification of free thiols from all three methods were comparable and showed similar trends even though the peptide mapping approach generally gave a higher free thiol content.
The authors conclude that compared to Ellman’s assay, the subunit approach is more sensitive, requires less sample and provides domain-specific information of the free thiol content. Compared to peptide mapping, the subunit method is faster, less labour intensive and lacks dependence on labelling efficiency. Finally, it demonstrated promise in the quantification of free thiols in a high throughput manner with domain specific information available.
The developed method has successfully been applied to several in-house IgG1 mAbs with different hydrophobicity and isoelectric points.
*V. Faid Y. Leblanc N. Bihoreau G. Chevreux Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls, J. Pharm. Biomed. Anal. 149 (2018) 541-546, https://doi.org/10.1016/j.jpba.2017.11.046
For more information on FabRICATOR and FabALACTICA please visit the following pages:
- FabRICATOR Product Page
- FabALACTICA Product Page
- Generation of Antibody Fragments
Interview with Valegh Faid at LFB Biotechnologies in France
Unique enzymatic digestions in study of antibody disulphides
Valegh Faid and colleagues at LFB Biotechnologies in France have developed and published an assay to study antibody disulphide bonds using middle-up LC-MS (Faid et al., 2017). The combination of FabRICATOR® for digestion below the hinge and FabALACTICA™ for digestion above the hinge, generated three fragments from a human IgG1 antibody; the hinge peptide, Fab and Fc/2 fragments. These fragments were resolved using RP-HPLC and mass spectrometry and enabled analysis of antibody disulphide bridges and other quality attributes.
Interview with Valegh Faid, Scientist at LFB and first author of the paper:
Why are antibody disulphide bonds important?
Disulphide bonds are highly important because of their critical role in the stabilization of protein conformations. Breaking and/or scrambling of disulphide bond occur during manufacturing and storage of biotherapeutics which is a concern in terms of safety and efficacy. The monitoring of these product-derived impurities is mandatory during development operations in order to minimize these forms.
How did you come up with the idea to combine FabRICATOR (IdeS) and FabALACTICA (IgdE)?
We have been using IdeS for many years in order to cleave IgG’s below the hinge; following DTT reduction, more amenable fragments for RP-HPLC/MS analysis are generated as previously published by our laboratory (Chevreux et al., 2011). This middle-up analysis is fast and very informative regarding the protein sequence integrity and post-translational modifications. However, investigating the oxidative state of disulfide bridges is tricky and often involved a time-consuming peptide mapping in non-reducing conditions.
In this context, IgdE is an interesting enzyme that cleaves specifically IgGs above the hinge and without requiring reducing conditions as papain do. The combination of IdeS and IgdE in non-reducing conditions presents the advantage to generate specifically three fragments i.e. hinge, Fc/2 and Fab that are both easily separated by RP-HPLC and analysed by MS.
How does the new enzymatic assay compare to previous methods to study antibody disulphide bonds?
Peptide mapping in non-reducing condition is the gold standard to investigate disulphide bonding of biotherapeutics. However, data interpretation is time consuming even if dedicated software to improve the treatment of data has largely improved. Although being slightly less informative than peptide mapping, this combined IdeS/IgdE middle-up approach increases the throughput for the investigation of free thiols and disulphide scrambling. Considering that other CQAs can also be monitored in the same experiment, it should be more applicable to routine use in process optimization, formulation screening and stability studies.
Would the assay be used in a QC setting relying solely on liquid chromatography separation?
The analytical workflow is robust and requires mere handlings of the antibody samples. Once the identification of each peak of the chromatogram is confirmed by MS, quantitation based on the UV detection is a current practice. Such analytical configuration involving an HPLC and a UV detection is actually common in most of QC labs and thus easily and robustly implementable.
How are you implementing this assay at LFB Biotechnologies?
This assay is integrated in our portfolio of analytical approaches for the analysis of mAbs currently in development, for process optimisation, batch characterization and stability studies.
Read more about FabALACTICA and FabRICATOR.
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