Articles tagged ”FabALACTICA”
Join Us for Our Next GenovisWebinar – Solve Analytical Challenges with SmartEnzymes™!
We are happy to welcome Angela Capolupo from Merck Serono to talk about how implementing Genovis SmartEnzymes in their workflows has allowed her and her team to resolve complex analytical and structural problems!
Unravelling Structural Conformers within a Therapeutic Multispecific mAb using FabALACTICA
Scientists at the University of Strasbourg and Sanofi describe the global structural characterisation of a trispecific antibody which exhibits two distinct structural isoforms when analyzed by size-exclusion chromatography (SEC). FabALACTICA proved to be a valuable tool in this study as above hinge digestion of this trispecific antibody allowed for middle-level characterisation of the isomeric Fab regions and facilitated cyclic-ion mobility (cIM) differentiation of the isomers, which was not possible in the intact molecule without prior SEC separation.
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FabALACTICA® in a Quantitative IgG1 Clone Profiling
Our immune system protects us from pathogens and various diseases, and the immune response is dependent on a person’s repertoire of immune cells, antibodies and circulating plasma proteins. A detailed characterization of these plasma components is important for the understanding of how they affect each individual’s immune response.
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FabALACTICA Facilitates the Structural Insight into SARS-CoV-2 Neutralizing Antibodies
The first steps of infection with SARS-CoV-2 is binding of a viral Spike protein to a host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Antibodies that block this interaction are emerging as early COVID-19 therapies, however, the neutralization potencies of the antibodies are less studied.
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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.
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C-terminal lysine clipping and Fc receptor binding using SmartEnzymes
Researchers at LFB Biotechnologies in Paris, France have carried out a thorough analysis and characterization of the impact of C-terminal lysine clipping to Fc-receptor binding using a range of SmartEnzymes from Genovis.
The scientists separated an IgG1 antibody using SCX separation and purified the fractions without C-terminal lysines K0, with 1 C-terminal lysine K1 and with both lysines intact K2. The purified fractions were characterized for any further differences using FabRICATOR digestion and middle-level analysis. This approached enabled the researchers to study multiple post-translational modifications such as charge variants, oxidations and Fc glycosylation in a simple and robust way. The characterization revealed that the lysine heterogeneity was the main differentiator and all other PTMs were distributed between the fractions.
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SmartEnzymes in Targeted Sequencing of Heavily Glycosylated IgA1
The use of IgG-based antibodies in various clinical fields have increased over that past decades, continuing the development of better biopharmaceuticals. The use of other immunoglobulins including IgA, characterized with the ability to recruit effector cells, is progressively being considered a useful alternative. The complexity of the heavily glycosylated IgA does however pose analytical challenges and no method currently exist that allows unraveling of the human repertoire of this subclass.
Scientists at Utrecht University present a mass spectrometry method using electron capture dissociation (EDC) to obtain sequence ladders of the variable regions on the heavily N- and O-glycosylated anti-CD20 IgA1 antibody. Using SmartEnzymes and a native top-down approach, the scientists compared the IgA1 antibody to its anti-CD20 IgG counterpart, and their Fabs.
SmartEnzymes in de novo Sequencing of Antibodies
Two complementary determining regions (CDR3) are considered major determinants of antigen-binding specificity that give rise to the human immunoglobulin repertoire with billions of unique antibodies. For de novo sequencing of the human repertoire, circulating antibodies can be analyzed by mass spectrometry after proteolytic cleavage. Complex mixtures such as plasma derived samples are however challenging to analyze due to the increased complexity that may prevent accurate assignments. Read more »
GlyCLICK® and Middle-up LC-MS Enables Robust ADC Development
Scientists at the University of Geneva and CNRS present site-specific ADCs generated using the GlyCLICK technology and an analytical middle-up LC-HRMS workflow as a potential core module for ADC development.
Antibody-drug conjugates (ADCs) are efficient therapeutic agents that possess the cell-targeting properties of monoclonal antibodies combined with the potency of cytotoxic drugs. Early generation ADCs were predominantly obtained through non-selective conjugation methods by incorporation of a drug payload at randomly distributed sites. Such methods result in highly heterogenous subpopulations of varying antibody-drug ratio (DAR) leading to potential loss of efficacy and impaired pharmacokinetics. While alternative strategies exploring genetic engineering have emerged for conjugation at non-natural amino acids, challenges related to both production and analytical characterization persist.
Glycan-mediated bioconjugation using the GlyCLICK technology is an attractive option to overcome the challenges of conventional bioconjugation without the need for genetic engineering to produce custom ADCs. By utilizing a unique combination of enzymes, the conserved Fc-glycans are remodeled and site-specifically conjugated using click chemistry for ADCs carrying two payloads per antibody (DAR=2.0) having controlled drug stoichiometry and preserved immunoreactivity. In this paper, Duivelshof et al. developed a site-specific ADC by coupling trastuzumab to DM1 using the GlyCLICK technology and evaluated the quality of the conjugation process using complementary reversed phase (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS).
The trastuzumab antibody was site-specifically conjugated to DBCO-functionalized DM1 (DBCO-PEG4-Ahx-DM1) using the GlyCLICK technology. To reduce sample complexity, the antibodies were digested with FabRICATOR® (Ides) or FabALACTICA® (IgdE) and reduced for comparison of native and GlyCLICK conjugated trastuzumab at the subunit level. The complementary HILIC and RPLC workflow allowed the authors to observe the significant shift in retention between the lipophilic drug payloads on the ADC and the hydrophilic N-glycans on native trastuzumab. These results enabled the scientists to confirm site-specific conjugation at the Fc-glycans sites, while hyphenation to HRMS detection allowed accurate determination of a DAR of 2.0 for GlyCLICK conjugated trastuzumab, which was not possible at the intact ADC level.
“Most ADCs are produced with non-selective bioconjugation of drug payloads to lysine or cysteine residues creating a wide variety of drug-antibody ratios (DAR). In the frame of new ADC product development, we believe that having control over the DAR and drug load distribution (DLD) is of crucial importance, as is the ability to accurately monitor these two CQAs. Therefore, the combination of the GlyCLICK technology to create homogeneous site-specific ADCs with the middle-up LC/HRMS approach to rapidly determine both the DLD and DAR has a great potential for ADC development.”
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.
