Articles in the Category ”References”

Automated Biotransformation Analysis of ADCs using FabRICATOR

The development of antibody-drug conjugates (ADCs) has evolved from first generation formats prepared by random conjugation technologies to next generation ADCs generated by site-specific conjugation. While significant improvements in overall efficacy and safety is displayed by site-specific formats, bioanalysis remains challenging due to complex in vivo biotransformation events including deconjugation, linker-payload cleavage and payload metabolism.

 

In this work, scientists at Bristol-Myers Squibb describe the development of an automated and fast affinity capture method using a cartridge-based platform combined with LC-HRMS analysis for biotransformation assessment of site-specific ADCs.

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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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FabRICATOR used to Locate Modification Sites of IgG Caused by Reducing Agents

A charge heterogeneity is an unfavorable phenomenon observed for mAbs and is considered as a critical quality attribute since it can alter the efficacy and pharmacokinetics of biopharmaceuticals. Acidic and basic species of an IgG are due to various chemical modifications on the molecule. The origin of acidic species has previously been reported to be formed by deamidation, oxidation of side-chains, cysteinylation, glycosylation, glycation, sialylation and fragmentation while the basic species comes from C-terminal lysine clipping, pyro-glu cyclization, succinimide formation and aggregation. Scientists at Boehringer Ingelheim together with scientists at NMI at University of Tübingen recently published a study characterizing the root cause of charged species of an IgG1 mAb.

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C-terminal lysine clipping and Fc receptor binding using SmartEnzymes

February 19, 2021 | References |

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.

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Antibodies Conjugated with GlyCLICK for Super Resolution Imaging

Super resolution microscopy techniques such as stimulated emission depletion microscopy (STED) improves imaging resolution compared to conventional light microscopy. In STED microscopy, super resolution is achieved using photoactivatable dyes that are excited and de-excited selectively with a laser to restrict fluorescense to a specific focal point. While such super resolution methods in combination with immunostaining advances the quality of imaging, limitations related to dyes and conjugation strategies remain. Read more »

Charge Heterogeneity Analysis of Antibody Subunits Generated by FragIT

Charge heterogeneity of monoclonal antibodies is an important critical quality attribute that requires close monitoring due to its potential impact on antibody efficacy and immunogenicity. Since the heterogeneity is mostly caused by post translational modifications such as C-terminal lysine clipping, deamidation, glycation, sialic acid or adduct formation, these modifications can pose significant challenges to the analytical scientists. Read more »

Generating Antibody Mimetics with GingisKHAN

Antibodies formulated as solid-state depots can potentially be used for local treatments and minimize the need for large systemic doses. Bevacizumab may for instance be administered locally to control post-operative scarring following glaucoma filtration surgery. A solid-state form would however be required in order to obtain a proper slow release of the antibody. Read more »

Antibody Mixtures Digested using GingisREX

The formulation of antibodies in mixtures has revealed significant clinical advantages but causes increased analytical challenges. Long-term studies of formulated antibody mixtures over time are both difficult and time consuming. An example of a post-translational modification that could occur during storage is the oxidation of tyrosine that may induce conformational changes of an antibody. Read more »

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 »