Articles tagged ”Stability testing”

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.


In order to study the possibility of such a solid-state formulation, scientists at the School of Health in London developed antibody mimetics and compared the stability after lyophilization to that of bevacizumab. Consisting of two Fabs linked together by a flexible polyethylene glycol (PEG) scaffold, the mimetics functionality is similar to that of IgG, but without the Fc-mediated effector functions. In addition, the mimetic format enhances binding affinity while reducing the propensity for aggregation.


To prepare the antibody mimetics, Fab fragments from bevacizumab were obtained by digestion above the hinge using either papain or GingisKHAN®. While both enzymes were able to produce Fabs without impacting the binding site, LC-MS analysis showed that the unspecific papain digestion resulted in three different isomers, and a free light chain due to disulfide reduction. In contrast, the GingisKHAN enzyme generated a homogenous pool of Fab fragments as was indicated by one single mass peak.


Antibody mimetics produced with the different Fab fragments were then subjected to freeze-drying for a head-to-head comparison with lyophilized bevacizumab. The results showed that only the mimetic produced with GingisKHAN generated Fabs was unsusceptible to aggregation and remained intact without heavy or light chain dissociation. In short, the scientists concluded that the antibody mimetic was more stable than IgG when subjected to lyophilization, and that pure homogenous Fabs could play an important part for the stability of the final product.


Khalili. 2020. Using different proteolytic enzymes to digest antibody and its impact on stability of antibody mimetics. Journal of Immunological Methods. doi: 10.1016/j.jim.2020.112933





Generation of human IgG1 Fab fragments.


GingisKHAN Fab kit
Generation and purification of human IgG1 Fabs.



SmartEnzymes™ in Quality Control of Commercial Antibodies

In a recent paper, Sokolowska and colleagues at Janssen Research and Development qualified and covalidated a subunit LC-MS method for quality control and stability testing of the oxidation status of commercial antibodies.


LC-MS is commonly used for therapeutic antibody development and characterization within the biopharmaceutical industry due to the inherent strengths to provide site-specific identification and quantitation of post-translational modifications. However, the implementation of LC-MS methods to commercial QC labs is challenging, since there are not many options for fully GMP compliant systems. In addition, the methods often require extensive MS expertise and suffer from time-consuming sample preparation and lack of robustness. To counter these obstacles, Sokolowska et al. have developed an LC-MS method that requires minimum analyst training. It uses validated GMP compliant software and is based on subunit analysis, which is proved to be faster and more robust compared to peptide mapping.


The assay uses FabRICATOR® (IdeS) and  IgGZERO® (EndoS) enzymes to generate deglycosylated IgG subunits suitable for MS analysis. FabRICATOR digests the antibody below the hinge and IgGZERO hydrolyzes the Fc N-glycans. The subunits are analyzed using reversed phase-ultraperformance liquid chromatography coupled to a quadrupole time-of-flight (RP-UPLC-QTOF) MS to monitor antibody oxidation for stability testing and commercial product release.


The developed subunit LC-MS assay was covalidated in three laboratories and showed comparable performance. The robustness was tested by varying both the LC-MS settings and the sample preparation. The enzymatic conditions included variations in protein concentration, enzyme lots, enzyme-to-protein ratio, digestion time and temperature, reduction time and temperature, and reagent concentrations. Minor variations in sample preparation all led to measured Fc oxidation within the method variation +/- 0.9%.


Figure 1. mAb subunit oxidation assay using FabRICATOR and IgGZERO (Sokolowska et al., 2020.)

The approval of this method opens the door for implementing other subunit LC-MS and multiattribute methods in QC laboratories to modernize commercial QC and stability testing.


Learn more by reading the full paper, follow the link below.