Due to their mode of production, therapeutic glycoproteins present complex and heterogeneous glycosylations. Their analysis usually requires the use of many orthogonal analytical techniques in order to fully characterise their nature and location. In this application note, the use of mass spectrometry as a powerful tool for Etanercept glycosylation characterisation is presented.

Using a comprehensive workflow, combining several analytical methods at the subunit, peptide, and released glycan levels, we extensively characterised the N- and O-glycosylations of Etanercept.

Profiling of the N-glycans was performed within a day using the RapiFluor-MS labeling kit (Waters), and is fully consistent with results from the literature. N-glycans identity was confirmed using an orthogonal mixed-mode AEX-RP column, which also gave access to the sialylation profile. This was further assessed using a short gradient on an AEX-HILIC column, yielding a single peak for each sialylation level.

O-glycan profiling was successfully achieved by carrying out a reductive β-elimination reaction that provided high release yields together with negligible peeling. Two core-1 glycans carrying one or two sialic acids were detected after PGC LC/MS, with high sensitivity and accuracy.

The overall sialylation was quantified by straightforward acidinduced release and DMB-labeling, from small amounts of Etanercept. The results were fully consistent with the N- and O glycosylation profiles determined previously.

Profiling of each of the three N-glycosylation sites was performed by widepore HILIC/MS, after triple-digestion of Etanercept, yielding short peptides. This stationary phase allows a complete resolution of glycosylated peptides vs. aglycosylated peptides, and CID MS/MS data provides a confirmation of the presence of glycans. We evidenced significant differences in the glycosylation of these three asparagines, the overall profile being consistent with what was found by released glycans analysis.

Discovery of the O-glycosylated peptides of Etanercept was performed following a modified protocol including a N deglycosylation and desialyation step, in order to generate short peptides carrying naked core 1 O-glycan exclusively. Targeted ETD fragmentation was subsequently performed to determine the O-glycosylation sites at the amino-acid levels. Out of the 89 putative sites, 13 were found to be glycosylated, with occupancies ranging from 1.6 to 100%.

Analysis of Etanercept subunits, generated by the IdeS enzyme, followed by N-deglycosylation and reduction, confirmed the presence of a maximum of 13 O-glycans, all on the TNFα receptor moiety. It is also a way to confirm the molecular weight of these subunits, which is normally challenging because of the important heterogeneity of Etanercept.

This comprehensive workflow is fully applicable to other glycoproteins, such as mAbs, ADCs, and other proteins produced by the recombinant DNA technology, using small quantities of sample, and in a reduced amount of time. Other analytical methods are available at Quality Assistance to complete the study of therapeutic proteins glycosylation, such as fucosylation relative quantification, and MALDI-TOF N- and O-glycans profiling.

In the recent years, monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) have emerged as potent and specific anticancer agents. However, due to their size, complexity, and heterogeneity, their physico-chemical characterisation requires a large number of assays aimed at verifying their sequence identity, post-translational modifications, and – in the case of ADCs – range/average number of conjugated drugs (Drug-to-Antibody Ratio: DAR). To this end, we have developed a range of analytical workflows employing mass spectrometry, in a regulated environment, to support pharmaceutical companies in the development and control of their mAbs and ADCs.

Access this 45 minute webinar to learn how state-of-the-art LC/MS technologies can be implemented for the efficient study of ADCs using analyses optimised for each level of information you need. 

Analytical performance of the different solutions proposed will be discussed, together with the presentation of numerous experimental results (with advantages and drawbacks) obtained during method development and optimisation.

We presented efficient and optimised approaches for the characterisation of ADCs by mass spectrometry, based on different levels of analysis: 

• Intact protein for Drug-to-Antibody Ratio determination
• Substructures (after IdeS / FabRICATOR digestion) 
• 2D-LC/MS (HIC-RP) for DAR determination and identification of the different isoforms 
• Peptide mapping for determination of conjugation sites and site-occupancy 

Applications of these methods to KADCYLA^® and/or ADCETRIS^® were also presented. 

The performances and limitations of the different analytical approaches were also discussed.

This application note presents the results obtained during a study performed to evaluate the use of human Platelet Lysates (hPL) as an alternative to Fetal Bovine Serum (FBS) in culture media for Mesenchymal Stem Cells (MSC).

Since decades, the supplementation of cell culture media with Fetal Bovine Serum (FBS) is a well-accepted routine practice in cell culture. FBS provides hormones, cytokines, growth factors and attachment factors that are required for growth and proliferation of cells in vitro. However, FBS is a cocktail of undefined qualitative
and quantitative composition with an animal origin.

During the last years, human Platelet Lysates (hPL) have been proven a valuable xeno-free alternative to FBS in culture media for cells, including Mesenchymal Stem Cells (MSCs). hPL is generated from human platelets by disruption of the platelet membrane after repeated freeze and thaw cycles. The plasma components of hPL require addition of anticoagulants such as heparin to prevent gelatinisation of hPL medium.

hPL is a powerful alternative to FBS for MSCs propagation by increasing proliferation rates and cell densities, with a good viability and phenotype stability.

Biotherapeutics are a class of complex molecules that require the use of innovative techniques for their characterisation. Biacore technology offers label-free assays that provide precise and reliable concentration and kinetic measurements. This can be applied throughout all stages of product development, from discovery to quality control, including stability studies and batch-to-batch consistency evaluation.

Biacore technology was used to develop and validate assays allowing precise characterisation of several types of biotherapeutics.

First, Biacore was used to determine the active concentration of a mAb by CFCA analysis. Concentrations were calculated from the measured binding rates. CFCA enables active concentration determination without the need for calibration standards. An application of quantification of influenza virus concentration in vaccine samples was developed. The Biacore technique appears to be a suitable method for accurate and precise quantification of HA in vaccine samples. The method was validated according to ICH Q2 requirements in the range 0.0625 – 4 μg/ml and shows good accuracy, precision, linearity and specificity.

The second SPR application was the determination of affinity and kinetic parameters of a monoclonal antibody with Fc receptors expressed on the effector cells. We showed that adalimumab exhibited complex kinetic characteristics and that the binding mechanisms differed between Fcγγ receptors. Binding to FcγγR1 was modelled as a 1:1 reaction while FcγR2a and FcγR3a assumed a steady-state affinity independent of polymorphisms. Our assays showed very good repeatability (intra-run precision) and intermediate precision. Two binding assays were also developed to study the kinetics of adalimumab-TNFa interaction. Similar kinetic and affinity parameters were obtained.
Finally, the Biacore comparability tool allowed the comparison of sensorgrams in terms of kinetics. This technology is very useful in the case of stability studies, batch-to-batch consistency evaluation and biosimilarity determination.

Access this 45 minute webinar to learn how state-of-the-art LC/MS technologies can be implemented for the efficient study of monoclonal antibodies using analyses optimised for each level of information you need (from intact protein to peptides/amino acids). Analytical performance of the different solutions proposed have been discussed, together with the presentation of numerous experimental results (with advantages and drawbacks) obtained during method development and optimisation.

Mass spectrometry is a very powerful technique widely used for the characterisation of biopharmaceuticals. 
Thanks to innovations in both hardware and software, this technique can now be implemented routinely. 

We presented versatile and efficient approaches for monoclonal antibodies, based on three levels of analysis: Intact protein, mAb substructures (after IdeS / FabRICATOR digestion), Peptide mapping after trypsin digestion

These were optimised in terms of sample preparation, LC separation (1D and 2D-LC), data processing / reporting

for mAbs isoform characterisation (batch-to-batch consistency, assessment of critical quality attributes, PTMs, glycosylation) and stability monitoring (mainly degradation by deamidation and oxidation). 

The performances and limitations of the different analytical approaches have been discussed, together with the advantages and drawbacks of the different LC columns and enzymes used. Particular attention will be paid to low pH trypsin digestion to minimise deamidation and oxidation for peptide mapping experiments. 
Thanks to the use of state-of-the-art analytical technologies, these reliable, sensitive and reproducible analyses can be done in a GMP regulated environment for QC and stability studies of biotech products.

Non-enzymatic chemical modifications such as deamidation, oxidation and disulfide bond scrambling can affect the stability and efficacy of biotherapeutic proteins. 

Access this 30 minute webinar to learn how the Q3D guideline for EIs can be efficiently implemented for all drug substances, excipients and drug products. 

Drug product manufacturers will soon require Elemental Impurities (EIs) specifications and/or batch analysis for all APIs and Excipients! 

Quality Assistance has developed a unique analytical strategy to reduce the costs linked to these regulation changes. Our concept consists of quantifying all elemental impurities using a fast, cost-effective generic ICP-MS method. Using our methodology will:

• save your customers a lot of time for their risk assessment
• avoid any specific method development to quantify elemental impurities

Such an analytical assessment will very soon become a market requirement and will therefore be a key selling point for you!

Quality Assistance used Biacore T200 technology to develop and validate assays allowing precise mAbs characterisation.

Due to their mode of production, monoclonal antibodies are complex and heterogeneous molecules. Their analysis usually requires the use of many orthogonal analytical techniques in order to fully characterise the different variants. In this application note, the use of mass spectrometry as a powerful tool for adalimumab characterisation is presented.

Humira^® (adalimumab) is an anti-TNFα antibody approved by FDA and EMA for different inflammatory diseases, among which rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, psoriasis and Crohn’s disease. It was the best-selling product worldwide in 2014, with global sales over $13 billion.

adalimumab is a fully human recombinant antibody expressed in CHO cells. As for all proteins produced via recombinant DNA technologies, the final product is a mixture of different variants. The resulting heterogeneity has to be thoroughly characterised as it can impact the safety and efficacy of the product.

Mass spectrometry is a technique that is now widely used for biopharmaceuticals characterisation. Thanks to innovations in both hardware and software, this technique can now be used routinely. We present in this application note the use of mass spectrometry to characterise Humira® at different levels.