Scientific Library

The discovery of human pluripotent stem cells 10 years ago turned the spotlight on the potential of pluripotent stem cells for personalised cell therapy. The scientific interest then quickly shifted towards the use of these cells for safety pharmacology, drug discovery and disease modelling. For all these purposes, in the mid to long term, properly characterised cell banks will be necessary. 

The characterisation of embryonic (ESC) and induced pluripotent stem cells (IPSC) used for manufacturing requires the development and validation of analytical methods (e.g. flow cytometry, microscopy, QPCR and bioassays). Cell characterisation includes the testing of cell product identity, determination of impurities, and assessment of biological activity and viability. Among the techniques available, flow cytometry is widely used to assess the expression of cell markers. Our laboratory has developed flow cytometry panels dedicated to the characterisation of extracellular and intracellular markers of ESC and IPSC, and to the detection of cell-related impurities. We proposed a method for the validation of flow cytometry panels according to the recommendations of international guidelines on the validation of analytical methods.

IPSC differentiated into cardiomyocytes and MSC-like cells were also used to test the performance of our flow cytometry panels to accurately monitor the manufacturing process of cell products.

In addition to the technical tips, this webinar aims at presenting a critical view on the use of flow cytometry platform for cell characterisation. 

Access the slides of this 45 minutes webinar to learn how to characterise pluripotent stem cells in terms of identity and purity. The analytical performance of the methods developed have been discussed along with the presentation of experimental results obtained during method development, optimisation and/or validation.

Access the slides of this 45 minute webinar to learn how to characterise human mesenchymal stem cells in terms of identity, purity and biological activity. The analytical performance of the methods developed have been discussed along with the presentation of experimental results obtained during method development, optimisation and/or validation.

Human mesenchymal stem cells are widely used in the field of cell therapy, a technology used for the replacement and regeneration of dysfunctional cells by healthy functional cells.

The characterisation of human MSCs used for manufacturing cell therapy products requires the development and validation of analytical methods which meet regulatory requirements. Cell therapy products emerge from a long process of cell culture, differentiation and purification where appropriate cell identity, purity and biological activity (potency) monitoring is mandatory. According to minimal criteria proposed by the International Society for Cellular Therapy (ISCT), MSCs must have the ability to adhere to cell culture plastic surfaces, express some phenotypic markers and differentiate into adipogenic, osteogenic and chondrogenic lineages.

Identity and purity tests can be based on the analysis of one or more specific markers by flow cytometry, fluorescent microscopy and/or qPCR. Biological activity (potency) can be measured using enzymatic assays or immunoassays linked to the relevant biological properties of the cell therapy product. We will present some efficient, optimised approaches developed at Quality Assistance for the characterisation of human mesenchymal stem cells

Access the slides of this 45 minute webinar to learn:

• How to overcome the challenges usually associated to Multiplex assays
• How to select the right technology for your needs

Cytokine profiling is a powerful tool to link the host immune system with disease pathogenesis and/or treatment efficacy. 
The analysis of cytokine expression is frequently required for the characterisation of the status of the immune system, to demonstrate the function of the cells of interest and as an analysis of surrogate markers for in vitro potency testing. 

Classically, the analysis of cytokine expression is performed by ELISA for each separate analyte. 
Multiplex assays allow the simultaneous measurement of multiple cytokines in a sample, increasing the efficiency (less time-consuming and less expensive) and considerably reducing the sample volumes needed.

In the context of a 9 month internal R&D project, we have compared three multiplex technologies for the measurement of cytokines:

• Electrochemiluminescence (ECL, MSD)
• Magnetic beads (Luminex, Merck Millipore) 
• Cytometric Bead Array (FACS Verse, BD)
• The performance of the three assays was evaluated in terms of their accuracy, repeatability, intermediate precision, quantification range and ability to detect cytokines in human PBMC samples. 

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

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. 

We presented efficient and optimized approaches for the characterisation of protein glycosylation by UPLC/mass spectrometry, based on different levels of analysis: 

• Fast and highly sensitive profiling of released N-glycans, with use of the RapiFluor-MS^® technology, 
• Specific release and profiling of O-glycans,
• Orthogonal released glycans analysis and sialylation profiling by mixed-mode chromatography, 
• Unambiguous determination of N- and O-glycosylation sites by widepore-HILIC peptide mapping, 
• High-resolution intact and subunit analysis. 

Applications of these methods to mAbs (Humira^®, Erbitux^®) and highly glycosylated proteins (Enbrel^®) will be presented. 

The performances and limitations of the different analytical approaches will be discussed. 

Access the slides of this 45 minute webinar to learn how Surface Plasmon Resonance can be implemented in a GMP regulated environment for the comprehensive study of monoclonal antibodies and other Biologics. 

The biological activity of monoclonal antibodies and their pharmacokinetics are highly dependent on their binding to Fcg and FcRn receptors and to their target antigen. 

An adequate control of these binding properties is therefore required for characterisation, batch release, batch-to-batch consistency and stability studies. 

Based on the example of Humira^® (Adalimumab), we will present the use of Surface Plasmon Resonance technology on a Biacore T200 system for the characterisation of monoclonal antibodies as regards: 

• Binding to Fcg and FcRn receptors 
• Binding to target antigen (TNFa) 
• Determination of active concentration by CFCA (Calibration-Free Concentration Analysis)
• Comparison of sensorgrams in the context of stability or comparability studies.

All these reliable, sensitive and reproducible analyses were developed by Quality Assistance and can be used for QC and stability studies of biotech products. 

Access the slides of 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.

Access the slides of 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.

Access the slides of 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!

Access the slides of this webinar which presented how the Q3D guideline for EIs can be efficiently implemented for all drug substances, excipients and drug products.

On 1st June 2016, all new drug products will have to comply with the ICH Q3D guideline for elemental impurities (EIs) and on 1st January 2018, all existing products will also have to fulfill the same requirements. Although this guideline sets specifications for drug products only, the risk assessment approach also involves the determination of metallic impurities in APIs and excipients.

Considering the huge number of samples which will have to be tested, Quality Assistance has developed and validated a generic cost effective ICP/MS procedure for the determination of the EIs listed in Q3D.
Our staff of experts is ready to help you to design an appropriate risk-based strategy and to carry out the necessary analytical determinations with our dedicated instruments (5 ICP-MS).

Although drug products issued from biotechnology have now been on the market for many years, accurate protein quantification remains a challenge. There are many colorimetric methods for protein quantification (Lowry, Bradford, BCA) but they all lack precision, and suffer from interferences and matrix effects, moerover, the accuracy is highly dependent of the protein used for calibration (...)