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Protein analytics

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Protein analytics

Icosagen has a dedicated department for protein analytics to ensure meticulous product characterization for optimal quality of both antibodies and recombinant proteins.

For customized solutions we have a comprehensive list of analytical options available to cover all the needs for proteomic characterization.

Different proteins produced and purified in 2023

In process control

Involves standard methods to analyze molecular weight, check molecule expression and secretion during production as well as confirm that produced antibodies are functional and bind to the target antigen.

Analytics play a key role in protein production in order to get functional proteins with high purity. Most commonly used methods are:

Coomassie stained SDS-PAGE and LabChip capillary electrophoresis — a standard test to analyze:

molecular weight
check molecule expression and secretion during production
estimate product purity during purification.

Western Blot is used to detect proteins from different samples.

ELISA test:

confirm that produced recombinant proteins are functional
determine EC50 values for antibodies
perform competitive binding assays.

Final product quality control

Analytical Size-Exclusion Chromatography (analytical HPLC-SEC):

characterize final product or purified fraction by molecular weight to monitor the purity of the product and detect the presence of aggregates or low molecular weight contaminants.

Analytical Ion-Exchange chromatography (analytical HPLC-IEX):

determine protein charge variant distribution
control final fraction of bispecific antibodies to assure no monospecific antibody versions are present, in case there is sufficient charge difference between these two.
This is usually supplemented with mass-spectrometry.

LAL-test (Charles River Endosafe® nexgen-PTS™):

determine the level of bacterial endotoxins in purified recombinant protein samples.

cGE by LabChip GXII Protein Characterization System (PerkinElmer) — provides the complete solution for product quantitation and molecular weight sizing or impurity and homogeneity analysis.

Product characterization

For customized solutions we have a comprehensive list of analytical options for glycosylation pattern analysis, stability studies in different stress conditions as well as formulation studies.

All-in-one protein stability analysis with the Uncle platform (Unchained Labs) — a stability platform that uses scanning fluorimetry (nanoDSF), static light scattering (SLS) and dynamic light scattering (DLS) detection methods to characterize protein stability. Allows to:

rank the stability of your products based on thermal melting and aggregation (Tm and Tagg)
compare different formulations to find the most favorable conditions for analyzed molecules
find excipients that prevent or delay the onset of aggregation
identify the presence of aggregates or impurities
isothermal stability.

Biolayer interferometry (BLI) binding analysis — binding and affinity analysis with Octet platform (Sartorius):

kinetics and affinity of
intermolecular interactions
assessing binding specificity
epitope mapping
rates of association and dissociation.

Surface plasmon resonance (SPR) binding analysis – antibody kinetics analysis with Carterra LSA instrument:

High-throughput kinetic profiling (ka, kd, KD + cross-selectivity)
- up to 384 x 1152 interactions per run with non-covalent capture (from purified samples and/or supernatants)
- up to 384 x 384 interactions per run with covalent coupling (from purified samples)
- analysis of mono-and multispecific antibodies.
High-throughput epitope binning

Mass spectrometry analysis (Exactive 240 with Vanquish Neo LC, Thermo Fisher Scientific):

determination of intact mass,
determination of intact mass (deglycosylated sample),
protein identification by peptide mapping,
protein N- and O-glycosylation profiling,
protein O-glycosylation site identification
protein N-glycosylation identification and quantitative determination of modification site occupancy.

Glycosylation pattern analysis:

Hydrophilic interaction liquid chromatography (HILIC) — an alternative high-performance liquid chromatography (HPLC) that can be used for identification and quantification of N-glycans or to determine fucosylation level in antibodies.

Capillary isoelectric focusing (cIEF) with Maurice platform (ProteinSimple).

Lunatic (Unchained Labs) – high-throughput concentration measurements with UV absorbance.

Antibody
development

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Protein
production

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Cell line
development

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Analytics

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