Detection of Protein Aggregation by Enzyme Immunoassay
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1 Detection of Protein Aggregation by Enzyme Immunoassay Protein Aggregation Detection (PAD) Technology Microsens Biotechnologies
2 Protein Aggregation Detection (PAD) Technology 1. The Seprion TM ligands 2. Background to the technology 3. Application to analysis of therapeutic protein aggregates 3. Summary
3 The Seprion TM ligands Seprion TM ligands are linear polymers with multiple, charged pendant groups Molecular weight >500 kda Big and flexible
4 The Seprion TM ligands Seprion TM ligands are immobilised to surfaces of 96-well microplates This provides a generic aggregated protein capture surface
5 Seprion TM ligands in an ELISA-like format - protocol The Seprion ligand is provided coated onto 96-well microplates Protocol
6 Seprion-coated microwell Seprion TM ligands in an EIA format Aggregated protein HRP Protein-specific conjugate Seprion TM ligand
7 Seprion TM ligand applications Aggregated proteins detected (so far): prion (human, cow, sheep, deer) β amyloid (human, mouse) tau (human) huntingtin (human, mouse) recombinant human monoclonal IgG human-mouse chimeric monoclonal IgG Research applications Applications in therapeutic protein manufacture. suggests universal binding mechanism
8 Track record in protein aggregation diseases - prions Animal health and food safety licensee for Seprion TM ligand technology: Idexx Laboratories Inc (Westbrook, Maine) Herdchek TM EIA kit for BSE, CWD and scrapie detection Fully validated by EU and USDA Worldwide market leader >3.75 million tests per annum in EU, Japan, US >15 million tests sold since launch in 2007 Largest market for detection of aggregated proteins
9 Application to the analysis of therapeutic protein aggregates The problem Large subvisible aggregates >1-10 µm can induce an innate immune response similar to that induced by bacteria There is regulation against the presence of >10 µm particles but no regulation against the presence of particles <10 µm However, all micron sized particles are derived from oligomer precursors. Arakawa et al* have identified nm particles in a number of therapeutic proteins that act as critical intermediates for the eventual formation of >10 µm particles i.e. bad batches can be predicted by monitoring nm sized particles or oligomers. *Arakawa et al., Bioprocess International. April 2007.
10 Seprion TM ligands in an ELISA-like format Two kits are available; one for monoclonal antibodies which contains a detection conjugate and one for other aggregated proteins Seprion for therapeutic protein aggregates PAD-Mab TM for monoclonal aggregates
11 Seprion TM ligands in an EIA format Seprion for therapeutic protein aggregates PAD-Mab TM for monoclonal aggregates
12 Application to the analysis of therapeutic protein aggregates recombinant human monoclonal Limit of detection of aggregates Method Various amounts of thermally aggregated antibody was spiked into 1 µg of monomeric starting material Results The mean absorbance at 450nm generated in the EIA is shown for each sample, error bar represents S.D, n=3. Conclusion The assay could detect less than 1 ng of aggregated antibody in the presence of a large excess of monomeric antibody.
13 Absorbance 450 nm Application to analysis of protein aggregation - rituximab (Rituxin TM, Mabthera TM ) Thermal aggregation time course - Seprion TM ligand EIA Thermal aggregation time coursestatic light scattering C 52.5 C 55 C Time (min) Andersen et al. Protein Science 19 (2009)
14 Application to analysis of protein aggregation - rituximab (Rituxin TM, Mabthera TM ) Method Thermally aggregated rituximab (120 min at 60 o C) was separated on a Superdex S200 size exclusion column. The trace measuring the absorbance of fractions at 214 nm is shown together with the absorbance at 450 nm following analysis of the aggregates present in these fractions using the Seprion PAD-Mab detection kit (incubation in Seprioncoated microplate well followed by wash and detection using an HRP conjugate) ( ).The inset box shows the first peak at the column void volume at 10 x expanded scale. Results and discussion From the size exclusion column it can be seen that most of the material elutes from the column in a single peak of unaggregated material at min. A small peak of aggregated material can be seen eluting at about 8.38 min. The Seprion based PAD-IgG assay which is specific for aggregated IgG, detects a large signal from aggregated material from min and the large quantity of unaggregated material eluting after this time is not detected.
15 OD Effect of aggregation method on rituximab aggregate size and the effect of different capture buffers Native protein not filtered. Buffer A capture Native protein not filtered. Buffer B capture Native protein filtered. Buffer A capture Native protein filtered. Buffer B capture Heat aggregated not filtered. Buffer A capture Heat aggregated not filtered. Buffer B capture Heat aggregated filtered. Buffer A capture Heat aggregated filtered. Buffer B capture Shaken aggregate not filtered. Buffer A capture Shaken aggregate not filtered. Buffer B capture Shaken aggregate filtered. Buffer A capture Shaken aggregate filtered. Buffer B capture Filtered samples were filtered through a 0.2 micron filter after aggregation and prior to assay Buffer A, not selective. Buffer B, selective for large aggregates > 200 nm. Heat aggregation produces smaller (<200 nm) aggregates that are not removed by filtration. Shaking produces larger (>200 nm) aggregates that can be removed by filtration. Buffer A is not selective for detection of aggregate size. Buffer B is selective for the detection of larger aggregates.
16 Methods for Detection and Characterization of Visible and Sub-visible Particles - these methods cannot differentiate between particles and aggregated proteins Method Principle Observable Size rangea Advantages Disadvantages Visual inspection Visualization, manual or automated Optical microscopy Microscopy-aided visualization of particles Assessment of clarity, opalescence, turbidity; visible particles Size and morphology of particles Light obscuration Blockage of light by particles Concentration and size of micron-sized particles Flow imaging Microscopic imaging Concentration, size and morphology of micron-sized particles Fluorescence microscopy Conductivity based particle counter Laser diffraction Dynamic light scattering Detection of induced fluorescence Electrical sensing zone method (Coulter method) >50 μm mm Easy to perform, information on particle size and shape >1 μm mm Easy to perform, information on particle size and shape μm Rapid analysis; counting and (size class) clustering of particles μm Potentially allows differentiation between protein aggregates and non-proteinaceous particles; shape information also obtained; may detect translucent particles Particles, amyloid proteins >1 μm mm High sensitivity; selective for protein aggregates; information on particle size and shape Concentration and size of (sub)micron-sized particles Laser light scattering/reflection Laser light scattering relative to particle size Fluctuations in scattered light intensity due to Brownian motion 0.4 1,200 μm Single particle detection, counting and characterization 20 nm 2 mm Single particle detection, counting and characterization Hydrodynamic size 1 nm 5 μm Easy to perform (batch mode); nondestructive; high sensitivity; low sample consumption Low resolution; limited particle discrimination; probabilistic nature; subjective; trained personnel or expensive equipment needed Limited resolution; sample preparation may create artifacts Large sample volume; no morphological information; may miss translucent particles; very sensitive to sample contamination (e.g. artifacts by air bubbles) Limited particle characterization; only part of sample is analyzed; high data volume; emerging technique Restricted to labeling/dyes/filter sets; no quantification possible Dilution of low conductivity samples into an electrolyte solution Requires high sample dilution Complicated data analysis; not quantitative; low resolution (weak differentiation between particle species); less suitable for polydisperse samples; very sensitive to contamination (e.g. dust) Nanoparticle tracking analysis MALLS Microscopic visualization by laser light scattering of Brownian motion of particles Time-averaged light scattering intensity of particles, detected at multiple angles Hydrodynamic size Molar mass, size (radius of gyration) 20 nm 1 μm Single particle detection and characterization; very useful for polydisperse samples kda MDa range High sensitivity; absolute determination of size; commonly used as online detector Low sample throughput; visualization, no imaging; trained personnel needed; emerging technique Solute concentration must be known; complicated data analysis; not quantitative; limited use in batch mode; very sensitive to sample contamination Turbidimetry, nephelometry Time-averaged light scattering intensity of particles Particle size/concentration dependent optical density or light scattering intensity From Engelsman et al Pharm Res April; 28(4): N/A Easy to perform; various designs and Observed signal depends on both size and methodologies concentration; no information on individual particle properties (number, size distribution); suitable only for comparative measurements
17 Methods for Detection and Characterization of Visible and Sub-visible protein aggregates Method Principle Observable Size range Advantages Disadvantages Electron microscopy Visualization Atomic force microscopy Topographical scanning SAXS/SANS Scattering of X- ray/neutron beam Native mass spectrometry Macro-ion mobility spectrometry Analytical Ultra Centrifugation Mass/charge detection of ionized molecules in a field Ion mobility and counting Sedimentation velocity (SV) Size and morphology of protein aggregates Size and morphology of nm-sized particles Size and shape of molecules/aggregates in solution Mass/charge ratio nm mm nm range, vertical resolution 0.01 nm Large size range; high resolution; detection of chemical composition of a particle (energy dispersive X-ray analysis) Molecular resolution; morphological particle properties From Engelsman et al Pharm Res April; 28(4): Sample preparation may create artifacts; limited representativeness of selected image area; not quantitative; time consuming; expensive; expert personnel needed Particle isolation and measurement itself may create artifacts; time consuming; expensive; limited representativeness of selected image area; expert personnel needed nm range High resolution Time consuming; expert personnel needed Atomic resolution up to MDa range Mass 3 65 nm or 5 kda 100 MDa Molecular weight and/or shape Very high resolution; high Gas phase analysis; requires accuracy and precision exchange into volatile buffer; high sensitivity detailed not quantitative; expensive structural information equipment and expert online detection possiblities personnel needed High resolution; high Gas phase analysis, requires molecular weight precision; exchange into volatile buffer; broad molecular weight requires dilute samples; range; no matrix interaction emerging technique during separation 1 nm 0.1 μm Absolute method; measurement of molecule size, shape; quantification of protein complexes; high resolution; applicable for wide conc. range Strongly dependent on quality of instrument components (e.g. centerpieces); complex data analysis; time consuming; expensive instrumentation; expert personnel needed
18 Summary Competing technologies Most methods measure particles, not protein aggregates Most methods not very sensitive Many require specialist skills and equipment Most methods are restricted in size of particles measured Not high-throughput The Seprion technology has unique properties compared to the other approaches Specifically detects protein aggregates not dust or other particles Sensitive (<1ng aggregate) Can detect a wide range of aggregate sizes nm to >µm Does not require a high skill for use or interpretation Convenient format - microplate for simple high-throughput testing Does not require expensive equipment
19 Summary Recommended process for protein aggregation detection in biopharmaceuticals..use Seprion TM ligand technology throughout a development/manufacturing process to detect presence of any aggregates research development production
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