Colour coding changes: Abvance Biotech, Richard, GSK, Lilly, MedImmune, Novartis

Impact of production process:

Likely (define cause of potential impact or reason), or

Unlikely

Likelihood of comparability definitions (assumption that individual assay within the same organisation is reproducible):

High - possible for direct comparison of absolute values across assay clusters which observe the same phenomenons e.g. DSF & DSC

Medium - possible for direct comparison of absolute or relative (wrt appropriate controls) values between individual assays

Low - possible to compare individual assays only using RAG flags

Parameter measured	Assay name	Assay abbreviation	Assay description (link to primary citation)	Purpose	Tech platform/aka (e.g. NanoPro)	Used by <company>	Stage	Measure of (units)	Data needed for BASECASE	Impact of production process	Likelihood of comparability - inc. assay clusters which observe the same phenomenons	Additional information or comments
INTRINSIC MOLECULAR PROPERTIES
Chemical stability	Capillary Isoelectric Focusing	cIEF	High throughout, capillary-based isoelectric immunoassay to determine charge profile and pI of molecule Michels et al., Anal. Chem 2012 dx.doi.org/10.1021/ac3008847	Measure of pI and charge heterogeneity - used in conjunction with LC-MS.	NanoPro Beckman Proteinsimple	GSK (check if used as control to determine pI) Lilly MedImmune (check)	Early selection Final profiling Endpoint data	pI	Capture buffer conditions Record pI of main isoform Record proportion of main isoform to acidic and basic species	Likely - Glycan composition will affect charge profile, therefore antibodies produced by different processes may show different profiles Low for antibodies (May be significant for other proteins in view of glycosylation impact)	High Should be comparable even with subtle differences in method and antibody formulation	Parameter of low value - can be predicted in silico reliably enough from sequence (published)
Chemical stability, Immunogenicity	Liquid chromatography-Mass spectrometry	LC-MS		Intact molecular mass and 'standard' glycosylation pattern and obvious fragmentation.		Abvance GSK Lilly MedImmune Novartis (check)	Final profiling Endpoint data	MW % intact species % of respective glycovariants	Capture buffer conditions	Likely - Glycan composition will affect 'standard' pattern	High	Parameter of low value - intact molecular mass and fragmentation can be assessed by SDS-PAGE. Ranking of molecules by LC-MS and CE-SDS comparable Re. Immunogenicity, need to define what a 'standard' glycosylation pattern is and how this is linked to immunogenicity
Structural stability	Differential Scanning Fluorimetry (including Thermofluor) (including Nano DSF)	DSF	DSF is a fast and economical method to determine the temperature at which a whole antibody or a Fab unfolds (Tm), i.e. the temperature at the mid-point of the unfolding transition. The Tm is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which become progressively more exposed as the antibody unfolds. The Tm is assigned using the first derivative of the raw fluorescence emission data.	Rank Fabs and antibodies according to their thermal stability as measured by the Tm. Screen for favourable buffer and/or additive conditions.	iQ5, CFX96 Real-Time Systems (Bio-Rad) Prometheus (Nanotemper)	GSK Lilly (rarely) MedImmune Abvance Novartis (Jain et al)	Early selection	Thermal stability (ºC)	Capture buffer conditions Record number of unfolding transitions Capture which dye is used as this can affect Tm (e.g SPYRO Orange) Capture conc of sample as will affect Tm Capture temp gradient used during run (e.g. A temperature ramp from 40 ºC to 95 ºC at a rate of 0.5 ºC/2 min) Capture whether Tm is for full IgG or Fab	Unlikely	High (assuming similar buffer conditions)	Ranking of molecules by DSF and DSC comparable
Structural stability	Differential Scanning Calorimetry	DSC		Rank Fabs and antibodies according to their thermal stability as measured by the Tm. Screen for favourable buffer and/or additive conditions.		Lilly MedImmune Novartis	Final profiling	Thermal stability (ºC)	Capture buffer conditions Record number of unfolding transitions Capture conc of sample as will affect Tm Capture temp gradient used during run (e.g. A temperature ramp from 40 ºC to 95 ºC at a rate of 0.5 ºC/2 min) Capture whether Tm is for full IgG or Fab	Unlikely	High	Ranking of molecules by DSF and DSC comparable
Structural stability, (self) Aggregation	Analytical Size-Exclusion Chromatography	aSEC	Antibodies are flowed through chromatography media consisting of Non-aggregated dimeric and monomeric antibodies elute as defined, well-separated peaks. Aggregated antibodies flow through the column faster and elute before the non-aggregated antibody peaks. Percentage aggregation can be worked out from the ratio of peak surface areas under the aggregated vs non-aggregated antibody peaks.	Verify structural integrity, oligomeric state, percentage aggregation of antibody samples.	GE Healthcare Agilent	GSK MedImmune Lilly Abvance Novartis (Jain et al)	Early selection Final profiling	Elution time, peak width, peak asymmetry relative to a well-behaved control antibody (% monomeric species, retention time per unit volume or time)	Capture buffer conditions Capture column type (e.g beads 25-45 µm in size - GE Superdex 200). Capture Flow rate Relative size (MW)	Unlikely	High
Chemical stability (integrity), Structural stability (integrity)	Capillary electrophoresis	CE-SDS		Verify chemical and structural integrity, quantify fragmentation	Beckman Caliper	GSK Lilly MedImmune (check) Novartis	Final profiling Endpoint data	Approx. MW Impurities	Capture sample prep conditions: reduced, non-reduced, denaturation	Unlikely	High	Ranking of molecules by LC-MS and CE-SDS comparable
(self) Aggregation (Hydrophobicity)	Hydrophobic Interaction Chromatography	HIC	HIC separates antibodies on non-biological surfaces based on the distribution and nature of the antibody hydrophobic patches. This approach assumes that the increased retention of antibodies on hydrophobic columns at moderate to high salt concentrations (e.g. ammonium sulphate, ammonium phosphate) correlates with increased antibody hydrophobicity. Antibodies are eluted in a mobile phase without added salt, i.e. only in buffer.	Assess non-specific interactions. Rank antibodies according to their relative hydrophobicity	GE Healthcare Agilent	GSK Lilly MedImmune Abvance Novartis Jain et al	Early selection	Retention time (retention time per unit volume or time)	Capture buffer conditions Capture column type (e.g beads 25-45 µm in size - GE Superdex 200). Capture salt gradient	Unlikely	High should be comparable (relative retention time rather than absolute values)	Retention time used for ranking of molecules.
Structural stability, (self) Aggregation	Dynamic Light Scattering	DLS	Dynamic Light Scattering (DLS) measures the translational diffusion coefficients Dt of nanoparticles and colloids in solution by quantifying dynamic fluctuations in scattered light due to Brownian motion of the molecules in liquid. Sizes and size distributions, in turn, are calculated from the diffusion coefficients in terms of hydrodynamic radius rh or hydrodynamic diameter dh. Technique can be used with high concentration samples (cf SEC)	Measure hydrodynamic radius (characterise/quantify aggregation)	Wyatt	Lilly (rarely) MedImmune	Final profiling Endpoint data	hydrodynamic radius (nm)	Capture buffer conditions Sample concentration Molecule type	Unlikely	High should be comparable for same buffer conditions and sample concentrations	Ranking of molecules by DLS and AC-SINS comparable
(self) Aggregation	Self-interaction by light scattering	K-Diff	DLS experiment which measures the propensity for molecule to self-associate under particular conditions. K-diff = Association constant defined by diffusion.	Rank antibodies according to their propensity to self-assemble	DLS	Lilly MedImmune (check) Novartis	Final profiling	(mL/g)	Capture buffer conditions Capture concentration range K-diff	Unlikely	Probably High
(self) Aggregation	Affinity-Capture Self-Interaction Nanoparticle Spectroscopy	AC-SINS	Sule et al., Mol. Pharmacuetics, 2013 dx.doi.org/10.1021/mp300524x High-throughput, low protein screen for self-association propensity, where antibodies are captured onto gold nanoparticles. Self-association is determined if the nanoparticles with captured antibody clump together resulting in a red-shift.	Assess propensity of antibody to self-associate / RSA at high concentrations	Gold nanoparticles Scanning plate reader	MedImmune Jain et al	Early selection	Plasmon wavelength redshift in nm	Capture buffer conditions Record redshift in nm, relative to non-capture / buffer only controls	Unlikely	Should be largely comparable if buffer conditions are the same
(self) Aggregation	Multiangle laser light scattering	MALLS	MALLS describes a technique for measuring the light scattered by a sample into a plurality of angles. It is used for determining both the absolute molar mass and the average size of molecules in solution, by detecting how they scatter light. (wikipedia)	Determine symmetry or presence of multiple aSEC peaks and shape independent measure of MW		Abvance						Does not provide much additional information over aSEC experiment
Chemical stability, (Immunogenicity)	In silico predictions			Predict pI and PTM sites in CDR		GSK MedImmune Novartis	Early selection					Not of high interest for comparisons. Can be applied to all selected molecules easily if wished for.
human, cyno, mouse (GSK), rat (Novartis) PK		FcRN interaction (SPR)	Binding to FcRn contributes to the long half-life of IgGs and albumine. Properties of antibodies to bind FcRn receptors is assessed for candidate ranking and risk minimizing.	Assessment of FcRN binding properties to identify risk of unfavorable PK.	Biacore EPIC	GSK Lilly Novartis	Final profiling Endpoint data	Apparent affinity at pH 6.0, 7.4 to FcRn (M) % Residual binding to FcRn observed	Capture buffer conditions Capture assay setup (ratio Ab:receptor, fitting model) and readout (ratio Ab:receptor)?	Unlikely	Medium/Low Comparability vs control, not absolute numbers as dependent on conditions used	Not necessarily predictive - unusual activity for FcRN signifies potential issue. However, in some cases no FcRN activity results in abnormal PK.
(human, cyno) PK	Heparin Sulphate Chromatography			Rule out proteoglycan interactions affecting PK	HPLC (Agilent, Waters, Thermo, etc)	Lilly	Early selection Final profiling	Retention time (or salt concentration at elution), relative to control antibodies	Column details Chromatography conditions (buffer, salt gradient details)	Unlikely	High	Not standard use. Data comparable to heparin binding (in cells)
(human, cyno) PK	ELISA Baculovirus particles	BVP ELISA	ELISA on baculovirus particles and non coated ELISA plates under low stringency blocking conditions to assess nonspecific binding / polyspecificity of lead antibody candidates. Binding observed to either BVPs or plastic indicates that the antibody has a risk of faster nonspecific clearance in vivo. Hotzel et al., mAbs, 2012 https://www.tandfonline.com/doi/abs/10.4161/mabs.22189	Polyspecificity / nonspecific binding / aberrant PK risk	ELISA	MedImmune Jain et al	Early selection Final profiling	Absorbance at 450nm if using HRP detection	Capture buffer conditions Capture concentration range of antibodies tested Record absorbance on both BVPs and non-coated wells Record binding ratio relative to controls (sticky and non-sticky)	Unlikely (providing there is high monomer purity)	Whether an antibody flags or not, should be comparable. Actual numbers may not be	Low use across industry - possibly used by Adimab and Genentech
(human, cyno) PK	Nonspecific HEK or CHO cell binding by flow cytometry (HyperCyte) or non-wash cytometry (Mirrorball)	HEK/CHO binding	Providing the target for an antibody panel is not expressed on a certain cell type (e.g. HEK293 or CHO), then assay systems using this cells are effective in determining the polyspecificity and therefore PK risk.	Polyspecificity / nonspecific binding / aberrant PK risk	Flow cytometry, Mirrorball (TPP Labtech)	MedImmune	Early selection Final profiling	Fluorescence counts	Capture assay conditions Capture concentration range of antibodies tested Record total fluorescence counts Record fluorescence ratio relative to controls (sticky and non-sticky)	Unlikely (providing there is high monomer purity)	Whether an antibody flags or not, should be comparable. Actual numbers may not be
Viscosity	Viscosity			Assess viscosity of unformulated vs formulated antibody	Malvern Rheometer Malvern Viscosizer	GSK (not routine so limited data) Lilly Novartis MedImmune (check)	Endpoint data		Capture buffer conditions Capture concentration	Unlikely	Low High if measured in similar buffer conditions	Very condition dependent so we need to get a sense of what comparisons are possible
ACCELERATED STABILITY (AS) PROPERTIES Stressed samples will be compared to non-stressed samples to determine changes due to accelerate stability conditions. Capture stress conditions (sample treatment) applied ahead of running each assay: stress stimulus, buffer conditions, storage temperature, sampling timepoints (incubation time)
Chemical stability	Capillary Isoelectric Focusing	cIEF	High throughout, capillary-based isoelectric immunoassay to determine charge profile and pI of molecule Michels et al., Anal. Chem 2012 dx.doi.org/10.1021/ac3008847	Change in acidic and basic variants. Effect on binding (SPR) of changing pI.	NanoPro Beckman Proteinsimple	GSK Lilly MedImmune	Early selection Final profiling Endpoint data		Capture buffer conditions Record pI of main isoform Record proportion of main isoform to acidic and basic species Record change in main isoform upon stress to acidic or basic species			As above, except stressed samples will be compared to non-stressed samples to determine changes in charge profile due to AS conditions.
Chemical stability	PTM by Liquid chromatography-Mass spectrometry	LC-MS (PTM)	Description (citation)	Identification and approx. quantification of post-translational modifications by peptide mapping	ESI-MS	Lilly Novartis	Final profiling Endpoint data	Relative amount of modification	Capture buffer conditions Capture digest conditions (pH)	Medium	High	Detailed experiment carried out on AS samples
Structural stability, (self) Aggregation	Analytical Size-Exclusion Chromatography	aSEC (AS)	Antibodies are flowed through chromatography media consisting of Non-aggregated dimeric and monomeric antibodies elute as defined, well-separated peaks. Aggregated antibodies flow through the column faster and elute before the non-aggregated antibody peaks. Percentage aggregation can be worked out from the ratio of peak surface areas under the aggregated vs non-aggregated antibody peaks. (citation)	Verify structural integrity, oligomeric state, percentage aggregation of antibody samples.	GE Healthcare Agilent	GSK MedImmune Lilly Abvance Novartis (Jain et al)	Early selection Final profiling Endpoint data	Elution time, peak width, peak asymmetry relative to a well-behaved control antibody (% monomeric species, retention time per unit volume or time) (% high molecular formation per unit time)	Capture buffer conditions Capture antibody concentration Capture column type (e.g beads 25-45 µm in size - GE Superdex 200). Capture Flow rate Relative size (MW)	Unlikely	High	Same as aSEC but using stressed samples
Chemical stability (integrity), Structural stability (integrity)	Capillary electrophoresis	CE-SDS		Verify chemical and structural integrity, quantify fragmentation	Beckman Caliper	GSK Lilly MedImmune (check) Novartis	Final profiling Endpoint data	Approx. MW Impurities	Capture sample prep conditions: reduced, non-reduced, denaturation	Unlikely	High	Used in AS Cluster with LC-MS
Structural stability, (self) Aggregation	Dynamic Light Scattering	DLS	Dynamic Light Scattering (DLS) measures the translational diffusion coefficients Dt of nanoparticles and colloids in solution by quantifying dynamic fluctuations in scattered light due to Brownian motion of the molecules in liquid. Sizes and size distributions, in turn, are calculated from the diffusion coefficients in terms of hydrodynamic radius rh or hydrodynamic diameter dh. Technique can be used with high concentration samples (cf SEC)	Measure hydrodynamic radius (characterise/quantify aggregation)	Wyatt	Abvance GSK	Final profiling Endpoint data	hydrodynamic radius (nm)	Capture buffer conditions Molecule type Sample concentration	Unlikely	High - should be comparable for same buffer conditions and sample concentrations	Cluster with AC-SINS
Chemical stability	Cation Exchange Chromatography	CEC (IEC)	Description (citation)	Change in acidic and basic variants.		Novartis (GSK)			~~Capture buffer conditions~~
Chemical stability	Capillary Zone Electrophoresis	CZE	Description (citation)	Change in acidic and basic variants.		Novartis	Final profiling		~~Capture buffer conditions~~
Structural stability, (self) Aggregation	Analytical Ultra Centrifugation	AUC	Low throughput method used to characterise and quantify aggregation (especially at high concentrations). Two types - sedimentation velocity or sedimentation equilibrium. Sedimentation velocity uses centrifugal force to measure sedimentation coefficient, diffusion coefficient and mass of sample.	Characterise and quantify aggregation	Beckman Coulter XLI-1	GSK Lilly Abvance	Final profiling	mass, distribution, sedimentation coefficient	Capture buffer conditions Molecule type concentration rotor speed		should be comparable for same buffer conditions
Chemical stability, Structural stability	Surface Plasmon Resonance	SPR	SPR measures changes in refractive index at a gold sensor chip surface which are proportional to changes in mass. It is used to measure binding kinetics and affinities of molecules. SPR can be used in conjunction with other techniques to look at the consequence of chemical or structural changes on the affinity of an antibody (an intrinsic property) to either to its antigen of interest or Fc receptors e.g. FcRn. SPR can also be used to measure the active concentration of a molecule (i.e. the proportion able to bind).	Measure changes in active concentration or kinetics. Change in target binding upon stress	Biacore ProteOn	GSK Novartis	~~Final profiling~~? Endpoint data	Ka (1/Ms), Kd (1/s), KD (M), active concentration (%)	Capture buffer conditions chip/surface used assay format (i.e. ligand and analyte) concentrations	technique measures impact of chemical/structural changes Low (define)	should be comparable if 1:1 assay format used High (define)	Probably not of high interest for comparison and prediction
	Capillary Gel Electrophoresis	CGE				GSK	Endpoint data		Capture buffer conditions
	PEG-induced precipitation					Lilly	Final profiling		Capture buffer conditions
	Reversed phase HPLC					GSK	Endpoint data		Capture buffer conditions
	Rheometry			Turbidity		Novartis	Endpoint data		Capture buffer conditions

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