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Big Molecule PK Profiling
Bioanalysis Platform Capability
- Detection: Fc, Fab, ADC, TAG, cytokine, and VHH
- AH50/CH50 hemolysis complement assay
- Species: Mouse, Monkey, Rat and human
- Anti-ds DNA Ab detect
- Data analysis with WinNonlin, interpretation and trouble shooting
- Receptor Occupy assay in mouse, rat, and monkey
- MDCK-hFcRn transcytosis for PK assay in vitro
- ADA & Nab detections with standardized methods
- Application: IgG ,Fc fusion protein, Fc mutation, ADC, and YTE etc.
- Multiple assay platform: ELISA, MSD, FACS, FRET, qPCR and HTRF
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IgG PK Assay Strategy
Coat antibody Detect antibody Drug Detection range Instrument Mouse Anti-Human
IgG（from AB MAX)HRP-Goat Anti-Human
IgG (from Jackson)Trastuzumab
(In house)320-40960
ng/ml384Plus
-SpectraMax -
IgG PK StandardizationDetermine the detection rangeDetermine the coating and detection antibody concentrations and incubation conditionDetermine Dilution Ratio（MRD)Determine Standard Curves and Quality Control Values （A&P Run）Method Validation:Reproducibility, Recovery, Selectivity, Stability, Dilution LinearitySample Analysis, troubleshooting and report
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Platform for Antibody PK Capability（Mouse）
Parameter Unit WT RAGKO Cmax μg/ml 13.6 11.3 Tmax h 0.25 0.417 AUC 0-t μg/ml*h 1300 1100 t1/2 h 344 223 MRT 0-inf_obs h 485 317 Validated PK methodologies (ELISA, MSD, CBA):Fc, Fab, TCR, ADC, His, cytokine, and NanobodyMultiple PK parameter analyses with PK WinNonlin softwareExperienced with mouse, rat and monkey PK studies for hundreds of antibodies. -
Monkey Antibody PK Profiling
Parameter Unit WT RAGKO Cmax μg/ml 191 134 Tmax h 115 35.2 AUC 0-t μg/ml*h 46500 12600 t1/2 h 126 56.6 MRT 0-inf_obs h 233 80.2 Validated PK methodologies (ELISA, MSD, CBA):Fc, Fab, TCR, ADC, His, cytokine, and NanobodyMultiple PK parameter analyses with PK WinNonlin softwareEstablishment for monkey PK studies and pre-tox studies. -
IgG ADA StandardizationDetermining Conditions:Detection range, coating and detect antibody concentrations and incubation, SCP(Screening Cut Point), CCP(Confirmatory Cut Point）and TCP(Titer Cut Point)Method Validation:Reproducibility, Drug tolerance, Selectivity, Sensitivity and StabilityControls:Positive and negative sample ConfirmationSample Analysis:ELISA or MSDTiter Determination and Trouble Shooting
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Bioanalysis Platform for ADA AnalysisADA analysis in Mouse Dosed Trastuzumab
Sample WT-1 WT-2 WT-3 RAGKO-1 RAGKO-2 RAGKO-3 WT-1 WT-2 WT-3 RAGKO-1 RAGKO-2 RAGKO-3 Dosing Pre-dose Pre-dose Pre-dose Pre-dose Pre-dose Pre-dose IV2+SC IV2+SC IV2+SC IV2+SC IV2+SC IV2+SC Inhibition Ratio -24.8 -22.1 -6.4 -1.3 6.0 5.3 29.8 26.6 -3.2 -4.9 0.0 -20.9 Result - - - - - - + + - - - - Titer - - - - - - 1.5 1 - - - - None ADA could been detected in RAGKO mouse for its immunodeficiency -
FcRn Crucial Role for Antibody Transcytosis into Tissue and Analyses in vitro
Coat antibody Detect antibody Drug Detection range Instrument Donkey Anti-Human
IgG（from Jackson)Goat Anti-Human
IgG (from Jackson)IgG
(In house)15-32805
pg/mlMSD -
MDCK-hFcRn/B2M Transcytosis Assay
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Generation for MDCK-hFcRn/B2M Stable Cell Line
Gene：Human FCGRT (UniProtKB-P55899, FCGRTN_HUMAN),and B2M (UniProtKB – P61769, B2MG_HUMAN)MDCK cell line：Madin-Darby Canine Kidney cellsCell transfection with lentiviral vector inserted interested human genesSelect single clone with high/moderate/low expressionFunction analyses with Transwell for a variety of antibody transcytosis -
MDCK-hFcRn Function: Transcytosis Assay
Sample Transcytosis ng/ml
(n=3)MDCK_IgG 0.94 MDCK-FcRn_IgG 19.40 MDCK_IgG-DXD 0.42 MDCK-FcRn_IgG-DXD 28.90 MDCK_IgG-AAA 1.14 MDCK-FcRn_IgG-AAA 3.09 MDCK_IgG-R-YTE 0.67 MDCK-FcRn_IgG-R-YTE 83.14 MDCK_FC-LALA-YTE 3.44 MDCK-FcRn_FC-LALA-YTE 182.81 MDCK_Albumin 0.38 MDCK-FcRn_Albumin 7.78 Add 40ug/Ml test articles in buffer A and incubate with cell for 18h,then detect buffer B Test articles:IgG:human IgGIgG-Dxd:deruxtecan linked human IgGIgG-R-YTE:FcRn binding enhnced human IgGIgG-AAA: FcRn binding deficient human IgGFC-LALA-YTE:FcRn binding enhanced hFc fusion proteinAlbumin:recombinant human albumin
IgG ,FC fusion protein, ADC, albumin could been specified transferred by MDCK-hFcRnYTE mutation could enhance transcytosis, while AAA mutation could inhibit -
Strategy for Receptor Occupation
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RO Calculation for IgG on N87 CellRO Calculation according to PE value:
Secondary Ab Normalization accordina to AF-647 value and RO
Final total receptor calculation ratio according to normalized secondary Ab
Based on MFI siangl to calculation RO and secondary Ab value deducted from calculation
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Immunology in-vitro Assay Capabilities
Immune Cells activationT cell activationImmune checkpointsProliferationCytokine secretionNK cell activation (IL-2)ProliferationMarker: IFN-γ, CD107aCytokine (single/multi-plex)Intracellular/ExtracellularSignal pathwayp-STATCell differentiation/polarizationT cell polarizationTh1, Th2Th17TregMonocyte derived DCImmature/MatureMonocyte derived MacrophageM1/M2 macrophageCell cytotoxicityT cell mediated cytotoxicityBsAbTCENK cell mediated cytotoxicityTumor cell killing after co-cultureAntibody related cytotoxicity assayADCCADCPCDCHemolysisAssay platform: FACS (up to 13 colors), FACS sorting, ELISA (single-plex), CBA,and MSD；
Proliferation (CTG, CFSE labeling, Ki67), Killing (CTG, LDH, CFSE labeling), HTRF, AlphaLISA -
Platforms of Efficiency, PK, PD and Safety
