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SELECTED CASE STUDIESprotein and antibody engineering
challenging proteins productionphage display • stability • biophysical
analyses • activity testing
NON CONFIDENTIAL
CASE STUDY I (PROTEIN ENGINEERING)
CHALLENGE
APPROACH
RESULT
PRODUCTION OF MEMBRANE PROTEIN ANTIGEN FOR VACCINE GENERATION
low yield expression, protein difficult to purify, unstable
pure and stable membrane protein prep for immunizations
SEQUENCE ENGINEERINGBI Sup Pell
Customer’s constructno expression
Sequence tweakingand optimization
Purification from inclusion bodiesand subsequent refolding
Modified constructexpression 10mg/L
Refolded proteinyield 70% of starting material
BI Sup Pell
EXPRESSION OPTIMIZATION PURIFICATION AND REFOLDING
signal sequence alterations to direct the protein to the secretion system; C-terminal sequence tweaks to enhance folding
CASE STUDY II (BIOPHYSICAL ANALYSIS)
CHALLENGE
APPROACH
RESULT
ANALYSIS OF CHARGE VARIANTS (ION EXCHANGE CHROMATOGRAPHY)
poor separation of different protein variants in IEX
enhanced resolution of protein species allowing for their effective separation
CUSTOMER’S CONDITIONS
Optimisation
Band- a- b- c- d- e- f
ISOELECTROFOCUSING OF SELECTED PEAKS
PURE BIOLOGICS CONDITIONS
optimization of chromatographic conditions
CASE STUDY III (PROTEIN REFOLDING)
CHALLENGE
APPROACH
RESULT
PRODUCTION OF A FUSION SIGNALING PROTEIN AS A DRUG LEAD
too toxic to be expressed in mammalian cells, cysteine-rich
2g of active protein (5mg/ml) delivered for animal study
EXPRESSION SCREEN PURIFICATION FROM INCLUSION BODIES
PROPRIETARY REFOLDING SCREEN
DENTIFICATION OF ACTIVE PROTEIN SPECIES
(1 OF 3 ACTIVE SPECIES)
SEPARATION&
FORMULATIONRECEPTOR BINDING, FUNCTIONAL OLIGOMER FORMATION & CYTOTOXICITY
ANALYSES COMPLETED
proprietary HTS of refolding conditions performed
SEC SPR DLS MTT
CASE STUDY IV (PROTEIN PRODUCTION)
CHALLENGE
APPROACH
RESULT
NATIVE, TWO STEP PURIFICATION OF A NON-TAGGED DRUG LEAD PROTEIN
customer had tight timelines, no tag preference
3.5g at 7mg/ml of active protein delivered for animal study in 4 weeks, >98% purity
STEP 1 : ION EXCHANGE CHROMATOGRAPHY STEP 2 : SIZE EXCLUSION CHROMATOGRAPHY
IEX resins and buffers scouting in mini scale and scale up
CASE STUDY V (ANTIBODY GENERATION BY PHAGE DISPLAY)
CHALLENGE
RESULT
SELECTION OF ANTIBODIES CAPABLE OF DIFFERENTIATING CANCER CELL LINES THAT ARE DRUG SENSITIVE AND INSENSITIVE
no biomarker known
cell subtype specific mAb found
DRUG
-INSE
NSITI
VE C
ELL L
INE
DRUG
-SENS
ITIVE
CEL
L LIN
E
PATENT PENDING
CASE STUDY VI (ANTIBODY GENERATION BY PHAGE DISPLAY)
CHALLENGE
APPROACH
RESULT
DISCOVERY OF BIOMARKERS USING TISSUES AND PATIENT DERIVED SAMPLES
no biomarker known, poor performance of commercial Abs
cancer specific clones identified
PBmAb#H386
PBmAb#C242
comm
ercial antib
ody
comm
ercial antib
ody
Negative control
Negative control
PBmAb#C242
PBmAb#H386
LIBRARY PRESELECTION ON TISSUE LIBRARY SELECTION ON PATIENT SAMPLES
naïve tissue preselection, selection against clinical samples
SCREENING
CASE STUDY VII (PEPTIDE APTAMER LIBRARY SCREEN BY PHAGE DISPALY)
CHALLENGE
APPROACH
RESULT
SELECTION OF PEPTIDE APTAMERS AGAINST DEFINED EPITOPE
selection against defined epitope; identification of site-specific clones
identification of clones specific only for the required epitope
ELISA WITH NON-MODIFED EPITOPE (WHOLE PROTEIN BINDERS)
ELISA WITH CHEMICALLY MODIFIED EPITOPE(POSITIVE CLONES ARE EPITOPE NON-SPECIFIC BINDERS)
modification of the epitope for ELISA screening of clones
Cont
act U
s
HeadquartersPure Biologics sp. z o.o. [Ltd.]Dunska 11, 54-427, Wroclaw, Poland+48 783 341 [email protected]. PureBiologics.com
German branchRudower Chaussee 2912489 Berlin, Germany+49 30 374 334 [email protected]
US Contact:Cheni Kwok, PhD, CLPBusiness [email protected]+1 650 318 1828
UK Contact:Walis Jones, Ph.D.Business [email protected]+44 770 313 2295
