e,po-OG '\
`i o. , 'l.. 2oos
`\@
`
`The Waterside Monoclonal Conference
`
`April 22-25, 1996
`
`Presented by: The Williamsburg BioProcessing Foundation
`
`The Omni Waterside Hotel
`
`Harborside - Norfolk, Virginia
`
`Process De"Velopment And Production Issues
`
`for !'1-~noclonal Antibodies
`
`1st Annual Meeting
`
`Pfizer Ex. 1005
`Page 1 of 7
`
`
`i o. , 'l.. 2oos
`\@
`
`The Waterside Monoclonal Conference
`
`April 22-25, 1996
`
`Presented by: The Williamsburg BioProcessing Foundation
`
`The Omni Waterside Hotel
`
`Harborside - Norfolk, Virginia
`
`Process De"Velopment And Production Issues
`
`for !'1-~noclonal Antibodies
`
`1st Annual Meeting
`
`Pfizer Ex. 1005
`Page 1 of 7
`
`
`
`The Waterside. Monoclonal Conference
`
`April 22-25, 1996
`
`Sunday. April :zl
`
`4:00- 10:00 pm:
`
`Check Ia - Jar:. Conn«lian
`
`Monday. Aprll 22
`
`7:00 • 8:00 am:
`
`Check Ia· Coltec I Bru.ldast
`
`8:00 - 8:30 MO:
`
`loO'Oductian
`Keilh L.. ~on· Confacncc Chaimlm
`
`8:30 • 9: IS .ni:
`
`9: IS • \0:00 am:
`
`Monodo11il Aotibodies • All lodusrty Pen pectin
`Anthony S. LllbWcclci, Sc.D. • SniichJ<liAo Be«ham
`
`Charactcrintion of Monoclonal A111ibodles
`.·
`Maurcca Costello • Snlithl<linc Bcc:cham
`
`10:00 • 10:30 1111:
`
`Break
`
`10:30 • I \:IS -.m;
`
`Devclopmcnr of Polei:iCy Au1179 ror Me>0oclonal Anribodia
`
`·---------------~Tho,:::::mas::::.R~y~slcatap::::~·~ID:.::,EC::..:.:Pharma.ce:::.::::::=utical~·=s:.....----------........,
`
`ll: IS • 12:00 noon:
`
`Chraaiatographic Techniques for !he Chsncruizalion af
`HamaoMAbs
`Recd J. limi$ • Ocncn1cc:h, Inc.
`
`ll:OO • 1:30 pm:
`
`Luocb • EJhiblr Ara
`
`1:30 - 2:15 pm:
`
`Manipubtion of the HaJt ln:imunc SysftlJI for ltcha11ccrucot
`of Rybridoma/MooodonaJ A4rtbody Gcnel'lltioO
`v3l)' 1... Milburn, Ph.D. • Pel-Frccz Molceular Diagliostias
`
`2:15 - 3:00 pll\:
`
`·
`
`Optimizalioo of Pcotela. £xprusioo by Flow Cyronurry a.od
`Media Supplcmcamrion
`Kclincth Karey, Ph.D. • Genzyme Corporation
`
`3:00 • J:JO pm:
`
`Break - Exhibit Arn
`
`j:JO • 4;15 pm;
`
`Etrccta o!HHD~elobi.o and Perf'luorourbo11 Bued Media
`Addl11ves on Cdl Mcrsbowm :aad Aalibody Yield.I
`Owla A. S2rdonini, Pb.D. • Ua.isyn Technologi..s
`
`Pfizer Ex. 1005
`Page 2 of 7
`
`
`
`Chromatographic Techniques for
`the Characterization of
`Human Monoclonal Antibodies:
`rhuMAb HER2
`
`Reed Harris
`Analytkal Chemistry Dept.
`Genentech, Inc.
`South San Francisco, CA 94080
`
`rhuMAb HER2 Background
`
`• HER2: Human Epidermal growth factor Receptor-2, also
`called neu or C-erbB-2.
`
`• Encodes 185 kDa membrane-spanning receptor p18511ER2.°
`• Overexpression of HEA2 = poor prognosis in breast cancer.
`• Antibodies vs. extracelluar domain of p185 11~:
`- renders HER2-overexpressing cell· lines cytostatic.
`- halts growth of implanted HE82• tumors.
`- increases chemotherapeutic susceptibility.
`
`•
`
`• Now in Phase Ill clinical trials (breast cancer).
`
`Pfizer Ex. 1005
`Page 3 of 7
`
`
`
`rhuMAb HER2 Structure
`
`._,
`
`• Humanized (CDR-grafted) version of a muri~e antibody.
`
`• 450-residue lgG, heavy chains, 214-resique k light chains.
`
`• Expressed in Chinese hamster ovary cells.
`
`• 12,000L production scale.
`
`• One glycosylation site in C112 dom~in (Asn-300).
`
`MonoS Cation Exchange Chromatography ofrhuMA~ HER2
`.
`.
`
`E c ...
`o:; ..
`t c
`..3 c
`~ ..
`~ a:
`
`~
`
`mllllllU
`
`+
`
`Pfizer Ex. 1005
`Page 4 of 7
`
`
`
`sel IEX-3
`
`"10
`
`20
`
`0
`
`. ] ::i IBX-4
`
`20
`e
`
`-;.
`~ s0l IEX-5
`
`i
`
`'10
`20
`0J====::::::__.,...._;:::::.._:::::;::::..~-=-__:=-.....:...-.-~~--.
`39
`.
`Ji' .
`39
`36
`Time
`'''"''- l
`
`Peak a: ~C:442-450 SLSLSPGK (+ Lys4SO)
`Peak b: HC:442-449 SLSLSPG
`(- Lys450)
`
`CpB Processing: Mammalian Cell Lines
`
`• C-terminal Lys processing:
`- Plasma·-derived antibodies
`- rCD4-lgG (transfected CHO)
`- MAb OKT3 (murine hybridoma; ascites or cell culture)
`- CEM231 MAb (mur;ne hybridoma; ascites or cell culture)
`
`• C-terminal Arg processing:·
`- Two-chain tPA (transfected CHO, Bowes melanoma)
`- huEPO (urine, transfected CHO) ·
`'
`• Carboxypeptidase not characterized, yet.
`
`Pfizer Ex. 1005
`Page 5 of 7
`
`
`
`iO
`'31
`&.rpea•d lgG1 huvy c.. 4\ C-tt:nl\li\us:-CSVM,H2>.l.HNliYTQICSl.'.il.Sl'vlC
`
`KE>.LHMMQJGLSl.SPQ: (+ L~SOJ
`Hl!ALHNHYTQXSLSLSPC (- Ly,450)
`.
`.
`
`I 'Ira
`
`120
`
`8
`
`s
`1 100
`E ..
`.. ,..
`era
`"' :;
`ti
`j
`~
`<
`
`60
`
`10
`
`lormylated
`poaptides
`
`2e
`
`0
`
`44
`
`46
`
`Ti.IM (min.)
`
`49
`
`sa
`
`IEX-l
`
`E c 30
`
`c
`
`; ·:1,v_, I
`: s ra i (~~-=-:/
`i ~;1~:.._ . ..:=::::::==-.....::.........::::;::::..._:=::::;::::::::__:;__~--~~
`
`'16
`15
`T t ""e Cm•,.,. )
`
`4?
`
`Peak C: LC:25-42 ASQOVNTAV~WYQOKPCK IA.snlO)
`Peak d: LC:25-42 ASQOVDTAVAWYQQKPGK
`IAsplO)
`11
`
`Pfizer Ex. 1005
`Page 6 of 7
`
`
`
`Deamidation of Light C~ain Asn-30
`
`• p18SHER2 binding assay:
`
`peak 1 82% specific activity
`peak 3 100% specific activity
`
`ft<f ··~
`• 25% of pool has deamidated Asn-30:
`25% @ 82% specific activity
`75% @ 100% specific activity
`pool @ 95% specific activity
`
`• Decided not to remove the deamidated material.
`
`• Deamidation increases when l:fCCF i5i held. even at 2·8°C;
`so harvests are taken straight through to purification.
`
`·7
`
`Tryptic M·ap Peak Assignments
`
`• S-carboxymethylation, trypsin digestion (no HC/LC separation).
`
`• RP-HP LC: Vydac C18, 4.6" x 250 mm, 300A, 30°C
`(A) o. 1% TFA in water, (8) 0.1% TFA in acetonitrile
`O - 40% solvent B in 80 minutes, 1 mUmin., HP 1090
`
`• Collect peak fractions by hand ..
`
`• Quantitation and peptide identification by amino acid analysis.
`Sequencing if not assigned by AAA alone, or shows mixtures.
`Mass spectrometry on modified or aberrant fractions.
`
`• Yields were 40-95% of 2 nmol of HG+ LC (150 µg) inje·cted.
`
`• Assigned 653/664 residues {98%).
`
`7
`--------------------· ····---------
`
`Pfizer Ex. 1005
`Page 7 of 7
`
`
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