DECLARATION op RONALD WETZEL
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`‘
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`BEFORE THE BOARD OF PATENT APPEALS AND CE
`
`Interference No. 102.572
`
`Examiner-in-Chief
`Mary P‘. Downy
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`))
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`2 )
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`)
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`Cahllly or at
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`v-
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`3082 at 81
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`1.
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`1. Ron Wezzal. deeluc sad am: am I an a citizen of the United Sum
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`raiding in Phoealxville. Pennzyl-unis. My Curriculum Vitae is enacted as Cnbilly
`Exhibit No. 5.
`I In a co-inventor of the United States Punt 4.316.551 canned
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`‘Recombinant
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`Immznoglobulin Preparations".
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`2.
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`Promblovernherl. l978.mApti11S.l989.Iwue3enierSeientisttt
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`Genemech.
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`Inc.
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`(Oeuenteeh)
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`located at 460 PL San Bruno Blvd" Scum San Francisco.
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`time 1 wt: neepennible for conducting cape:-ixuents.
`Culfomin 94080. During that
`supervising others
`in conducting experiments.
`investigating ruched:
`to produce
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`proteins by recombinant means. charneurizing protein structure:
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`and protein
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`structure/function
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`relnionrhipt.
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`-
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`.
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`3.
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`I am currently empxoyed by Sxnithlcnne Beeehm Phlruaeeudel-13.
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`in
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`Kins 01' Prussia. Pennsylvania :3 a Research Fellow in the Macromolecular Science:
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`Department.
`4.
`development!
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`_
`In me tax of 1932. Dr. Herb Heynekcr approached me to am...
`in a run:-eh yr-eject
`to express immunoglabuline in B.eoli.
`At um.
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`um I knew that such 8 project was going on u Gcnemech.
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`I was awn-e lhat Dr. An
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`Rig:-S mm ‘ht City of Rape.
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`in Dunn. Cmtcmia. had spent a sabbntiehl ‘period It
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`Geneateeh doing initial work in the atoning at the cDNA from I hybridum: cell
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`line
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`I-Ieyneker had been working with
`I also was aware that Dr.
`producing an antibody.
`him 4'-‘V11!!!
`that
`time. und that Dr. Heynuker: group was lnvoived in the project
`Iflur
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`Dr. mu; setumed to the City of Hope.
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`5.
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`Dr.
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`I-leynekcr told me that his group. working wlth Shmuel Cabilly. a
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`poet-gloetorial reuow in Dr. Art Riggs laboratory at
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`the Cfity oi Hopt. had expressed
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`DECLARATION OF RONALD WETZEL
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`irnmunoglobullna
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`chain: directed against human careinoembryonic antigen" in
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`E.coli.
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`Since a major hurdle of tho eapresaien or imxnunoglobulin was expected to be
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`in the folding of the protein. and since I had neatly had some success in folding
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`other recombinant proteins in my lab.
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`it seemed to me that In! 31'“? man: be able to
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`contribute to the project.
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`6.
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`Joanne Perry. a reaearch associatl
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`in my lab. and I began work on the
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`E..eoli
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`lmnatnoglobulla project on or about January. 1983.
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`I have rwoaxtxuexed the
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`course of our experimonta on lmmnnoglobulin club:
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`then 5630* by er-Iinininl
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`Jeanne Perry‘: and my notebook: from this time (cahiliy Exhibit: No. 6. 8 and 9).
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`'7.
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`I began won: on January 16. 1983. by atmnptin;
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`to isolate and purify
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`the imtntmozlobulln heavy chain and light chain produced "in
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`two different B.eell
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`strains from cell pastes which i
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`received from Mike Mumford (cahllly Exhibit No. 18.
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`Bates Non. 00701 and 00727). We selubillzed the inclusion body material
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`in a
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`guanidine hydrochloride solution followed by 5300 gel pomaation chromatography-
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`Jeanno Parry and I continued to try to mate pm-itiad preparation of
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`immttaeglobulin chains. alter eonvenlng them to their S-aelfenate derivatives.
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`DBAB chromatography in urea was tried as a follow-trp atop to the 8-300 column. to.
`flu-that purify the protein‘,
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`01: Jlnutrx 25. 19:3. ccaemy Exhibit No. 6. Bates No. 0042-0044. 0047.
`3-
`0050. and 0064).
`I began a refolding exper-lures: using our purified preparations of
`Nth!
`Ind baa?! chaina.
`However. analysis of SDS-polyacrylamide pl alactophoreala
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`(PAGE) alerted us that by dialyzlng our heavy ehaia into nativa better it became
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`susceptible to proteolyaia thus compromising the refolding reaction. we found a
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`means to eliminate the proteua by DEA; chromatography. We also found that
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`it could
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`this point we decided to continue our I-abiding experiments
`inhibilw 33‘ PMSP. At
`198
`working with either donaturant-soiubiliaed inclusion body preparations. 9:
`total
`lysatcs. without any further purification. and with PMS? added to the refolding.
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`buffers andler
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`to buffer: and to preplrt
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`lyaatea.
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`'
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`I recorded in my notebook the remit: from a
`on February 24. 1983.
`9.
`western blot of an SDS-PAGE gel by Jeanne Perry (Cabilly Exhibit No. 8. Bates Nea.
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`—
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`00223-00331).
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`From this Western him we noted lhl production of heavy and light
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`_chain protein prooueed in the co-trannfor-mad E.eoll eella.
`Furthennore. we were
`able to estimate the level of their Cxprusioat
`from cell pane which we received fromv
`Mike Mumford (Cabilly Exhibit No. 18. Bates No. 00730). The null: of the Western
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`blot analysis of the production levels of
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`irrrmrmoglebulln chains in B.eoli were used
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`DECLARATION OF RONALD WETZEL
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`to calculate the theoretical maximum eoaaible yield in refolding experiments.
`later
`which in turn was used to calculate *5 yicm.
`10.
`After a number at refolding reactions using E.coii Vmatcxfiai. we decided
`to use the authentic monoclonal. antibody. denatured and dct-iv
`t
`as the 8-
`sulionate. as a substrate for optimization of the refolding conditions.
`According to
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`¢~J
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`these conditions a mixture of the irnrnunogiobuiin S-suifonatca were incubated with
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`rnereaptoathanol and EDTA at pH 8.5 in ccneentratad urea for about
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`two hour: at 37°C.
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`then this solution was diaiyzcd against a nitrogen-saturated buffer cenxiating of 0.5
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`M urea in 0.! M sodium glyvcinate. pH 30.8. 1mM EDT!» Sm)-4 reduced giutathlonco
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`0.lmM exldiwed giutathiono. and iomhl glycine ethyl ester at 4' C. Reactions were
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`runner dialyaed against PBS (phoxphare-buttered eaiine) bafere being assayed.
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`The
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`Few‘! SEW 8 “M7 Significantly higher than that for an untreated aarnplc (Cabilly
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`Exhibit No. 6. Bates Nos. 006i. oo‘r7—oo:t).
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`We then used the above refolding condition: on the E.ceti derived
`11.
`material. Between March 18. 1983 and March 24. 1983. we conducted an experiment
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`The reaulta show a
`in which CBA-binding activity was generated after refolding.
`refolding yield of 0.7695 starting from a cetnnefomed cellular extract. and a yield of
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`0.32% starting from a mixture of a heavy chain S-suifonate and the urea-soiubilized ’
`crude extract cf light chain producing cells.
`The value of i580 nglrni
`in the
`cotnneforzned refolding reaction was
`significantly higher
`than the background
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`levels oi‘ apparent activity obtained from controls of either heavy chain alone (44!
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`gym!) or
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`iixht chain alone (108 nglmi):
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`these latter value: arise from non-xpacifio
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`binding to CEA in the assay.
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`‘mi: data shows that heavy chain and light chain
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`recombine in the retoiding reactiun to genome antigen binding activity (Cabiiiy
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`Exhibit No. 6. Rate: Not. 0087-0088).
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`12.
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`I
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`further declare that all statements made of my own knowledge are true
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`and than all statements made on information and belie!‘ am believed to be true: and
`further
`that
`these statements were made with the knowledge lhlt willful flit;
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`statements and the like so made are punishable by fine or
`under Section 1001 of Title 18 of the United sat
`a.
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`imprieonneont, or both.
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`A
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` Dated: 2‘? /qw
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`Ronalti Wctzel
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`»
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