`
`PRODUCTION OF PLASMA PROTEINS
`FOR THERAPEUTIC USE
`
`Ecilcd b}
`
`JOSEPH BERTOLINI. PhD.
`(NI. Biulhrmlnrs
`Mt‘lhnunw, Albumin
`
`NE". CONS. PhD
`Furlhu Upliolh; Ply. Ltd.
`Eugchm-ul. Auxuulia
`
`JOHN (.‘URLING. B‘Sc
`Juhn (’u my ( m will"; AH
`Uppmlm waucn
`
`WWI LEY
`AJ()HT\ \\l|.l:\ A; SUN). ISL. I'L‘UHK -\ l |( W
`
`
`
`
`
`Page 1 0f 20
`
`CSL EXHIBIT 1028
`
`

`

`Copyright I't‘ 20H by John Wiley XL Sunni Inc. All rights res-awed
`Published by John Wiley & Sum, lnct, Hubuken. New Icrxy.
`Published srmultuncousl) in Camdu
`
`No part m‘ this publication miy be reproduced. .sturcrl in it retrieval Wstem. or transmitted In any torm or by any means. electronic. mechanical. photocopying.
`recording. scanning. or titlirrwisc. except as permitted under Section I07 ui I03 til the I070 United Swtex (‘upyight Art Without cidler the prior wnnen
`permission of the Publisher, ur authorization through payrnentof the appropnnte perrcopy fee to the Copyright Clearance Center. lnc., 222 Rosewood Drive.
`Danvers. MA 0l023. (97S) 750-8400. fax (978) 750—4470. or nn the web tit www.cnpynghmnm Requem tn the Publisher fr‘r permission fihuuld he
`addrexwd to the Pcnnisoiau Department. John Wiley & Suns, 1mm.
`1 l
`l River Street. Humken, NJ 07030. (201 J 748—00“. fax (:01) 7425-0006. at nuhnc at
`htlp:l/www.wilcy.:(>l‘rt/go'pclrliissiori.
`[mm m Liability/Mmlnmcr 0t" Warranty- While the publisher and author have used their best ct’torts In preparing this 3005:. thcv make no representntmns or
`warranties with respect to the accuracy or completeness oi the cuntrn's (if this book and >pccilicully disclaim .iny implied wmuntica ut~ lllL'lLlIiullBltillly or
`fitness for a partial." purpose. No warranty may be created or extended by sales representutlves or written sales mutteriils. The ntlvice and strategies
`contained herein may not he surtable fur your situation. You should consult with a profcwunal where uppropnaie. Neither the publisher nor author fihall be
`hub]: fur my hm ut‘ pmlit m ml) uthcr counnetuul danugcx. including but lint limited to hpccidl. imidental. sonscqucnliul. or UtltCI danmgca.
`
`For general information on our nther prllltlt‘tx and services or for technical support, plum.- contact our Customer (‘nre Department within the United State;
`at (800) 702-2974 t‘ulSldC the United States at Hi 1') 372-1995 urlax (.ll7)572—-1002.
`
`Wiley .ilm thlishc: its books in a variety of electronic tunnats. Sonic content that appeals in print may not be available in elcrrtrnnic fonnuts. Forinorc
`infor'mnm about Wiley products. Visit nur web site it mu» wiley mm
`
`library of Congress Catalaging-in-I’uhlicariun Dara:
`[’rcduction of plasma proteins for therapeutic incl stiller: by Joseph Ucnolmt. Neil (jam. .ind John (lirlmg.
`p . urn
`Includes bibliographical references .ind index.
`ISBN 9380470024514) tcloih)
`lll. Curling.) M. (.lUlll1 Ni.)
`l. Bcrtulini. Joseph,
`ll
`(Ems. Neil.
`[DNLM 1. Blood Proteinvthempeutlc use. 2. Blmxi Proteins chemical synthesis
`filfifi'q-ttcil?
`
`3. Chemical Fractionation. WH ADO]
`
`lttlZUJUh‘V
`
`Printed in the United State.» ut‘Amcnc-i
`Illlllt'lhiJlT'l
`
`Page 2 of 20
`
`
`
`

`

`
`
`CEJNHIBITOR
`
`.lAN Oven. CHKisrtNe KRAMER. AKKY KUENDERMAN, DlANA WOUTEKS, AND SAtrt-iA ZEHRI.E1)ER
`
`17.1
`
`INTRODICTION
`
`inhibitor or C1-
`(formerly Cl—esterase
`Cl-inhibitor
`inactitator). a heat-labile plasma protein that
`inhibits the
`esterolytic activity 01‘ complement factor 1 (Cl), was dis-
`covered in the 19505 [1]. Soon after, a deficiency of C1-
`inhibitor was recognized as causing hereditary angioedema
`tl-lAE). a disabling mid even potentially life-threatening
`disease that had been described as early as 1888 by Osler
`[3-4]. Cl—inhihitor has now been characterized as a scrinc
`protease inhibitor ("serpin"). present in blood plasma, capa-
`ble of inhibiting a number of pt'utcascs that play an impot-
`taiit role in several physiological systems. This chapter
`focuses oii a nunibct of aspects of Cl-inliibitor and HAE,
`including the inanv nliysiolugical roles that Cl-inhibitot‘
`t‘XC'lb in “"wa the plottitctiun of Cl -inhibitor concentrates
`troni humw "lasina. and the treatment 01‘ “AB and poten—
`tially other diseases. with Cl inhibitor concentrates.
`
`17.2 BIOCHEMISTRY
`
`(‘l-inhibitor is primarily synthesi'It-d in the liver but also in
`other sites such as monocytes. skin tibroblasts and endothe-
`lial cells [5—8].
`It
`is a blllgleL‘huln glycoprotein with an
`apparent molecular mass of approximately 100 105 kDa as
`determined by analytical ultracentrifugation and sodium
`dodccyl sulfate polyaciylamide gel electrophoresis (SDS
`PAGE) [9 12|. The MW of the deglycosylated form of the
`protein as analyzed by SDS PAGE “as reported to be
`78 kDa [12]. When based on the amino acid composition
`(the molecule consists of 478 amino acids). a molecular
`
`mass ot‘53 kDa was calculated without the glycans [13]. The
`
`set-pin domain. located on the (‘-terminus. consists of 366
`amino acids while the nonscrpin, N—terminal domain com—
`prises of 112 amino acids [14]. Cl-inhibitor has 21—27%
`sequence homology with other serpins, although this homol-
`ogy does not extend to the nonscrpin domain. The structure
`of the aniinovtemiinal part of the Cl-inhibitor molecule
`makes it unique among the serpins as it contains a highly
`repetitive region comprising 14 tandem repeats of the
`tetrapeptide Gln-ProThr-Thr and variations thereof [15].
`Figure 17.1 shows a theoretical
`thrcc-dimcnsional (3D)
`model of the actiyc form of the scrpin domain of Cl-
`iiihibitoi, and a model of the latent l‘Ullll dCl'lVCLl from crystal
`stiucture analysis sourced from the Research Collaboratory
`[or Stiuctut‘al Bioinl‘orinatics (RCSB) protein data bank
`[16.17]. The respective access codes to view the structures
`are IMOQ and ZOAY.
`Cl—inhibitor is heatily glyeosylated. Ten of the 13 gly-
`cosylation sites are present on the N-terminal part of
`the protein. with the other three on the serpin domain
`IH, The molecule contains two distiltide bridges connect—
`ing the ainino-terminal and the (‘~termmal parts [13]. There
`are no free sullhydryl groups, The tsoelectric pomt was
`repottcd to be 2.7 2.1% |0.lt)|_ The molecule is denatured in
`the presence 01‘ methanol or ethanol
`(e 3., during cold
`ethanol fractionation of plasma). ether. at (LZDC in plasma,
`at
`low pH. and by heating to 60C in the absence of
`stabilizers [1.19 21].
`The gene for C1 inhibitor is located on chromosome 1 1,
`more specifically on locus llqll q13.l [13] and is denomi
`nated SERPINGI. SERPING/
`is known to be highly susr
`ccptible for mutations [22}:
`to date over 250 different
`mutations hate been described in the special Cl—inhibitor
`gene mutation database [23].
`
`Prod!“ lion 0} l’/.l.wiu Prulema‘ [or 'IIIt‘I‘J/willtr‘ Use. First Edition. Edited by Juaepn Bertolini. Neil (ioss. and John Curling.
`© 2015 John Wiley .5: Sons. lnc. Punished 21115 by John Wiley 54 Sons. Inc,
`
`241
`
`
`
`Page 3 of 20
`
`

`

`
`
`
`
`
`
`Factor X113 (FXIla). the active fragment of Factor XII (FXI lt‘t‘
`kallikrein. and Factor Xla (FXla) [3137]. In the classical
`complement activation pathway. Cl—inhibitor can regulate
`activated CIS and Clr. while in the lectin pathway 1'0,
`complement activation the mannan binding lectin~associared
`serine proteases, MASP-l and MASP-Z are targeted [38—42].
`Insufficient
`inhibition of the contact system by C1.
`inhibitor results in uncontrolled release of the vasoactrvc
`nonapeptide bradykinin [43]. As is discussed fully in Section
`17.7. HAE is the best-known clinical condition caused by
`Cl—inhibitor deficiency. leading to uncontrolled bradykinin
`generation [44]. Treatment is now possible by replacement
`with plasma—derived concentrates of Cl-inhibitor [447 46]_
`The key regulatory role of Cl-inhibitor in preventing
`unregulated bradykinin production is its action on FXIIa
`and kallikrein activity [44].
`Initiation of Factor Xll
`activation can occur through trauma, infection and other
`inflammatory responses, and by binding of Factor Xll to
`negatively charged surfaces originating from bacterial lipo~
`polysaccharide, oligosacchan'dcs, connective tissue proteo-
`glycans, damaged basement membranes, extracellular RNA.
`or platelet polyphosphatcs [44.47.48]. In the presence of
`FXlla, prekallikrein is cleaved to plasma kallikrein that acts
`upon high molecular weight kininogen (HMWK) to release
`the vasoactive nonapeptide bradykinin. Plasma kallikrcin
`also converts inactive Factor Xll to active FXlla. generating
`a positive feedback loop. Amplification of kallikrein pro—
`duction results in augmented hradykinin production and
`local vasodrlatatron. The cleavage of hradykrnin from
`HMWK rs highly localized since the prekalhkrern and the
`HMWK substrate circulate as a complex. Bradykinin will
`bind to its bradykinin B2 receptor that
`is expressed on
`endothelial cells. This induces an increase in the vascular
`
`permeability that allows fluid to move into the interstitial
`space resulting in edema. Cl-inhibitor, through its modula-
`tion of FXIIa and kallikrein activity, is a key component of
`homeostatic mechanisms regulating the function of the
`contact activation system.
`C1 -inhibitor also has an important role in the regulation of
`the complement activation pathway. In fact it is the only
`plasma protease inhibitor with an identified regulatory role
`in the classical and lectin pathway of complement activation
`[49]. In addition. an inhibitory effect on the alternative
`pathway of complement activation has been shown, albeit
`the mechanism of action has not yet been elucidated [50].
`Art
`in vilru study using supraphysiological levels of Cl-
`iriltibitor showed that the inhibiting effect was significantly
`different for the three complement activation pathways and
`depended upon the applied concentration of the Cl-inhibitor‘
`[5|]. Cl-inhibitor also targets other proteases such as plas-
`rttirt [9,38,52,53] and tissue plasminogen activator [54].
`Thus.
`it may also have a role in the regulation of the
`fibrinolytic system. Furthermore. Cl-inhihitor is an acute
`phase protein, the. concentration of which may rise at least
`two-fold during an inflammatory reaction [55]. Figure 17.2
`
`
`
`Reactive site
`Pt Atgm
`
`
`
`\ N-termlnus
`
`3D-modcls of the scrpin domain of Cl-irthibrtor
`FIGURE 17.]
`(derived from the Protein Database [16]). (21) Theoretical model of
`the active form [l7].
`(b) Model of the latent form based on the
`crystal structure [14]. The glycan structures are not shovm
`
`17.3 PHYSIOLOGY
`
`The mean Cl-inhibitor concentration in plasma is approxi~
`mately 0.2 mg/mL [9.10.12.24.25I, equivalent to 1 plasma
`unit (PU) per milliliter, with individual values as low as
`0.14mg/mL [24] and as high as 0.38 mg/mL [25].
`Varying values have been reported for the in viva half-1i fc
`of Cl-inhibitor. In normal subjects (and HAE patients under
`normal physiological circumstances), a half-life of 64—68h
`was found [26]. However. a fractional catabolic rate of
`2.5%lh has been reported in normal individuals equivalent
`to a half—life of approximately 28 11 [27,28]. In transfused
`l'lAE patients a wide range of half-lives for Cl-inhibitor
`clearance have been measured (7—90h). with a rnediart (or
`meant of 3040b [29.30] This variation may represent
`individual differences in the biotransformation and clear—
`
`ance of the administered Cthibitor and a sensitivity to a
`variation in the administered dose.
`
`Ct-inhibitor is the major inhibitor of proteases of the
`contact system and of the classical and lectin pathways ot
`complement activation. In the contact system these include
`
`Page 4 of 20
`
`
`
`ilIl
`
`

`

`
`
`HANUFACTURE
`
`243
`
`Bradykimn l
`
`
`
`l
`
`Contact system
`FXlla kalliktein
`
`Plasmrn l,
`l
`
`«
`"
`Regulation system
`
`
`
`7/, -
`
`/
`
`«\
`
`\
`
`l Fibrinolysis system
`Iasmln
`TPA
`
`
`.v_d
`
`LP.
`
`FXlla
`L— FXIa
`-
`‘
`—"V\\§ ‘#_ _ ‘
`7—7/
`\‘l Cl-inhibitor P 7"!”
`, \\ (/11, —R. _,
`_
`\_
`itiASP-r MASP-Z
`
`FXllt crs C1r
`
`Complement system.
`classwalp\athwaryw
`\
`\.
`
`Complement system.
`"Bligtflwiy,
`//'
`
`//
`
`t
`
`? (unidentified)
`
`Complement system,
`
`
`alternatlve pathway
`——~—
`
`
`\t
`4/
`\ 63a 1 7/ /
`05a ]
`MAC 1
`
`FIGURE 17.2 The central role of Cl-iohibitor in the regulation 0f Various physiologic systems:
`contact system, complement system. coagulation system. and tihrinolytrc \ydcm, M M? membrane
`attack complex; MASP: mannan binding lectrn—associated protease.
`
`
`
`..._..Mm“,.w...«t..v..~.u.....wm
`
`presents an overview of the central role ofCl—lnhibitor in
`various physiological systems. For detailed reviews of the
`role 01‘ Cl-rnhibitor in the coagulation, complement, and
`tibrinolytic systems, the reader is referred to Refs [55,56].
`The inhibition of CluRflt‘fll complement activation by
`Cl—inhibitor cart be potentintcd in vitro by highly sulfated
`glyeosaminoglycans such as heparin and sulfated dextrans
`through a resultant
`increased inhibition of (_‘1s and Clr
`[57,58].
`1n a rat model.
`this effect lasted for 60—90 min
`[50]. (ilycosnminoglyciins do not hrue such an effect on the
`contact system components FXlla and kallikrein [60.61], but
`FXTa mhibitron wtrs increased more than lOOl'old in the
`
`presence of dcxtran sulfate [61]. In principle, this selective
`potentiation mechanism offers new ways. for therapeutically
`modulating the biological effects of C1 inhibitor on contact
`activation. However. although recently more insight into the
`meclwiivr z r' heparin notcntiutict‘ of C1 ~inhibitor has been
`"t‘l“t".' " '.\ s‘l'l‘ t'nktromi whether the potentiation by this
`EL‘=."'.'
`'~ {Yr pltvsiolugrca' sigtriliwttcc it:
`i'rvu [14,621].
`
`the protease and results in cleavage of the
`substrate b),
`exposed (“l-inhibitor reactive site by the protease. leading
`to a covalent. SDS—stable complex between the protease and
`(‘l-inhihitor and :r
`rrtpid confonnational change of the
`(‘i-inhihitor
`protein
`that
`inactivates
`the
`protease
`(“mousetrap" ell’cct) |(15],
`In this process,
`in addition to
`the protease, Cl-inhibitor rs also inactivated due to the
`covalent binding to the target proteases. 'l'hus, (‘l-inhthttor
`is called a suicide inhibitor. The mechanism of action is
`
`described in more (161.111 by thgenztar—Bos and Hack [18].
`Following the inactivation reaction. the Cl-inhibitor—prote-
`ase complexes are cleared from the circulation through
`binding to scrpinspccrtlc receptors in the liver (low-density
`lipoprotein receptor-related protein)
`[64] or uptake by
`ncutrophils and tnonocytcs [65].
`Figure 17.3 shows a 3D model of the binding and
`inactivation of MASPZ by C1 inhibitor. The serpin domain
`of Cl'lnllthUl' was modeled using the crystal structure
`tZOAYl PICViUUsly reported [14.16].
`
`17.4 MECHANISM OF ACTION
`
`17.5 MANUFACTURE
`
`The Cl-inllthiltil‘ inactivates the target proteascs Cls. Ctr.
`FXIla. FXln. MASP-l, and MASP-Z by exposing a loop
`containing the active sir: Pt—P!‘ peptide bond (Arg444—
`'l‘hr445i of the. inhibitor [13.18.50] This is recognized as a
`
`Plasma-derived Cl—inhibitor concentrates have been used
`
`Clinically to treat ”AB in Europe, Canada. and Japan for
`many decades. However. only ti few rrranufacturcrs have
`implemented a production process for C1~inhibitor in their
`
`
`
`Page 5 of 20
`
`

`

`
`
`244
`
`('l-lNHlBlTOR
`
`Protease MASPQ
`
`
`
`
`
`center
`l’leactrve
`loop((ECU—-
`
`/l"1 A'9
`éN-g-Ivcans
`[3SheetA«fig
`
`f‘:} ‘52-.
`
`N-terminal)
`domain
`
`’
`
`Serpln Ci-inhibitor
`
`Michaelis cornuex
`
`Covalent complex
`
`FIGURE 17.5 Mechanism oi’inactivation of the target protease MASP-Z by (ll-inhibitor. 'l'he left
`and middle model show the binding of MASP-l to the reactive eenter loop of Cl-inhtbitor and the
`formation of the noneovalcntly bound complex. Cleavage of the Pl—l’l' peptide bond by MASP~2 is
`followed by a rapid conformational change of C l-inlribitor tmousetrap effect) by which the protease
`is buried in the central B-slrect of Cl-inhibitor and is covalently bound to it (right model). Only a
`portion of MASP 2 is shown. The N-terminal domain and glycans were modeled from neutron
`diffraction and electron microscopic data. (Modeling and pictures courtesy of Dr. L Beiruobr,
`lnetitute of Enzymology, Budapest. Hungary.)
`
`plasma fractionation plant. One of the lirst successful
`attempts to purify Cl—inhibitor with relatively high purity
`and yield was reported by Pensky in lObl. Starting from
`serum. contaminating proteins were precipitated with 40%
`saturated ammonium sulfate (SAS) and further purification
`was achieved by ion-exchange chromatography [2”.
`In
`1970, another procedure was reported from Behringwerkc
`AG (Marburg. Germany) that described the purification of
`Cl-inlribitor'
`for biochemical characterimtion from [:2
`
`diluted recalcilied acid citrate dextrose (ACD) plasma by
`the use ofclu'otrratograplry on DL’AE—cellulosc, followed by
`fractionated 5A3 precipitation (50—65% SAS fraction; and a
`SephadenB G-l50 gel filtration I9]. lit 1982. Nilsson and
`Wimarr [1]] published a purification procedure consisting of
`precipitation ol~ Cl-irrhibitor from plasma by polyethylene
`glycol
`(PEG).
`followed by chromatography on DEAF.-
`cellulose and hydrophobic interaction chromatography on
`hcxyI—Sepharose‘“. Table
`17.1 summarizes these and a
`number of other published procedures for the purification
`of (‘ l-rnhibitor. Many of these activities- were for research
`purposes and synthetic protease inhibitors were often added
`to the starting material
`to prevent degradation of (‘L
`inhibitor. As these substances can be toxic. these purification
`procedures are not suitable for manufacturing (ll-inhibitor
`for clinical application.
`
`A variety of virus reduction steps have been applied in
`the published Cl-inhibitor manufacturing processes. These
`have included pasteurization, using appropriate stabilization
`conditions (3M potassium or ammonium citrate [83] or
`60% sucrose [67]), dry-heat
`treatment (68 100°C) [71].
`“vapor heat” treatment
`[74,84,851, S/D treatment
`[79].
`and nanofiltration [79,80,82]
`Based on the purification approaches detailed above. a fev.
`plasma product manufacturers embarked on developing a
`large-scale procedure for producing C | ~iuhibitor for clinical
`use. One of the lirst products manufactured involved Cl»
`inhibitor captured from ctyoprecipitate-depleted plasma by
`adsorption to [)EAE-Sephadex A—SO equilibrated at a slightly
`lowered ionic strength. Funher purification was achieved by
`incremental precipitation steps using ammonium sulfate [66].
`In later versions of this product, as produced by the Central
`Laboratory of the Netherlands Red Cross Blood Transfusion
`SCFVICC (now Sanquin Blood Supply Foundation. Amsterdam.
`The Netherlands), the safety for transmission of hepatitis B
`virus was improved by addition of a small amount of anti—
`hepatitis Bs immrrnoglohnlin and tor HIV by the introduction
`of a dry-heat treatment step (72b. oil”(') in With [86}. This
`concentrate was succeeded by a next-generation product,
`launched in 1997. called Color”. and was based on purihea-
`tion principles published by Wickerhauser and Benny [7 L72]
`
`Page 6 of 20
`
`

`

`
`TABLE 17.] Principles of Published Purification Methods of (‘Llnhlliitur from Human Plasma or Serum
`
`Purification" Factor
`Source Material
` Yicld ('5‘)
`
`and Atltlitiu's
`
`Purification Steps Puntv and/m Sat .,
`.
`aoaoomnumny
`
`236 ~ . 90 Uni; N
`
`148 \
`
`100 150.-
`
`300 . . 295%
`
`35 (activit) I
`
`i66l- I974
`
`33—42 (activity)
`
`l10jl981
`
`299592 on SDS-PAGE
`
`50 (activity)
`
`lll]:1982
`
`610 a
`Homogeneous on SDS PAGE
`
`72 (antigen)
`
`[I2]1‘)83
`
`90%. 40—60 Ufnig
`
`20 (actn'ity)
`
`(67.08% I986
`
`Humugcneous on SDS-PAGF
`
`735 ~
`Homogenenuc on SDS~PAGE
`
`6.0 PUnig (antigen)
`b5 PL'lmg (dawn);
`
`4W) . >90%
`6 7 PU/mg
`
`40 (antigen)
`
`55 (antigen)
`fil (activity)
`
`20 (antigcn)
`2-1 (actwtty)
`
`16 (activityl
`(160PU/kg)
`
`[69;1987
`
`(7012us7
`
`[711-1987
`
`[721* 1988
`
`((‘nnrmueri)
`
`
`
`Rcl‘t‘t‘cnt‘rs and Year
`of Publication
`
`[23": 196]
`
`[qit‘iQ7n
`
`3.1 (nonvit) l
`
`(l 1409} SA_S piccipitdliuti
`(2) DownR Z—XlC‘ anion-exchangc chmniamgraph)
`(l ) DEAE cellulose DEJZ" chromatography
`(2) ‘0—65‘}? S A? plt‘t‘lpllfllitlll
`(3) Scphadcx G- [50 gel filtration
`(4)1.one electrophorcsu
`(l ) DEAE—Szplimlcx A-SO clitmimtugrapliy
`(2) 50 65% SAS precipitation
`
`( l) 6% PEG 6000 pt‘ccipttation
`(2') DEAE~ccllulosc DEBUDE 52 " uhtuniatogiapliy
`(3) Concanaval in A—Sephamsc chromatography
`(1)69? PEG 6000 precipitation
`(2) DEAE-Scphuccl"3 chromatography
`(3) llexyl-Sepharose chromatography
`(l) 5% PEG 4000 precipitation
`(2) LysincScpharose chromatography
`(3) DEAE-Sephzidcx A~SO chromatography
`(4) Scphadcx G-lSO gcl filtration
`(5) Hydroxylapmte chromatography
`(UQAE-Sephadrtx chromatograph)
`(2) 60% SAS prccipitation
`(3) Pasteunzauon ( Y 60% Sucrosei
`(4) 60°?- SAS precipitation
`(S) l‘hcnyl—Scphamsc chromatography
`(1) Monoclonal anti-Cl-inhibitor chroniamgraphy
`(2) FPLC Muntv Q HR Llu‘utnatugraph)
`(l) 16 45% PEG 4000precip1tation
`(2) DEAE-Sephacel chromatography
`(3)2n2'1chclatc agaros: chromatography
`(4 ) Anticontaminant immunoadmrption
`chromatography
`(1) DEAE-Scphadcx A-SO clirmnatogi aphy
`(2) 20% PEG 4000 precipitation
`(1)(‘M—Srpha(lex (150 (hit)llltlltlglupi)‘
`(4) 50—65%- SAS precipitation
`(5) Dry-heat treatment
`(I) DFAE-Scphadcx 4-50 chmmamgrapliy
`(2) 20% PEG 4000 precipitation
`(3) (lM-Scpharosc H“ chromatography
`
`Scrum
`
`Rcualcrhcd AC1) plasma
`
`Ptnthrutnltitt rompin-
`dcplctcd citrutcd
`cryosupcrnatant plasma
`ACDot (‘PDplthnIa
`»
`liDTA. polvhrcnc. DFP
`
`l’lasmintigcn~dcp|ctcd
`EDTA plasma
`~ EDTA, polyhronc
`Fi‘csli EDTA plasma
`~ bcnzznnidinc
`
`Prnthromhtn complex-
`dcplulcd gitiulcd
`cryosupcmatant plasma
`
`Scrum or plasma
`->!A llupaiiii. DH’
`ACD plasma
`- PMSF, NPUB
`
`(‘rymupcrnatant pltmtia
`
`Factor lX-(lcplctcd plasma
`
`Id
`
`Page 7- Fr 20
`
`

`

`
`
`9%
`
`
`TABLE 17.1 (Cami/med)
`Source. Material
`and Additives
`Purification Stops
`
`Cryosupcmatant of fresh-
`trozen plasma
`
`Cryosupematant plasma
`
`Citt'atcd plasma
`1 EDTA, NPBG, SBTI
`
`Preadsorbed fresh-frozen
`placma
`— bcnzamidmc.’ ’PMSF
`Citrated plasma. depleted of
`cryoprecipitatc.
`prothrombin complex and
`anttthrom bin
`Citratetl plasma
`
`Prothrombin complex-
`tlepletctl cryosuper-
`natartt citratcd plasma
`
`( ll DEAE-SCphadex A-SO chromatography
`(2) 20% PEG 4000 precipitation
`(3) CM-Sepharose FF chromatography
`(4) Dry-hcut treatment (80‘C. 72 h)
`( l) DEAE-Scphadex chromatography
`(2) PEG precipitation
`(3) SAS precipitation
`(41) "Vapor" heating
`(1) 21.4-45‘7r PEG 3350 precipitation
`(2) .lacalin-agarosc cinematography
`(3) Phenyl—Sepharose chromatography
`(1)21 .4—45% PEG 3350 precipitation
`(2) Jacaltn—agarosc chromatography
`(3) DEAE-Sepharose FF chromatography
`(4) Phonyl-Scphartise chmmatogt‘aphy
`(l) Cibocron‘KrBluo Scpharosc chromatography
`(2) Mono Q FPLC chromatography
`
`(1) DMAE»Fractogel‘B EMD 650M chromatography
`(2) $11) treatment
`(3) SOrFractogcl EMD 650M chromatography
`(4) 35 and 15 nm filtration
`(I) QAE-Sephadex A-St) chromatography
`(2) 12% PEG 4000 precipitation
`(3) ’l'ween'm 80 treatment and QAE-Sephadex
`adsorption
`(’4) 15 nm filtration
`(l) DbAbSepharose H<‘ chromatography
`(2) 20% PEG precipitation
`(3) CM-Scphatosc FF chromatography
`(4) Pasteurizatiort (10h, 60C)
`(5) 15 nm filtration (two filters in series)
`
`
`
`
`
`Purification” Factor from Plasma
`Purity and/or Specific Activity
`795x, >95%
`20 PU/mg
`
`'t'ieltl (9'0)
`
`33 (activity)
`(179 PU/kg plasma)
`
`References and Year
`of Publication
`
`[731* 1992
`
`30.8 PU/mg
`
`Not mentioned
`
`l7~‘l.75lix 1998
`
`7 78x ”(anti gen)
`2955 x "(activityfl
`Homogeneous on SDS-PAGE
`39 U/mg
`‘
`Honiogencom on SDS-PAGE
`
`39 (antigen and activity)
`
`[76) 1989
`
`Not mentioned
`
`1771 1993
`
`Homogeneous on SDS-i‘AGE
`
`23 (activity)
`
`[78] 1990
`
`>80%
`65. PU/mg
`
`6.0 PLi/mg
`
`>90%
`'34 PU/mg
`
`45 t antigcnl
`58 (activity)
`
`I79]A 1994
`
`50 (activity!
`
`[80p 2001
`
`35 {activity}
`
`l81.82h1999.2007
`
`ACD: acid citrate dextrose; CPD: citrate phosphate dextrose; DFP; diiboptopyl fluoroplrusphatc. NPGB;p-ttitrupltcnylf/I'-guanidinu bcnmalc; PMSF. plietiyltncthylsulfuttyl fluoride;
`SB'l‘l: soybean trypsin inhibitor; N: nitrogen; PEG: polyethylene glycol; PU: plasma unit of Clvinhibitor activity; SDS-PAGE: sodium dodecyl sulfate polyacrylztmidc gel
`electrophoresis. Dowex is a trade mark of Dow Chemical Company; DEAE-ccllulose DE'32 and DES2 are trade marks of Whatman; Sephadex, Sephace] and Sepharosc are trade
`marks of GE Healthcare Ltd; Fractogel is a trade mark of Merck Chemicals; Cibacron is a trade mark of Ciba-Geigy Corp; Tween 80 is a trade mark of 1C1 Atitcricas. Inc.
`“Specrhc activities tU/mg or PU/mg) cannot be compared directly, because of ditterences in assays and standards. Assuming a plasma Cl~inhtbttor protein concentration of 0.207
`0.25 mg/mL and an activity of l PU/mL, 100% pure CI-inhihitor has a specific activity of aL-S PUr’mg. Axsuming a total protein concentration in plasma nmo mgjtnl , the theoretical
`llliiltillldlly purification factor is 240—300 timer.
`”The asterisk indicates that the method is designed for largerscttlc production.
`“Depends on the nutty used.
`
`
`
`Page 8 of 20
`
`

`

`
`
`CHARACTERIZATION
`
`247
`
`1t exhibited a higher purity (_>90% homogeneity), a higher
`concentration t I00 U/mL), a higher process yield, and higher
`safety margins with respect to transmission of viruses [81.82].
`The fractionation process for this product in The. Netherlands
`still comprises capturing (Tl-inhibitor from prothrombin
`complex-depleted
`cryosupematant
`plasma
`by
`anion-
`exehange chromatography,
`followed by purification by
`PEG precipitation, pasteurization (10h, 60°C), and cation—
`exchange chromatography. PEG precipitation and pasteuriza
`tion in this process are talidated steps for virus removal [82].
`In a later product version, a 15 nm vims removing filtration
`step (nanofiltration) using two filters in series was imple-
`mented [82]. This had no effect on the pharmacokinetics and
`allowed the anti—hepatitis Bs irnmunoglobulin [82,87] to be
`omitted from the product. A similar product produced front
`US—sourced plasma is now also marketed iii the United States
`by ViroPharnra Inc. under the trade name of CinryzeTM.
`The on ly other planner—derived C l —iuhibitor product that at
`present has a marketing license for treating HAE patients is
`Berinert‘". produced by CSL Behring (Marburg, Germany).
`The first-generation product was introduced in Germany in
`1979 [88.89]. Since then Berinert has been launched in a large
`number of countries worldwide, including Japan andrecently
`also the United States. The present generation product,
`introduced in 1985, incorporates pasteurization and an addi—
`tional purihcntion step in the manufacturing process. The.
`sartivg material
`for the rnnnulacture ot'
`the product
`is
`c‘rg‘oprec'tpi :t‘-3~;‘ep|etcti
`plasma
`the
`process
`involves
`rev-town ol' the prothrt'imbrn complex proteins with DEAE-
`Seam-fer. ”lt‘ capture oi
`the ('l-inhihitor trorn the (low
`\
`if“
`wit" f'h‘ 9-Ser‘icdex and further purification by
`pl'P'v‘ilF'Ltlit'Ji‘ witl‘ 60”.; SAS followed by hydrophobic inter
`action chromatography on PherrylSepharose. As a virus
`inactivation step, pasteurization is performed for th at
`60 ’C in the presence of sucrose and glycine [67,68]. Besides
`the pasteurization, the hydrophobic chromatography step is
`also claimed as a virus reduction step [89].
`It‘ the past, Inimttno AG (Vienna. Austria; now Baxter
`BioScicnce) also produced a plasma—derived C l-irrhibitor‘
`pt'yfiuct [C l—esteruse inhibitor TIM 3) but marketing of this
`product ceased iii
`the 20003. The production process
`consisted. of adsorption of Cl-inhibitot onto DEAE-
`Sephedex and purification by consecutive precipitations
`using PEG and ammonium sulfate [75]. Virus inactivation
`was achieved by the so-calted “vapor heat“ treatment.
`consisting of heating the moist.
`lyOpliitized product at
`60°C for
`ltth at slightly elevated pressure [74,8435];
`Another Baxter process comprised capturing of ('i l -inhibi-
`tor on QAE-Sephadex, followed by PEG precipitation,
`treatment of the supernatant solution with a detergent
`(Tween 80), and subsequent capture of the protein onto
`an anion~exchange resin. The recovered eluate was then
`tiltered through a 15nm virus removal tilter. Finally, the
`finished product was treated with dry heat (37211, 380°C)
`
`[80]. A C l-inhibitor product based on this process has not
`been launched on the market.
`
`The Centre Regional de Transfusion Sanguine (CRTS) in
`Lille, now part of LFB (laboratoire Franqats du Fractionne—
`meat et des Biotechnologies, Paris, France) has also devel-
`oped a large-scale production scheme for Cl—inhibitor but
`this product was never taken into clinical trials and it was
`never marketed [79]. The process made use of anion-
`exchange chromatography on DMAE Fractogel EMD and
`. adsorption to SOJ Fraetogel EMD. As virus reduction steps.
`SID treatment and sequential 35 and 15 nm filtration were
`applied.
`
`17.6 CHARACTEITIZATION
`
`-
`
`Therapeutic Cl—inlribitor products are tested by a variety of
`analytical methods before release. Because of its limited
`worldwrde use and the small number of manufacturers.
`
`currently no pharmacopocia requirements exist for C1-
`mhibitor concentrates. Therefore. manufacturers analyze
`their C l-inhibitor products using their own specific analyti-
`cal methods. However, the European Pharmacopoeia and the
`United States Pharmacopoeia monograph requirements for
`other plasma proteins are used as a basis. Like all phannn-
`centical products for human use, the product needs to he
`tested tor sterility, absence of pyrogens and endotoxtns,
`excipients, and osmolalit)‘. Biological activity, usually meas-
`tired in a chromogenic assay as the capacity to inhibit Cl s, as
`well as antigen levels are used as relatively simple means to
`characteriwe the product [90]. Prompted by the large varia-
`tion in assay results between laboratories worldwide
`reported by WagenaarvBos, a program has been run by
`the National Institute of Biological Standards and Control
`tNlBSC, London, UK) to raise lntcmational Standards for .
`measuring C l-inhibitor in plasma and concentrates [90,91].
`These Standards (08/262 for plasma and 08/256 for con—
`centtates') have now been approved by the WHO Expert
`Cotruriittee on Biological Standardimtion, and represent a
`great advance in the standardization of testing requirements
`and for the adoption of measures to enhance the quality of
`therapeutic Ct-inhibitor concentrates.
`During the development of the Cl-inhibilor products
`extensive characterization studies are performed using com-
`monly used methods for protein characterization. In addition,
`specific tests suited for the analysis of glycoproteins are used
`including electrophoresis. isoelectric focusing. glycosylalion
`analysis, mass spectrometry. molecular mass analysis, and
`2D-fiuorescence difference gel electrophoresis (Zl)-DIGE).
`This data is used to verify the native state of the molecule.
`Methods are employed to measure the amount of active
`C 1 -inhibitor, tlu‘ough the use of a so-called functional ELISA
`[59,69,92]. The amount of inactivated Cl-inhibitor can also
`
`
`
`Page 9 of 20
`
`

`

`
`
`248
`
`(fl-INHIBITOR
`
`be measured using a radioimmunoassay with an antibody
`specific for neoepitopes that arise after cleavage [93].
`It is imponant that the Cthibitor molecule in thera-
`peutic preparations has a native conformation. Ibere should
`be minimal aggregation or degradation or" the protein as
`detected by SDSJ’AGl-i or sr'le exclusion chromatography.
`This minimizes any possrhle immunogenicity ot’the product.
`The preparations should also have a high purity to avoid side
`effects that can be caused by copun'fying plasma proteins.
`For instance, although Cl-inhibitor products are usually
`manufactured from prothrombin complex-depleted plasma,
`it is still necessary to show that any remaining coagulation
`factors are removed during the manufacturing process to
`exclude the risk of possible thromboembolic complications
`following in viva administration.
`
`17.7 CLINICAL ISSUES
`
`17.7.] Hereditary Angioedema
`
`HAE is a rare autosornal dominant disorder affecting
`approximately l:10,000 up to 1:50.000 people [94.95].
`HA5 is caused by a significantly reduced capacity to inhibit
`kallikrein [2] that is caused by a deficiency of C twinhihitor
`[3]. This can be either quantitative (HAE type I. 85% of the
`cases) or qualitative (HAE type II. 15% of the cases). These
`two types are indisttngurshable in clinical presentations.
`
`The disease occurs equally in men and women. and no
`difference between ethnic groups has been demonstrated.
`Although HAE is a congenital disease. as many as 25% of
`the mutations are de nova mutations [22.96]. Cl-lnlllbilor
`mutations that cause HAE type 1 occur throughout the gene
`and result in truncated or misfolded proteins that are not
`efficiently secreted, resulting in decreases in both antigenic
`and functional levels of Cl-inhibitor. Mutations that cause
`HAE type II usually involve exon 8 at or near the active site,
`and result in a mutant protein that is secreted. but that is
`dysfunctional. In addition a type [11 has been described with
`similar clinical features as HAE types I and [1, but with
`normal C l-inhibitor functionality. This syndrome is rather
`heterogeneous, but many cases are caused by a genetic
`defect