Glyeoproteinpharmaceuticals: entificand regulatoryconsicleratisns, and the US OrphanDrugAct* Darrell Teh-YungLiu_ Many pnrtcin phart~~;cccuticals b&g dcvclopcd for humar~ USC xc glycoproteins, with oligosnccharidcs covnlcntly attached to chc polypcptidc side chains. Thr propLWi.illy covcrcd by oligos~icclxlridc - chc molccul;ir cqLiiv;llcnt of a sugx-contcd pill’. lncrcnsing cvidcncc suggests tlut oligosxcharidcs i:;lil PI;:): .I11 import:mt role in glycoprotcin function. In rvaluatit~g the thuapcutic c&x :md s:ifcty of ;I gtycoprotcin t’liarlnacctiticnl, the intrinsic propcrtici of the oliRtrsaccli:tridcs ;is well :~s tlicir niodul;~ting ctfcct on the protciu must lx cnnsidcrcd. This xticlc provides ;m intcrprctntivc ovcrvi~w of the scientific alid rcgul3tory considcr.~tions pcrtincnt to glycoprotein ph.irmaccLLticals, with emphasis on issues of rnicrc~llctcro~ci~c~~ Wiociatcd with glycoprotcins, ,ind including a discussion of the US Orphan 13rLig Act with regard to glyroprotein pliamln~cuticals. It is not irltcwicd. howc~u, to lx comprchuisive trc;ltmcnt ofxicntific 2nd regulatory issues that 22; common to 411biol~~yic;~lprodilcts produced by bio?cctinolo~~. A nunibcr ofdocLimct;ts IXW: been issued over the past by narional control aaehoritics ;md interdcc& n:ltional orgnizations r&cd to this sL$cct (‘T:LblcI), nnd thcrc npp~zus to be 3 gc~md co~~sctw~~samong dl documents rbout how to &al with the ln;lin prtr!4m~~ il
itrvolvcdt. General properth ofglycoproteins: microheterogeneity Althougtl gly~::xylation is :I c01mmi protcin--modit?.r%ioii process i:; eukaryotic cells, the r;ingc of strwturcs for prcttcin-linked glycnns is cscccdingly large, utrd rhc biologic:J signiticancc of this hctcrogcncity is
nL>t well undrrsttrod. In addition to their intrinsic propertics, oligosaccharidcs CM influcncc the physicochcmic:il nnd biologicnl propcrtics nf proteins to which tlxy src attached, due to their location on the
glycoprotcin su&cc and their hydrodynnrnic activity. Glycosylation can affect solubility, rcsistancc to protcolytic attack and thcnnal inactivation, qu~tcrnxy structure, xtivity, targeting, sntigrnicity, and functional activity half2ifc of the protein conccracd. Most glycoprotcins, whcthcr isolntcd from ndtrn2; (i-DNA) soLmxs or produced by rccombin;~nt-l)NA tcch,loloq, arc structurally hctcrogcncous, Hctcrowith mitny gcxity is the rule rather than the cxccption biologic:11 produc:s, c.g. bactcrinl :lnd vimI vaccines, polyclonnl antibodies. The hctcroallcrgcns, 4 gincity c;i thssc 12uninplcs rcscllts froni the prcscncc of miscur0 ofprorcins, carbohydr;ltcs tmd lipids, wlicrcns the hctl:ropcncity ;tssoci;~ccd with glycnprotrin ph:,rl~~:Lc~utic~Ilsis due to glycosylation of identical poltrpcptidcs with ditfcring oiigosaccharidc strLuxLmx This hns Icd to the conccp~ of’glycofurms’, proposed by D+vck and his collc~~gucs to dcscribc the ‘dcfincd’ hctcrogcneity of glycoprotcinsJ. Giycoforn~s frcq!:cg:tly h~c di&rcnt physical and chcmicsl propcrtics lcdding to poecntial thrlctiorinl diversity. of different cell types on glycosylation hetcrogcnt+y can nrisc tium the prcscncc or nbscncc of iv- or O-ylycosylatiou ;ts wvll 2s glycosykbn with difl?rcut oligx~cclmidc strilctur:rl moti&. Thr cell type :iiid thr ctrlbditions Lmdcr whicli the cells :lrc grown 31~) dctcmiinc ttnc glyc3n strLlcttirc of3 glycoprotcin. Since prok;lryotcs do not carry oLlt protein glycosyl;ttion, human proteins synthcsizcd ill prokuyotic host cclls SUCIIas E, ti~li xc not glycosylxcd, l60
e&ire
Monkejr)I”
(Rabbit)” (Rabbit)‘* (Monkey)13 (%bbit)?5
Figure 1 In viva functional activity of t-PA and modified t-PA molecules. Tissue plasminogen activator (t-PA) is a 65 kDa glycoprotein which may be cleaved in vitro or in vivo by limited proteolysis into its A and B chains, that have binding and catalytic activities, respectively. Tne A chain consists of four distinct domains: finger-like (F), growthfactor like (G), Kringle 1 (Kl) and Kringle 2 (K21 domains. The fulldength t-PA cloned and expressed m CHO cell systems (&PA) has a comparable glycoform profile and functional activity with that of the natural t-PA (Arepresents glycosylation). The halflife of &PA and of t;PA derived from a human me!anoma call line b&PA) for radiolabeled and for fibrinolytic activity was -3 min in rabbit@. and was 5.6 min (nearly twice as long) for the unglycosylated, full-length t-PA produced in E. coliil. Removal of F, G, and Kl domains further prolonged the half-life of the unglycosylated t-PA. This modified t-PA, consisting of K2, and the &chain catalytic domains, had an activity halflie of 11.6-15.4 min in rabbits.12. Another ag!ycosylated variant of t-PA, also consisting of K2 and the &chain, was shown to have a funcbonal activity half-life of 20-25 min in monkeys compared with the functional acttvity half-life of 3.3 min of rt-PA’s, These studies confirm that the structures responsible for the rapid elimination of t-PA from plasma reside in the amino-terminal of the A chain of I-PA14. A hybrid molecule (in which the A chain is derived from plasmin [shaded!, and the B thain from t-PA) was studied in mouse and rabbit models. Plasmin has a domain structure very similar to that of t.PA but has a much longer half-life activity in viwo because of the absence of a liver receptor signal in its A chain. The hybrid protein so constructed was shown to have a functional activity half-life in excess of 60 min. like plasmin, but it functioned as a t-PA in mice and rabbits’s,
clinical &Is. Factitious proteins can brr m..dc cnsily. but the physiological and ciinical conscqurnces of the ihangcs made in the mnlcculc arc hard to predict. Asrcssing the contributiorl ofoligosacchnridcs to the biological activity of a glycoprotcin is usually pcrfwucd using partially ar complctcly dcglycosylatcd matcdals. Enzymatic or chemical trcatmcnt of giycoproteins to rcmovc oligosaccharides is of&n pcrformcd in the prcscncc of denaturing agent, as is the rccovcry of nail-hhglycosylatcd protein from timicamycintrcntcd mammalian cells and agiycosyl protein from E. r~li cells. For bioactivity mcasurcmcnt of these marcrials, it is csscntinl to cnsurc that the natural conformations of the proteins arc properly rcstorcd. The cvatuntion of the biological activity of aglycosyl huolan protein producrd in E, 131/ircquircs particular cart. Wlicn liim3ii proteins with multiple halfcystincs arc synthcsizcd in E. rclli that lacks the ability ho form disulfidc bonds oxidativcly intraccllu!arly. the resulting proteins arc often found in the inso!*.!b!e inclusion bodies of cytoplasm where the half-cystincs remain in the reduced form. The isolation al1.d purification of such protein oken rcquirc
i _...._.___ __.. __..____ TBTECH APRlL 1992:VOL
101
the use of dctcrgent and reducing agents. if suitable coxzditions arc not devised for the spontaneous regcncration of native protein conformation in the absence ofthe fkilitating effect of oligosaccharidcs for protein foldin ?: and subunit assembly, the resulting protein may not possess proper biologkal activity due to th: failure ofthc pi-otcin to assume correct conformation, rnthcr thau to the lack of glycosylation. However, many aglycosyl hunlan proteins. including the fulllength rt-IJAl I, trlmcatcd rt-PA”, IL-Z’“, K-517, and macrophagc colony stimulating fktor (CSF) (type B)‘” have been produced in E. adi with full biological activity. albeit with alter& i/r IW functional acti+ half-l&i. Thcsr findings suggest that ti,csc particular aglycosyi proteins contain sutficient intrinsic information to urldcrgo spontaneous rc-folding of the molcculcs, and thus assume the correct conformation. Thcyalso support the proposal by Anfinscn”’ that the spcnhc tbrce-dilncnsiorlal structure of proteins is dic:ltcd primarily by the linear scqucncc rJ its amino acids. Effects of glycosylation on immunogeneity and pharmacokinetics The nbsrncc of p!ycosylation for human proteins produced in E. mii may rcvcal nex antigenic sites, or
ma‘; cause altcrcd immunogcnicky due to the tendency of aglycosyl proteins to aggrcgatc. In addition, glycoprotcins with ‘unnatural carbohydrate struckms (e.g. those produced in yeast or certain mousr cells) may bc ant++: most humans hnvc circulating antibodies against K-h&cd yeast mamxm chains”‘, and approximately 1% of circulating hmnan IgG is specific for the terminal Gala(l,3)Gal cpitope gcncratcd by mouse Cl27 ~~11s”‘ Howcvcr, . it is not yet known whcthcr the prcscncc of Gnla(l,3)Gal Qtructurc, or thr antigcnic mannan structure in every glycoprotcin will ncccssarily lcad to more rapid clcnrancc due to antigen-antibody complex formation followed by phagocytosis. Although pharmaceutical glycoproteins produced in hctcrologou> z;~ammalinn cells may differ fi-om their natura’r counterparts with respect co detailed oligosaccharidc structures, cxp+cncc to dxc indicates that, in general, immune responses due to niicrohctrrogcnrit of glycan structures arc not a major conccr~. Howcvcr, the question of which molecular char-~rcrisdcs ir: a glycoprotcin actually a&ct its potential immunogcnicity cannot, as y-t. bc nnswercd My. To some extent, the tcndcncy of some recombinant glycoproteins to be immunogcoic 111ayreflect ditkrcnccs among individuals rccciving thr drug (c.g. hurnau lcukocytc associated [HLA] tissue tvp: and hisrory of nntigcn csposurc). as much as .it rctlccts significant d8crcnccs in the drug n~Jcculcs thcmsclvcs”. It remains to be seen whether mduciug microhcterogcncic atficts the irmxunc response. Proteins without appropriate carbohydrate nmictics lllay have altered phnnnacokinetics. The specific in rrjtro activity of El’0 is incrtascd upon desialylation, but the ir: r4*~~activity is abolished due to rapid irl lain>
117 J
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clearance”+ glycoproteina lacking the tcrnrinal sialic acid rcsiducs&e rapidly taken up-by hepatic cells and catabolized”. Likc\sise, the loss of ifr V~IUJ biological activity ofdcsialylatcd granulocyt: macrophagc colony stimulating factor (CM-CSF) also apparently results from rapid hcpatic clcarance2s. Reliable quantitative analysis of sialic acid will be an important -spcct of quality control for glycoproccin pharmaceuticals.
protein is intIucn4 not only by the cell type, but alto by the conditions under which the cells ‘u-c grown. The particular process uscci for the puriticdtion ~nav also favor sclcction of one g~ycoforln. Evaluation if glycoprotcin consistency would necessarily invol\:c both in-process control and car&l prr&ct analyris. Poor control of a production proccrs can !cad to (1) the introduction of an adventitious agent such as a virus for which 110 reliable assay is available. or Regulatory concerns (3) altered glycosylation and changes in properties There is an imuressivc list of orotcin drum derived that escape detection. For thcsc reasons, both the prodfrom cloned gcnis currently being used or”evairrated uct and the process of biologicals’ production are in humans, or soon to bc available for clinical trials. regulated. The list includes insulin. human Mowth hornlow, Both physicochcmical and biological methods arc various cytokincs, albumin, clotting factors, .mononecessary to establish the identity. potency and purity clonnl antibodies, andthc surface ,mtigcns ofche hepaof pharmaceutical proteins produced by rDNA techtitis, herpes and AIDS viruses. This new wave of nology. Tests for identity should include con~parkocc biological products has nccessita;ed a rc-asscssmcnt of of the products with refcrcncc matctials, conslctlng, the historical concerns over Froduct purity, safcq, whcrc possible, of the n:tural human substmcc. The potcocy and cficacy. National control authorities and specific tests that will ac. .,uatccIy characterize any parinternational organizations have responded by dcticular product on a lot-to-lot basis will depend on the veloping a series of regulatory documems. Table 1 nature of the product. Es.~r,@cs of tests which may summarizes documents relevant to glycoprotcin be useful during product dciclopmcnt are hstcd in the pbarmaccuticals issued by the USA, the European regulatory documents (Table 1). Comtnur~ity (EC), Japan, and the World Health In addition to the conventional analysis ofthc identOrganization (WHO). In recent years, there has been ity and integrity of the protein, the chromatographic an effort :o harmonize any significant differences that fingerprinting and characterization of the liberated might exist by USA, EC, and Japan but, as yet, a ohgosaccharidcs will be mosr valuable. Compositional analvsis ofthc carbohvdrate, prrsc, is less valuable since ‘unified’ document has not been dcvelopcd. Ncvcrrheless, there appczrs to be a gcncral consensus among it will not rcvcal glycosylation sites or site hctcroregulatory agencies about how to deal with the major gcncity. Quantitative analysis of sialic acid. and the issues involved. With regard to glycoprotcins, there is presence or absence ofthe Galo(l,S)Gal cpitopc, will be important for reasons already mentioned. Sire not n document that deals spccificaiIy with giycosylexclusion, ion-cxchangc, lcctin affinity chromatograation. Glycoprotein pharmaceuticals arc also subject to phy of pronasc glycopcptidcs and r~~,o-djl,ie,,sional HPLC, high-pH anion-exchange chromatography for convcsntional regulatory issues applying to all bioliberated Minkcd oligosaccharidcs have been used in logical products. Even though many glycoprotcin pharmaceuticals will be produced by mammalian cells evaluating rhe consistency ofrccombinanc IILMXW glycoprotcin intended for thcmpcutic use. The ability to that can properly glyco;ylaLc, and can oxidarively form separate and isolate each individual glycoform and the correct disulfide bonds and allow 5: glycoproteins characterize it as complctcly as possible by a cpmbito bc secreted, mammalian ceils may con&ill viruses, host-cell cotnponents that may be antigcnic in nation of methods based on di&xiug physicc::n~micat principles and biological principles (both ,!v !:i!*o humans, or pot*ntially oncogcnic DNA. It is, thercand irr vim)) will certainly be hclptid in dctiGng apfore, important to ensure that giycoprirteins derived from mammalian cells are significantly purified to propriatc means tc control and ensure lot-to-:.;t consistency of the product. ensure removal and/or inactivation of cellular DNA, Both it; vil~o and irr V~PObioassa?s for appropriate host-ccl] antigens, and adventitious agents. properties of the material arc cssermal to co&inn that The issue of glycoprotein microheterogeneity prcthe same material is being made iii each batch. SOI~Csents a challenge to industry and rrlgulators alike. The concern is not only how microhctcrogeneity is times the mechanism ofaccion ofa product is not well established. Ncverthc-Icy in vifro evidcncc ofproduct generzted, but also how to develop ;nalytical appotency may correlate with product action and proaches for detecting it and evaluating its signiticancr provide a database for dewmining what mcasurab!c with regard to the safety and efficacy or the product. properties of the product will predict therapeutic Glycoproteins cannot bc adcqua:ely d&cd by efticacy. The extent, frcqucncy and mctho& of composition analysis alone and there is, as yet, no smanimal testing must be detcnnincd on a cast-by-ca$c pie and rapid means to conduct a complete srructural in&ding tissue analysis of a complex glycoprotcin. Experience has basis to establish phamracokinetics, shown that the way in which a biological product is distribution and clearance mcchar&ms of a glycoprotein pharmaceutical. prcparcd is very important: in-process control is thcreThe nature and extent of aninlal and clinical tosfore ati important aspect of defining and charactetizing the product. The glycosylation pattern of a g;lyco- icity testing required for new protein product, --~
TWECH AWL
1
Box 1. The US Orphan Drug Act and gfycoprotein pharmaceuticals
The Orphan Drug Act was enacted by the Congress in :983 to prcmote the development of drugs that are needed by, but not availabfe to, people in the United States with ‘rare diseases or conditions’. Critical to the definition of an ‘orphan drug’ is the prevalence figure of 200000 affected oeoole in the USA as a ceiling for a specified rare disease or conditibnzd. The central purpose ofihe Orphan Drug Act was to stimulate innovation in developing treatmstts for patients with rare diseases and conditions. An important component of this Act is the seven-year market exclusivity provision of Section 527(a). This provision essentially provides non-patent monopoly protection for orphan drugs. It does so by pre venting the Food and Drug Administration FDA) from approving another ‘same’ drug for the ‘same’ indication for seven years after the approval of the first orphan product. Since the Orphan Drug Act was enacted, 53 orphan drugs have been approved for 59 indications and there have been over 400 orphan drug designationz.27. Despite this record of success, there has also been considerable controversy. Congressional hearings have been held; numerous amendments proposed, enacted and vetoed, and one court decision handed downz7. There are a number of scientific, policy, and legal aspects of the law that are subject to further refinements.
produced by rDNA technology is a ,x3jor concern. In general, the more dissimilar a prclduct is from its natural analogue. the more stringem the requirement for testing needs to be. The rcquircmcnt for prechnical acute toxicology in anima!:;, and long-tern1 animal testing (including tests for carcinog&city, tcratogcnicity and cffccts 011 fertili$, will dcpcnd on the availability of animal models, anJ should be based on the intcndcd use of the product. its mode of action and mctabo!ic fate. Specific pre-clinical toxicity cvaluations arc best considered on a cnsc-by-case basis. In tl3c final analysis, howcvcr, only carefully dcsigncd clinical trials and long-term sur&liancc can to the rclcvnnce of heterogeneity provide mswc~~ to the safety and efficacy of glycoprotcin phannaccutica!s. Two pharmaceutical protein Frcparations that have the same primary protein structure may have diff?rent oligosaccharidc strlucttircs 0’ di&rcnt proportions of each glycofor~n; however, dcmollstration of ditErrnccs in clinical cfticacy or safety would be critical to support a finding th:lt thr two preparations are clinically di&xcnt. Such a consideration has been proposed as the major criterion for awarding csclusivity under the ‘Orphan Drug Act’ (set Box 1). In the rontcxt cf glycoprotcin phnrlnaceuticals, I would like to focus On 013easpect: what constitutes ‘same’ or ‘differcnt’ in glycoprotcin pharmaceuticals f;,r the purposes of the exclusivity provisia:,! How to be ‘difFerent’ Gcncral criteria that hnvc been conridcrcd bv the FlIA are (I) the chcmicsl structure ofan orpha~r :irug, and (2) its clinical cficcts; or both’“. The nl+r problem with the use of the chemical structure alone as a mcasurc ofdiffcrcllcc is that its strict application would 133canthat minor ;hcn3ical altcratior3s could result in two drugs being diffcrznt under the law white being crsct3tially biologically and clinically the same. The -“_.___._“__._
TIBTECH APRIL 19%
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P/O1 101
second criterion, which the FDA proposes to adopt, gives considerable protection to the first approved orphan drug product against a second spoosoj’s bid to dcfcat exclusive marketing rights by introducjng minor molecular changes. It would con:tit:lte the best-available mechanism to protect the integrity of the chief incentive for orphan drug development that Congress created, yet also cnablc more effective thcrapics to bc developed without penalty. For traditional drugs (small molrculcs), any difiercnce in the chemical structure of the drug’s active moiety may render the drug a new molecular entity. The mcldified drug has a high probability of being diRerent from the original in its action or toxicity. It would, thcrcfore, need to undergo full clinical and toxicological testing since it is not possible to tell from csamining the structure of the two molc;ules or pcrforming simple iri 11ir~or irr tpifrc,tests whether they would behave identically. For macromolecular drug such as phannaccutical glycoproteins, some degree of hctc;ogcncity ahnost always mists aud it is possible to ndx c!m?gcs in n~acromolcculcs that arc very likely to have littlc or no pharmacological or chnical effect. The search for clinical superiority The definition of differences in clinical effect that would be suffkient to support a couclusion that two drug were ditrercnt is critical. On what basis would a new phnm3nceutical glycoprotein be considered differcnt? What sort ofclinical advantages would have to be dcmolxtrated? Clinical superiority would have to be shown by at lcast one of the following criteria: (I) Grcap:r cffectiveneS~ based on direct comparative clinical trials: (2) Substantially greater safety, for instance, by the elimination of a contaminant or ;m ingrcdicnt associated with advclyr cGcts: (3) Make a major cormibution to patient care, for instance, dViiC?OpiiiUlt of a n oral vs parentcral dosage form. III any cvc-ot, the burden ofproof will bc on the sponsur ofthc subsequent drug who is contending that the new drug is ditfcrcnt and superior. An optional proposal by FDAzH as a possible approach for dctermining difkrcnccs between two drugs is as follows:
This ‘option gives considcrablc ‘conceptual’ cxclusivity, pi-otccting the first product to treat an orphan discast, against a second sponsor’s attcn3pts to break cs&sivity through introducing minor molecular rhanges. It wou!d also bc reasonably simple to implement. Most chemical dif&renccs simply would not cause a drug to be considered different unless they Wcrr shown to create a significant clinicai dL&;cnce.
119 J
feature
Wndcr thisoption, two pharmaceutical glycoprotein products would be dcemcd to be the same ifthc. .~sqly diffcrcnces in chemical structures are due to post,translational events, infidelity of :ranslation, infidelity of transcription, or minor differences in amino acid sequence. Structural di&renccs in &~co.~ylatlon patterns or tertiary structu-f*c are also presumed not to result in d&c-rent drugs. This presumption oisameIIL'SS can be overcome by evidence of ‘clinical supcriority’
as
outlined
above.
Genentech, Eli Lilly and hGH The scope and nature oTthe ‘sa_mc’dmg debate can be gauged in the context of two macromolecular pharmaceuticals approved as orphan products, humau growth hormone (hGH) and EPO”. Two rUNA hGH pmducts are currci,tly mandated for the same patient population. Gcncntcch’s 197~amii10 acid vcrsion ofhGH was approved first as an orphan drug, and contains a terminal Met residue not present in the natural product. Eli Lilly’s rl>NA-derived 191 -amino acid version was subsequently approved as an orphan drug and is identical to the natural product, The FDA’s reason for approval of the Lilly’s hGH is based partly on the &mica1 diffcrcncc ofthc two products, in this IllSt311CC, by one amino acid residue at the amino termiuus, and additionally on the findiugs that the Lilly’s product. which is identical to the natural product, is less antigenic than Gcncntech’s product. This should lead to increased saZety and the posslblhty of a Iowcr risk of therapeutic failure. Amgen, Chugai-Upjohn and EPO The cast much tnorc akin to glycoprotcin pharmacectic.ils involves ElJO. Amgcn is in patent litigation to prevent marketing of a competitor rDNA-derived EPO product produced by C!:iigai-iipjohn. Amgcn has rrceived scvcn years of marketing exclusivity from the FDA under the Orphan Drug Act for its EPO. The two products are identical in their amino acid sequences, but exhibit some variation in glycosylation. Chugai-Upjohn has claimed that the glycosylation ditrerencc can produce a biologically significant difference making the drug different for purposes of the Orphan Drug Act. Chugai-Upjohn’s claim has so far not been favcraily considered. Defining the parameters that make a drug the same or different for purposes of the exclusivity provision is not an easy problem to resolve. The approach taken by the FDA, based on a chemical structure test. involving thr active rnoirty of a drug in conjunctio? with consider;ltions of clinical superiority, is deemed a significant step in the right direction towards solving the ‘same’ drug issue”.
mammalian cells: (1) the site ofglycosylation is dictated to a large extent by the primary amino acid scqucncc of the protciu and the host ccl1 typ&s; (2) the type of glycan structure observed at a specific glycosylation site is grcszly preserved’: (3) microhcterogcneity is rcquently due to the prcscncc or absence of t;rminal f’ucosc or sialic acid, and the extent of site microheterogeneity due to other sources is oficn minimal’; (4) there is a rcmarkablc dcgrec of similarity in the oligosaccharidc structures of a particular gIycoprotcin isolated from human sources and its rccombinnnt counterpart produced in specific hctcrologous mammalian cells, such as CHO cells”. For rDNA-dcrivcd therapeutic glycoprotcins, it would seem to bc advantageous and, in many instances, i.ssential. for human glycoprotcins containing multiple disulfidc bonds to be synthrtizrd il: niammzlian cells (that carry out correct glycosplation, disulfide bond formation and secretion of the product), rather than in illsect, yc;lst or prokaryotic cclis. Although human glycopro&is synthcsizcd in hcterologous mammalian cells may dit%r in their detailed glycan stmcture, it n~ay well be easier to rccogkc and characterize dit;fcrent glycoforms than to dctcrminc the biological roles of such structural ditf;-renccx, or to conclude that one glycoform (or one *et of glycofonns) is clinically superior to another. Acknowledgements The author wishes to express appreciation to Drs T. Bull, K. Scamon and 1’. Noguchi for their critical review of this manuscript, and to Dr E. Esbrr for providingregulatory documents from WHO. EC. and Japan. The cspcrt assistance of S. Unger in the preparation of this paper is deeply appreciated. References
Conclusions Protein-linked oligosaccharides can display enorHOLE diversity in structure’“T3”, creating consldcrable microheterogeneity for a 2, en glycoprotein. There do, however, appear to be identifiable paramccers which determine glycosylation characteristics in recombinant proteins produced from traosfccted TIBTfCii 4FRtL 1992 NOF 101
Recombinant inbred mouse strains: models for disease study Monica J. Justice, Nancy A. Jenkins and Neal G. Copeland Recombinant inbred (RI) strains of mice and the closely related reclsmbinant congenie strains offer considerable promise for identifying and characterizing genes causally associated with many different diseases. Loci associated with diseases such as heart disease, autoimmune disease and leukemia have already been identified through the use of these unique strains. The laboratory mouse is well-suited 2s a model organism for the study of human diseases’. For example, many spontaneous mouse mutations have been idcnti&d that mimic human disease. Thcsc include some of the earliest-studied mouse variants such as rodlcss retina, shaker, hyperglycemia and hypopituitary dwarfism’, as well as more-recently identified mutations such as the mouse r& mutation (a mod4 for human muscular dystrophy)“. Unlike humans, the mouse can be manipulated genetically as well as experimentally. One can perform controlled matings, can specifically alter genetic traits, and can control the genetic background influencing specific diseases. Likewise, one can cxpcrinirntally manipulate various fattars that may have an influctlcc on the dcvelopmcnt of disease.
In 1916, C. C. Little reported that ‘hereditary &ctars play an extremely important part in determining the incidence of cancer iu micc’J. However, at that time, mice were not well-suited for studying the mode ofinheritancc ofsusceptibiiity to cancer bccausc ofthe lack ofhontogencity in the genetic background. Consequently, many inbred strains were derived in order to study the genetics of cancer. An inbred strain of mouse is created by brothersister mating for over 20 generations. Thus, all members of an inbred strain exhibit a high degree ofsimilarity both genetically and physically. Studies ofinbrcd strains of mice led to the knowledge that many genes, including oncogcncs (see Box l), gcncs affecting immune function (c.g. histocompatibility loci), and many unidentified genes play a role in the development of, and susceptibility to, cancers.“. It became obvious that cancer was not a Elscvier acknowledger the tight of the US Govrmnwnt and its agcnrr and contracton to retain a non-e?iclusivc royalcpficc license in and to any copytight coveting thr articic.
--.__I____ TIBTECH APRIL 1992 lvOL10)
itrvolvcdt. General properth ofglycoproteins: microheterogeneity Althougtl gly~::xylation is :I c01mmi protcin--modit?.r%ioii process i:; eukaryotic cells, the r;ingc of strwturcs for prcttcin-linked glycnns is cscccdingly large, utrd rhc biologic:J signiticancc of this hctcrogcncity is
nL>t well undrrsttrod. In addition to their intrinsic propertics, oligosaccharidcs CM influcncc the physicochcmic:il nnd biologicnl propcrtics nf proteins to which tlxy src attached, due to their location on the
glycoprotcin su&cc and their hydrodynnrnic activity. Glycosylation can affect solubility, rcsistancc to protcolytic attack and thcnnal inactivation, qu~tcrnxy structure, xtivity, targeting, sntigrnicity, and functional activity half2ifc of the protein conccracd. Most glycoprotcins, whcthcr isolntcd from ndtrn2; (i-DNA) soLmxs or produced by rccombin;~nt-l)NA tcch,loloq, arc structurally hctcrogcncous, Hctcrowith mitny gcxity is the rule rather than the cxccption biologic:11 produc:s, c.g. bactcrinl :lnd vimI vaccines, polyclonnl antibodies. The hctcroallcrgcns, 4 gincity c;i thssc 12uninplcs rcscllts froni the prcscncc of miscur0 ofprorcins, carbohydr;ltcs tmd lipids, wlicrcns the hctl:ropcncity ;tssoci;~ccd with glycnprotrin ph:,rl~~:Lc~utic~Ilsis due to glycosylation of identical poltrpcptidcs with ditfcring oiigosaccharidc strLuxLmx This hns Icd to the conccp~ of’glycofurms’, proposed by D+vck and his collc~~gucs to dcscribc the ‘dcfincd’ hctcrogcneity of glycoprotcinsJ. Giycoforn~s frcq!:cg:tly h~c di&rcnt physical and chcmicsl propcrtics lcdding to poecntial thrlctiorinl diversity. of different cell types on glycosylation hetcrogcnt+y can nrisc tium the prcscncc or nbscncc of iv- or O-ylycosylatiou ;ts wvll 2s glycosykbn with difl?rcut oligx~cclmidc strilctur:rl moti&. Thr cell type :iiid thr ctrlbditions Lmdcr whicli the cells :lrc grown 31~) dctcmiinc ttnc glyc3n strLlcttirc of3 glycoprotcin. Since prok;lryotcs do not carry oLlt protein glycosyl;ttion, human proteins synthcsizcd ill prokuyotic host cclls SUCIIas E, ti~li xc not glycosylxcd, l60
e&ire
Monkejr)I”
(Rabbit)” (Rabbit)‘* (Monkey)13 (%bbit)?5
Figure 1 In viva functional activity of t-PA and modified t-PA molecules. Tissue plasminogen activator (t-PA) is a 65 kDa glycoprotein which may be cleaved in vitro or in vivo by limited proteolysis into its A and B chains, that have binding and catalytic activities, respectively. Tne A chain consists of four distinct domains: finger-like (F), growthfactor like (G), Kringle 1 (Kl) and Kringle 2 (K21 domains. The fulldength t-PA cloned and expressed m CHO cell systems (&PA) has a comparable glycoform profile and functional activity with that of the natural t-PA (Arepresents glycosylation). The halflife of &PA and of t;PA derived from a human me!anoma call line b&PA) for radiolabeled and for fibrinolytic activity was -3 min in rabbit@. and was 5.6 min (nearly twice as long) for the unglycosylated, full-length t-PA produced in E. coliil. Removal of F, G, and Kl domains further prolonged the half-life of the unglycosylated t-PA. This modified t-PA, consisting of K2, and the &chain catalytic domains, had an activity halflie of 11.6-15.4 min in rabbits.12. Another ag!ycosylated variant of t-PA, also consisting of K2 and the &chain, was shown to have a funcbonal activity half-life of 20-25 min in monkeys compared with the functional acttvity half-life of 3.3 min of rt-PA’s, These studies confirm that the structures responsible for the rapid elimination of t-PA from plasma reside in the amino-terminal of the A chain of I-PA14. A hybrid molecule (in which the A chain is derived from plasmin [shaded!, and the B thain from t-PA) was studied in mouse and rabbit models. Plasmin has a domain structure very similar to that of t.PA but has a much longer half-life activity in viwo because of the absence of a liver receptor signal in its A chain. The hybrid protein so constructed was shown to have a functional activity half-life in excess of 60 min. like plasmin, but it functioned as a t-PA in mice and rabbits’s,
clinical &Is. Factitious proteins can brr m..dc cnsily. but the physiological and ciinical conscqurnces of the ihangcs made in the mnlcculc arc hard to predict. Asrcssing the contributiorl ofoligosacchnridcs to the biological activity of a glycoprotcin is usually pcrfwucd using partially ar complctcly dcglycosylatcd matcdals. Enzymatic or chemical trcatmcnt of giycoproteins to rcmovc oligosaccharides is of&n pcrformcd in the prcscncc of denaturing agent, as is the rccovcry of nail-hhglycosylatcd protein from timicamycintrcntcd mammalian cells and agiycosyl protein from E. r~li cells. For bioactivity mcasurcmcnt of these marcrials, it is csscntinl to cnsurc that the natural conformations of the proteins arc properly rcstorcd. The cvatuntion of the biological activity of aglycosyl huolan protein producrd in E, 131/ircquircs particular cart. Wlicn liim3ii proteins with multiple halfcystincs arc synthcsizcd in E. rclli that lacks the ability ho form disulfidc bonds oxidativcly intraccllu!arly. the resulting proteins arc often found in the inso!*.!b!e inclusion bodies of cytoplasm where the half-cystincs remain in the reduced form. The isolation al1.d purification of such protein oken rcquirc
i _...._.___ __.. __..____ TBTECH APRlL 1992:VOL
101
the use of dctcrgent and reducing agents. if suitable coxzditions arc not devised for the spontaneous regcncration of native protein conformation in the absence ofthe fkilitating effect of oligosaccharidcs for protein foldin ?: and subunit assembly, the resulting protein may not possess proper biologkal activity due to th: failure ofthc pi-otcin to assume correct conformation, rnthcr thau to the lack of glycosylation. However, many aglycosyl hunlan proteins. including the fulllength rt-IJAl I, trlmcatcd rt-PA”, IL-Z’“, K-517, and macrophagc colony stimulating fktor (CSF) (type B)‘” have been produced in E. adi with full biological activity. albeit with alter& i/r IW functional acti+ half-l&i. Thcsr findings suggest that ti,csc particular aglycosyi proteins contain sutficient intrinsic information to urldcrgo spontaneous rc-folding of the molcculcs, and thus assume the correct conformation. Thcyalso support the proposal by Anfinscn”’ that the spcnhc tbrce-dilncnsiorlal structure of proteins is dic:ltcd primarily by the linear scqucncc rJ its amino acids. Effects of glycosylation on immunogeneity and pharmacokinetics The nbsrncc of p!ycosylation for human proteins produced in E. mii may rcvcal nex antigenic sites, or
ma‘; cause altcrcd immunogcnicky due to the tendency of aglycosyl proteins to aggrcgatc. In addition, glycoprotcins with ‘unnatural carbohydrate struckms (e.g. those produced in yeast or certain mousr cells) may bc ant++: most humans hnvc circulating antibodies against K-h&cd yeast mamxm chains”‘, and approximately 1% of circulating hmnan IgG is specific for the terminal Gala(l,3)Gal cpitope gcncratcd by mouse Cl27 ~~11s”‘ Howcvcr, . it is not yet known whcthcr the prcscncc of Gnla(l,3)Gal Qtructurc, or thr antigcnic mannan structure in every glycoprotcin will ncccssarily lcad to more rapid clcnrancc due to antigen-antibody complex formation followed by phagocytosis. Although pharmaceutical glycoproteins produced in hctcrologou> z;~ammalinn cells may differ fi-om their natura’r counterparts with respect co detailed oligosaccharidc structures, cxp+cncc to dxc indicates that, in general, immune responses due to niicrohctrrogcnrit of glycan structures arc not a major conccr~. Howcvcr, the question of which molecular char-~rcrisdcs ir: a glycoprotcin actually a&ct its potential immunogcnicity cannot, as y-t. bc nnswercd My. To some extent, the tcndcncy of some recombinant glycoproteins to be immunogcoic 111ayreflect ditkrcnccs among individuals rccciving thr drug (c.g. hurnau lcukocytc associated [HLA] tissue tvp: and hisrory of nntigcn csposurc). as much as .it rctlccts significant d8crcnccs in the drug n~Jcculcs thcmsclvcs”. It remains to be seen whether mduciug microhcterogcncic atficts the irmxunc response. Proteins without appropriate carbohydrate nmictics lllay have altered phnnnacokinetics. The specific in rrjtro activity of El’0 is incrtascd upon desialylation, but the ir: r4*~~activity is abolished due to rapid irl lain>
117 J
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clearance”+ glycoproteina lacking the tcrnrinal sialic acid rcsiducs&e rapidly taken up-by hepatic cells and catabolized”. Likc\sise, the loss of ifr V~IUJ biological activity ofdcsialylatcd granulocyt: macrophagc colony stimulating factor (CM-CSF) also apparently results from rapid hcpatic clcarance2s. Reliable quantitative analysis of sialic acid will be an important -spcct of quality control for glycoproccin pharmaceuticals.
protein is intIucn4 not only by the cell type, but alto by the conditions under which the cells ‘u-c grown. The particular process uscci for the puriticdtion ~nav also favor sclcction of one g~ycoforln. Evaluation if glycoprotcin consistency would necessarily invol\:c both in-process control and car&l prr&ct analyris. Poor control of a production proccrs can !cad to (1) the introduction of an adventitious agent such as a virus for which 110 reliable assay is available. or Regulatory concerns (3) altered glycosylation and changes in properties There is an imuressivc list of orotcin drum derived that escape detection. For thcsc reasons, both the prodfrom cloned gcnis currently being used or”evairrated uct and the process of biologicals’ production are in humans, or soon to bc available for clinical trials. regulated. The list includes insulin. human Mowth hornlow, Both physicochcmical and biological methods arc various cytokincs, albumin, clotting factors, .mononecessary to establish the identity. potency and purity clonnl antibodies, andthc surface ,mtigcns ofche hepaof pharmaceutical proteins produced by rDNA techtitis, herpes and AIDS viruses. This new wave of nology. Tests for identity should include con~parkocc biological products has nccessita;ed a rc-asscssmcnt of of the products with refcrcncc matctials, conslctlng, the historical concerns over Froduct purity, safcq, whcrc possible, of the n:tural human substmcc. The potcocy and cficacy. National control authorities and specific tests that will ac. .,uatccIy characterize any parinternational organizations have responded by dcticular product on a lot-to-lot basis will depend on the veloping a series of regulatory documems. Table 1 nature of the product. Es.~r,@cs of tests which may summarizes documents relevant to glycoprotcin be useful during product dciclopmcnt are hstcd in the pbarmaccuticals issued by the USA, the European regulatory documents (Table 1). Comtnur~ity (EC), Japan, and the World Health In addition to the conventional analysis ofthc identOrganization (WHO). In recent years, there has been ity and integrity of the protein, the chromatographic an effort :o harmonize any significant differences that fingerprinting and characterization of the liberated might exist by USA, EC, and Japan but, as yet, a ohgosaccharidcs will be mosr valuable. Compositional analvsis ofthc carbohvdrate, prrsc, is less valuable since ‘unified’ document has not been dcvelopcd. Ncvcrrheless, there appczrs to be a gcncral consensus among it will not rcvcal glycosylation sites or site hctcroregulatory agencies about how to deal with the major gcncity. Quantitative analysis of sialic acid. and the issues involved. With regard to glycoprotcins, there is presence or absence ofthe Galo(l,S)Gal cpitopc, will be important for reasons already mentioned. Sire not n document that deals spccificaiIy with giycosylexclusion, ion-cxchangc, lcctin affinity chromatograation. Glycoprotein pharmaceuticals arc also subject to phy of pronasc glycopcptidcs and r~~,o-djl,ie,,sional HPLC, high-pH anion-exchange chromatography for convcsntional regulatory issues applying to all bioliberated Minkcd oligosaccharidcs have been used in logical products. Even though many glycoprotcin pharmaceuticals will be produced by mammalian cells evaluating rhe consistency ofrccombinanc IILMXW glycoprotcin intended for thcmpcutic use. The ability to that can properly glyco;ylaLc, and can oxidarively form separate and isolate each individual glycoform and the correct disulfide bonds and allow 5: glycoproteins characterize it as complctcly as possible by a cpmbito bc secreted, mammalian ceils may con&ill viruses, host-cell cotnponents that may be antigcnic in nation of methods based on di&xiug physicc::n~micat principles and biological principles (both ,!v !:i!*o humans, or pot*ntially oncogcnic DNA. It is, thercand irr vim)) will certainly be hclptid in dctiGng apfore, important to ensure that giycoprirteins derived from mammalian cells are significantly purified to propriatc means tc control and ensure lot-to-:.;t consistency of the product. ensure removal and/or inactivation of cellular DNA, Both it; vil~o and irr V~PObioassa?s for appropriate host-ccl] antigens, and adventitious agents. properties of the material arc cssermal to co&inn that The issue of glycoprotein microheterogeneity prcthe same material is being made iii each batch. SOI~Csents a challenge to industry and rrlgulators alike. The concern is not only how microhctcrogeneity is times the mechanism ofaccion ofa product is not well established. Ncverthc-Icy in vifro evidcncc ofproduct generzted, but also how to develop ;nalytical appotency may correlate with product action and proaches for detecting it and evaluating its signiticancr provide a database for dewmining what mcasurab!c with regard to the safety and efficacy or the product. properties of the product will predict therapeutic Glycoproteins cannot bc adcqua:ely d&cd by efticacy. The extent, frcqucncy and mctho& of composition analysis alone and there is, as yet, no smanimal testing must be detcnnincd on a cast-by-ca$c pie and rapid means to conduct a complete srructural in&ding tissue analysis of a complex glycoprotcin. Experience has basis to establish phamracokinetics, shown that the way in which a biological product is distribution and clearance mcchar&ms of a glycoprotein pharmaceutical. prcparcd is very important: in-process control is thcreThe nature and extent of aninlal and clinical tosfore ati important aspect of defining and charactetizing the product. The glycosylation pattern of a g;lyco- icity testing required for new protein product, --~
TWECH AWL
1
Box 1. The US Orphan Drug Act and gfycoprotein pharmaceuticals
The Orphan Drug Act was enacted by the Congress in :983 to prcmote the development of drugs that are needed by, but not availabfe to, people in the United States with ‘rare diseases or conditions’. Critical to the definition of an ‘orphan drug’ is the prevalence figure of 200000 affected oeoole in the USA as a ceiling for a specified rare disease or conditibnzd. The central purpose ofihe Orphan Drug Act was to stimulate innovation in developing treatmstts for patients with rare diseases and conditions. An important component of this Act is the seven-year market exclusivity provision of Section 527(a). This provision essentially provides non-patent monopoly protection for orphan drugs. It does so by pre venting the Food and Drug Administration FDA) from approving another ‘same’ drug for the ‘same’ indication for seven years after the approval of the first orphan product. Since the Orphan Drug Act was enacted, 53 orphan drugs have been approved for 59 indications and there have been over 400 orphan drug designationz.27. Despite this record of success, there has also been considerable controversy. Congressional hearings have been held; numerous amendments proposed, enacted and vetoed, and one court decision handed downz7. There are a number of scientific, policy, and legal aspects of the law that are subject to further refinements.
produced by rDNA technology is a ,x3jor concern. In general, the more dissimilar a prclduct is from its natural analogue. the more stringem the requirement for testing needs to be. The rcquircmcnt for prechnical acute toxicology in anima!:;, and long-tern1 animal testing (including tests for carcinog&city, tcratogcnicity and cffccts 011 fertili$, will dcpcnd on the availability of animal models, anJ should be based on the intcndcd use of the product. its mode of action and mctabo!ic fate. Specific pre-clinical toxicity cvaluations arc best considered on a cnsc-by-case basis. In tl3c final analysis, howcvcr, only carefully dcsigncd clinical trials and long-term sur&liancc can to the rclcvnnce of heterogeneity provide mswc~~ to the safety and efficacy of glycoprotcin phannaccutica!s. Two pharmaceutical protein Frcparations that have the same primary protein structure may have diff?rent oligosaccharidc strlucttircs 0’ di&rcnt proportions of each glycofor~n; however, dcmollstration of ditErrnccs in clinical cfticacy or safety would be critical to support a finding th:lt thr two preparations are clinically di&xcnt. Such a consideration has been proposed as the major criterion for awarding csclusivity under the ‘Orphan Drug Act’ (set Box 1). In the rontcxt cf glycoprotcin phnrlnaceuticals, I would like to focus On 013easpect: what constitutes ‘same’ or ‘differcnt’ in glycoprotcin pharmaceuticals f;,r the purposes of the exclusivity provisia:,! How to be ‘difFerent’ Gcncral criteria that hnvc been conridcrcd bv the FlIA are (I) the chcmicsl structure ofan orpha~r :irug, and (2) its clinical cficcts; or both’“. The nl+r problem with the use of the chemical structure alone as a mcasurc ofdiffcrcllcc is that its strict application would 133canthat minor ;hcn3ical altcratior3s could result in two drugs being diffcrznt under the law white being crsct3tially biologically and clinically the same. The -“_.___._“__._
TIBTECH APRIL 19%
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second criterion, which the FDA proposes to adopt, gives considerable protection to the first approved orphan drug product against a second spoosoj’s bid to dcfcat exclusive marketing rights by introducjng minor molecular changes. It would con:tit:lte the best-available mechanism to protect the integrity of the chief incentive for orphan drug development that Congress created, yet also cnablc more effective thcrapics to bc developed without penalty. For traditional drugs (small molrculcs), any difiercnce in the chemical structure of the drug’s active moiety may render the drug a new molecular entity. The mcldified drug has a high probability of being diRerent from the original in its action or toxicity. It would, thcrcfore, need to undergo full clinical and toxicological testing since it is not possible to tell from csamining the structure of the two molc;ules or pcrforming simple iri 11ir~or irr tpifrc,tests whether they would behave identically. For macromolecular drug such as phannaccutical glycoproteins, some degree of hctc;ogcncity ahnost always mists aud it is possible to ndx c!m?gcs in n~acromolcculcs that arc very likely to have littlc or no pharmacological or chnical effect. The search for clinical superiority The definition of differences in clinical effect that would be suffkient to support a couclusion that two drug were ditrercnt is critical. On what basis would a new phnm3nceutical glycoprotein be considered differcnt? What sort ofclinical advantages would have to be dcmolxtrated? Clinical superiority would have to be shown by at lcast one of the following criteria: (I) Grcap:r cffectiveneS~ based on direct comparative clinical trials: (2) Substantially greater safety, for instance, by the elimination of a contaminant or ;m ingrcdicnt associated with advclyr cGcts: (3) Make a major cormibution to patient care, for instance, dViiC?OpiiiUlt of a n oral vs parentcral dosage form. III any cvc-ot, the burden ofproof will bc on the sponsur ofthc subsequent drug who is contending that the new drug is ditfcrcnt and superior. An optional proposal by FDAzH as a possible approach for dctermining difkrcnccs between two drugs is as follows:
This ‘option gives considcrablc ‘conceptual’ cxclusivity, pi-otccting the first product to treat an orphan discast, against a second sponsor’s attcn3pts to break cs&sivity through introducing minor molecular rhanges. It wou!d also bc reasonably simple to implement. Most chemical dif&renccs simply would not cause a drug to be considered different unless they Wcrr shown to create a significant clinicai dL&;cnce.
119 J
feature
Wndcr thisoption, two pharmaceutical glycoprotein products would be dcemcd to be the same ifthc. .~sqly diffcrcnces in chemical structures are due to post,translational events, infidelity of :ranslation, infidelity of transcription, or minor differences in amino acid sequence. Structural di&renccs in &~co.~ylatlon patterns or tertiary structu-f*c are also presumed not to result in d&c-rent drugs. This presumption oisameIIL'SS can be overcome by evidence of ‘clinical supcriority’
as
outlined
above.
Genentech, Eli Lilly and hGH The scope and nature oTthe ‘sa_mc’dmg debate can be gauged in the context of two macromolecular pharmaceuticals approved as orphan products, humau growth hormone (hGH) and EPO”. Two rUNA hGH pmducts are currci,tly mandated for the same patient population. Gcncntcch’s 197~amii10 acid vcrsion ofhGH was approved first as an orphan drug, and contains a terminal Met residue not present in the natural product. Eli Lilly’s rl>NA-derived 191 -amino acid version was subsequently approved as an orphan drug and is identical to the natural product, The FDA’s reason for approval of the Lilly’s hGH is based partly on the &mica1 diffcrcncc ofthc two products, in this IllSt311CC, by one amino acid residue at the amino termiuus, and additionally on the findiugs that the Lilly’s product. which is identical to the natural product, is less antigenic than Gcncntech’s product. This should lead to increased saZety and the posslblhty of a Iowcr risk of therapeutic failure. Amgen, Chugai-Upjohn and EPO The cast much tnorc akin to glycoprotcin pharmacectic.ils involves ElJO. Amgcn is in patent litigation to prevent marketing of a competitor rDNA-derived EPO product produced by C!:iigai-iipjohn. Amgcn has rrceived scvcn years of marketing exclusivity from the FDA under the Orphan Drug Act for its EPO. The two products are identical in their amino acid sequences, but exhibit some variation in glycosylation. Chugai-Upjohn has claimed that the glycosylation ditrerencc can produce a biologically significant difference making the drug different for purposes of the Orphan Drug Act. Chugai-Upjohn’s claim has so far not been favcraily considered. Defining the parameters that make a drug the same or different for purposes of the exclusivity provision is not an easy problem to resolve. The approach taken by the FDA, based on a chemical structure test. involving thr active rnoirty of a drug in conjunctio? with consider;ltions of clinical superiority, is deemed a significant step in the right direction towards solving the ‘same’ drug issue”.
mammalian cells: (1) the site ofglycosylation is dictated to a large extent by the primary amino acid scqucncc of the protciu and the host ccl1 typ&s; (2) the type of glycan structure observed at a specific glycosylation site is grcszly preserved’: (3) microhcterogcneity is rcquently due to the prcscncc or absence of t;rminal f’ucosc or sialic acid, and the extent of site microheterogeneity due to other sources is oficn minimal’; (4) there is a rcmarkablc dcgrec of similarity in the oligosaccharidc structures of a particular gIycoprotcin isolated from human sources and its rccombinnnt counterpart produced in specific hctcrologous mammalian cells, such as CHO cells”. For rDNA-dcrivcd therapeutic glycoprotcins, it would seem to bc advantageous and, in many instances, i.ssential. for human glycoprotcins containing multiple disulfidc bonds to be synthrtizrd il: niammzlian cells (that carry out correct glycosplation, disulfide bond formation and secretion of the product), rather than in illsect, yc;lst or prokaryotic cclis. Although human glycopro&is synthcsizcd in hcterologous mammalian cells may dit%r in their detailed glycan stmcture, it n~ay well be easier to rccogkc and characterize dit;fcrent glycoforms than to dctcrminc the biological roles of such structural ditf;-renccx, or to conclude that one glycoform (or one *et of glycofonns) is clinically superior to another. Acknowledgements The author wishes to express appreciation to Drs T. Bull, K. Scamon and 1’. Noguchi for their critical review of this manuscript, and to Dr E. Esbrr for providingregulatory documents from WHO. EC. and Japan. The cspcrt assistance of S. Unger in the preparation of this paper is deeply appreciated. References
Conclusions Protein-linked oligosaccharides can display enorHOLE diversity in structure’“T3”, creating consldcrable microheterogeneity for a 2, en glycoprotein. There do, however, appear to be identifiable paramccers which determine glycosylation characteristics in recombinant proteins produced from traosfccted TIBTfCii 4FRtL 1992 NOF 101
Recombinant inbred mouse strains: models for disease study Monica J. Justice, Nancy A. Jenkins and Neal G. Copeland Recombinant inbred (RI) strains of mice and the closely related reclsmbinant congenie strains offer considerable promise for identifying and characterizing genes causally associated with many different diseases. Loci associated with diseases such as heart disease, autoimmune disease and leukemia have already been identified through the use of these unique strains. The laboratory mouse is well-suited 2s a model organism for the study of human diseases’. For example, many spontaneous mouse mutations have been idcnti&d that mimic human disease. Thcsc include some of the earliest-studied mouse variants such as rodlcss retina, shaker, hyperglycemia and hypopituitary dwarfism’, as well as more-recently identified mutations such as the mouse r& mutation (a mod4 for human muscular dystrophy)“. Unlike humans, the mouse can be manipulated genetically as well as experimentally. One can perform controlled matings, can specifically alter genetic traits, and can control the genetic background influencing specific diseases. Likewise, one can cxpcrinirntally manipulate various fattars that may have an influctlcc on the dcvelopmcnt of disease.
In 1916, C. C. Little reported that ‘hereditary &ctars play an extremely important part in determining the incidence of cancer iu micc’J. However, at that time, mice were not well-suited for studying the mode ofinheritancc ofsusceptibiiity to cancer bccausc ofthe lack ofhontogencity in the genetic background. Consequently, many inbred strains were derived in order to study the genetics of cancer. An inbred strain of mouse is created by brothersister mating for over 20 generations. Thus, all members of an inbred strain exhibit a high degree ofsimilarity both genetically and physically. Studies ofinbrcd strains of mice led to the knowledge that many genes, including oncogcncs (see Box l), gcncs affecting immune function (c.g. histocompatibility loci), and many unidentified genes play a role in the development of, and susceptibility to, cancers.“. It became obvious that cancer was not a Elscvier acknowledger the tight of the US Govrmnwnt and its agcnrr and contracton to retain a non-e?iclusivc royalcpficc license in and to any copytight coveting thr articic.
--.__I____ TIBTECH APRIL 1992 lvOL10)
