SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 25, SUPPL. 3, 1999
`
`Production and Chemical Processing of
`Low Molecular Weight Heparins
`
`ROBERT J. LINHARDT, PH.D. AND NUR SIBEL GUNAY, M.S.
`
`ABSTRACT Heparin is an animal tissue extract
`that is widely used as an anticoagulant drug. A num(cid:173)
`ber of low molecular weight heparins (LMWHs), in(cid:173)
`troduced in the past decade, are beginning to displace
`pharmaceutical (or compendial) grade heparins as
`clinical antithrombotic agents. This article desocibes
`the chemical properties of the glycosaminoglycan
`(GAG) heparin and how it is prepared and processed
`into pharmaceutical grade heparin. There are several
`commercially produced LMWHs that are prepared
`through the controlled depolymerization of pharma(cid:173)
`ceutical grade heparin. The chemistry of the com(cid:173)
`mercial processes used for manufacturing LMWHs is
`discussed. Structural differences are found in the
`LMWHs prepared using different commercial
`processes. Careful control of process variables has
`generally resulted in the reproducible preparation of
`LMWHs that are structurally uniform and of high
`quality. The specifications, however, remain different
`for each LMWH. Thus, LMWHs are a group of simi(cid:173)
`lar but different drug agents. As the structural prop(cid:173)
`erties of LMWHs vary significantly, the bio-equiva•
`lence or
`inequivalence of
`these agents must
`ultimately be established by the pharmacologists and
`the clinicians.
`
`Keywords: Low molecular weight heparin,
`analysis, structure, process, production
`
`Heparin, a clinical anticoagulant, has been one of
`the most effective and widely used drugs of this
`century. 1•2 As one of the oldest drugs currently stN.l in
`widespread clinical use, heparin is unique as it is among
`the first biopolymeric drugs and one of only a few carbo-
`
`Department of Medicinal and Natural Products Chemistry, University
`of Iowa, College of Pharmacy, Iowa City, Iowa.
`Reprint requests: Dr. Linhardt, Professor of Pharmacy and Chem(cid:173)
`ical Engineering, University of Iowa, College of Pharmacy, PHAR
`303A, University of Iowa, Iowa City, IA 52242.
`
`hydrate drugs. Indeed, heparin's introduction predates
`the establishment of the United States Food and Drug
`Administration. 2 Low molecular weight heparins
`(LMWHs ), also referred to as low molecular mass he(cid:173)
`parins (LMMHs ), are a group of heparin-derived antico(cid:173)
`agulant/antithrombotic agents that began their develop(cid:173)
`ment during the last quarter of this century. 3-7
`The introduction of LMWHs primarily resulted
`from an improved understanding of the molecular basis
`of the biochemistry associated with the coagulation cas(cid:173)
`cade. 8-10 The isolation of the serine protease inhibitor,
`antithrombin III (AT), and the characterization of coagu(cid:173)
`lation factors (serine proteases), such as thrombin and
`factor Xa (inhibited by AT), were critical in driving the
`development of LMWHsJ,8 Heparin accelerates the in(cid:173)
`hibition of these coagulation factors by AT, preventing
`the generation of a fibrin clot. In the coagulation cas(cid:173)
`cade, one factor activates the next until prothrombin
`(factor II) is converted to thrombin (factor Ila) by factor
`Xa. It is thrombin that acts on fibrinogen to form a fibrin
`clot.8 The very nature of this cascade suggested a thera(cid:173)
`peutic opportunity to develop an agent that was more
`specific than heparin (which acts at many points in the
`cascade) and that might provide more subtle regulation
`of coagulation and reduce the major hemorrhagic side
`effects associated with heparin. LMWHs were originally
`developed based on this rationale. Various laboratories
`observed that when heparin is fractionated based on size
`or broken down chemically or enzymatically, its activity
`against thrombin is decreased to a much greater extent
`than its activity against factor Xa. 11 - 15 The separation of
`activities result from differences in their molecular re(cid:173)
`quirements for inhibition. Factor Xa interacts directly
`with AT bound to a specific pentasaccharide sequence in
`heparin (the AT binding site) and requires a short heparin
`chain comprised only of these saccharide units for its in(cid:173)
`activation.5 In contrast, thrombin must also bind adja(cid:173)
`cent to AT on a flanking sequence in heparin, thus re(cid:173)
`quiring a longer heparin chain with 18 or more
`saccharide units for its inactivation.3,8 As factor Xa lies
`at the convergence of the extrinsic and intrinsic path(cid:173)
`ways of the coagulation cascade, it was speculated that a
`
`Copyright ©1999 by Thieme Medical Publishers, Inc. 333 Seventh Avenue, New York, NY 10001. Tel: + 1(212) 760-0888, ext. 132.
`0094-6176/1999/E1098-9064(1999)25:S3:0005-0016:STH00547X
`
`5
`
`Luitpold Pharmaceuticals, Inc., Ex. 2027, P. 1
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`

`
`6
`
`SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 25, SUPPL. 3, 1999
`
`LMWH having an enhanced anti-factor Xa/anti-factor
`Ila ratio would facilitate more subtle regulation of coag(cid:173)
`ulation and an improved therapeutic index.8-10
`Our understanding of the precise mechanism of ac(cid:173)
`tion of LMWHs through biochemical, pharmacological,
`and clinical studies have suggested that the initial ratio(cid:173)
`nale for their development may have been naive and not
`entirely correct.7·8 Nevertheless, LMWHs have been
`successfully introduced as new effective and improved
`antithrombotic/anticoagulant agents
`throughout
`the
`world. This review focuses on the chemical processing
`and production of LMWHs and how the various process(cid:173)
`ing routes result in structural differences among these
`pharmaceutical agents.
`
`WHAT IS HEPARIN?
`
`Within a decade of its discovery in 1916, heparin
`was identified as an anionic polysaccharide containing a
`uronic acid residue2 (Fig. 1). Early researchers showed
`that heparin also contained 0-sulfate esters and N-sul(cid:173)
`fated glucosamine residues. By 1970, iduronic acid was
`shown to be the major uronic acid component in heparin
`and a generalized structure of heparin could be drawn.
`Over the past two decades, the structure of the AT pen(cid:173)
`tasaccharide binding site has been discovered with much
`of heparin's fine structure elucidated, and an improved
`understanding of its conformation2l-23 and interaction
`with proteins was established.16-18,20,24-27
`Heparin is prepared by extraction from the tissue of
`slaughter house animals (i.e., porcine intestine, bovine
`lung). Like all other natural polysaccharides, heparin is a
`polydisperse mixture containing a large number of chains
`having different molecular weights (MWs).28,29 Heparin
`is composed of a major trisulfated disaccharide repeating
`unit (Fig. 1 ), but it also contains a number of additional
`disaccharide structures.18-20 It is these additional disac(cid:173)
`charide units that make heparin's structure complex and
`that also comprise the AT pentasaccharide binding site,
`important for heparin's anticoagulant activity.
`
`The heparin family of glycosaminoglycans (GAGs)
`includes both heparin and the related undersulfated poly(cid:173)
`saccharide heparan sulfate.30.31 While heparin and he(cid:173)
`paran sulfate GAGs are biosynthesized through a com(cid:173)
`mon pathway, structural studies clearly indicate that the
`structures of heparin and heparan sulfate are distinctly
`different.32,33 All the disaccharides found within heparin,
`including those comprising the AT pentasaccharide bind(cid:173)
`ing site34, are also found within heparan sulfate but in
`different proportions.30 Heparin and heparan sulfate,
`both found in tissues commonly used to prepare pharma(cid:173)
`ceutical grade heparin, differ substantially in their anti(cid:173)
`coagulant activity.3o Extraction methods that focus on
`the high specific anticoagulant activity required to meet
`United States Pharmacopeia (USP) specifications serve
`to eliminate much (but not all) of the heparan sulfate
`GAG from pharmaceutical grade heparin.
`Pharmaceutical grade heparin is a purified tissue
`extract comprised primarily of polydisperse GAGs con(cid:173)
`sisting primarily of heparin but containing other GAGs,
`such as heparan sulfate. Small amounts of dermatan sul(cid:173)
`fate, once present in some pharmaceutical grade he(cid:173)
`parins, have now been virtually eliminated.18,28,35 Chains
`of molecular weight from 5000 to over 40,000, making
`up polydisperse pharmaceutical grade heparin, also dis(cid:173)
`play significant sequence heterogeneity.' For example,
`many fully sulfated heparin chains are simply composed
`of uniform repeating sequences of trisulfated disaccha(cid:173)
`ride (Fig. 1). Alternatively, heparin chains having an in(cid:173)
`termediate level of sulfation are comprised of long seg(cid:173)
`ments of fully sulfated sequences with intervening
`undersulfated domains, such as that comprising the AT
`pentasaccharide binding site (Fig. 1). Finally, undersul(cid:173)
`fated heparin chains ( <2 sulfate groups/disaccharide)
`may simply be contaminating heparan sulfate.
`Not all heparin chains contain an AT pentasaccha(cid:173)
`ride binding site. Only 20 to 50% of the polysaccharide
`chains comprising pharmaceutical grade heparin contain
`an AT binding site and are called "high affinity hepa(cid:173)
`rin."1 No difference has been reported in the overall
`charge.or the average size of high affinity and low affin-
`
`OH
`
`Trisulfated disaccharide Disulfated disaccharide Antithrombln Pentasaccharide Binding Site
`
`Trisulfated disaccharide
`
`n+m= 16
`for MW 12,000
`FIG. 1. Chemical structure of a representative chain of pharmaceutical heparin. Clusters of trisulfated disaccharides (n and m, where
`n + m = 16 for MW 12,000), flank disulfated disaccharides and AT pentasaccharide binding site (ABCDE). Some structural variability
`both within and outside the AT binding site is indicated by multiple substituents. The attached substituents correspond to the major AT
`binding site structure found in porcine intestinal heparin.
`
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`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`

`
`PRODUCTION AND CHEMICAL PROCESSING OF LMWHS-LINHARDT, GUNAY
`
`7
`
`TABLE 1. Composition of Heparins from Different Species and Tissues*
`
`Species
`
`Porcine
`Bovine
`Bovine
`Ovine
`Hen
`Clam
`
`Tissue
`
`intestine
`lung
`intestine
`intestine
`intestine
`
`N-acetylAT
`Binding Site
`
`0.5 (0.3-0.7)
`0.3
`0.3
`0.7
`0.3
`0.5
`
`Average Number in One Heparin Chaint
`
`N-sulfoAT
`Binding Site
`
`Trisulf ated
`Disaccharide
`
`Disulfated
`Disaccharide
`
`0.1
`0.3
`0.3
`0.4
`0.2
`0.4
`
`10 (10-15)
`14
`10
`11
`6.7
`5.0
`
`1.2(1-2)
`1.0
`1.7
`1.4
`1.7
`1.9
`
`*adapted from Loganathan et al.19
`tThe numbers shown in parentheses indicate a range of values typically observed.
`
`ity heparin. Some high MW heparin chains may contain
`more than a single AT binding site and thus display an
`enhanced level of anticoagulant activity. 36
`Heparins obtained from different tissues and differ(cid:173)
`ent species also differ structurally (Table 1).19 The most
`widely used tissue for the preparation of pharmaceutical
`grade heparin is porcine intestine. Heparin prepared from
`bovine lung differs substantially from porcine intestinal
`heparin. Bovine lung heparin has a higher sulfation level
`and slightly higher MW than porcine intestinal heparin,
`increasing its affinity for thrombin (factor Ila). Porcine in(cid:173)
`testinal heparin contains an AT binding site primarily hav(cid:173)
`ing an N-acetyl group in residue A (Fig. 1), while bovine
`lung heparin primarily has an N-sulfo group at residue A,
`resulting in their slightly different affinities for AT.19
`The disaccharide composition of porcine intestinal
`heparins can also differ substantially from each other.18
`The two mainly used raw materials (intestinal mucosa
`and whole intestine) contain differing amounts of conta(cid:173)
`minating heparan sulfate that can carry over into the fi(cid:173)
`nal pharmaceutical product. There are different sub(cid:173)
`species of hogs and the mast cell content of intestinal
`tissue can vary based on the diet and environment in
`which the animals are raised. These variables potentially
`contribute to the already complex structure of pharma(cid:173)
`ceutical grade heparin.
`
`HOW IS HEPARIN PREPARED?
`
`Methods of commercial production of pharmaceuti(cid:173)
`cal grade heparin are tightly guarded industrial· secrets
`and few publications or patents describe most commonly
`used pharmaceutical processes. The process of preparing
`pharmaceutical grade heparin has been altered some(cid:173)
`what over time as the primary tissue source has changed
`from dog liver to beef lung and finally to porcine intes(cid:173)
`tine.37 The methods used today for the commercial
`preparation of heparin involve five basic steps: (1)
`preparation of tissue; (2) extraction of heparin from tis(cid:173)
`sue; (3) recovery ofraw heparin; ( 4) purification of hep(cid:173)
`arin; and (5) recovery of purified heparin.
`
`The preparation of the tissue begins with the collec(cid:173)
`tion of the appropriate animal organ tissue at the slaugh(cid:173)
`terhouse and its preparation for processing. The whole
`intestine is either used to prepare "hashed pork guts" or
`processed into casings, which requires removal of the
`endothelial lining from the intestinal lumen. Crude hepa(cid:173)
`rin extraction typically takes place at the hog slaughter(cid:173)
`ing facility itself. Additional high potency heparin may
`be recovered by saving the waste brine solution of the
`hog casings operation.38
`In the second step, heparin is separated from the
`tissue. The use of elevated temperatures and pressures39
`and/or proteases ensures the solubilization of all GAGs.
`Currently, commercial crude heparin extraction processes
`involve hydrolysis at alkaline pH aided by proteolytic en(cid:173)
`zymes. Optionally, the digested tissue may be filtered or
`screened to remove any large particles yielding a deeply
`colored solution containing GAGs, peptides, and nucleic
`acids. At this point, the enzyme is often inactivated by
`heating the filtrate for 15 minutes at 90°C, at the same
`time serving as a sanitary step.
`The third step is the recovery of raw heparin. Cur(cid:173)
`rently, anion exchange resin is added, enabling the hepa(cid:173)
`rin-like GAGs to selectively adsorb onto the resin4o ac(cid:173)
`cording to the charge-density of the different GAGs.
`After complete adsorption of the heparin, the resin is de(cid:173)
`livered to a crude heparin manufacturing facility where
`it is washed and subsequently eluted. The concentrated
`crude heparin solution is usually filtered, precipitated,
`and vacuum dried (stage 12 heparin).
`The purification of crude heparin is typically per(cid:173)
`formed under good manufacturing practices conditions
`and deals with potential impurities originating from the
`starting material or introduced during crude heparin extrac(cid:173)
`tion. Generally, the crude heparin is dissolved in purified
`water, filtered at low pH to remove residual protein, and
`oxidized at alkaline pH to sanitize, decolorize, and depyro(cid:173)
`genate the material. This is often followed by cation ex(cid:173)
`change chromatography to remove extraneous cations,
`ethanol precipitation to reduce nucleotides, and sometimes
`by a chemical treatment to inactivate any viruses that
`might be present. The purified heparin is precipitated and
`
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`
`

`
`8
`
`SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 25, SUPPL. 3, 1999
`
`either vacuum-dried or redissolved in purified water, fol(cid:173)
`lowed by various filtration steps and freeze-drying. The
`yield of porcine intestinal heparin is typically 10-25 mg/g
`wet tissue corresponding to 30,000 to 50,000 U/animal.
`
`WHAT ARE LMWHS?
`
`LMWHs are defined as salts of sulfated GAGs hav(cid:173)
`ing an average MW of less than 8000 Da and for which
`at least 60% of all molecules have a MW of less than
`8000 Da. These are obtained by fractionation or depoly(cid:173)
`merization of heparin and have a potency of greater than
`70 units/mg of anti-factor Xa activity and a ratio of anti(cid:173)
`factor Xa activity to anti-factor Ila activity of::=:: 1.5.41
`Before any LMWHs had been approved for human
`use, the implicit goal of pharmaceutical scientists was
`that the composition of these LMWHs should closely re(cid:173)
`semble the structure of heparin in all aspects except MW
`and ratio of anti-factor Xa to anti-factor Ila activity. An
`ideal LMWH might be simply a LMW subfraction of
`heparin prepared by sizing, using gel permeation chro(cid:173)
`matography (GPC). Direct size fractionation has been
`used to prepare a LMWH on a laboratory bench scale
`that exhibits the appropriate MW and activity properties,
`and contains the same disaccharide composition and se(cid:173)
`quences as heparin. Such methods, however, are rarely
`used on the scale required for the commercial manufac(cid:173)
`ture of heparin.42 Pharmaceutical chemists have relied
`on a number of chemical or enzymatic depolymeriza(cid:173)
`tion methods to manufacture commercial quantities of
`LMWHs. These depolymerization methods were se(cid:173)
`lected to give a product with: (1) suitable average MW
`and low polydispersity; (2) anti-factor Xa/anti-factor Ila
`activity >1; (3) structure similar to a LMWH prepared
`through fractionation and with few structural artifacts
`resulting from the depolymerization method used; ( 4) no
`residual toxic reagents;43 and (5) a cost-effective repro(cid:173)
`ducible and scalable process having a minimum number
`of process steps, little if any required purification, nei(cid:173)
`ther labor, reagent nor capital intensive, and high yield(cid:173)
`ing. While no current manufacturing process meets all of
`these goals, the currently used processes have afforded a
`first generation of clinically useful LMWHs.
`
`METHODS FOR PREPARING LMWHS
`
`Many years of experience in the manufacture of
`pharmaceutical grade heparin have shown that it exhibits
`a surprisingly high level of physical and chemical stability
`with a shelf life approaching a decade. Numerous
`processes have been used to prepare pharmaceutical grade
`heparins involving the use of harsh conditions including
`elevated temperature, pressure, shear, high ionic strength,
`acid, base, and organic solvents. These processes have re(cid:173)
`producibly afforded a uniformly high quality product. The
`
`success of the manufacturers represented the only avail(cid:173)
`able data demonstrating the physical and chemical stabil(cid:173)
`ity of heparin. Recently, an accelerated stability study un(cid:173)
`der elevated temperatures and under acidic and basic
`conditions confirmed the surprising stability of heparin.44
`A decomposition pathway for heparin under these stressed
`conditions has been proposed. Elevated temperatures can
`result in substantial damage to functionality within the
`heparin molecule (i.e., loss of sulfation) that occurs con(cid:173)
`currently with depolymerization. Neutral and acidic path(cid:173)
`ways result in a similar formation of small desulfated
`products, while the basic pathway terminates in a Canniz(cid:173)
`zaro reaction and de Bruyn van Eckenstein rearrange(cid:173)
`ment.42 Other physical parameters such as agitation result
`in no structural alterations, as the heparin molecule is not
`sufficiently large to be shear-sensitive.
`The oxidative instability of heparin had been
`widely observed by heparin manufacturers. Indeed, an(cid:173)
`tioxidants (bisulfite and metal chelators) have been
`added at various stages in heparin's manufacture to en(cid:173)
`hance stability.42 These observations suggested the pos(cid:173)
`sibility of utilizing oxidative methods
`to prepare
`LMWHs. Manufacturers have also observed microbial
`degradation of heparin. A bacterial enzyme, heparin
`lyase I (heparinase), is known to act on heparin.45,46 This
`enzyme acts in a random endolytic fashion through a 13-
`eliminative cleavage mechanism.46 This enzymatic reac(cid:173)
`tion can be mimicked chemically by esterifying the car(cid:173)
`boxyl group of the uronic acid residue and treating the
`resulting heparin ester with base.47 Thus, enzymatic or
`chemical 13-eliminative cleavage offers a second possi(cid:173)
`ble method for heparin depolymerization and the manu(cid:173)
`facture of LMWHs. It is interesting to note that many of
`the processes to prepare LMWHs started out as analyti(cid:173)
`cal tools to degrade heparin in an effort to understand its
`structure.
`Heparin can be oxidatively broken down using a
`variety of oxygen containing reagents like hydrogen per(cid:173)
`oxide or by ionizing -y-irradiation (Fig. 2).11.48-50 Each of
`these methods relies on the generation of oxygen radi(cid:173)
`cals that !ife believed to act by oxidizing sensitive sac(cid:173)
`charide residues within the heparin polymer. Nonreduc(cid:173)
`ing sugars are essentially inert to aqueous hydrogen
`peroxide except in the presence of alkali or in the pres(cid:173)
`ence of a metal catalyst. Both of these conditions lead to
`the generation of the hydroxy 1 radical that will react with
`sugar residues and degrade them to 1-, 2- and 3-carbon
`fragments without modifying the residues on either side
`of the point of attack. The most susceptible residues ap(cid:173)
`pear to be those that are unsubstituted at positions 2 and
`3 in the sugar ring. Studies of the composition of disac(cid:173)
`charides that result from oxidative depolymerization of
`LMWHs suggest that nonsulfated uronic acid residues in
`heparin are selectively oxidized to volatile acids (i.e.,
`formic acid).51 Under controlled conditions (tempera(cid:173)
`ture, pressure, time, oxidant), LMWHs having appropri(cid:173)
`ate MWs and activity can be obtained in reasonable
`
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`
`

`
`PRODUCTION AND CHEMICAL PROCESSING OF LMWHS-LINHARDT, GUNAY
`
`9
`
`HO
`
`OX
`
`O
`
`o
`
`~HY
`
`0
`
`H
`
`OH
`
`OH
`
`OX
`
`m
`
`NHS03-
`
`f:{-
`~2ox Q }2 - CH,OS03-
`0xid~
`co:;on ~
`
`Chemic~) - _
`/
`~-elimin/
`
`~ ~-~1hmatic
`~mination
`
`CH,OS03-
`
`~0"
`
`NHS03-
`
`£;-0"
`
`NHS03-
`
`m
`m
`FIG. 2. Depolymerization of heparin to prepare LMWHs. The heparin chain in the center can undergo depolymerization by each of
`the four processes shown. Heparin chain size is reduced (n > m), affording a LMWH.
`
`yields. 11 •4s,s2 Of these oxidative methods, only hydrogen
`peroxide has been utilized to commercially prepare
`LMWHs (ardeparin sodium and parnaparin sodium) for
`clinical use (Table 2).
`In addition to oxygen radical processes, it is possi(cid:173)
`ble to oxidatively depolymerize heparin through deami(cid:173)
`nation (Fig. 2). In these reactions, heparin is N-ni(cid:173)
`trosated, using either nitrous acid or another nitrosating
`reagent such as isoamyl nitrite, at the amino group of its
`N-sulfoglucosamine residues. The resulting unstable N-
`
`nitrososulfamide loses nitrogen and sulfate and gener(cid:173)
`ates a carbocation at C-2 of the saccharide residue. Sub(cid:173)
`sequent ring contraction of this residue and hydrolysis of
`the adjacent glycosidic bond affords a LMWH. Each
`product chain resulting from this process contains an an(cid:173)
`hydromannose residue (bearing a terminal aldehyde) at
`the reducing terminus. This residue can subsequently be
`converted to anhydromannitol using a reducing agent,
`such as sodium borohydride. Controlled deaminative
`cleavage is possible by controlling the process condi-
`
`TABLE 2. Commercially Avaifable LMWHs
`
`LMWH
`
`Trade Name
`
`Manufacturer
`
`Preparation Method
`
`Ardeparin sodium
`Certoparin sodium
`Dalteparin sodium
`
`Normiflo
`Sandoparin
`Fragmin
`
`Enoxaparin sodium
`
`Lovenox
`Clexane
`
`Wyeth-Ayerst
`Novartis
`Pharmacia-U pjohn
`Kissei
`Rhone-Poulenc Rorer
`Avantis
`
`Oxidative depolymerization with H20 2
`Deaminative cleavage with isoamyl nitrite
`Deaminative cleavage with nitrous acid
`
`B-eliminative cleavage of the benzyl ester of heparin
`by alkaline treatment
`
`Nadroparin calcium
`
`Fraxiparin
`
`Sanofi-Winthrop
`
`Deaminative cleavage with nitrous acid
`
`Parnaparin sodium
`Reviparin sodium
`
`Fluxum
`Clivarin
`
`Alfa Wassermann
`Knoll
`
`Oxidative depolymerization with Cu+ and Hz02
`Deaminative cleavage with nitrous acid
`
`Tinzaparin sodium
`
`Innohep
`Logiparin
`
`Braun
`Novo/Leo/Dupont
`
`B-eliminative cleavage by heparinase
`
`Approved
`Markets
`
`USA
`Germany
`USA, Japan
`UK, Germany
`USA
`Germany
`Spain
`France
`Germany
`Italy
`Canada
`Germany
`Germany
`Denmark,
`USA
`
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`

`
`10
`
`SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 25, SUPPL. 3, 1999
`
`tions (temperature, pH, time) or by limiting the amount
`of nitrosation reagent.53,54 The LMWH product formed
`using these controlled conditions is obtained in high
`yield and has the appropriate chemical and biological
`properties. Several LMWHs prepared through deamina(cid:173)
`tive cleavage are currently used clinically (Table 2).
`Two 13-eliminative methods, one enzymatic and the
`other chemical, are used to commercially prepare
`LMWHs. In the enzymatic method, heparin lyase I (or
`heparinase) is used to depolymerize heparin.is The ex(cid:173)
`tent of this reaction can be conveniently monitored by
`measuring the change in absorbance associated with the
`unsaturated uronate residue formed in each product mol(cid:173)
`ecule.46,55 Cleavage takes place only at the 2-0-sul(cid:173)
`foiduronic acid residue.46 The depolymerization is
`stopped by removing or inactivating the enzyme. After
`recovery of the GAG from the enzyme and very low
`MW byproducts (i.e., disaccharides and tetrasaccha(cid:173)
`rides), a LMWH is obtained that has the desired MW
`and activity properties. This method is used to prepare
`the clinically used LMWH product, tinzaparin sodium
`(Table 2). Chemical 13-elimination can involve the direct
`treatment of heparin or its quaternary ammonium salt
`with base.56,57 Alternatively, the benzyl ester can be pre(cid:173)
`pared by treatment of the benzethonium salt of heparin
`with benzyl chloride and base with heating.58 Under
`these conditions, chemical 13-elimination takes place, af(cid:173)
`fording LMWH that contains an unsaturated uronate
`residue in the nonreducing end. Cleavage occurs specifi(cid:173)
`cally at iduronic acid without preference for the presence
`or absence of a 2-0-sulfo group. Proper control of these
`process conditions affords the clinically used LMWH,
`enoxaparin sodium (Table 2).
`
`ANALYSIS OF HEPARIN AND LMWHS
`
`The analysis of pharmaceutical grade heparin is
`governed by the USP and other national pharmacopeia.
`Bioanalyses include a routine in vitro coagulation assay
`and a test for bacterial endotoxins (pyrogens). The hepa(cid:173)
`rin assay, when expressed in units/mg or international
`units/mg, is the amount of heparin that will cause 1 ml of
`sheep plasma to half-clot when kept for 1 hour at 20°C
`compared to a USP reference standard (K-4) or the Fifth
`International Standard for Unfractionated Heparin
`(WH0-5), respectively. The analysis of LMWHs is' gov(cid:173)
`erned by the European Pharmacopeia and proposals
`have also been made to amend the USP monograph to
`cover LMWHs.41 Such analyses include in vitro ami(cid:173)
`dolytic assays in a purified system for anti-factor Xa and
`anti-factor Ila activities, and structural identification by
`nuclear magnetic resonance and size exclusion chro(cid:173)
`matography.
`Less routine analyses rely on in vitro protein (i.e.,
`AT) binding assays and protein interaction studies, so(cid:173)
`phisticated ex vivo or in vivo animal assays and finally,
`
`human clinical evaluation.3-7,36,59 Refined chemical,
`chromatographic, electrophoretic, and spectroscopic as(cid:173)
`says have been developed that are often used by research
`laboratories studying heparin. The presence of unsubsti(cid:173)
`tuted glucosamine can be measured in heparin and
`LMWHs by chemical modification with an amine reac(cid:173)
`tive fluorescent reagent.60 While chemical assays for
`uronic acid glucosamine have been used in heparin
`analysis, these have been largely displaced by rapid and
`more reliable spectroscopic analysis.61 Chromatographic
`analysis of heparin and LMWHs primarily involves
`high-pressure liquid chromatography (HPLC), such as
`gel-permeation chromatography (GPC) to determine
`MW and polydispersity.28,29 This method, once requiring
`the tedious production of MW standards, can now be
`performed using a heparinase-prepared LMWH standard
`through the clever use of dual mass and absorbance de(cid:173)
`tection. 20,62 Ion exchange chromatography, following
`sample pyrolysis, can be accurately used to determine
`the level of sulfation or the presence of contaminating
`metals or anions. 63 Disaccharide compositional analysis
`(Table 3) and oligosaccharide mapping of heparin and
`LMWHs have in the past relied on HPLC analysis fol(cid:173)
`lowing the complete or partial heparinase catalysis of ni(cid:173)
`trous acid depolymerization of heparin or LMWHs.51,64
`Capillary electrophoresis (CE) has gained popularity,
`making disaccharide analysis and oligosaccharide map(cid:173)
`ping extremely rapid and sensitive.65,66 Oligosaccharide
`mapping by CE can provide data on the content of AT
`binding sites in different lots of a given LMWH.67 Poly(cid:173)
`acrylamide gel electrophoresis has been used to deter(cid:173)
`mine the MW and polydispersity of heparin and
`LMWHs.68 Spectroscopic analysis, while requiring ex(cid:173)
`pensive instrumentation, can facilitate the high resolu(cid:173)
`tion analysis of heparin and LMWHs, often providing
`information that is not available using any other tech(cid:173)
`niques. Proton nuclear magnetic resonance (NMR) spec(cid:173)
`troscopy has been used to determine the ratio of iduronic
`to glucuronic acid, the content of an N-acetylglu(cid:173)
`cosarnine, and position of sulfation.20,3o,59,60 Since this
`method is· quantitative and nondestructive, it can be con(cid:173)
`veniently used to afford a disaccharide analysis.30 Proton
`NMR has also replaced more tedious methods for the de(cid:173)
`termination of contaminating GAGs, such as dermatan
`sulfate,35 or the contaminating organic molecules that
`might be used in the manufacturing of a pharmaceutical
`grade heparin or LMWH.69 Finally, proton NMR is ex(cid:173)
`tremely valuable in the conformational analysis of hepa(cid:173)
`rin.21,22 Carbon NMR spectroscopy has also been used in
`heparin and LMWH analysis. While requiring substan(cid:173)
`tially more sample than proton NMR, this method re(cid:173)
`sults in higher signal resolution and offers abundant
`structural information.69 By measuring the intensity of
`signals resulting from the reducing end anomeric carbon
`and all other anomeric carbons, this method offers a so(cid:173)
`phisticated standard-free determination of the number(cid:173)
`averaged MW ofheparin7o and LMWH.71 While modern
`
`Luitpold Pharmaceuticals, Inc., Ex. 2027, P. 6
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`

`
`PRODUCTION AND CHEMICAL PROCESSING OF LMWHS-LINHARDT, GUNAY
`
`11
`
`TABLE3. Disaccharide Composition of LMWHss1
`
`Trisulfated Disaccharides
`
`Disulfated Disaccharides
`
`Monosulfated Disaccharide
`
`Sample
`
`Heparin
`Fractionated
`LMWH
`Ardeparin
`Sodium
`Dalteparin
`Sodium
`Enoxaparin
`Sodium
`Nadroparin
`Calcium
`Parnaparin
`Sodium
`Tinzaparin
`Sodium
`
`2SNS6S
`
`NS3S6S
`
`NS6S
`
`2SNS
`
`51.9
`34.7
`
`86.1
`
`49.3
`
`71.3
`
`17.2
`
`48.4
`
`88.9
`
`1.4
`1.2
`
`1.7
`
`2.6
`
`1.9
`
`0.8
`
`0.4
`
`1.9
`
`4.1
`4.0
`
`7.0
`
`3.2
`
`11.4
`
`1.2
`
`3.6
`
`7.5
`
`2.6
`1.6
`
`6.1
`
`3.1
`
`2.5
`
`1.8
`
`4.4
`
`5.4
`
`6S
`
`1.1
`0.9
`
`1.3
`
`2.0
`
`1.4
`
`0.7
`
`0.3
`
`1.4
`
`soft ionization techniques available in mass spectrome(cid:173)
`try (MS) offer some intriguing possibilities for heparin
`and LMWH analysis, the only successful application of
`MS has been the characterization of heparin-derived
`oligosaccharides. Fast atom bombardment (FAB),72
`MS/MS-FAB,73 electrospray ionization (ESI),74 and ma(cid:173)
`trix-assisted laser desorption ionization (MALDI)75 have
`all proved effective in such analyses. CE or HPLC/MS
`interfaces also offer possibilities for improved, standard(cid:173)
`free analysis of heparin disaccharides and oligosaccha(cid:173)
`rides. The primary limitation for the direct MS analysis
`of heparin or LMWHs remains their high polydispersity
`and microheterogeneity. Other spectral techniques, such
`as las