Protein Formulation
`1 and Delivery
`Second Edition
`
`'
`
`Edited by
`Eugene J. McNally
`Gala Biotech, a Catatent Pharma Solutions Company
`Middleton, Wisconsin, USA
`Jayne E. Hastedt
`ALZA Corporation
`Mountain View, California, USA
`
`informa
`
`healthcare
`
`Nl!W York London
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`Bausch Health Ireland Exhibit 2010, Page 1 of 38
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`Library llt Congress Cala loging-in -Puhlica llon Da la
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`Protein fonnulution 1md delivery' I edited by EugcncJ, McNally, Jayne E. Hnstedt. - 2nd ed.
`p.: cm.-·
`(Drugs and the pharmaccu1ical sciences : 175)
`Includes bibliographical references and index.
`ISBN-13: 978-0-8493-7949-9 (hardcover : alk. paper}
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`I. Pro1ei n drugs--Oosagc forms.
`I. McNa lly. Eugene J., 1961- IL HRslcdf. Jayne E.
`Ill. Series: Drugs and 1hc pharmaceutic.ii sciences : v. 175.
`[DNLM: I. Protei n Conformation. 2 Drug Del ivery Sys1cms. 3. Drug Design.
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`Bausch Health Ireland Exhibit 2010, Page 2 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01104
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`2
`
`Chemical Considerations in Protein
`and Peptide Stability
`
`Paul M. Bummer
`University of Kentucky, Lexington, Kentucky, U.S.A.
`
`0EAMI0ATION
`Introduction
`
`The dcamidarion reactions of asparagi ne (Asn) and glutamine (G in) side-chains
`are among the most wldely studied noncnzymatic covalent modifications to
`proteins and peptides ( 1-7). Considerable research efforts have been extended
`to elucidate the details of the dcamidation reaction in both in vitro and in vivo
`systems, and a number of well-written, in-depth reviews are available ( 1- 5,8,9).
`This work touches only on some of the highlights of the reaction and on the roles
`played by pH, temperature, buffer, and other formulation components. Possible
`dcamidation-associated changes in the protein structure and state of aggregation
`also are examined. The emphasis is on Asn deamidation, since Gin is significan tly
`less reactive.
`
`Reaction Mechanism
`
`The primary reaction mechanism for the deamidation of Asn in water-accessible
`regions of peptides and proteins al basic or neutral conditions is shown in Figure I.
`For the present, discussion is confined to the intramolecular mechanism, uncom(cid:173)
`plicated by adjacent ami no acids at other points in the primary sequence. Under
`alkaline conditions, the key step in the reaction is the formation of a deprotonatcd
`amide nitrogen, which cruTies out the rate-determining nucleophilic allack on tl1e
`side-chain carbonyl, re~ulting in a tetrahedral intermediate and fina lly the formation
`of the five-member succinimide ring. For such a reaction, the leaving group must be
`
`7
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`Bausch Health Ireland Exhibit 2010, Page 3 of 38
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`8
`
`Bummer
`
`·~.l::~-R,--
`
`0
`Aspa raglnyl Residue
`
`·~ ~~ N/y R1
`
`0
`
`Reactive rion
`
`Aspartyl Residue
`
`lsoaspartyl Residue
`
`Figure 1 PrnpuscJ rtt1etion rncchanb10 for de.amidation of asparaginyt residue. Note the
`fonnation of the S\1CtinlinhJyl mtctmcJinte and the two possible final products.
`
`easily protonuted, and in this-ca.,e, it is responsible fo,· the characteristic formation of
`ammonia (NH,). The succinimide ring intermediate is subject to hydrolysis, result(cid:173)
`ing in either the corresponding aspartic acid or the isoaspartic acid (~-aspartate).
`Often, the ratio of the products is 3: I, isoaspartate to aspartate ( I 0- 12). In the case
`of acid catalysis (pH < 3), a tetrahedral intermediate is also fom1ed, but breaks down
`with the loss of NH, without going through the succinimide (13- 17). The reaction
`also appears to be sensitive to racemization at the (X--carbon, resulting in mixtures of
`o- and 1.-isomers ( IO, 13-15). The rate of degradation of the parent peptide in aque(cid:173)
`ous media often follows pseudo-first-order kinetics (16,17).
`
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`Chemical Considerations in Prorein and Pepride Stab/lily
`
`9
`
`A number of mher nltema1ive reac1ions arc possible. The mos1 prevalent
`r.,action appe;ir, m be a nuclcophilic u11ack o r 1hc Asn sicle-chain amide nitrogen
`on the pcp1ide carbonyl. resulting in main-chain ckavagc ( 10,16,18). This reac-
`11nn (Fig. 2) i~ slower 1han 1!101 of cyclic imide formauon and l• most frequi,nily
`observed when A,n is followed by pl'lllinc. a re~iduc incapahlc of forming an ion(cid:173)
`ized pcp1ide-bond nitrogen.
`
`pH Dependence
`
`Under conditions of strong acid (pH 1-2), deamidaLion by direct hydrolysis of the
`amide side-chain becomes more favorable than fonnation of cyclic imide ( 16, 19).
`Under 1hese excrcme conditions, the reaction is oOen complicated by main-chain
`cleavage and denaturation. Deamidation by this mechanirn1 h not likely to pro(cid:173)
`duce isoasparta1e or signilicant mcemlza1ion (16).
`Under more moderate conditions, the effect or pH is 1hc resull of two
`opposing reactions: (i) deprotonation of the peptide-bond nitrogen. promoting
`
`+
`
`R1 = Amino end of protein
`
`R2 = Carboxyl end of prolein
`
`figure 2 Proposed reaction mechanism for mam•chain cleavage by asparaginyl residues.
`
`Bausch Health Ireland Exhibit 2010, Page 5 of 38
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`10
`
`Bummer
`
`the reaction and (ii) protonation of the side-chain- leaving group, inhibiting the
`reaction. In deamidation reactions of short chain peptides uncomplicated by
`structural alterations or covalent dimcri1.ation (20) , the pH-rate profi les exhibit
`the expected "V" shape, with a minimum occurring in the pH range of3 to 4 ( 16).
`Computation studies by Peters and Trout (2 1) have been helpful in shedding light
`on the effect of pH . These authors have suggested that under mildly acidic condi(cid:173)
`tions (3 < pH, 4), the rate-limiting step is the attack of the deprotonated nitrogen
`on the side-chain. The rate-l imiti ng step at neutral pH is the hydrogen transfer
`reaction , while under basic conditions (pH > 7), it is the elimination of NH; from
`the tetrahedral intennediate. Experimental studies have shown that the increase
`in rate on the alkaline side of the minimum docs not stricUy correlate with the
`increase in deprotonation of the amide nitrogen, indicating that the rate of reac(cid:173)
`tion is not solely dependent on the degree of the peptide-bond nitrogen deprot(cid:173)
`onation (16,19). The pH minimum in the deamidution reaction measured in vitro
`for proteins may (22) or may nOt (23) fa ll in the same range as that of simple
`peptides. Overall pH-dependent effects may be modified by structure-dependent
`factors, such as dihedral angle Aexihility, water accessibility, and proximity of
`neighboring ami no acid side-chains (see section Peptide and Protein Structnre).
`
`Effect ofTemperature
`
`The temperature dependence of the deamidation rate has been studied in a variety
`of simple peptides in solution ( 16,24,25). Small peptides are easily designed to
`avoid competing reactions, such as oxidat ion and main-chain cleavage, and arc
`thus usefu l to isolate attention directly on the deamidation rate. Jn solution, deain(cid:173)
`idation of small peptides tends to fo llow an An11enius rclarionship. Activation
`energies of the reaction do tend to show pH dependence, and a discontinuity in the
`Arrhenius plot is expected when the mechanism changes from direct hydrolysi
`(acid pH) to one of cyclic imide (mildly acidic to alkaline pH).
`The deamidation rate of proteins al.so hows temperature dependence
`(23,26,27) under neutral pH. For deamidation reactions alone, temperature(cid:173)
`associated rate acceleration in proteins may be due to enhanced flexibility or the
`molecule, allowing more rapid formation of the cyclic imide (28). or it may occur
`by catalysis by side-chains brought into the vicinity of the dcarnidation site (5).
`The avai lability of water appears to be an important determinant in
`temperature-a~sociated effects. In studies of lyophilized formulation of Val-Tyr(cid:173)
`Pro-Asn-Gly-Ala, the deamidation rate constant was observed to increase about an
`order of magnitude between 40°C and 70°C (29). ln contrast, in the solid state, the
`Arrhenius relationship was not observed. Further, the deamidation in the solid state
`showed a marked depende nce upon ~1e temperature when the peptide was lyopbi(cid:173)
`lized from a solution of pH 8, while little temperature dependence was observed
`when lyophili1,ation proceeded from solutions at either pH 3.5 or pH 5. The authors
`related this temperature difference to changes in the reaction mechanism that may
`occur as a function of pH.
`
`Bausch Health Ireland Exhibit 2010, Page 6 of 38
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`Chemical Considerations in Protein and Peptide Stability
`
`11
`
`Adjuvants and Excipients
`
`Tbe influence on deamidation by a variety or buffer ions and solvents has been
`examined. As po inted oul by Cleland el al. (4) and reinforced by Tomizawa et
`al. (13), many of these additives are unli kely 10 be employed as pharmaceuti(cid:173)
`cal excipients for formulation, but they may be employed in protein isolation
`and purification procedures (30). Important clues to stabilizatioo strategies can
`be gai ned from these studies, In the follow ing, it is fruitful to keep in mind the
`importance of the auack of the ionized peptide-bond nitrogen on the side-chain
`carbonyl and the hydrolysis of the cyclic imide (Fig. I).
`
`Buffers
`
`Bulfer catalysis appears to occur in some but not all peptides and proteins studied
`(5). Bicarbonate ( 16) and glycine (12) buffers appear to accelerate deamidatioa. On
`one hand, the phosphate ion has been shown to catalyze deamidation, both in pep(cid:173)
`tides and in proteins ( 12, 13, 16.31-34), generally in the concentration range of 0 to
`20 mM . Capasso et al. (35,36) observed the acceleration of deamidation by acetate,
`carbonate, Tris, morpholi ne, and phosphate buffers only in the neu!ral 10 basic pH
`ranges, On the other hand, Lura and Schrich (37) found no influence on the rnte of
`deamidation of Val-Asn-Gly-Ala when buffer components (phosphate, carbonate, or
`imidazole) were varied from Oto 50 mM. A general acid-base mechanism by which
`the phosphate ion catalyzes dearnidation was challenged in 1995 by Tomizawa et al.
`( l 3), who found that the rate of lysozyme at 100°C did not exhibit the expected linear
`relationshi p of deamidation rate on phosphate concentration. Although not linked 10
`deamidation, it is worthwhile to note that at pH = 8 and 70°C, tris(hydroxymethyl)
`aminomethane buffer (Tris) has been shown to degrade to liberate high.ly reactive
`fomialdehyde in forced stability studies of peptides (38).
`
`Io nic Stre ngth
`
`The effects of ionic strength appear to be complicated and not open to easy gen(cid:173)
`e ralizations. Buffer and ionic strength effects on dearnidation are evident in pro(cid:173)
`ceins at neutral to alkaline pH (5). lo selected peptides and proteins, the catalytic(cid:173)
`activity of phosphate has been shown lo be reduced mode ra tely in che presence
`of salts NaCl. LiCI, and Tris HCI (12, 13). Of these alts, NaCl showed the least
`protective effecc against dcam ida1ion ( I 3).
`In the peptide Gly-Arg-Asn-Gly al pH 10, 37°C, the half-life t,n of deami(cid:173)
`dation dropped from 60 hours to 20 bouts when the ionic strength was increased
`from 0 .1 to 1.2 (22). However, in the case of Val-Ser-Asn-Gly-Val at pH 8, 60°C,
`!here was no observable difference in the 111, o( deamidation when solutions with(cid:173)
`out salt were compared 10 those containing l M NaCl or LiCl ( 12). Interestingly,
`for lyso1.yme at pH 4 and I 00°C, added sale sh<Jwed a pro1ec1ive effect against
`deamidation, but only in the presence of the phosphate ion (13).
`ln reviewi ng the data above, Brennan and Clarke (17) tentatively attributed
`the promotion of deamidation by elevated levels or ions to enhanced stabil ization
`
`---
`
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`

`72
`
`Bummer
`
`of the ionized peptide-bond nitrogen, promoting attack on the side-chain amide
`cnrbonyl. Other mech:misms would inc lude di 111p1i n of teniary s1111crure in pro(cid:173)
`teins that may have stabilized Asn residues. in some us-yet unknown fn~hi n. Thal
`promotion of denmidation is observed ins me cases o f peptides, and inhibi1iu11 in
`others docs suggest rather complex and competing effects. Clearly, the stabi lizing
`effects, when observed at all, are often at levels of salt too concentrated for most
`pharmaceutical formulations.
`
`Solvents
`
`The effect of various organic solvents on the rate of deamidation has not received
`much attention; it would be expected, however, I hat in the presence of a reduced
`diele tr!c medium. the peptide-
`nd nitroge n would be les, likely 10 ionize. Since
`the anionic peptide-bond nitrogen is ncces,ary in the rorma1ion or the cyclic
`imide, a low dielectric medium woul<l retard the prolJ,ress of the re1tction and be
`reflected in the free energy difference for ionizat ion of the peptide-bond nitrogen
`(17). Following thi_s hypo1hesis, Brcnnun and Clnrke (3\1) unnly'l.cd succi nimidc
`formation of 1he peptide Val-Tyr-Pro-Asn-Gly-Al:l l1he .:ime peptide employed
`by Patel and Borchardt (16) in studies of pH effects in aqueous soluUonJ as a func(cid:173)
`tion of organic cosolvent (ethanol. glycer 1, and dioxin) at constant pH and ionic
`strength. The lower dielectric constant media resulted in a significantly lower rate
`of deamidation, in agreement wi lh the hypothesis. II was .irguci.l 1h01 the similar
`rates of deamidation for different cosolvent systems or the sa me effecti ve dielec(cid:173)
`tric constant indicated that changes in vis ·osity and wuter content or the medium
`did not play a significant role.
`The effect of orga nic cosolvents o n deamidation in proteins is even less
`well characterized than that of peptides. Trifluoroethanol (TFE) inhibits dcami(cid:173)
`dation of lysozyme at pM 6 and l00°C (13), and of the dipeptide Asn-Gly. but
`does not inhibit the deamidation of free ami no acids. The mechanism of protec(cid:173)
`tion is not clear; direct interaction of the TFE with the peptide bond was postu(cid:173)
`lated. hut 1101 demonmnted. An nllemati ve hyp111he. is is 1h111 TFE induces greater
`structural rigfd ity in lh protein , producing a stru,·turc somewhat resistant to lhe
`formaii ,, of the yclic imide in1cr111cdin1e. Orher. pharmaceu1ically accepiable
`solvent~, ethanol and glycerin, did not exhibit the same protective effects as TFE
`on lysozyrnc.
`Of course, in dosage form design, organic, solvents such as TFE are not use(cid:173)
`ful as pharmaceutical acljuva.n1 . TI1e effects of low dielectric may still supply a
`rationale for the solubiliz.ation of peptides in aqueous surfactant systems, where
`the hydrophobic region of a micelle or liposome could potentially enh, nee 1he
`stabili,..ation oftheAsn residues from deamidation. As pointed out by Br~nnan and
`Clarke ( 17), 1he results of experiments in organic solvents can have implications on
`the prediction of points of deamidu1ion in proteins as well. For Asn residues neur
`lhe s urface of the protein, where tile dielectric constant is expected to approach that
`of water. the deamidution rate would be expected to be high. For Asn residues bur(cid:173)
`ied in more hydrophobi regions of the protein. where pulariUes arc lhought to be
`
`Bausch Health Ireland Exhibit 2010, Page 8 of 38
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`

`Chemical Considerations in Protein and Peptide Stability
`
`13
`
`more in line with that of ethanol or dio,rnne (40), reaction rates would be expecte<l
`to be considcrnbly slower.
`Computational s1udie, un the effects 11f ,olvent on the reaction were e arned
`out rccc,11ly by Caiak Cl ul (41 ). Th"Y rcp,,r1 1ha1, in the absence of waler, 1J1c
`overall acliva1ion energy banfor is on lbe order of 50 kcal/mo!, and lha1 this drups
`10 a value of about 30 kcal/mol in the pn:sence of water. fn all, about three water
`molecules participate directly in 1be reaction, assisting in hydrogen transfer and in
`the cyclization outlined in Figure I .
`
`Polymers and Sugars
`
`Considerable interest has developed in I.he stabilization of proteins and peptides in
`solid matrices, either polymeric or sugar based. In most solid polymer matrices,
`the primury role is to improve pht1nnncokine11c aml pharmcodynnmic propenies
`c,f the nctive by modifying release drnractcristics nnd most studies arc designed
`with this intention in mind (42). Sugars are usuaUy employed us un aid to lyophi(cid:173)
`lization of proteins, with the intent of maintaining the teninry structure and pre(cid:173)
`venting aggregation (43).
`The state of the polymer and the activity of water appear lo be critical factors
`in the stabilization of the peptide against deamidation. In general, the observed
`degrndu1ion rate constants exhibit 1he following rank order: solution > rubbery
`polymer> glassy polymer (38.44-46). 1-lowever. this observation doe~ nornppcar
`10 be valid in every case (44). It has been propo,cd that up 10 30% or a peptide
`may bind to polyvinylpyrrolidone (PVP) in the solution stale, complicating the
`kinetic anulysis (47).
`Peptide s1abili1y in polymer matrice, that are them<elv~ also undergoing
`dcgr:idation provide, a unique chnllenge. For example. it has been observed that
`PVP mny fmm adduct, with the N-terminu, nr peptid1,s (4~). Systemntic Sludic, of
`the deamidation of a model peptide in nhm, or the copolymer polylactic-glycolic
`acid (PLGA) have , hown 1ha1 the rc.iclion i~ the primary route of' de~rada1ion
`only after longer storage times ut hig her water content (49). The delay in 1hc
`onset of' deamidation of peptide in PLGA may be relnted to the time necessary Lo
`establish an "acidic microclimate" that arises from the hydrolysis of the polymer
`(50). In support of this acid-catalyzed deamidation hypothesis in PLGA films, the
`reaction product. isonspona1c was nut round.
`Computational , 1udies nmy supply additional insight. Computer simula(cid:173)
`tions or the mobility of peptide. water. NHi, and polymer in PVP matrix have
`been carried out by Xiang and Anderson (5 1). They observed that the diffusiv(cid:173)
`ity of water, NH3• and peptide were hctween two and three orders of magnitude
`slower in PVP compared to aqueous solution. lmponantly, the conformational
`dynamics of the peptide in the glassy polymer exhibited a higher energy barrier
`between states than seen for the peptide in wmcr. Thus, two of the crilicnl events
`in the process of deamidation, the conformational chnnges necessary to form the
`cyclic intermediate in the glassy polymer and the dlffusioo away of the NH, after
`release, are both slowed considerably in the solid state.
`
`Bausch Health Ireland Exhibit 2010, Page 9 of 38
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`

`14
`
`Bummer
`
`The effect of sugars on the deamidation of a model peptide has been exam(cid:173)
`ined. At pH= 7, a solution of peptide in a 5% sucrose or mannitol reduced the
`deamidation rate to about 16% of that found in the absence of sugars (52). When
`stored in the solid state, the rate of reaction was even slower, although sucrose
`appeared to stabilize the pept ide to o grcnter extent than did mnnnitol. lt was
`observed that sucrose remamed amorphous during the lest period w hile mannitol
`crystal lized, complicating the interpretation or 1hc dmu (53). Cle land ei al. (43)
`determined that 360: I was the optimal sugar-antibody molar ratio necessary to
`inhibit aggregation and deamidation over a three-month period. Sugars sucrose,
`trehalose, and mannitol were able to stabilize the protein so long as Jess than 8.4%
`moisture was present.
`Our understanding of the stabilization of peptides and proteins in polymer
`and sugar matrices is far from complete, and additional insight into the molecular
`mechanism might benefit frmn the bounty of studies cu1Tied out with small mol(cid:173)
`ecules in sm1ilnrsystems. faperiments must be designed carefully and interpreted
`with caution so as to clearly separate the solvent effects of water and perhaps even
`NH3 on the reaction from the plastisizing effects on the matrix.
`
`Peptide and Protein Structure
`
`The abi lity to identi fy whi h Asn or Gin residues in u thernpeuti protein or
`p pticlc 111:iy be vu lnernble 10 dea111idn1ion wou ld have grenl prac1ic:1 l appli :t•
`tio11 m prefnrmulut.ion und form ul utioo s tudies. ll1c effects of various level> o(
`ll'Ucrure-primnry, secondary, anJ tcriiaiy-arc believed to be comple,c aud
`varied. At present. only primary structure effects have b ·en charncte rizcd in u
`systematic man ner.
`
`Primary Sequence
`
`The primary sequence of amino acids in a peptide or protei n is often tbe first
`piece of structural data presented lo the formulation scientist. Considerable effort
`has been spent to elucidate the influence of flanking amino acids on the rates of
`deamidation of Asn and Gin residues. The potential effects of flanking amino
`acids are best elucidated in simple peptides, uncomplicated by side reactions or
`secondary and tertiary structure effects.
`
`Effect of amino acids preceding Asn or Gin:
`In an extended series of
`early studies, Robinson and Rudd (24) examined the inOucnceof primary sequence
`on the deamidation of Asn or Gin in the middle of a variety of pentapeptides. Mild
`physiologic conditions (pH 7.5 phosphate buffer at 37°C) were employed. A few
`general rules can be extracted from this work:
`
`1.
`
`In practically every combination tested, Gin residues were less prone
`to deamidation than Asn. For the two residues placed in the middle of
`otherwise identical host peptides, the half-life of the reactions differed
`by u factor ra ngi ng from two- to threefold.
`
`Bausch Health Ireland Exhibit 2010, Page 10 of 38
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`

`Chemical Considerations in Protein and Peptide Stability
`
`1S
`
`2.
`
`In peptides Gly-X-Asn-Ala-Gly, steric hindrance by unionized X side(cid:173)
`chains inhibits dcamidarioo. The rank order of deamidation rate found
`was Gly >Ala> Val > Leu > Ile, with the t,n ranging from 87 to 507
`days. It remains unclear why bulky residues inhibit the reaction, but
`reduced flexibility of the sequence may be a facror. A similar effect was
`noted when Gin replaced Asn. In this case,t1n ranged from 418 to 3278
`days, in accordance with the diminished reactivity of Gin.
`3. For the same host peptide, when the X side-chain was charged, the deam(cid:173)
`idation rate of Asn followed the rank order of Asp> Glu > Lys > Arg.
`
`Effect of amino acids following Asn or Gin: Early experiments on di pep(cid:173)
`tides under extreme conditions indicated a particular vulnerability of the Asn-Gly
`sequence to deamidation (54 ). More recent studies of adrenocorticotropic hor(cid:173)
`mone (ACTH)-like sequence hexapeptide Yal-Tyr-Pro-Asn-Gly-Ala under physi(cid:173)
`ologic conditio ns (55) have verified that deamidation is extremely rapid (r,12 of 1 .4
`days at 37°C). The formation of the succinimide intermediate is thought to be the
`basis for the sequence dependence (I 0 ) of deamidalion. It is genera Uy believed
`that bulky residues following Asn may inhibit sterically the formation of the suc(cid:173)
`cinimjde intermediate ia the dearuidalion n:aclion.
`Stcric hindrance of the cyclic imide formation is not tbe o nly possible
`genc,,;l, of sequence-dependent deamidation. The resistance to cyclic imide
`t'onntulon in the presence of a carboxyl-flanking prcline peptide may be related
`to the inability o f the prolyl amide nitrogen to allack the Asn side-chain ( IO).
`The computational studies of Radkiewicz et al. (56) suggest that the effect of
`the adjacent residue may largely be attributed to electrostatic/induc tive effects
`inAuencing the ability of the peptide nitrogen atom to ionize (as seen in F ig. I).
`In the case of glycine, the inductive effect is insufficient 10 explain the results,
`and the authors argue that the ability of glycine to sample more conformational
`space compared to other amino acids may help stabilize the nitrogen anion.
`Experime ntally, the replacement of the glycyl residue with the more bulky
`leucyl or prolyl residues resulted in a 33- to 50-fold (respectively) decrease in
`the rate of deamidation ( I 0). Owing to the highly flexible nature of the di pep(cid:173)
`tide, the deamidation rate observed in Asn-Gly is though t to represent a lower
`limit.
`ln more recent studies, deamidation of Val-T'yr-X-Asn-Y-Ala. a peptide
`sequence derived from ACTH, was examined with d ifferent residues in both
`flanking positions (57). When X was histidine (and Vis glycine), no acceleration
`of deamidation was found relative lo a peptide where X is praline. Placing a His
`following the Asn wos found to re,o;;ult in similar rntes of deamidation when X was
`phenylalanine. leucine. or valine. The rate when X was histidine was slower than
`that of alanine. cysteine, serine, or glycine. These results indicate that histidine
`does not have unique properties in fac ilitating succinimide formation. Of inter(cid:173)
`est was the observation that h.istidine on the carboxyl side of the Asn did seem to
`accelerate main-chain cleavage products.
`
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`16
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`Bummer
`
`Some of the general rules for peptides may also show higher levels of
`depenJcnce on primary sequence.1.'ylcr-Cross und Schrich ( 12) rn1died the influ(cid:173)
`ence or different amino acids on 1he udjncem amino end of 1he pcntopcptide Val(cid:173)
`X-Asn-Ser-Val at pH 7.3. For X = His, Ser, Ala, Arg, und u:n, dcnmidmion rates
`were essentially constant and approximately seven times slower than the Val(cid:173)
`Ser-Asn-Gly-Val standard peptide. Of special interest to the investigators was the
`observation of no difference in deamidation rates between those amino acids with
`and wi1hm11 ~-branching (such as voline ror glycine). This Is in direct contrast 10
`the ondlngs of Rohinson and Rmld (24) ol' !!Hold d ifference., In deamidat,on
`for valine .,11b,1i1u1ion for g lycine in Gly-X-Asn-Ala-Oly, shown enrlier. Under
`the mild alkaline conditions of Patel ond Borchardt ( 16), Val-l)'r-X-Asn-Y-Ala,
`no difference in the deamidation rate constants was observed when proline was
`substituted for glycine in the X position.
`
`Data mining: Data-mining approaches have been employed to formulate
`a semiquantitative means of predicting the effect of primary structure on rate of
`deamidation. Capasso (58) proposed the extrathermodynamic relationship shown
`in Equation I :
`
`Logk,=X,+Z,,.+ Y,
`Herc k1 is the observed rnte consrun1 for deum1dntiun. X, i.s the ave.rage contribu-
`11011 of the specific amino acid thnl precedes Asn. Y, is 1he avemge contribution
`due to the amino acid that follows A,n. and z.,, is the value when both the preced(cid:173)
`ing :tnd followi ng amino acids are glycine. Over 60 peptides were included in 1he
`dm ubnse. As expected. 1hc greatest influence 011 1hc deamidmion rote in peptides
`was round 10 arise from 1he iden1i1y of the following nmino acid. Some of lhe
`values for Y, are listed in Table I . A, suggested previou~ly. relmive 1n 1he effect
`of g lycine. bulky hydrophnbic amino acids such as vnline, lcucine, and is<1leucinc
`
`(I)
`
`Table 1 Rate Constants Reported for the Reacdoo• of
`0 1-1" with the Side Chains of Selected Amino Acids (JO I)
`
`Amino acid
`
`Cystcinc
`Tyrosim:
`rryptopban
`Histidine
`Methionine
`Phenylalanine
`Arginine
`CySline
`Serine
`Alanine
`
`k (L/mole-s)'
`
`4.7 X 10'0
`1.3 x 10•0
`1.3 X l010
`5 X 10'
`8.3 X lO'
`6.5 X lO'
`3,5x 10'
`2.1 X 109
`3.2 l( ID'
`7.7 X 101
`
`•Most volu~ determined via radiolysi~.
`"The pH values of m,my nf these studies have not been listed.
`
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`Chemical Considerations in Protein and Peptide Stability
`
`17
`
`appear to show the slowest deamidation rate, while the sma ller, more polar histi(cid:173)
`dine and serine show a rate closer to that of glycine. There do appear 10 be some
`discrepancies between these results and those mentioned earli~r ( 12, 16.57). in
`particular with respect to the experimentally observed e ffect of histidine. Clearly,
`different databases may give different results. At best, Equation I may be viewed
`as a first approximation for estimating deamidation rates in fomllllation studies.
`Robinson el al. (59- 61) have taken a different approac h 10 mining by
`including means to account for the three-dimensional structure of the side-chains
`a nd by avoiding the use of data gathered in the presence of the known catalyst,
`phosphate buffer. A method has been proposed to estimate the deamidation reac(cid:173)
`tion half-life al 37°C and pH = 7 .4 based on the primary sequence (61 ). The
`extent to which this method may accurately predict the deamidation rate of pep(cid:173)
`tides in pharmaceutical systems has not yet been rigorously tested experimen(cid:173)
`tally, but if proven valid , it would supply a rnlher usefu l tool i11 guiding early
`formulation studies.
`
`Secondary and Tertiary Structure
`
`X-ray or nuclear magnetic resonance (NMR) data can provide a detailed map of
`the three-dimensional structure of the protein or peptide. The role of secondary
`and tertiary structures in intramolecular deamidation of proteins has been dis(cid:173)
`cus ed by Chazin and Kossiakoff (62). II is beyond the scope of this work lo
`present a comprehensive review of the details of deamidation reactions in spe(cid:173)
`cific proteins. Excellent reviews of a variety of specific proteins exist (6). For the
`most part, detailed mechanisms relating the secondary and tertiary st ructures of
`proteins to enhancement of rates of deamidation are not yet avai lable.
`Clear differences in the deamidat ion rates of some proteins are evident when
`native and denatured states are compared ( 13,63). Denaturation is thoughtto enhance
`main-chain flexibility and water accessibility (62), Sufficient conformational flex(cid:173)
`ibility is required for the Asn peptide to assume the dihedral angles of <fl= -I 20°C
`and 'I' =+ l 20°C necessary for succinimide formation . ln as much as such angles
`tend to be energetically unfavorable (64) in native proteins, it may be expected that
`As,, residues in the midst of rigid secondary structures, such as helices, may be
`res