11
`
`Protein Formulation
`
`and Delivery
`
`Second Edition
`
`Edited by
`
`
`Eugene J. McNally
`
`
`Gala Biotech, a Catalent Pharma Solutions Company
`
`
`Middleton, Wisconsin, USA
`
`Jayne E. Hastedt
`ALZA Corporation
`
`Mountain View, California, USA
`
`informa
`
`healthcare
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`NewVork loodon
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`Bausch Health Ireland Exhibit 2010, Page 1 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01105
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`Bausch Health Ireland Exhibit 2010, Page 2 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01105
`
`

`

`2
`
`Chemical Considerations in Protein
`and Peptide Stability
`
`Paul M. Bummer
`y, Lexington, Kentucl<.y, U.S.A.
`University of Kentuck
`
`DEAM I DATION
`
`Introduction
`
`The dcamida1ion reactions of asparagine (Asn) and glulamine (Gin) side-chains
`are among Lhe most widely studied nonenzymnlic covalent modifications lo
`proteins and peptides ( 1 -7). Considerable research efforts have been exlendcd
`to elucidate the detai.ls of the dcamidation reaction in both in vitro and in vivo
`systems. and a nu mber of well-wrillen, i n-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 formula1ion components. Possible
`dcamidation-associated changes io the protein structure and state of aggregation
`also are examined . The emphasis is on Asn deamidation, since Gin is significantly
`less reactive.
`
`Reaction Mechanism
`
`The primary reaction mechanism for the deamidation of Asn in water-accessible
`regions of peptides and proteins at basic or neutral conditions is shown in Figure I ,
`For the present, discussion i s confined to the intramolecular mechanism, uncom­
`plicated by adjacent amino acids at other points in the primary sequence. Under
`alkaline conditions, the key step in the reaction is the fommtion of n deprotonatcd
`amide nitrogen, which carries out the rate-determining nucleophilic attack on the
`side-chain carbonyl, resulling in a tetrahedral intermediate and finally the formation
`of the five-member succinimide ring. For such a react.ion, the leaving group must be
`
`7
`
`Bausch Health Ireland Exhibit 2010, Page 3 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01105
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`

`

`8
`
`�J>,-R-,---+
`
`0
`
`Asparaglnyl Residue
`
`Bummer
`,, l� N�
`R1
`0
`
`Reactive r ion
`
`Figure 1 Proposet.l reuclion 111cchanbm for dcamidntion of asparaginyl residue. Note the
`formation of the �uc<:inl1oldyl mtetmcdi:ite and the two possible final products.
`
`Aspartyi Residue
`lsoaspartyl Residue
`
`easily protonaled, and in this-case, it is responsible for the characteristic fonnation of
`
`
`
`
`
`
`
`
`result­hydrolysis, (NH3). The succinilrude ring intermediate is subject to
`ammonia
`
`
`
`
`ing In either the corresponding ospartic acid or the isoaspartic acid ('3--aspartate),
`
`
`
`
`
`
`Often, the ratio of the products is 3: I, isoaspartaie to aspartate (10-12). In the case
`
`
`
`
`
`of acid catalysis (pH < 3), a tetrahedral intermediate is also fom1ed, but breaks down
`
`with the loss of NH3 without
`
`
`
`going through the succinimide ( 13-17). The reaction
`
`
`
`
`
`
`also appears to be sensitive to racemization at the IX-carbon. resulting in mixture1; of
`
`
`
`
`
`
`o- and 1,-isomers ( 10, 13-15). The rate of degradation of the parent peptide in aque­
`
`
`
`ous media often follows pseudo-first-order kinetics (16,17).
`
`Bausch Health Ireland Exhibit 2010, Page 4 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01105
`
`

`

`
`
`
`
`Chemic;i/ Considerations in Protein and Peptide St;ibility
`
`
`
`
`
`9
`A number of other alternative reactions arc possible. The most prevalent
`1•eaction appears to be. 3 nuclcophilir m111ck of the Asn sic1(N!hain amide nitrogen
`on 1hc pt:p1ide carbonyl, rc,ul1lng in mnin-chain cl�avage ( 10, 16, 18). Thisreac­
`uon (Fig. 2) is slower than 1.ha1 of cyclic imide formation and L" mos1 frequemly
`observed when Asn is followed by proline. a residue incapable of forming an ion•
`ized peptide-bond nitrogen.
`Under conditions of s.trong acid (pH l-2), deamidalion by direct hydrolysis of the
`amide side-chain becomes more favorable than formation of cyclic imidc (16, 19).
`Under these extreme conditions, the react.ion is: often complicated by main-chain
`cleavage and denaturation. Deamidation by Lhis mechanism is not likely Lo pro­
`duce isoaspartare or significant racemization (16).
`Under more moderate condjtions, the effect of pH is 1.hc result of two
`opposing reac1ions: (i) deprotonation of the peptide-bond nitrogen, promoting
`
`pH Dependence
`
`+
`
`R1 = Amino end of protein
`Figure 2 Proposed reaction mechanism for rnam-chain cleavage by asparaginyl residues.
`R2: Carboxyl end of protein
`
`Bausch Health Ireland Exhibit 2010, Page 5 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01105
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`

`

`1 0
`
`Bummer
`
`the reaction and (li) protonation of the side•chain-leaving group, inhibiting the
`reaction. ln deamidation reactions of short chain peptides uncomplicated by
`structural alterations or covalent dimcri1.ation (20), the pH•rate profiles exhibit
`the expected "V" shape, with a mi nimum occurring in the pH range of3 lo 4 ( 1 6).
`Computation studjes 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
`tions (3 < pH, 4), the rate-limiting step is the attack of the dcprotonated nitrogen
`on the side-chain. The rate-li miting step at neutral pH is the hydrogen transfer
`reaction, while under basic conditions (pH > 7), it is the elimination of NH; from
`the tetrahedral iotennediate. Experimental studies have shown that 1·he increase
`in rate on the alkaline side of the minimum docs not .strictly correlate with the
`increase in deprotonation of the amide nitrogen, indicating that the rate of reac­
`tion is not solely dcpendenl on the degree of the peptide..bond ni1rogen deprot­
`onation ( 1 6, 1 9). The pH minimum in the dcamidution reaction measured in vitro
`for proteins may (22) or may not (23) fal l in the same range as that of simple
`peptides. Overall pH-dependent effects may be modified by structure-dependent
`factors, such as dihedral angle flexibility, water accessibil ity, and proximity of
`neighboring amino acid sjdc-chains (see section Peptide and Prmein Structure).
`
`Effect of Temperaiure
`
`The temperature dependence of 1hc dcarnidation rare has been studied in a variety
`of simple peptides in solution ( 1 6,24,25). Small peptides arc easily designed to
`avoid competing react.ionS', such as oxidation and main-chain cleavage, and arc
`thus useful to isolate auentioo directly on the dcamidation rate. In solution, deam­
`idation of small peptides tends lo follow an Arrhenius rclarionship. Activation
`energies of the reaction do tend to show pH dependence, and a discontinu ity in the
`Arrhenius plot is expected when 1he mechanism changes from direct hydrolysis
`(acid pH) to one of cyclic imide (mildly acid.ic to alkaline pH).
`The deamidation rate uf proteins also hows temperature dependence
`(23,26,27) under neutral pH. For deamidation reactions alone, temperature­
`associated rate acceleration in proteins may be due lo enhanced flexibility of the
`molecule, allowing more rapid formation of the cyclic imide (28). or it may occur
`by catalysis by side-chains brought into the v icinity of the deamidation site (5).
`The availability of water appears to be an iir1portant determinant in
`temperature-associated effects. In studies of lyophilized formulation of Val-Tyr­
`Pro-Asn-G1y-Ala, the deamidation rate constant was observed to increase about an
`order of magnitude between 40°C and 70°C (29). In contrast, in the solid state, the
`Arrhenius relationship was not observed. Funher. the deamidation in the solid stale
`showed a marked dependence upon the temperature when 1he peptide was Jyop.hi­
`li1..ed from a solution of pH 8 1 while little temperature dependence was observed
`when lyophilization proceeded from solutions al 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
`Mylan v. Bausch Health Ireland - IPR2022-01105
`
`

`

`
`
`
`
`Chemical Considerations in Protein and Peptide Stdbilily
`
`1 1
`
`Adjuvants and Excipients
`
`The influence on deamidation by a variety of buffer ions and solvents has been
`examined. As pointed out by Clelapd et al. (4) and reinforced by Tomizawa et
`al. ( 1 3), many of these additives are unlikely to be employed as pharmaceutj­
`cal excipiems for fonnu laLion, but they may be employed in protein isolation
`and purification procedures (30). I mportant clues to stabilization strategies can
`be gained from these studies, In the follow ing, il is fruitful to keep in mind the
`importance of the attack of the ionized peptide-bond nitrogen on the side-chain
`carbonyl and the hydrolysis of the cyclic imide (Fig. I ),
`
`B u ffers
`
`Buffer catalysis appears to occur in some but not all peptides and proteins studied
`(5). Bicarbonate ( 1 6) and glycine ( 12) buffers appear to accelerate deamidatioo. On
`one hand, the phosphate ion has beeo shown to catalyze deamidation, both in pep­
`tides and in proteins ( 12, 1 3, 16,3 1 -34), generally in the concentration range of O to
`20 mM. Capasso et al. (35,36) observed the acceleration of deamidation by acetate,
`carbonate, Tris, morpholine, and phosphate buffer., only in the neutral to basic pH
`ranges. On the other hand, Lura and Schrich (37) found no influence on the rate of
`deamidation of Yal-Asn-Gly-Ala when buffer components (phosphate, carbonate, or
`imidazole) were varied from O to 50 mM. A general acid-base mechanism by which
`the phosphate ion calalyws deamidalion was challenged in l 995 by Tomizawa et al.
`( 1 3), who found lhat lhe rale of lysozymc at 1 00°C did not exhibit the expected linear
`relationship of deam1dation rate on phosphate concentration. Although not linked to
`deamidation, il is worthwhile to note that at pH � 8 and 70°C, tris(hydroxymethyl)
`ami nomethanc buffer (Tris) has been shown to degrade to liberale highly reaclive
`fom1aldehyde in forced slabilicy studies of peptides (38).
`
`I o n i c Slrength
`
`The effects of ionic strength appear to be complicated and nor open to easy gen­
`eralizations. Buffer and ionic strength effects on deamidation are evident in pro­
`teins at neutral to alkaline pH (5). In selected peptides and proteins, the catalytic
`activity of phosphate has been shown to be reduced moderately in 1he presence
`of salts NaCl. Li Cl, and Tris HC I ( I 2, 1 3 ). Of these sails, NaCl showed the least
`protective effect against dcamidation ( 1 3).
`In the peptide Gly-Arg-Asn-Gly at pH 10, 37 °C, the half-life ,,,, of dcami­
`dation dropped from 60 hours to 20 bou,s when the ionic strength was increased
`from 0.1 lo 1 .2 (22). However, in the case of Val-Ser-Asn-Gly-Va! at pH 8, 60°C,
`there was no observable difference in the 1 112 of deamidation when solutions with­
`out salt were compared to those containing I M NaCl or LiCI ( 1 2). Interestingly,
`for lysozyme at pH 4 and 1 00°C, added salt showed a protective effect against
`deamidation, but only in the presence of the phosphate ion ( 1 3) .
`In reviewing the data above, Brennan and Clarke ( 1 7) tentatively attributed
`the promotion of deam.idation by elevated levels of ions to enhanced stabilization
`
`Bausch Health Ireland Exhibit 2010, Page 7 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01105
`
`

`

`12
`
`Bummer
`
`of the ionized peptide-bond nitrogen, promoting attack on the side-chain amide
`carbonyl. Other mechanism� would include dis11.1p1io11 or rer1 k1ry st mcrurc in pro­
`teins that may have stahillled Asn residues. in some us�yet u nknown fashion. That
`promotion of de:1midation is observed in some cases of peptides. and inhibi1iun in
`others doc� suggest rather complex and competing effects. Clearly, the stabilizing
`effects t when observed at all, are often at levels of sa1t too concentrated for most
`pharmaceutical formulations.
`
`Solvents
`
`The effect of various organic solvents on the rate of dcamidation has not received
`much altention; it would be expected, however, that in the presence of a reduced
`dielectric medium. the pept ide-bond nitrogen would be le» likely to ionize. Si nc.e
`the anionic peptide- bond nitrogen is ncccs�ary in the format ion of t he cyclic
`imide, a low dieicctric medium would retard the progress of 1he reaction and be
`reflected in the free energy difference for ionization of the peptide-bond n itrogen
`( 1 7). Fol lowing this hypothesis, 13rcnnun and Clnrke (3\1) anuly1.cd succini midc
`formation of the reptitle Vnl -Tyr-Prc,-Asn-Gly-Ala I t he ,ame peptide employed
`by Patel ilnd Borchardt ( 16) in studies of pli effects in aqueous solutionJ as n func­
`tion of org;mic cosolvent (ethanol, glycerol, and dioxin} at constant pH and ionic
`strength. The lower dielectric constant media resulted in a significantly lower rate
`of deamid:ition, in agreement with the hyp(llhesis. II was ;,)rgucll lhOI 1 hc �imi lar
`rates of deamidation for different cosoJvenl systems of the same effective die lec­
`tric constant indicated that changes in viscosity and water content or the medium
`did not play a significant role.
`The effect of organic cosolvents on deamidation in proteins is even less
`well characterized than that of peptides. Trifluoroethanol (TFE) inhibits deami­
`dation of lyso,.yme at pli 6 and l 00°C ( 1 3), and of the dipeptide Asn-Gly. but
`does not inhibit Lhe deamidation of free amino acids. The mechanism of protec­
`tion is not c1car; direct interaction of the TFE with the peptide bond was postu­
`l attd, hut nm demomarnted. A n n l !emat ive hyporhesi� i!i t hut TF'E induces �reatt:r
`struct ural rigid ity in 1 hc prolei n , producing a strut·turc somewhat resis1nnt to the
`fonm.ui n of the cyclic imide intcrmcdime� 01her. ph:m11:1ceu1 ical ly ncceptable
`solvents, ethanol and glycerin, did not exhibit the same protective effects as TFE
`on lysozyme.
`Of course. in dosage form design, organic solvents such as TPE are not use­
`ful as pharmaceutical adjuvants. The effects of low dielectric may srill supply a
`rationale for t 11e solubi.Hzntion of peptides in aqueous surfactant systems, where
`the hydrophobic region of a micelle or liposome could potentially enhance the
`stabilization of the Asn residues from deamidation. As pointed out by Brnnmm and
`Clarke ( 17), the results of experiments in organic solvents can have implications on
`the prediction of points of deamjdulion in proteins tlS well. For Asn residues near
`the surface of the protein, where the dielectric constant is expected to approach th:tl
`of water, the deumidntion rate would be expected to be high. For Asn residues bur­
`ied in more hydrophobic regions of the protein. where polarities are thought to be
`
`Bausch Health Ireland Exhibit 2010, Page 8 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01105
`
`

`

`...--
`
`Chemicdl Considerations ;n Protein and Peptide Stability
`
`13
`
`to be coosidcrnbl y slower:
`
`more in line with that of ethanol or dio.xane (40), reaction rates would be expected
`
`Compututional �1udie!'I un the cffec1� uf solvent on !ht: reaetion were carried
`out recently by Cata� c1 al.. (41 ). 11,ey rcp1.1r1 rhat, in the absence of waler, 1J1e
`overall activation ei1�rl:l'.y barrier is on 1he ordcr of50 kcal/11101, �nd thlit this drop�
`to a value of about 30 kcal/mo) in the presence of water. fn all, about three water
`molecules participate directly in the reaction, assisting in hydrogen transfer and in
`the cyclization outlined in Figure I •
`
`
`Polymers and Sugars
`Considerable in1erest has developed in the stabilization ofproteirtS and peptides in
`solid matrices, either polymeric or sugar based. Jn mosl solid polymer matrices.
`the primnry role is to improve phammcokinellc anti pharmcodynamic properties
`of the nclive by modifying release clmr.ictcrislics nnd most studies arc designed
`
`lization of proteins, with the intent of maintaining the tertiary structure and pre­
`
`The state of the polymer and the activity of water appear to be critical factors
`in the stabilization of the peptide against deamidation. In general, the observed
`
`Peptide- stabili1y in polymer sn0:1riccs 1ha1 :ue 1hemseh1c-s also undergoing
`dcgrodation provide� a unique chnllenge. For exafnple. i1 has been observed that
`PVP rnay form adtl11c1� with 1lw N-terminu, or peplides (48). Sysremnlic sludics of
`
`with this intention in mind (42). Sugars are usually employed as un aid to lyophi­
`venting aggregation (43).
`degmdution rote constants exhibit the following mnk order: solution> rubbery
`polymer> glassy polymer (38.44-46). �lowever. this observation docs not appeur
`to be valid in every case (44). It has been proposed that up to 30% of a peptide
`may bind to polyvioylpyrrolidone (PVP) in the solution state, complicating the
`kinetic analysis (47).
`the deaniidution of a model peptide in films of the copolymer polylactic-glycolic
`acid (PLGA) have ,hown that the rcuclion is the primary roule of degradation
`only Qfter longer storngc limes at higher water content (49). The delay in the
`onset of deamidution of peptide in PLGA may be relttted to the time necessary to
`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
`tions of the mobility of peptide, water, NHi, and polymer in PVP matrix have
`been carried out by Xiang and Anderson (51). They observed that the diffusiv­
`dynamics of the peptide in the glassy polymer exhibited a higher energy barrier
`cyclic intermediate in the glassy polymer and the diffusion away of the NH, after
`
`reaction product. isonsportutc was not foundA
`Computational i.ludies may 1'Upply udditional insight. Computer simula•
`
`ity of water, NH3, and peptide were between two and three orders of mugnilude
`slower in PVP compared to aqueous solu1ion. Importantly, the confonnmional
`
`between states than seen for the peptide in wmcr. Thus, two of the cri1jcal events
`in the process of deamidation, the conformational chnnges necessary to form the
`
`release, are both slowed considerably in the solid state ..
`
`Bausch Health Ireland Exhibit 2010, Page 9 of 38
`Mylan v. Bausch Health Ireland - IPR2022-01105
`
`

`

`1 4
`
`Bummer
`
`The effect of sugars on the deamidation of a model peptide has been exam­
`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 peptide to n gr1''11er extent t han did mnnnitol. lt was
`observed that sucrose remained amorphous during the lest perio(I wl1ile mannitol
`crystal lized, complicating the interpretatil)n or the daw (53). Cleland et al. (43)
`determined that 360: I was the optimal sugar-antibody molar ratio necessary to
`inhibit aggregation and deamidation over a th1·ee-n'lonth period. Sugars sucrose,
`trehalose, and mannitol were able to stabil ize 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 lnto the molecular
`mechanism rnigh1 benefil frnm the bounty of stud ies cutTied out with small mol­
`ecules in similnr sy ·terns. Ex periments must be de$igncd carefully and interpreted
`with caution so as ro clearly separate the solvent effects of water and perhaps even
`N H3 on the reaction from the plastisizing effects on the matrix.
`
`Peptide and Protein Structure
`
`The abi lity to identify whi h Asn or Gin residues i n u thempeutic protein r
`pept ide may be vulnerable 10 dcamidm ion would have grem pract icnl ltpi)l ic:,­
`tlon in preformuJntion and formulution .studies. 111c effects nr various level� or
`1ruc1ure-pri mary1 secondary, and 1cr1 iary-are believed 10 be complex n11d
`varied. Al prese111, only primary structure effect� hnvc been charat·terizcd i n u
`systematic manner.
`
`Primary Sequence
`
`The primary sequence of amino acids i n a peptide or protein is often tbe tirs1
`piece of structural data presented to the formulation scientist. Considerable effort
`has been spent to elucidate the influence of Hanking amino acids on the rates of
`dea.mida.tion or Asn and Gin residues. The potential effects of flanking amino
`ac ids are best elucidated in simple peptides, uncomplicated by side reactions or
`secondary and teniary structure effects.
`
`In an extended series of
`Effect of amino acids preceding Asn or Gin:
`early studies, Robinson and Rudd (24) examined the innucncc of primary sequence
`on the dearnidation 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:
`
`I.
`
`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 di ffered
`hy a factor rnnging from two- to threefold.
`
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`15
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`Chemical Com;derdtions in Protein omd Peptide SlabUity
`
`2. ln peptides Gly-X-Asn-Ala-GJy, steric hindrance by unjonizcd X side­
`chains inhibits dcamidarioo. The rank order of deamidation rale found
`wa, Gly >Ala> Val> Leu> lie, with the tin ranging from 87 to 507
`days. It remains unclear why bulky residues inhibit the reaction, but
`reduced flexibility of the sequence may be a fact0r. A similar effect was
`noted when Gln replaced Asn. ln this case, t112 ranged from 418 to 3278
`days, in accordance with the di_mjnished reactivity of Gin.
`3. Forlhe same host peptide, when the X side-chain was charged, the deam­
`idation rate of Asn followed the rank order of Asp> Glu > Lys > Arg.
`Effect or amino acids followingAsn or Gin: Early experiments on dipep­
`tide.c. under extreme conditions indicated a particular vulnerability of the Asn-Gly
`sequence to deamidation (54). More recent studies of adrenocorticotropic hor­
`mone (ACTH)-like sequence hexapeptide Val-Tyr-Pro-Asn-Gly-Ala under physi­
`ologic conditions (55) have vetified thnt deamidation is extremely rapid (t,n of 1.4
`days at 37°C). The formation of the succinimide intennediate is thought to be the
`basis for the sequence dependence ( I 0) of deamidation. It is genera Uy believed
`that bulky residues following Asn may inhibit sterically the formation of the suc­
`cinimidc ini.ermediare iu 1..he dearu.idation react.ion.
`Stcric hindrance of the cyclic imide formation is not tbe onJy possible
`gcne-s.is of sequence-dependent deamidation. The resistance to cyclic imlde
`formation in the presence of a carboxyl-H:1.nking praline peptide may be related
`to the inability of the prolyl amide nitrogen to attack the Asn side-chain (10).
`The computational studies of Radkiewicz et al. (56) suggest that the effect of
`the adjacent residue may largely be allributed lo electroslalic/inductive effects
`intluencing the ability of the peptide nitrogen atom 10 ionize (as seen in Fig. I).
`In the case of glycine, the inductive effect is insufficient to explain the resuJts,
`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.
`Experimentally, 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 (10). Owing to the highly flexible nature of the di pep­
`tide, th_e deamidation rate observed in Asn-Gly is thought to represent a lower
`limit. In mane recent studies. deamidation of Val-Tyr-X-Asn-Y-Ala. a peptide
`sequence derived from ACTH, was examined with different residues in both
`flanking positions (57). When X was histidine (and Y is glycine), no acceleration
`of deamidation was found relative to a peptide where X is proline. Placing a His
`following theAsn was found to re...,ult in similar rates of deamidation when X was
`phenylalanine, leucine, or valine. The rate when X was histidine was slower lhan
`that of alanine, cysteine, serine, or glycine. These results indicate that histidine
`does not have unique properties in facilitating succinimide formation. Of inter­
`est was the observation that histidine on the carboxyl side of the Asn did seem lo
`accelerate main-chain cleavage producl"·
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`16
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`Some of the general rules for peptides may also show higher levels of
`dependence on primary sequence. Tyler-Cross :ind Schrich ( 12) smdied the influ­
`ence of diffcrcm amino acids on 1he udjncem amino end of the pcnrnpcptide Val­
`X-Asn-Ser-Val at pH 7.3. For X; His, Ser, Ala, Arg, and Leu, dcamidmion rates
`were essenLially constant and approximately seven times slower than the Val•
`Ser-Asn-Gly-Val standard peptide. Of special interest to the investigators was the
`observation of no djfference in deamidation rates between those amino acids with
`and withnm �-branching (such as vo..line ror glycine). This is ill di reel contrast 10
`the findings of Robinson and Rudd (24) of 10-fold differences lu deamidat,on
`for valine substitution for glycine in Gly-X-Asn-Ala-Gly, shown earlier. Under
`the mild alkaline conditions of Patel nod Borchardt (16), Val-,Yr-X-Asn-Y-Alu,
`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 10 formulate
`a semiquantitative means of predicting the effect of primary s1ructure on rate of
`deamidation. Capasso (58) proposed the extrnthermodynamic relationship shown
`in Equation I :
`
`(I)
`l-lerc k1 is the observed rnte c:onsmn1 for denmidntion, Xr i.� the ave.rage contribu­
`tion of the specific umino acid 1hn1 precede� A.�n. Y11 i� the nverage cnntribution
`due to the amino acid that follows A,n. and z,.._... is the v:iluc when both the preced­
`ing und following amino ncids nre glycine.. Ovc.r 60 pc.prides were included in the
`darubase. As expected. rhc grcate�t influence on 1hc de,11nidn1ion rate in peptides
`wa� found to oris:e frorn 1he idcn1i1y of the following ninino acid. Soinc of the
`values for Y1, ore listed in Tobie I. A., suggested previnusly. relative 1n 1he effect
`of glycine. bulky hydrophobic amino acids s:uch as vnnne. lcucine, and isolcucinc.
`
`Table 1 Rate Constants Reported for the Rcactiona of
`
`
`
`
`OW with the Slde Chains of Selected Amino Adds (IOI)
`
`5 >< IO'
`
`Amino acid k (Umole-s)>
`4.7 X 1016
`CyslCine
`1.3 >< IO'◊
`Tyrosint
`1.3 >< IO'°
`fryptopban
`Histidine
`Methionine 8.3x 10•
`Phenylalanine 6.5x 10•
`3.5 x JO'
`Arginine
`Cystine
`2.1 X 10"
`3.2 )c 10•
`7.7 X !01
`
`Serine
`Alanine
`
`•Most vnlues dcttonnined vi11 radiolysis.
`
`
`
`
`'The pH values of many of these studies have not been listed.
`
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`Chemical Considerations in Protein and Peptide Stability
`
`1 7
`
`appear t o show t h e slowest deamidation rate, w h i l e the smaller, more polar histi­
`dine and serine show a rate closer to that of glycine. There do appear to be some
`d iscrepancies between these resu l ts and those mentioned earliec ( 1 2, 1 6.57), in
`particular with respect to the experimentally observed effect of histidine. Clearly,
`different databases may give different result . At best, Equation I may be viewed
`as a first approximation for estimating deamidation rates in fonnulation studies.
`Robinson el al. (59-6 1 ) have taken a different approach to mining by
`including means to account for the three-dimensional structure of the side-chains
`and by avoiding the use of data gathered in the presence of the known catalyst,
`phosphate buffer. A method has been proposed to estimate the dcamidation reac­
`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 dcamidation rate of pep­
`tides in pharmaceutical systems has not yet been rigorously tested experime.n­
`tally, but if proven valid, it would supply a rather useful tool in guiding early
`formulation studies.
`
`Secondary and Tertiary Struct u re
`
`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 dcamidation of proteins has been dis­
`cussed by Chazin and Kossiakoff (62), It is beyond the scope of this work to
`present n comprehensive review of the details of deamidation reactions in spe­
`cific proteins. Excellent reviews of a variety of specific proteins exist (6). For the
`most part, detailed mechanisms relating the secondary and tertiary structures of
`proteins to enhancement of rates of deamidation are not yet available.
`Clear differences in the deamidation rates of some proteins a.re evident when
`native and denatured states are compared ( 1 3.63). Denaturation is though no enhance
`main-chain ftexibi1ity and water accessibility (62), Sufficienl conformational Hex­
`-l 20°C
`ibility is required for the Asn peptide to assume the dihedral angles of <I>
`and lf1 = + 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
`Asn residues in the midst of rigid secondary structures, such as helices, may be
`resistant to deamidation. Other reactions, such as cross-linking might also give rise
`to rigid regions of the protein and enhanced resista