A publication of the
`American
`Pharmaceutical
`Association
`and the
`American
`Chemical
`Society
`
`MINIREVIEW
`MINIREVIEW
`
`JOURNAL OF
`
`Pharmaceutical
`Sciences
`
`May 1999
`May 1999
`Volume 88, Number 5
`Volume 88, Number 5
`
`Solid-State Chemical Stability of Proteins and Peptides
`Solid-State Chemical Stability of Proteins and Peptides
`
`M. C. LAI' AND E. M. ToPP*
`M. C. LAI† AND E. M. TOPP*
`
`Contribution from Department of Pha►maceutical Chemistry, The University of Kansas, 2095 Constant Ave.,
`Contribution from Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave.,
`Lawrence, Kansas 66047.
`Lawrence, Kansas 66047.
`
`Final revised manuscript received March 5, 1999.
`Received September 16, 1998.
`Final revised manuscript received March 5, 1999.
`Received September 16, 1998.
`Accepted for publication March 11, 1999.
`Accepted for publication March 11, 1999.
`
`Abstract 0 Peptide and protein drugs are often formulated in the
`Abstract q Peptide and protein drugs are often formulated in the
`solid-state to provide stabilization during storage. However, reactions
`solid-state to provide stabilization during storage. However, reactions
`can occur in the solid-state, leading to degradation and inactivation
`can occur in the solid-state, leading to degradation and inactivation
`of these agents. This review summarizes the major chemical reactions
`of these agents. This review summarizes the major chemical reactions
`affecting proteins and peptides in the solid-state: deamidation, peptide
`affecting proteins and peptides in the solid-state: deamidation, peptide
`bond cleavage, oxidation, the Maillard reaction, (cid:226)-elimination, and
`bond cleavage, oxidation, the Maillard reaction, j3-elimination, and
`dimerization/aggregation. Physical and chemical factors influencing
`dimerization/aggregation. Physical and chemical factors influencing
`these reactions are also discussed. These include temperature,
`these reactions are also discussed. These include temperature,
`moisture content, excipients, and the physical state of the formulation
`moisture content, excipients, and the physical state of the formulation
`(amorphous vs crystalline). The review is intended to serve as an aid
`(amorphous vs crystalline). The review is intended to serve as an aid
`for those involved in formulation, and to stimulate further research on
`for those involved in formulation, and to stimulate further research on
`the determinants of peptide and protein reactivity in the solid-state.
`the determinants of peptide and protein reactivity in the solid-state.
`
`Introduction
`Introduction
`In the last two decades, proteins and peptides have
`In the last two decades, proteins and peptides have
`become an important class of potent therapeutic drugs.
`become an important class of potent therapeutic drugs.
`However, their susceptibility to chemical degradation in
`However, their susceptibility to chemical degradation in
`solution presents a challenge in the development of stable
`solution presents a challenge in the development of stable
`protein pharmaceuticals.1 As a result, many polypeptide
`protein pharmaceuticals.' As a result, many polypeptide
`drugs are formulated as lyophilized or freeze-dried products
`drugs are formulated as lyophilized or freeze-dried products
`to prolong their shelf life.2-4 While a “dry” formulation is
`to prolong their shelf life.2-4 While a adry° formulation is
`generally more stable than the corresponding aqueous
`generally more stable than the corresponding aqueous
`formulation, chemical degradation reactions can still
`formulation, chemical degradation reactions can still
`occur.2-4 In some cases, protein stability in the solid state
`occur.2-4 In some cases, protein stability in the solid state
`is less than or comparable to that in solution.5,6
`is less than or comparable to that in solution.5,6
`Factors that may impact the chemical stability of pro-
`Factors that may impact the chemical stability of pro-
`teins and peptides in the solid-state include residual
`teins and peptides in the solid-state include residual
`
`* To whom correspondence should be addressed. Phone: (785) 864-
`* To whom correspondence should be addressed. Phone: (785) 864-
`3644. Fax: (785) 864-5736. e-mail: topp@ ukans.edu.
`3644. Fax: (785) 864-5736. e-mail: topp@ukans.edu.
`† Current address: Bristol Myers Squibb, New Brunswick, NJ.
`Current address: Bristol Myers Squibb, New Brunswick, NJ.
`
`moisture and the excipient(s) used in a formulation.
`moisture and the excipient(s) used in a formulation.
`Excipients, such as polymers, are often included in protein
`Excipients, such as polymers, are often included in protein
`and peptide formulations to protect the drug from degrada-
`and peptide formulations to protect the drug from degrada-
`tion during processing and/or storage or to act as a matrix
`tion during processing and/or storage or to act as a matrix
`for controlled release. This review presents an overview of
`for controlled release. This review presents an overview of
`the chemical degradation reactions common to proteins and
`the chemical degradation reactions common to proteins and
`peptides in the solid state, and of our current knowledge
`peptides in the solid state, and of our current knowledge
`regarding the effects of formulation and storage factors on
`regarding the effects of formulation and storage factors on
`peptide and protein stability in these systems.
`peptide and protein stability in these systems.
`The degradation pathways of proteins in the solid state
`The degradation pathways of proteins in the solid state
`can be classified into two types: chemical and physical.
`can be classified into two types: chemical and physical.
`Chemical instability involves covalent modification of a
`Chemical instability involves covalent modification of a
`protein or amino acid residue to produce a new molecule
`protein or amino acid residue to produce a new molecule
`via bond cleavage, bond formation, rearrangement, or
`via bond cleavage, bond formation, rearrangement, or
`substitution. These chemical processes include such reac-
`substitution. These chemical processes include such reac-
`tions as deamidation at asparagine (Asn) and glutamine
`tions as deamidation at asparagine (Asn) and glutamine
`(Glu) residues,7 oxidation of sulfur atoms at cysteine (Cys)
`(Glu) residues,7 oxidation of sulfur atoms at cysteine (Cys)
`and methionine (Met) residues, disulfide exchange at Cys,
`and methionine (Met) residues, disulfide exchange at Cys,
`and hydrolysis of aspartate (Asp) and glutamate (Glu)
`and hydrolysis of aspartate (Asp) and glutamate (Glu)
`residues.8 Physical instability refers to changes in the
`residues.8 Physical instability refers to changes in the
`three-dimensional conformational integrity of the protein
`three-dimensional conformational integrity of the protein
`and does not necessarily involve covalent modification.
`and does not necessarily involve covalent modification.
`These physical processes include denaturation, aggrega-
`These physical processes include denaturation, aggrega-
`tion, precipitation, and adsorption to surfaces.1 Chemical
`tion, precipitation, and adsorption to surfaces.' Chemical
`instabilities, such as deamidation and disulfide bond
`instabilities, such as deamidation and disulfide bond
`cleavage, may lead to physical instabilities, and vice versa.
`cleavage, may lead to physical instabilities, and vice versa.
`The physical instabilities of proteins will not be discussed
`The physical instabilities of proteins will not be discussed
`in detail here, since the focus is chemical instability. The
`in detail here, since the focus is chemical instability. The
`reader may refer to a recent article on the physical
`reader may refer to a recent article on the physical
`instability of proteins for further discussion.9 Instead, this
`instability ofproteins for further discussion.9 Instead, this
`review will discuss reactions and factors that contribute
`review will discuss reactions and factors that contribute
`to the chemical instability of proteins and peptides in the
`to the chemical instability of proteins and peptides in the
`solid-state.
`solid-state.
`The different types of chemical reactions that contribute
`The different types of chemical reactions that contribute
`
`© 1999, American Chemical Society and
`© 1999, American Chemical Society and
`American Pharmaceutical Association
`American Pharmaceutical Association
`
`10.1021/js980374e CCC: $18.00
`10.1021/js980374e CCC: $18.00
`Published on Web 04/14/1999
`Published on Web 04/14/1999
`
`Journal of Pharmaceutical Sciences / 489
`Journal of Pharmaceutical Sciences / 489
`Vol. 88, No. 5, May 1999
`Vol. 88, No. 5, May 1999
`
`MYLAN - EXHIBIT 1016
`
`

`

`O% /NH,
`C
`
`CH2
`
`NH
`
` CH
`
`
`
` C NH -- CH2 --C-
`
`O
`Asn-Hexapeptide
`
`O
`
`O
`C
`
`--NH
`
`CH C
`
`N CH2-
`
`O
`Asu-Hexapeptide
`
`O
`
`C- NH-
`
`
`CH2-
`
`CH C OH
`O
`
`NH
`
`CT-- 2 L-
`
` C-
`I
`O
`
`IsoAsp- Hexapephde
`Scheme 1sDeamidation.
`Scheme 1—Deamidation.
`
`/ OH
`C
`
`CH2
`
`'NH
`
`CH - --- C NH CH2
`
`O
`
`Asp4lexapeptMe
`
`to protein and peptide instability in the solid-state are first
`to protein and peptide instability in the solid-state are first
`presented. These include deamidation, peptide bond cleav-
`presented. These include deamidation, peptide bond cleav-
`age, oxidation, the Maillard reaction, (cid:226)-elimination, and
`age, oxidation, the Maillard reaction, /3-elimination, and
`covalent dimerization or aggregation. In the next section,
`covalent dimerization or aggregation. In the next section,
`formulation factors that affect chemical stability, such as
`formulation factors that affect chemical stability, such as
`temperature, moisture, and excipients, are discussed. The
`temperature, moisture, and excipients, are discussed. The
`review concludes with a summary and a discussion of the
`review concludes with a summary and a discussion of the
`implications for future research.
`implications for future research.
`
`Solid-State Reactions of Peptides and Proteins
`Solid-State Reactions of Peptides and Proteins
`DeamidationsChemical instability in the solid state
`Deamidation—Chemical instability in the solid state
`due to deamidation has been observed for human growth
`due to deamidation has been observed for human growth
`hormone (hGH),4,10 recombinant human interleukin-1 re-
`hormone (hGH),4,1° recombinant human interleukin-1 re-
`ceptor antagonist,11 recombinant bovine somatotropin
`ceptor antagonist,11 recombinant bovine somatotropin
`(growth hormone),12 and insulin.13,14 While there have been
`(growth hormone),12 and insulin.13,14 While there have been
`numerous mechanistic studies of protein and peptide
`numerous mechanistic studies of protein and peptide
`deamidation in solution,7,15-19 few such studies have been
`deamidation in solution,7,15-19 few such studies have been
`reported for deamidation in the solid state. Two studies
`reported for deamidation in the solid state. Two studies
`which provide a mechanistic perspective on deamidation
`which provide a mechanistic perspective on deamidation
`in solids are summarized below.
`in solids are summarized below.
`The stability and mechanism of degradation of the Asn-
`The stability and mechanism of degradation of the Asn-
`hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) were studied in
`hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) were studied in
`solid formulations lyophilized from acidic solutions ranging
`solid formulations lyophilized from acidic solutions ranging
`from pH 3-5. The main degradation pathway for Asn-
`from pH 3-5. The main degradation pathway for Asn-
`hexapeptide in the pH 3 formulation is deamidation via
`hexapeptide in the pH 3 formulation is deamidation via
`hydrolysis of the Asn side chain to produce the Asp-
`hydrolysis of the Asn side chain to produce the Asp-
`hexapeptide, which is further hydrolyzed at the Asp-Gly
`hexapeptide, which is further hydrolyzed at the Asp-Gly
`amide bond to generate a small quantity of tetrapeptide
`amide bond to generate a small quantity of tetrapeptide
`(Scheme 1).18 As the pH of the solution prior to lyophiliza-
`(Scheme 1).18 As the pH of the solution prior to lyophiliza-
`tion increases from 3 to 5, intramolecular attack of the
`tion increases from 3 to 5, intramolecular attack of the
`carbonyl center of the Asn side chain by the amide nitrogen
`carbonyl center of the Asn side chain by the amide nitrogen
`of the succeeding amino acid to form a cyclic imide
`of the succeeding amino acid to form a cyclic imide
`intermediate becomes more prominent, as evidenced by an
`intermediate becomes more prominent, as evidenced by an
`increase in the cyclic imide in the product distribution
`increase in the cyclic imide in the product distribution
`(Scheme 1).18 In solution at pH 5, the cyclic imide is
`(Scheme 1).18 In solution at pH 5, the cyclic imide is
`hydrolyzed to form the isoAsp-hexapeptide, which is the
`hydrolyzed to form the isoAsp-hexapeptide, which is the
`dominant degradation product;15 however, the isoAsp-
`dominant degradation product;15 however, the isoAsp-
`hexapeptide was not observed in the solid state.18 The
`hexapeptide was not observed in the solid state.18 The
`absence of the isoAsp-hexapeptide in the product distribu-
`absence of the isoAsp-hexapeptide in the product distribu-
`tion may be due to the low level of water available for
`tion may be due to the low level of water available for
`hydrolysis in the solid state. The mechanism of deamida-
`hydrolysis in the solid state. The mechanism of deamida-
`tion for the Asn-hexapeptide in the solid state was found
`tion for the Asn-hexapeptide in the solid state was found
`to be similar to that in solution.18 In an extension of this
`to be similar to that in solution.18 In an extension of this
`work, we have recently investigated the deamidation of this
`work, we have recently investigated the deamidation of this
`
`490 / Journal of Pharmaceutical Sciences
`490 / Journal of Pharmaceutical Sciences
`Vol. 88, No. 5, May 1999
`Vol. 88, No. 5, May 1999
`
`peptide in solid poly(vinyl alcohol) and poly(vinyl pyrroli-
`peptide in solid poly(vinyl alcohol) and poly(vinyl pyrroli-
`done) matrixes.20-22 As in the lyophilized peptide, the
`done) matrixes."-22 As in the lyophilized peptide, the
`mechanism of deamidation appears to be similar to that
`mechanism of deamidation appears to be similar to that
`in solution, but the kinetics and product distribution are
`in solution, but the kinetics and product distribution are
`altered, particularly in matrices of low water content.
`altered, particularly in matrices of low water content.
`Human insulin has also been observed to undergo
`Human insulin has also been observed to undergo
`deamidation in the solid-state via a mechanism similar to
`deamidation in the solid-state via a mechanism similar to
`that in solution.13 In insulin formulations lyophilized from
`that in solution.13 In insulin formulations lyophilized from
`acidic solutions (pH 3-5), the rate-determining first step
`acidic solutions (pH 3-5), the rate-determining first step
`involves intramolecular nucleophilic attack of the C-
`involves intramolecular nucleophilic attack of the C-
`terminal AsnA21 carboxylic acid onto the side-chain amide
`terminal AsnA21 carboxylic acid onto the side-chain amide
`carbonyl to release ammonium and to form a reactive cyclic
`carbonyl to release ammonium and to form a reactive cyclic
`anhydride intermediate, which can further react with
`anhydride intermediate, which can further react with
`various nucleophiles.13 The cyclic anhydride intermediate
`various nucleophiles.13 The cyclic anhydride intermediate
`may react with water to form [desamidoA21] insulin,13 and
`may react with water to form [desamidoA21] insulin,13 and
`may also react with another molecule of insulin to form
`may also react with another molecule of insulin to form
`covalent dimers.13 While the cyclic imide intermediates
`covalent dimers.13 While the cyclic imide intermediates
`formed during Asn-hexapeptide deamidation in the solid
`formed during Asn-hexapeptide deamidation in the solid
`state were observed to accumulate,23 the cyclic anhydride
`state were observed to accumulate,23 the cyclic anhydride
`intermediate formed during insulin deamidation did not.13
`intermediate formed during insulin deamidation did not.13
`Strickley and Anderson were able to verify that insulin
`Strickley and Anderson were able to verify that insulin
`deamidation proceeds via formation of a cyclic anhydride
`deamidation proceeds via formation of a cyclic anhydride
`by using aniline trapping of the intermediate.13 Consistent
`by using aniline trapping of the intermediate.13 Consistent
`with these findings, Pikal and Rigsbee observed that the
`with these findings, Pikal and Rigsbee observed that the
`deamidation of human insulin occurs predominantly at
`deamidation of human insulin occurs predominantly at
`AsnA21, except at high relative humidity when deamidation
`AsnA21, except at high relative humidity when deamidation
`at AsnB3 is more prevalent.14
`at AsnB3 is more prevalent.14
`Similar to the degradation of the Asn-hexapeptide
`Similar to the degradation of the Asn-hexapeptide
`discussed previously, the solid-state degradation of a model
`discussed previously, the solid-state degradation of a model
`Asp-hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) is dependent
`Asp-hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) is dependent
`on the pH of the bulk solution prior to lyophilization. This
`on the pH of the bulk solution prior to lyophilization. This
`value is often referred to as the “pH” of the solid, since the
`value is often referred to as the apH° of the solid, since the
`true hydrogen ion activity is difficult to measure and pH
`true hydrogen ion activity is difficult to measure and pH
`is technically undefined in the solid-state. Under acidic
`is technically undefined in the solid-state. Under acidic
`conditions (“pH” 3.5 and 5.0), the Asp-hexapeptide mainly
`conditions (apH° 3.5 and 5.0), the Asp-hexapeptide mainly
`decomposes to produce a cyclic imide intermediate via base-
`decomposes to produce a cyclic imide intermediate via base-
`catalyzed intramolecular cyclization.23 Hydrolysis of the
`catalyzed intramolecular cyclization.23 Hydrolysis of the
`Asp-Gly amide bond also occurs but to a lesser extent.
`Asp-Gly amide bond also occurs but to a lesser extent.
`Under neutral and basic conditions (“pH” 6.5 and 8.0), the
`Under neutral and basic conditions (apH° 6.5 and 8.0), the
`Asp-hexapeptide degrades exclusively via intramolecular
`Asp-hexapeptide degrades exclusively via intramolecular
`cyclization to produce the Asu-hexapeptide, which is fur-
`cyclization to produce the Asu-hexapeptide, which is fur-
`ther hydrolyzed to form the isoAsp-hexapeptide.23 At “pH”
`ther hydrolyzed to form the isoAsp-hexapeptide.23 At apH°
`8, the isoAsp-hexapeptide is the dominant degradation
`8, the isoAsp-hexapeptide is the dominant degradation
`product,23 similar to that observed in solution.24
`product,23 similar to that observed in solution."
`Peptide Bond CleavagesA second common degrada-
`Peptide Bond Cleavage —A second common degrada-
`tion pathway for peptides and proteins involves cleavage
`tion pathway for peptides and proteins involves cleavage
`of the peptide bond. Representative pathways of peptide
`of the peptide bond. Representative pathways of peptide
`bond cleavage are shown in Scheme 2. Lyophilized human
`bond cleavage are shown in Scheme 2. Lyophilized human
`relaxin formulated with glucose can undergo hydrolytic
`relaxin formulated with glucose can undergo hydrolytic
`cleavage of the C-terminal serine (Ser) residue on the
`cleavage of the C-terminal serine (Ser) residue on the
`B-chain (Trp28-Ser29-COOH) upon storage at 40 °C.25 This
`B-chain (Trp28-Ser29-COOH) upon storage at 40 ° C. 25 This
`observation was supported by a reduction in molecular
`observation was supported by a reduction in molecular
`mass corresponding to the loss of Ser from fragment T5-
`mass corresponding to the loss of Ser from fragment T5-
`T9 of relaxin, as verified by liquid chromatography/mass
`T9 of relaxin, as verified by liquid chromatography/mass
`spectroscopy (LC/MS) and tryptic digest.25 Li et al. proposed
`spectroscopy (LC/MS) and tryptic digest.25 Li et al. proposed
`that this cleavage involved an initial reaction of the Ser
`that this cleavage involved an initial reaction of the Ser
`hydroxyl group with glucose followed by subsequent hy-
`hydroxyl group with glucose followed by subsequent hy-
`drolysis of the Trp-Ser bond via a cyclic intermediate.25
`drolysis of the Trp-Ser bond via a cyclic intermediate.25
`In the solid state, the major degradation pathway of
`In the solid state, the major degradation pathway of
`aspartame (R-aspartylphenylalanine methyl ester, APM)
`aspartame (a-aspartylphenylalanine methyl ester, APM)
`is intermolecular cyclization to form exclusively diketopip-
`is intermolecular cyclization to form exclusively diketopip-
`erazine (DKP) with the elimination of methanol.26 In
`erazine (DKP) with the elimination of methano1.26 In
`solution, the degradation of aspartame at neutral and basic
`solution, the degradation of aspartame at neutral and basic
`pH also occurs mainly via cyclization to form diketopip-
`pH also occurs mainly via cyclization to form diketopip-
`erazine (DKP) or hydrolysis at the ester linkage to form
`erazine (DKP) or hydrolysis at the ester linkage to form
`R-aspartylphenylalanine (AP) and methanol.27 Since water
`a-aspartylphenylalanine (AP) and methano1.27 Since water
`was absent in the aspartame solid-state study, no hydroly-
`was absent in the aspartame solid-state study, no hydroly-
`sis products were observed.26
`sis products were observed.26
`The instability of the undecapeptide substance P (SP)
`The instability of the undecapeptide substance P (SP)
`in the solid state also proceeds through diketopiperzine
`in the solid state also proceeds through diketopiperzine
`
`

`

`HO\ , O
`
`Vi,)
`CH - C
`
`v (
`CH C
`
`N
`
`H
`N CH
`
`R
`1
`
`O
`
`R2
`
`O
`H
`C-
`
`H
`
`O
`H
`CH C
`
`CH - C
`
`O
`
`- NH - CH - C
`
`
`
`N H
`
`CH
`
`O
`
`2
`
`R1
`
`O
`
`H
`C N
`
`CH C -
`
`R2
`
`CH C N
`H
`
`RI
`
`0'•
`
`H O
`2
`
`NH
`
`CH
`
`OH N
`
`O
`
`H
`N
`
`CH- C
`
`O
`
`R2
`
`O
`
`O
`
`NH
`
`CH C
`
`R1
`
`/
`NH
`
` H
`N
`2
`
`O
`
`O
`
`CH C
`
`R2
`
`O
`
`NH - CH -C OH
`R1
`
`HO//
`
`H
`N
`2
`
`O
`
`H
`C N
`
`CH C
`
`O
`
`R2
`
`OH
`
`O O
`I I
`
`N
`
`OH
`
`O
`
`NH
`
`R
`
`I
`
`O C
`
`H
`N
`2
`
`H
`
`CC
`
`Scheme 2sProteolysis.
`Scheme 2—Proteolysis.
`formation. The main pathway of decomposition consists of
`formation. The main pathway of decomposition consists of
`the sequential release of N-terminal dipeptides via their
`the sequential release of N-terminal dipeptides via their
`diketopiperazines, cyclo(Arg-Pro) and cyclo(Lys-Pro).28 Un-
`diketopiperazines, cyclo(Arg-Pro) and cyclo(Lys-Pro).28 Un-
`der the conditions studied, the release of N-terminal
`der the conditions studied, the release of N-terminal
`dipeptides dominates over other possible routes of spon-
`dipeptides dominates over other possible routes of spon-
`taneous modification, such as oxidation and deamidation.28
`taneous modification, such as oxidation and deamidation.28
`OxidationsThe side chains of His, Met, Cys, Trp, and
`Oxidation —The side chains of His, Met, Cys, Trp, and
`Tyr residues in proteins are potential sites for oxidation
`Tyr residues in proteins are potential sites for oxidation
`(Scheme 3).1 A major chemical decomposition pathway for
`(Scheme 3).1 A major chemical decomposition pathway for
`human growth hormone (hGH) in the solid state is me-
`human growth hormone (hGH) in the solid state is me-
`thionine oxidation at Met14 to form the sulfoxide.10 Even
`thionine oxidation at Met14 to form the sulfoxide.1° Even
`with minimal oxygen ((cid:24)0.05%) in the vial headspace,
`with minimal oxygen (—0.05%) in the vial headspace,
`decomposition via oxidation is comparable to or greater
`decomposition via oxidation is comparable to or greater
`than that due to the alternative reaction of deamidation.4
`than that due to the alternative reaction of deamidation.4
`Storage of lyophilized hGH in an oxygen atmosphere
`Storage of lyophilized hGH in an oxygen atmosphere
`results in greater decomposition than storage in a nitrogen
`results in greater decomposition than storage in a nitrogen
`atmosphere.29 As in solution, atmospheric oxygen can easily
`atmosphere.29 As in solution, atmospheric oxygen can easily
`oxidize Met residues in the solid state, leading to chemical
`oxidize Met residues in the solid state, leading to chemical
`instability and loss of biological activity.
`instability and loss of biological activity.
`Human insulin-like growth factor I (hIGF-I), lyophilized
`Human insulin-like growth factor I (hIGF-I), lyophilized
`from phosphate buffer, also undergoes oxidation at its Met
`from phosphate buffer, also undergoes oxidation at its Met
`residue.30 There were no significant differences in reaction
`residue.3° There were no significant differences in reaction
`rates (second-order kinetics) between solution and solid
`rates (second-order kinetics) between solution and solid
`states.30 However, Met oxidation in the solid-state consti-
`states.3° However, Met oxidation in the solid-state consti-
`tutes a greater fraction of the total protein modification
`tutes a greater fraction of the total protein modification
`than in solution.30 Both oxygen content and light exposure
`than in solution.3° Both oxygen content and light exposure
`affect the oxidation rate.30 Exposure to light increases the
`affect the oxidation rate.30 Exposure to light increases the
`oxidation rate by a factor of 30.30 This increase in oxidation
`oxidation rate by a factor of 30.30 This increase in oxidation
`rate with exposure to light and oxygen suggests that
`rate with exposure to light and oxygen suggests that
`photooxidation and molecular oxygen may be involved in
`photooxidation and molecular oxygen may be involved in
`the generation of radicals. However, no further experiments
`the generation ofradicals. However, no further experiments
`were conducted to determine the nature of the radicals
`were conducted to determine the nature of the radicals
`involved or the mechanism of Met oxidation in the solid
`involved or the mechanism of Met oxidation in the solid
`state.
`state.
`
`The oxidative deamidation of a cyclic hexapeptide, ace-
`The oxidative deamidation of a cyclic hexapeptide, ace-
`tylcysteine-asparagine-(5,5-dimethyl-4-thiazolidinecarbo-
`tylcysteine-asparagine-(5,5-dimethy1-4-thiazolidinecarbo-
`nyl)-(4-(aminomethyl)phenylalanine)-glycine-aspartic acid-
`ny1)-(4-(aminomethyl)phenylalanine)-glycine-aspartic acid-
`cysteine cyclic disulfide, in a lyophilized mannitol formula-
`cysteine cyclic disulfide, in a lyophilized mannitol formula-
`tion does not appear to depend on molecular oxygen as a
`tion does not appear to depend on molecular oxygen as a
`reactive species.31 The oxidation reaction occurs at the
`reactive species.31 The oxidation reaction occurs at the
`aminomethyl phenylalanine moiety to form a benzaldehyde
`aminomethyl phenylalanine moiety to form a benzaldehyde
`derivative.31 This oxidative degradant is not detected in
`derivative.31 This oxidative degradant is not detected in
`the neat solid drug stored under atmospheric oxygen,
`the neat solid drug stored under atmospheric oxygen,
`suggesting that oxygen is not involved in the reaction.31
`suggesting that oxygen is not involved in the reaction.31
`Instead, the decomposition of the heptapeptide may be due
`Instead, the decomposition of the heptapeptide may be due
`to a reaction with reducing sugar impurities in the man-
`to a reaction with reducing sugar impurities in the man-
`nitol excipient.31 The proposed mechanism involves (1)
`nitol excipient.31 The proposed mechanism involves (1)
`formation of a Schiff base from the peptide primary amine
`formation of a Schiffbase from the peptide primary amine
`reacting with the carbonyl of the aldehydic group on the
`reacting with the carbonyl of the aldehydic group on the
`reducing sugar, (2) tautomerization to a more stable
`reducing sugar, (2) tautomerization to a more stable
`configuration, conjugated with the phenyl group, and (3)
`configuration, conjugated with the phenyl group, and (3)
`hydrolytic cleavage of the new Schiff base to generate the
`hydrolytic cleavage of the new Schiff base to generate the
`observed aldehyde derivative.31 The first part of this
`observed aldehyde derivative.31 The first part of this
`proposed reaction, the formation of the Schiff base, pro-
`proposed reaction, the formation of the Schiff base, pro-
`ceeds via a mechanism similar to the Maillard reaction,
`ceeds via a mechanism similar to the Maillard reaction,
`discussed below.
`discussed below.
`Maillard ReactionsThe food industry has studied
`Maillard Reaction —The food industry has studied
`extensively the nonenzymatic browning of food due to the
`extensively the nonenzymatic browning of food due to the
`Maillard reaction, which results from reducing sugars
`Maillard reaction, which results from reducing sugars
`reacting with either amino or free amine groups in proteins,
`reacting with either amino or free amine groups in proteins,
`leading to changes in both the chemical and physiological
`leading to changes in both the chemical and physiological
`properties of the proteins (Scheme 4).32 The first phase of
`properties of the proteins (Scheme 4).32 The first phase of
`the Maillard reaction involves a condensation reaction
`the Maillard reaction involves a condensation reaction
`between the carbonyl of a reducing sugar and an amino
`between the carbonyl of a reducing sugar and an amino
`group to form an N-substituted glycosylamine, which then
`group to form an N-substituted glycosylamine, which then
`converts to a Schiff base and a molecule of water.33
`converts to a Schiff base and a molecule of water.33
`Subsequent cyclization and isomerization (Amadori rear-
`Subsequent cyclization and isomerization (Amadori rear-
`rangement) result in derivatives which cause discoloration
`rangement) result in derivatives which cause discoloration
`
`Journal of Pharmaceutical Sciences / 491
`Journal of Pharmaceutical Sciences / 491
`Vol. 88, No. 5, May 1999
`Vol. 88, No. 5, May 1999
`
`

`

`A. Met
`
`H
`N
`
`CH
`
`0
`
`C
`
`R2
`CH
`
`N
`
`H20 2
`
`
`
`HN
`
`CH
`
`O
`
`Perfonnic Acid
`
`S•
`
`H
`N
`
`CH
`
`O7,5=O
`
`0
`
`C
`
`O
`
`C
`
`R2
`CH
`
`R2
`
`CH
`
`N
`H
`
`N
`H
`
`B. Tm
`
`O
`
`H
`N
`
`CH —.
`
`- C
`
`12
`
`CH
`
`N
`H
`
`C
`
`O
`
`•.„ [0]
`
`H
`N
`
`<
`
`CH
`
`0
`
`C
`
`R2
`
`CH
`
`N
`H
`
`C
`
`OI
`
`0
`
`C
`
`0
`
`OH
`
`O
`
`R
`
`CH
`
`H + R' NH2 (Lys or Arg)
`
`OH
`
` R
`
`CH
`
`R'
`
`N
`
`C
`
`H
`
`unstable
`
`+H20
`
`- H2O
`
`0
`
`R'
`
`OH NH
`
`R
`
`C-
`
`CH2 NH
`
`R'
`
`
`
`
`
`R
`
`C
`
`C
`
`H
`
`Amadori Rearragement
`Scheme 4sMaillard Reaction.
`Scheme 4—Maillard Reaction.
`
`Enol
`
`HN
`
`1
`
`0
`
`H
`N
`
`CH
`
`C
`
`N
`H
`
`.R2
`CH - C —.
`
`0
`
`H
`N
`
`0
`
`1:2
`
`CH
`
`N
`H
`
`0
`
`R2
`
`H
`N
`
`C
`
`CI
`
`C
`
`R2
`
`N
`H
`
`;2
`
`CH
`
`N
`H
`
`If1+1
`
`H
`N
`
`I
`
`CH
`-
`
`SH
`
`H
`
`OH
`
`C. Cys
`
`H
`N
`
`H
`
`0
`
`C
`
`R2
`CH
`
`N
`H
`
`C
`
`0
`
`O
`
`,
`
`14
`
`CH
`
`C
`
`N
`
`R2
`CH
`
`0
`
`SOH
`
`H
`N CH
`
`0
`
`
`
`
`
`CH
`
`N
`H
`
`/
`011
`Scheme 5s(cid:226) Elimination.
`Scheme 5—fl Elimination.
`
`8
`
`O
`
`R2
`
`N
`
`C
`
`N
`
`C
`
`primarily due to the Maillard reaction.35 Lysine is usually
`primarily due to the Maillard reaction.35 Lysine is usually
`lost more rapidly than the other amino acids because of
`lost more rapidly than the other amino acids because of
`its free (cid:15)-amino group, which will react easily with the
`its free e-amino group, which will react easily with the
`carbonyl of reducing sugars.33 However, other residues,
`carbonyl of reducing sugars.33 However, other residues,
`such as arginine, asparagine, and glutamine, have been
`such as arginine, asparagine, and glutamine, have been
`observed to react with reducing sugars also.25,34
`observed to react with reducing sugars also.25,34
`(cid:226)-EliminationsLyophilized bovine insulin has been
`fl-Elimination —Lyophilized bovine insulin has been
`observed to degrade rapidly with increased water content
`observed to degrade rapidly with increased water content
`to produce both covalent and noncovalent aggregates after
`to produce both covalent and noncovalent aggregates after
`incubation at 50 °C at various relative humidities.36 Cos-
`incubation at 50 °C at various relative humidities.36 Cos-
`tantino et al. hypothesized that the reducible covalent
`tantino et al. hypothesized that the reducible covalent
`interactions were due to thiol-catalyzed disulfide ex-
`interactions were due to thiol-catalyzed disulfide ex-
`change.36 They speculated that these free thiol groups
`change.36 They speculated that these free thiol groups
`resulted from the (cid:226)-elimination of intact disulfide bonds
`resulted from the )3-elimination of intact disulfide bonds
`in insulin.36 The proposed mechanism for (cid:226)-elimination
`in insulin.36 The proposed mechanism for /3-elimination
`involves hy