Solution and Solid-State Chemical Instabilities of ... - ACS Publications

Chapter 3 S o l u t i o n a n d Solid-State C h e m i c a l Instabilities of A s p a r a g i n y l a n d A s p a r t y l Residues in M o d e l Peptides 1

CeciliaOliyai1,2and Ronald T. Borchardt

Downloaded by CORNELL UNIV on June 7, 2012 | http://pubs.acs.org Publication Date: August 19, 1994 | doi: 10.1021/bk-1994-0567.ch003

1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66045 The kinetics and mechanisms of the degradation of asparagine (Asn) and aspartic acid (Asp) residues in model hexapeptides were examined in aqueous and solid states. Specific chemical reactions that affect the stability of Asn and Asp residues in polypeptides include deamidation of the Asn side chain, Asp-X and/or X-Asp amide bond hydrolysis, and Asp-to-isoAsp interconversion. The exogenous parameters which influence the product distribution and the rates of degradation in solution and in solid state were studied. Under lyophilized conditions, the nature of the excipient most significantly affected the rate of decomposition of the Asp-hexapeptide, whereas the pH of the pre-lyophilized solution determined the product distribution. In contrast, the pH of the starting solution dictated both the rate and extent of degradation of the freeze-dried Asn-hexapeptide. It was observed that pH, temperature, and buffer concentration played important roles in determining the chemical lability and degradation routes in aqueous medium. Although the pharmacological properties of polypeptides have long been recognized, the realization of their use as therapeutic agents came only recendy with the advent of biotechnology, which permits the production of proteins and peptides on a commercial scale. As a result, the number of recombinant products is widespread and ever increasing (1). Unlike small molecules, polypeptides possess not only their primary sequence but also higher order structure (i.e. secondary, tertiary, and quaternary). Thus, the development of stable formulations for proteins as pharmaceuticals is difficult, and success often depends on an understanding of the physical and chemical instability of the molecule. This chapter is not intended to address the broad topic of physical and chemical instability of polypeptides since there are many appropriate reviews and books which provide comprehensive treatments of these subjects (2-4). The primary objective of this review is to focus on the effects of exogenous factors on the chemical instability of proteins, specifically, the instability of asparagine (Asn) and aspartic acid (Asp) residues in solution and in the solid state. 2

Current address: Formulation Research and Development, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608 0097-6156/94/0567-0046$08.00/0 © 1994 American Chemical Society In Formulation and Delivery of Proteins and Peptides; Cleland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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Asparaginyl and Aspartyl Residues in Peptides


Instability of Aspartic Acid Residues In solution Several studies have indicated that the chemical stability of Asp residues in proteins and peptides is dependent on the local conformation and primary sequence around the potentially reactive Asp residue (5, 6). Two chemical reactions that are known to affect specifically Asp-containing proteins and peptides include Asp-toisoAsp interconversion via cyclic imide formation and Asp-X and/or X-Asp amide bond hydrolysis. Several investigators have illustrated the magnitude of these nonenzymatic degradation pathways, which can substantially reduce the biological activ ity of Asp-containing proteins (5-12 and Oliyai, C.; Borchardt, R. T., University of Kansas, unpublished data). Asp residues in polypeptides tend to undergo cyclization to produce the cyclic imide. Such rearrangement has been observed in classical solution and solid phase syntheses, recrystallization (13), and hydrogenolysis (14) of peptides which contain β-alkylaspartyl and free β-carboxyl groups. In addition, peptide bonds of Asp resi dues are cleaved in dilute acid at a rate at least 100 times greater than other peptide bonds (15). The enhanced hydrolysis, occurring at Asp-X and/or X-Asp locations, is attributed to intramolecular catalysis by the carboxyl group of the Asp side chain. Our laboratory has attempted to delineate the relevant factors which modulate the chemical activity of Asp residues in polypeptides by studying the kinetics and mechanism of degradation of a model hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala; Asphexapeptide) whose primary sequence contains a single Asp residue. In solution, the apparent rates of degradation of this Asp-hexapeptide were determined as a function of pH, buffer concentration, and temperature (16). The major degradation pathways for the Asp-hexapeptide consisted of hydrolysis at the Asp-Gly amide bond and the isomerization of Asp to isoAsp via the cyclic imide intermediate, the Asu peptide. The extent and routes of degradation were pH-dependent. Under highly acidic conditions (pH 0.3-2.0), the Asp-hexapeptide decom posed predominantly via intramolecular hydrolysis of the Asp-Gly amide bond, forming a tetrapeptide, Val-Tyr-Pro-Asp, and a dipeptide, Gly-Ala (Figure la). Two plausible mechanisms of the Asp-Gly amide hydrolysis were postulated based on the kinetic and solvent isotope experiments (16). Both mechanisms, which possibly involve intramolecular nucleophilic and general base catalyses, are catalyzed by hydrogen ions (16). In addition, the starting peptide could also cyclize to generate an Asu-hexapeptide (cyclic imide) which remained stable under acidic conditions and constituted approximately 7% of the total degradation products. The ring closure is known to be catalyzed by both acids and bases (17-20). The Asp-hexapeptide favored cyclization over the peptide cleavage reaction as the pH was increased. The Asuhexapeptide formed in cyclization further degraded to give rise to isoAsp and Asp peptides at higher p H values (pH > 4) as a result of base-catalyzed hydrolysis. For example, at pH 4.0 the majority of the peptide (60%) rearranged to produce the Asuhexapeptide, which was further hydrolyzed to form the isoAsp-hexapeptide while approximately 10% underwent Asp-X hydrolysis (Figure lb). The overall kinetic scheme at pH 4.0 to 5.0 was complicated by this transition from predominantly amide hydrolysis to cyclic imide formation, which eventually led to isomerization. The Asp-hexapeptide exhibited apparent minimum chemical stability at pH 0.3 and pH 4.0 (Figure 2). In this pH-rate profile, the observed rate constants (k bs) for the disappearance of the Asp-hexapeptide at p H 0.3 to 3.0 were determined from the slopes of linear plots of the logarithm of peptide peak area versus time. At p H 4.0 and 5.0, k b were estimated by adding the pseudo-first-order rate constants for the parallel formation of the Asu-hexapeptide and -tetrapeptide. These individual rate constants were generated by fitting the data to Laplace MicroMath (Salt Lake City, UT) and nonlinear least-squares regression (MINSQ). For p H 6 to 10.0, the loss of the Asp-hexapeptide was characterized by pseudo-first-order reversible kinetics 0

0

s

In Formulation and Delivery of Proteins and Peptides; Cleland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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FORMULATION AND DELIVERY O F PROTEINS AND PEPTIDES

whereby kobs = kf (apparent rate constant for the formation of the isoAsp peptide) + k (apparent rate constant for the regeneration of the Asp peptide), where kf and k were generated from the best fit obtained. The contribution from the Asp-Gly amide hydrolysis to the overall degradation was negligible above p H 6.0 (Figure lc). Furthermore, since the Asu-hexapeptide was extremely unstable at neutral and alkaline pH, hydrolyzing rapidly to form the isoAsp-hexapeptide and to regenerate the Asp-hexapeptide, only the isoAsp hexapeptide product was observed (Figure lc). The apparent rate of degradation followed pseudo-first-order reversible interconversion kinetics (16). Buffer catalysis did not occur to any significant extent for the forward and reverse reactions, which were also essentially pH-independent at p H 8.0 and above (Figure 2). It may bereasonable to suggest that the formation of Asu-hexapeptide from the Asp-hexapeptide involves the nucleophilic attack by the deprotonated amide nitrogen on the free carboxylic acid species. Thus, as the pH of the solution is increased, the equilibrium concentration of the nucleophilic amide ion increases but the fraction of protonated species decreases. These two effects exacdy offset each other, and the observed rate becomes independent of pH.


r

r

(a) -

Val-Tyr-Pro-Asu-Gly-Ala

Val-Tyr-Pro-Asp-Gly-AlaVal-Tyr-Pro-Asp + Gly-Ala

( b )

Val-Tyr-Pro-Asu-Gly-Ala (cyclic imide)

Val-Tyr-Pro-Asp-Gly-Ala (Asp-hexapeptide)

Val-Try-Pro-isoAsp-Gly-Ala (isoAsp-hexapeptide

Val-Tyr-Pro-Asp + Gly-Ala (tetrapeptide) (c)

[Val-Tyr-Pro-Asu-Gly-Ala] (cyclic imide)

Val-Tyr-Pro-Asp-Gly-Ala

Val-Try-Pro-isoAsp-Gly-Ala

Figure 1· Degradation pathways for Asp-hexapeptide in solution at 37°C (0.5 M). (a) at pH 0.3 to 3 (pH 0.3,1.1, 1.5, and 2.0 H Q ; p H 3.0 formate); (b) at pH 4 to 5 (acetate); (c) at pH 6 to 10.0 (pH 6 and 7.4 phosphate; p H 8.0 tris; p H 9.0 and 10.0 borate). (Adapted from réf. 1).


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-2.4-,

0

2

4

6 pH

8

10

12

Figure 2. pH-rate profile for the degradation of the Asp-hexapeptide at 37°C (kob is the observed rate constant for the loss of the Asp-hexapeptide). (Adapted from réf. 1). S

In the Solid State. Proteins often have poor and erratic oral bioavailability (21) and may require lyophilization to achieve adequate shelf-life stability (22). However, development of freeze-dried formulations presents a new set of stability variables such as the effect of excipients, residual moisture content, and lyophilization cycle. The nature of the excipients used in pharmaceutical formulations has been reported to influence the general stability of freeze-dried drugs. Excipient crystallinity, which can be modulated by the choice of lyophilization cycle, can affect the stabilization of proteins during lyophilization. Inclusion of certain amorphous additives has been shown to minimize freeze-drying-induced aggregate formation (23-25). In another study, a partially amorphous excipient system provided the greatest protection against aggregation and chemical decomposition via methionine oxidation and Asn deamidation (26). Crystallization of excipients, which leads to a decrease in Tg (glass transition temperature), has been observed to occur when the formulations are stored at 25°C (27). As a result, the degradation rate increased sharply with increasing temperature and with increasing moisture level. Additives were also observed to preserve the native structure of proteins by acting as protectants during freezing and dehydration stresses (28-31). Optimal recovery of activity of proteins upon freeze-drying and rehydration was correlated with the use of such additives in the formulations. Water facilitates spontaneous chemical degradation by increasing molecular mobility and chain flexibility to encourage intermolecular and intramolecular rearrangements and by directly participating in chemical reactions. Moreover, water can interact with the excipient to render physical transformation of solids (i.e. excipients and drug), compromising the overall stability of the active drug (32). Thus, die residual water level in freeze-dried products represents another critical parameter in the chemical stability of drugs. While the concept of pH may not have meaning in the


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FORMULATION AND DELIVERY OF PROTEINS AND PEPTIDES

solid state, the pH of the bulk solution determines the extent of ionization of the peptide and buffer species both in solution and in the solid state (26). In some cases, the rate constant of degradation in solid state can be comparable to or higher than that in solution at a given pH (33, 34). Also, depending on the choice of buffers, the pH can change during the freezing process as a result of crystallization of buffers (35). Thus, it is important to confirm whether the same p H dependence for the rate of degradation generated from solution stability studies applies in the solid state. Assessment of these variables is imperative for designing the most stable dosage forms with minimal lot-to-lot shelf-life variability. We have recently conducted a study which evaluated the individual and inter active effects of formulation variables such as the p H of pre-lyophilized solution, moisture level, temperature, and type of bulking agent on the chemical stability of the model Asp-hexapeptide in the lyophilized state (Oliyai, C.; Patel, J. P.; Carr, L . ; Borchardt, R. T., University of Kansas, unpublished data). AH bulk solutions were prepared by dissolving appropriate amounts of peptide and excipients in 0.01 M of pH-adjusted dibasic sodium phosphate/citrate buffer solutions. These bulk solutions were then lyophilized and rehydrated with 0.8 μΐ (medium moisture level) and 1.6 ul (high moisture level) of h2o. The samples that were not rehydrated following lyoph ilization were designated as having the lowest moisture level. The moisture-loaded samples were then stored at temperatures ranging from 40° to 60°C. The vials were removed at designated time intervals to be analyzed by reversed-phase H P L C . The degradation products were identified by coinjecting authentic samples. The degradation pathways of the Asp-hexapeptide in the lyophilized state were dependent on the pH of the bulk solutions and the moisture content of the freeze-dried formulations. In general, the kinetics of the disappearance of Asp-hexa peptide followed pseudo-first-order reversible kinetic behavior under all experimental conditions (Figures 3a-b). This type of kinetic profile was justified by the product distribution observed. Under acidic conditions (pH 3.5 and 5.0), the lyophilized Asphexapeptide predominantly decomposed to generate the Asu-hexapeptide, irrespective of the type of excipient present in the formulation (Table I). The hydrolysis of the Asp-Gly amide bond constituted a much less significant pathway under these condi tions (Figure 4, Table I). At pH 6.5 and 8.0, the parent hexapeptide exclusively isomerized via formation of the Asu-hexapeptide to produce the isoAsp-hexapeptide (Figure 4, Table I). The extent of hydrolysis of the Asu-hexapeptide intermediate at pH 8.0 exceeded that at pH 6.5, rendering the isoAsp-hexapeptide the major product of degradation in the basic environment. Although the type of excipient (amorphous vs. crystalline) did not influence the degradation routes, the choice of excipient sub stantially affected the mean rate constant of peptide decomposition. Consistently, the amorphous lactose/peptide formulations were significantly more chemically stable at all temperatures, p H values, and moisture levels than formulations containing crystalline mannitol. These results are consistent with the view that an amorphous or partially amorphous matrix usually imparts more stabilization to the freeze-dried protein drugs than does the crystalline excipient system (23-26). Upon examination, the product distribution in the solid state was significantly different from that in solution. Evidently the hydrolysis of the Asu-hexapeptide inter mediate and the Asp-Gly peptide bond (formation of the tetrapeptide) was suppressed in a water-deficient environment. Thus, at lower pH values (3.5, 5.0 and 6.5), the major product observed was the Asu-hexapeptide, and only trace amounts of the tetrapeptide and isoAsp-hexapeptide were detected at pH 3.5 and 6.5, respectively (Figure 4, Table I). At p H 8.0, the base catalysis component of the hydrolysis of the Asu-hexapeptide intermediate compensated for the nearly inoperative water catalysis term. Consequently, the rate of decomposition of the Asu-hexapeptide was sufficient to afford the isoAsp-hexapeptide as a major degradation product at p H 8.0 (Figure 4, Table I).


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3.

Figure 3. Representative curve-fitted plots for the loss of lyophilized Asp-hexapeptide (50°C) in formulations containing (a) lactose (average moisture content = 2.7 % ± 0.2% h2o) and (b) mannitol (average moisture content = 0.4% ± 0.05% H 0). 2


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FORMULATION AND DELIVERY O F PROTEINS AND PEPTIDES

Pathway b, step 1

O^OH CH

Η

ο I II

Η ο C-N-C-C-N-C-C-OH ι ι n ι ι η ι ι Η Η Ο Η Η Ο Η CH 2

-N-C-C-OH ι ι Η CH,

w

3

Asu-hexapeptide

Asp-hexapeptide

Pathway b, step 1

Pathway a

Η Η Ο C — N - C - C - N - C - C1 - O H

Ο^ΟΗ

CH

2

+


~Ν-έΛ-ΟΗ Η Η Ο Α Α »

Tetrapeptide

Η

V 7-γ-ΪΓ7-γ-

3

Dipeptide

Pathway a ρΗ3.5 ρΗ5.0 ρΗ6.5 ρΗ8.0

Ηο

H-H-è-C-N-è-S-OH Η Δ Η η Ο ίΗ A CuH η -N-C-C-OH Η Η ο

minor undetected undetected undetected

IsoAsp-hexapeptide Pathway b Step 1 Step 2 major undetected major undetected major minor minor major

Figure 4. Degradation pathways for the Asp-hexapeptide in the solid state. Table I. Representative Peptide Distribution at 60°C for the Lyophilized Asp-Hexapeptide in the Presence of Mannitol and Lactose at Different pH Values (six months) % of Total Initial Concentration Mannitol Formulations Asp-hexa Average H2O peptide content (0.3% ± 0 . 0 8 % ) pH3.5 15 pH5.0 8 pH8.0 9 Lactose Formulations Average H2O content (1.9% ±0.7%) pH3.5 pH5.0 pH8.0 a

55 73 54

IsoAsphexapeptide ND ND 23

a

ND ND 17

Asu-hexa peptide 45 57 18

12 9 7

Not detectable


Tetra peptide 2.5 ND ND

4 ND ND

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OLIYAI & BORCHARDT

Instability of Asparagine Residues In Solution. Deamidation of Asn residues accounts for a high incidence of chemical instability in proteins. The prevalence of this degradation pathway is illustrated by the ever-increasing number of polypeptides which experienced deamidation and, in some cases, loss of their biological activity as a result of the chemical transformation (6,12,36-43). During the deamidation reaction of Asn residues, the side chain amide of the Asn residue either is hydrolyzed directiy to an Asp residue or proceeds through a five-membered cyclic imide intermediate to Asp and isoAsp residues (Figure 5). o CHj-C-NH

2


-~NH—CH—C-NH—CH — II

I

Ο

it

R

Ο

L-asparaginyl peptide

o II H ,

a/

c

? ~ >--CH--f> •*~NH — C H — C I II H R Ο Ο

L-cyclic imide (Asu peptide)

/

Ο U CHj—C-O"

•~vNH — C H — C - N H — C H — C — η

ι

^~NH—-CH—C-O"

il

Ο R L-aspartyl peptide

\

O R II ι CHj—C—NH—CH n

Ό

Ο

\

J*

Ο

L-isoaspartyl peptide

ο

II CHj—C-Ο" ~ Ν Η ~ έ Η - 0 - Ο Η

S Tetrapeptide

+

H

H

'*-? - Ç ~ R Ο

Dipeptide

Figure 5. Deamidation pathways for the Asn-hexapeptide via (a) direct hydroly sis of the Asn amide side chain or (b) formation of the cyclic imide intermediate. (Adapted from ref. 20). Previous studies have demonstrated that non-enzymatic deamidation is by no means a randomly programmed, post-translational modification. Several determinants such as primary sequence (44, 45), local conformation (40, 46, 47), and exogenous factors (38, 48) dictate the deamidation of native proteins. Therefore, it is important to understand the factors that affect deamidation in order to design rational strategies for protein stabilization.


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In our laboratory, we have studied the effects of exogenous factors such as pH, buffer concentration, and temperature on the kinetics of deamidation and on the degradation product ratio, isoAsp/Asp peptides. A model Asn-containing hexapeptide, whose primary sequence coincides with residues 22-27 of adrenocortico tropic hormone (Val-Tyr-Pro-Asn-Gly-Ala), was selected for this study since the degradation products are easily separated from the parent peptide. The solution degradation of this model hexapeptide, Asn-hexapeptide, was pH-dependent and followed pseudo-first-order kinetics (20). Under acidic conditions (pH 1-2) at 37°C, the Asn-hexapeptide degraded to produce the normal Asphexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) via direct hydrolysis (Figure 5, pathway a). This Asp-hexapeptide, in turn, underwent Asp-Gly amide bond hydrolysis to generate a tetrapeptide (Val-Tyr-Pro-Asp) (Figure 5, pathway a). The formation of the cyclic imide (Asu-hexapeptide; Figure 5, pathway b) was also detected at acidic pH, although its appearance was much slower than the direct hydrolysis reaction, constituting only 10% of the total product. The cyclic imide remained stable in acidic medium and, thus, did not break down further to form the Asp and isoAsp peptide products. From p H 5 to 12, it was shown that deamidation of the Asn-hexapeptide involved the formation of a cyclic imide intermediate followed by its subsequent rapid hydrolysis to form the isoAsp and Asp hexapeptides in a ratio of 4 to 1 (Figure 5, pathway b). Buffer catalysis was observed in the pH range 7-11, but little or no catalysis was observed from pH 5.0 to 6.5. The kinetics of deamidation of the Asnhexapeptide in solution obeyed the Arrhenius relationship within the temperature range studied (50°-90°C, pH 5.0; 25°C-70°C, pH 7.5). The pH dependence for the rate of deamidation of the Asn-hexapeptide is illustrated in Figure 6. Overall, the rate constant of deamidation was slower in acid than it was in the neutral to alkaline region. The Asn-hexapeptide experienced maxi mum chemical stability at pH 3.0 to 4.0. The pH-rate profile showed a unit negative slope on the acidic side (pH 1-2), indicating specific acid catalysis. From pH 5 to 12, the rate was catalyzed by hydroxide ions.

ι -ι o-1Ν

'

Ο

M

a

-2-3-4_ H—•—.—•—ι—·—«—'—'

•

5

0

2

4

6

8

ι — ' — ' — ' —

10

12

1

14

pH

Figure 6. pH-rate profile for the deamidation of the Asn-hexapeptide in aqueous solution (37°C, 0.5 M). (Adapted from réf. 20).



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OLIYAI & BORCHARDT


In the Solid State. While extensive research efforts have been devoted to understanding deamidation in aqueous medium, very little is known about this intramolecular chemical event in the solid state. Reports about proteins undergoing deamidation in freeze-dried formulations are limited (26). Often this chemical reaction is further complicated by the concomitant existence of other chemical or physical instability problems in a given system. Consequendy, the kinetics and mechanisms of degradation in such a system become extremely complex and, thus, it is not feasible to examine deamidation in a more thorough fashion. A more complete understanding of the influence of exogenous factors on the deamidation process was made possible through a study of the solid state instability of the model Asn-hexapeptide, Val-Tyr-Pro-Asn-Gly-Ala, whose kinetics and mechanism of degradation in aqueous solution are already well understood (Oliyai, C.; Patel, J. P.; Borchardt, R. T., University of Kansas, unpublished data). The objective of this study was to evaluate the chemical stability of the Asn-hexapeptide in lyophilized formulations as a function of pH of the starting solution, temperature, and residual moisture level. The disappearance of the Asn-hexapeptide appeared to fit empirically to the pseudo-first-order reversible kinetic model under all experimental conditions. Representative plots of the results along with the calculated lines from this model at different p H values and at 40°C and low moisture level are shown in Figure 7. The theoretical lines were generated using equation 1 (kl+k2)t

A = Ao[(k /k + k )e1

1

2

+(k lk + k )1 2

1

2

(1)

where k i and k2 are forward and reverse rate constants, respectively, A is the concentration of peptide remaining at any time t, and A o is the initial peptide concentration. Analysis of variance calculations indicated that the effects of formulation variables on the reverse rate constant k2 were not statistically significant irrespective of experimental conditions. 100

Figure 7. Representative curve-fitted plots for the disappearance of the Asnhexapeptide at various pH values (40°C) and low moisture level (0.3 % ± 0.09% water).


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At p H 3.5, the Asn-hexapeptide deamidated via direct hydrolysis to produce the Asp-hexapeptide which, in turn, was hydrolyzed at the Asp-Gly amide bond to generate a small quantity of tetrapeptide (Figure 8). Additionally, the parent hexapeptide cyclized to form the cyclic imide product in a quantity equivalent to the Asphexapeptide (Figure 8). However, the cyclic imide formation was favored over the


lOOl

H Β 0 0 Ο

% Asn-hexapeptide % Tetrapeptide % Asp-hexapeptide % Asu-hexapeptide % IsoAsp-hexapeptide

Figure 8. Peptide distribution vs. pH at 40°C and low moisture level (0.3 % ± 0.09% water) after one year. direct hydrolysis of the Asn side chain at higher temperatures, moisture levels, and pH. The formation of cyclic imide required not only the appropriate pH environment but also, more importantly, the chain flexibility suitable for adopting the necessary local conformation for cyclization. Increasing either temperature or residual moisture content would enhance chemical reactions by increasing chain flexibility. Thus, the amount of cyclic imide significantly increased at pH 3.5 as a function of temperature or moisture level. While the propensity of the Asn-hexapeptide to cyclize increased slightly when going from pH 3.5 to 5.0, the rate of hydrolysis of the Asn side chain decreased (Figure 8). The Asn-hexapeptide was most unstable at pH 8.0, such that about 73% of the Asn-hexapeptide deamidated after 12 months at 40°C, generating isoAsp-hexapeptide and Asp-hexapeptide as the major and minor products, respec tively (Figure 8). The overall rate of decomposition of the Asn-hexapeptide was minimal at pH 5.0 where the gradual transition from direct hydrolysis of the Asn side chain to deamidation via cyclic imide intermediate occurred. Conclusion Using small model peptides, we have illustrated that the chemical reactivities of Asn and Asp residues in solution and in the solid state are significantly affected by the exogenous environment. Degradation pathways and reaction mechanism(s) of selected chemical transformations can be elucidated by these studies. Undoubtedly, in an intricate system such as proteins, other factors (e.g. local conformation) would also influence the chemical instability of potentially labile Asp and Asn residues.


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This type of information on the influence of exogenous and endogenous factors on chemical degradation of proteins may lead to the option of site-directed mutagenesis in which the labile sites or adjacent amino acids which catalyze the instability are being replaced by more inert residues. Obviously, in a large complex protein, this type of stabilization strategy will need to avoid both disrupting the conformational state of the protein needed for biological activity and invoking an unwanted immune response. Often, more traditional formulation methods, which lead to the use of the optimal exogenous factors, will have to be employed to stabilize proteins.


Acknowledgments C O . acknowledges the financial support provided by the Parenteral Drug Association Pre-Doctoral Fellowship, Abbott Laboratories, and The National Institute of General Medical Sciences Biotechnology Traineeship. The authors like to thank Professors Richard Schowen (The University of Kansas) and Jeffrey Fox (The University of Utah) for their invaluable comments and discussions. We would also like to acknowledge the following individuals from Abbott Laboratories and the University of Kansas: Dr. Jitendra Patel, Dr. Madhup Dhaon, Linda Carr, Dr. Steve Krill, Dr. John Quick, Jerry Sutherland, Dr. Alex Puko, Christopher Smith, and Brian Moon. Literature Cited 1. Oliyai, C.; Schöneich, C.; Wilson, G. S.; Borchardt, R. T. In Topics in Pharmaceutical Sciences ; Crommelin, D. J. Α.; Midha, Κ. K . , Eds.; Medpharm Scientific Publisher: Stuttgart, 1992; pp. 23-46. 2. Manning, M. C.; Patel, K.; Borchardt, R. T. Pharm. Res. 1989, 6, 903-918. 3. Stability of Protein Pharmaceuticals Part A: Chemical and Physical Pathways of Protein Degradation; Ahem, T. J.; Manning, M. C., Eds.; Pharmaceutical Bio technology; Plenum Press: New York, 1992; Vol. 2. 4. Stability of Protein Pharmaceuticals Part B: In Vivo Pathways of Degradation and Strategies for Protein Stabilization; Ahern, T. J.; Manning, M. C., Eds.; Pharmaceutical Biotechnology; Plenum Press: New York, 1992; Vol. 3. 5. Ota, I. M . ; Clarke, S. Biochemistry 1989, 28, 4020-4027. 6. George-Nascimento, C.; Lowenson, J.; Borissenko, M . ; Calderon, M.; MedinaSelby, Α.; Kuo, J.; Clarke, S.; Randolph, A . Biochemistry 1990, 29, 9584-9591. 7. Marcus, F. Int. J. Peptide Protein Res. 1985, 25, 542-546. 8. Clarke, S. Annu. Rev. Biochem. 1985, 54, 479-506. 9. Tsuda, T.; Uchiyama, M . ; Sato, T.; Yoshino, H . ; Tsuchiya, Y . ; Ishikawa, S.; Ohmae, M . ; Watanabe, S.; Miyake, Y . J. Pharm. Sci. 1990, 79, 223-227. 10. Inglis, A . S. Methods Enzymol. 1983, 91, 324-332. 11. Kirsch, L. E.; Molloy, R. M.; Debono, M . ; Baker, P.; Farid, Κ. Z. Pharm. Res. 1989, 6, 387-393. 12. Johnson, Β. Α.; Shirokawa, J. M.; Hancock, W. S.; Spellman, M . W.; Basa, L . J.; Aswad, D. W. J. Biol. Chem. 1989, 264, 14262-14271. 13. Ondetti, Μ. Α.; Deer, Α.; Sheehan, J. T.; Kocy, O. Biochemistry 1968, 7, 40694075. 14. Perseo, G.; Forino, R.; Galantino, M . ; Gioia, B.; Malatesta, V.; DeCastiglione, R. Int. J. Peptide Protein Res. 1986, 27, 51-60. 15. Schultz, J. Methods Enzym. 1967, 11, 255-263. 16. Oliyai, C.; Borchardt, R. T. Pharm. Res. 1993, 10, 95-102. 17. Schön, I.; Kisfaludy, L. Int. J. Peptide Protein Res. 1979, 14, 485-494. 18. Bodanszky, M.; Kwei, J. Z. Int. J. Peptide Protein Res. 1978, 12, 69-74. 19. Blake, J. Int. J. Peptide Protein Res. 1979, 13, 418-425. 20. Patel, K.; Borchardt, R. T. Pharm. Res. 1990, 7, 703-711.



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