+
`
`+
`
`Effects of Reducing Sugars on the Chemical Stability of Human Relaxin in the
`Lyophilized State
`
`SHIHONG LI*, THOMAS W. PATAPOFF†, DAVID OVERCASHIER†, CHUNG HSU†, TUE H. NGUYEN†, AND
`RONALD T. BORCHARDT*,X
`Received October 23, 1995, from the *Department of Pharmaceutical Chemistry, Simons Research Laboratories, 2095 Constant Avenue,
`The University of Kansas, Lawrence, KS 66047, and †Pharmaceutical R&D, Genentech Inc., South San Francisco, CA 94080 .
`Final
`Accepted for publication May 8, 1996X.
`revised manuscript received April 1, 1996 .
`
`Abstract 0 Sugars and polyols have been used routinely with lyophilized
`proteins and peptides as bulking agents, cryoprotectants, and lyopro-
`tectants. However, reducing sugars may present a problem as excipients
`since they are potentially reactive with proteins.
`In this stability study of
`recombinant human relaxin (Rlx) with various sugars as excipients in
`lyophilized formulations, we observed rapid covalent modifications of the
`protein in the presence of glucose. Analysis of the protein by LC/MS
`and tryptic mapping indicated two major degradation pathways. Covalent
`adducts of glucose with amino groups on the side chains of the protein
`(i.e., Lys and Arg) formed via the Maillard reaction.
`In addition, a
`significant amount of Ser cleavage from the C-terminal of the B-chain of
`relaxin was also identified when glucose was used as the excipient. It
`was observed that the latter reaction occurred to a greater extent in the
`solid state than in solution. We proposed a mechanism for this reaction
`involving an initial reaction of the Ser hydroxyl group with glucose followed
`the Trp- Ser amide bond via a cyclic
`by subsequent hydrolysis of
`intermediate.
`In contrast to glucose, mannitol (polyhydric alcohol) and
`trehalose (nonreducing sugar) produced stable, lyophilized formulations
`of Rlx.
`
`Introduction
`Many pharmaceutical proteins are preferably prepared as
`lyophilized formulations in order to achieve adequate shelf
`stability. Although proteins are generally more stable in
`lyophilized solid forms, degradation and loss of activity may
`still occur during processing and storage. Lyophilized for-
`mulations invariably incorporate excipients such as bulking
`agents, buffers, tonicity modifiers, cryoprotectants, and lyo-
`protectants. The type and amount of excipient can be crucial
`factors in determining the overall stability of proteins and
`peptides in lyophilized formulations.1,2 Sugars and polyols are
`the most common excipients used in lyophilized or solution
`formulations of proteins or peptides.3 As cryoprotectants and
`lyoprotectants, they are effective in stabilizing proteins
`against denaturation and preventing aggregation during
`freezing and lyophilization, respectively.4-7 Previous work on
`lyophilized protein formulations has emphasized the effects
`of the physical state (crystalline vs amorphous) of excipients
`and moisture content on protein stability.2,8-10 For example,
`it has been reported that the cystallinity of the excipient can
`substantially affect the rate of chemical decomposition of an
`Asp-hexapeptide.9,10 On the other hand, excipients may also
`directly interact with proteins and peptides, which can cause
`their destabilization during processing and storage. It is also
`well recognized that reducing sugars may covalently react
`with the amino side chain of amino acid residues via the
`Maillard reaction.11-13
`Recombinant human relaxin (Rlx) is a protein hormone that
`plays a major role in biological responses of reproductive
`
`X Abstract published in Advance ACS Abstracts, July 1, 1996.
`
`BS
`
`Figure 1sRepresentation of the primary structure of human relaxin (Rlx). Arrows
`indicate potential trypsin cleavage sites and resulting peptide fragments.
`
`tissues during pregnancy. The primary structure of Rlx is
`illustrated in Figure 1.
`In the development of Rlx as a
`therapeutic agent, the stability of this protein has been
`evaluated in solution. Two major chemical degradation
`pathways of Rlx were identified: hydrolysis of the N-terminal
`Asp residue and oxidation of Met residues.14,15
`In this report, we studied how reducing and nonreducing
`sugars as excipients affected the chemical stability of Rlx in
`the lyophilized state. To our surprise, we did not observe
`oxidation to be a major pathway of chemical degradation in
`lyophilized formulations of Rlx.
`Instead, we observed the
`formation of glucose-Rlx adducts via the Maillard reaction
`and the parallel hydrolysis of the Trp28Ser29COOH peptide
`bond on the B-chain of Rlx. The latter reaction was totally
`unexpected and, to our knowledge, has not been described
`previously in the literature.
`
`Experimental Section
`MaterialssRlx was produced by Genentech, Inc. (South San
`Francisco, CA). The production, isolation, and purification procedures
`of the protein have been described in the literature.16-18 Sigma
`Chemical Co. (St. Louis, MO) supplied mannitol, glucose, trehalose,
`and L-1-(tosylamido)-2-phenylethyl chloromethyl ketone (TPCK)-
`treated trypsin. Sodium phosphate and sodium chloride were pur-
`chased from Mallinckrodt, Inc. (Paris, KY).
`Lyophilization and Storage of Rlx FormulationssRlx (0.1 mg/
`mL) was prepared in phosphate buffer (20 mM) with sodium chloride
`(100 mM) at pH 5. Mannitol, glucose, and trehalose were chosen as
`excipients, and the concentrations varied from 1% to 2% (w/v) in the
`solutions prior to lyophilization. Aliquots (0.4 mL) containing Rlx
`combined with aqueous solutions of various excipients were dispensed
`into 2-mL autosample vials. These vials were then labeled, loosely
`capped, loaded onto trays, and lyophilized.
`Lyophilization was carried out in a Leybold-Heraeus lyophilizer
`(Model GT20). Two different lyophilization cycles for Rlx samples
`were designed as illustrated in Table 1. The samples were then stored
`at 40 °C for up to 2 weeks and removed at designated intervals for
`HPLC analysis. Lyophilization of Rlx from solutions containing
`mannitol as an excipient formed better cakes than did lyophilization
`from trehalose- or glucose-containing solutions.
`
`© 1996, American Chemical Society and
`American Pharmaceutical Association
`
`S0022-3549(95)00456-4 CCC: $12.00
`
`Journal of Pharmaceutical Sciences / 873
`Vol. 85, No. 8, August 1996
`
`1 of 5
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`+
`
`+
`
`Table 1sLyophilization Cycles for Rlx Samples
`
`No. 1
`
`No. 2
`
`Loading and freezing
`Loading temp
`Freezing temp
`Cooling rate
`Primary drying
`Secondary drying
`
`5 (cid:176) C
`- 55 (cid:176) C
`0.5 deg/min
`Ramp up to - 20 (cid:176) C, 150 mTorr vacuum in 2 h, hold for 34 h
`Ramp up to 20 (cid:176) C, 150 mTorr vacuum in 2 h, hold for 6 h
`
`5 (cid:176) C
`- 55 (cid:176) C
`0.5 deg/min
`Ramp up to - 40 (cid:176) C, 50 mTorr vacuum in 2 h, hold for 40 h
`Ramp up to 20 (cid:176) C, 50 mTorr vacuum in 6 h, hold for 6 h
`
`Table 2sStability of Rlx in the Lyophilized Formulations with Various
`Sugars as Excipientsa
`
`RLX
`
`Excipient
`
`Glucose
`Mannitol
`Trehalose
`
`Remaining Rlx (%)
`
`52
`92
`92
`
`"'
`
`aThe lyophilized solid formulations contained 0.1 mg/mL Rlx, 20 mM phosphate
`buffer, and 100 mM sodium chloride (pH 5) with glucose, mannitol, or trehalose
`as excipient (2% w/v). The lyophilization conditions (cycle no. 1) are described
`in Table 1. The samples were stored at 40 (cid:176) C for 14 days and analyzed by
`RP-HPLC.
`
`the
`Solution Formulation of Rlx with Glucose as
`ExcipientsRlx (0.1 mg/mL) was prepared in phosphate buffer (20
`mM) and sodium chloride (100 mM) with glucose at pH 5. Glucose
`concentrations were 0, 10, and 20% (w/v) in the Rlx solutions. The
`samples were sealed and stored at 40 °C for up to 2 weeks, after which
`they were analyzed by HPLC.
`Analysis of Rlx and Its Degradation ProductssEach lyophi-
`lized sample was reconstituted into 0.8-mL Milli-Q water immediately
`before HPLC analysis. A HP-1090L HPLC system was employed for
`the analysis of Rlx. The system utilized a Vydac RP-C4 column (4.6
`(cid:2) 300 mm) which was equilibrated at 40 °C. Mobile phase A consisted
`of H2O (0.1% TFA); mobile phase B was 9:1 (v/v) ACN/H2O (0.1%
`TFA). Aliquots (20 (cid:237)L) of each sample were injected onto the HPLC.
`A linear gradient started with 20% B and increased to 40% B over 20
`min. The flow rate was 1 mL/min, and detection was achieved at
`214 nm.
`Characterization of the Degradation Products of RlxsLC/
`MSsThe molecular weights of Rlx degradants and tryptic peptides
`were determined by LC/MS. Native Rlx and its degradation products
`were purified by RP-HPLC in order to eliminate or dilute the high
`content of glucose in solutions. The samples were concentrated under
`vacuum in a Speed-Vac (Savant Instruments, Farmingdale, NY). A
`Sciex API III triple quadrupole instrument fitted with an ion spray
`(nebulized-assisted electrospray) was employed to collect mass spec-
`tral data. Molecular masses were calculated and analyzed by
`Hypermass software.
`Tryptic DigestionsDegraded Rlx samples were purified by RP-
`HPLC and concentrated under vacuum in a Savant Speed-Vac. The
`samples were then digested with TPCK-treated trypsin in the
`presence of 0.2 mM calcium chloride at 30 °C for 4 h in 10 mM Tris-
`acetate buffer, pH 7.0. For tryptic mapping by RP-HPLC, the initial
`equilibration mobile phase was 0.1% TFA in 3% ACN. A linear
`elution gradient was generated from 3-30% ACN over 1 h.
`
`Results
`Effects of Various Sugars on the Chemical Stability
`of Rlx during Storage at 40 °CsRlx formulated with
`mannitol and trehalose as excipients in the lyophilized state
`was fairly stable when stored at 40 °C (>90% of Rlx remained
`after 14 days of storage, Table 2). In contrast, when glucose
`was present as the excipient, a significant amount of degrada-
`tion of Rlx was observed under the same storage conditions
`(Table 2, Figure 2). This degradation appeared to be the
`result of storage at 40 °C rather than lyophilization, since
`formulations analyzed by RP-HPLC immediately after lyo-
`philization showed no significant degradation of Rlx (retention
`time ) 17.1 min). In glucose-containing samples stored at
`
`874 / Journal of Pharmaceutical Sciences
`Vol. 85, No. 8, August 1996
`
`X
`
`y
`
`15
`
`18
`
`Tlme(min)
`Figure 2sRP-HPLC chromatogram of Rlx with glucose as excipient
`in the
`lyophilized formulation. The formulation was lyophilized from the solution containing
`0.1 mg/mL Rlx, 2% (w/v) glucose, 20 mM phosphate buffer, and 100 mM sodium
`chloride at an initial pH of 5. The lyophilized sample was stored at 40 (cid:176) C for 2
`weeks.
`
`40 °C, two early-eluting peaks were observed: an unknown
`major peak (X) at 16.9 min and a minor peak (Y) at 16.7 min,
`which corresponds to the retention time of des-Asp Rlx.
`Characterization of the Degradation Products of Rlx
`with Glucose as ExcipientsThe degradation products of
`Rlx derived from the glucose formulation were analyzed by
`LC/MS. Results from LC/MS (Figure 3) indicated that the
`early-eluting peak X had a mass of 5876, which was 86 units
`(SD (cid:25) 1 unit) less than that of Rlx (5962), corresponding to
`the loss of a Ser residue. With careful analysis, a number of
`late-eluting peaks of Rlx were also identified by LC/MS. These
`degradation products were not well separated from Rlx on RP-
`HPLC (Figure 2). They were characterized as covalent
`adducts of Rlx with glucose on the basis of their mass values
`(Figure 4). It was shown by LC/MS that up to four glucose
`molecules were covalently attached to the protein. In order
`to identify the specific hydrolytic site and the locations of the
`amino acid residues involved in adduct formation, tryptic
`mapping of the degradants was conducted.
`Tryptic Digestion and LC/MS AnalysissLC/MS analy-
`sis of the tryptic peptides of degraded Rlx is shown in Figure
`5. Hydrolytic cleavage of the C-terminal Ser residue from the
`B-chain was confirmed by the mass reduction (1533 - 1446
`) 87) on fragment T5-T9 (Table 3). Lys and Arg residues
`are the potential sites for the Maillard reaction.11-13 Three
`adducts were identified on fragments T1 (containing one Lys),
`T8 (containing one Arg), and T6 (containing one Lys), respec-
`tively. The only fragment that could not be confirmed by mass
`spectrometry was T2-T7. However, it was observed that the
`T2-T7 fragment was a broadened peak on the tryptic map,
`indicating the possible involvement of glucose adduct forma-
`
`2 of 5
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`+
`
`+
`
`R1x
`5962
`
`des-Ser R1x
`
`5876
`
`100
`
`75
`
`25
`
`8000
`Mol1cula1 Wolghl
`Figure 3sLC/MS analysis of Rlx degradants (early-eluting peak X) derived from samples of Rlx lyophilized with glucose as excipient. The degradation product was
`produced during storage of the lyophilized protein at 40 (cid:176) C. The lyophilized formulation consisted of 0.1 mg/mL Rlx, 2% (w/v) glucose, 20 mM phosphate buffer, and
`100 mM sodium chloride at an initial pH of 5.
`
`6100
`
`6200
`
`5700
`
`6800
`
`5900
`
`100
`
`75
`
`i
`-f
`! .
`i a:
`
`50
`
`25
`
`Rix
`5982
`
`Rlx+Glucose-H20
`
`6125
`
`Rlx+301ucose-3H20
`
`6◄ 50
`
`Rlx+2Glucosc-2H20
`6207
`
`Rlx+40lucosc-41120
`
`6812
`
`Mot1c:uJ11 Waight
`Figure 4sLC/MS analysis of Rlx degradants (later-eluting peaks) derived from samples of Rlx lyophilized with glucose as the excipient: covalent adduct formation.
`The lyophilized formulations and the storage conditions are the same as those described in Figure 3.
`tion. T2-T7 contains one lysine and one arginine, both of
`procedure. The lyophilized samples from the second procedure
`(which had a lower freezing temperature) resulted in less
`which are potential reaction sites.
`Des-Ser Formation under Various ConditionssAs
`extensive degradation of Rlx during storage. This could be
`shown in Figure 6, the concentration of glucose (1-2% w/v)
`related to the residual moisture level originating from differ-
`ent freezing temperatures during lyophilization. However, we
`in the lyophilized formulations did not seem to affect the
`were not able to determine the moisture content because of
`amount of des-Ser Rlx formation. However, the extent of des-
`the small amounts of sample in each vial. Similar degradation
`Ser Rlx formation was likely dependent on the lyophilization
`
`Journal of Pharmaceutical Sciences / 875
`Vol. 85, No. 8, August 1996
`
`3 of 5
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`+
`
`+
`
`100
`
`75
`
`-f
`i
`.i
`~
`a:
`
`50
`
`25
`
`~
`
`j:;
`
`0
`
`f
`
`~
`
`f-,
`
`0
`
`~#
`§
`~
`
`0
`:fl
`ft
`" i
`
`~ tr
`~
`~
`
`o+-----..------.-----~--~~-.--------.---------.-------
`1 o.o
`
`201
`10.3
`
`◄OI
`20.6
`
`601
`30.8
`Sa1wllm1 (min)
`Figure 5sTryptic map of the degraded Rlx with glucose as excipient in the lyophilized state. The masses of the individual tryptic peptides were analyzed by LC/MS
`and are presented in Table 3.
`
`801
`◄ I.I
`
`1001
`51,5
`
`1201
`62.1
`
`Table 3sLC/MS Analysis of Tryptic Peptides Derived from Chemically
`Degraded Rlx with Glucose as the Excipient in the Lyophilized Solid
`Forma
`
`des-Ser Formation (%)
`0
`
`\;J
`0
`
`0
`
`Mass
`
`Theor
`
`1293.6
`1455.8
`990.5
`1152.7
`516.3
`678.5
`1136.5
`1298.7
`1533.7
`1446.7
`
`Obsd
`
`1293
`
`990
`1152
`516
`677
`1136
`1297
`1533
`1446
`
`Assignment
`T2- T7
`T2- T7 + glucose - H2O
`T1
`T1 + glucose - H2O
`T8
`T8 + glucose - H2O
`T6
`T6 + glucose - H2O
`T5- T9
`T5- T9 - Ser
`a The Rlx formulation was lyophilized from a solution containing 0.1 mg/mL
`Rlx, 2% glucose, 20 mM phosphate buffer, and 100 mM sodium chloride at pH
`5. The sample was incubated at 40 (cid:176) C for 14 days followed by tryptic digestion
`and LC/MS analysis.
`
`Potential Reactn Sites
`
`Lys, Arg
`
`Lys
`
`Arg
`
`Lys
`
`Ser
`
`products of Rlx were also observed when the protein was
`stored in concentrated glucose solutions, although the reaction
`occurred to a much lesser extent than in the solid form (Figure
`6).
`
`Discussion
`It has been reported that both the physical state and the
`chemical nature of the excipient could affect the overall
`stability of proteins in lyophilized formulations.2,8-10 There-
`fore, different types of sugars were selected as excipients in
`the stability study of the lyophilized formulations of Rlx.
`Glucose was chosen as a model reducing sugar, mannitol as
`a model polyhydric alcohol, and trehalose as a model nonre-
`ducing sugar.
`It was observed that only glucose induced
`significant chemical degradation of Rlx during storage at
`elevated temperature.
`In contrast, the protein was much
`
`876 / Journal of Pharmaceutical Sciences
`Vol. 85, No. 8, August 1996
`
`0%insol.
`
`10% in sol.
`
`20% in sol.
`
`1 % in Iyo. (#1 cycle)
`
`2% in Iyo. (#1 cycle)
`
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`Glucose Content (%,w/v)
`Figure 6sEffects of glucose on des-Ser formation in solution and the solid state.
`Des-Ser Rlx formed during storage with glucose as excipient in solution and the
`lyophilized solid state at 40 (cid:176) C for 2 weeks. Solution and lyophilized formulations
`contained 0.1 mg/mL Rlx, 20 mM phosphate buffer, and 100 mM sodium chloride
`(pH 5) with various amounts of glucose. Glucose concentration was 0, 10, or
`20% (w/v) in solution and 1 or 2% in the lyophilized solid state.
`
`more stable when mannitol or trehalose was used as excipient.
`It is likely that the different stabilities of Rlx are due to the
`distinct chemical nature of these excipients (reducing vs
`nonreducing sugars). Covalent adduct formation of glucose
`with the protein was identified as one of the degradation
`pathways. This modification was not unexpected as the
`nonenzymatic Maillard reaction has been well documented
`in the literature.11-13 The reaction involves the condensation
`of reducing sugars with amino groups of amino acids (Lys and
`Arg) in proteins to form glycosylamino acids, which may
`undergo subsequent rearrangement. With tryptic digestion
`of the protein, we have identified individual reaction sites on
`Rlx, including Lys-A(9), Lys-B(9), and Arg-B(17). The ad-
`
`4 of 5
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`+
`
`+
`
`OH
`I
`
`f"•
`(a) R-~-NH-CH-COO- +
`0
`
`O
`II
`R'- C-H -
`
`(
`
`R'CH
`'o
`I
`OH
`I
`CH,
`H,O
`I
`I
`I
`Rif•NH-CH-COO- --L--- R-C=N-CH-Coo·
`0
`
`R'CH
`/ 'o
`tu,
`O\
`
`the C-terminal Ser residue. It will be interesting to examine
`whether other C-terminal residues (e.g., Thr) can be cleaved
`in a similar manner by reducing sugars. The detailed
`mechanisms are currently under investigation.
`This study highlighted the significance of selecting ap-
`propriate excipients to optimize protein stability during
`storage. It is evident that nonreducing sugars are preferred
`to reducing sugars as excipients in protein formulations due
`to the propensity of reducing sugars to participate in various
`protein modifications shown by this study.
`
`References and Notes
`1. Hora, M. S.; Rana, R. K.; Wilcox, C. L.; Katre, N. V.; Hirtzer,
`P.; Wolfe, S. N.; Thomson, J. W. Dev. Biol. Stand. 1991, 74, 295-
`306.
`2. Pikal, M. J.; Dellerman, K. M.; Roy, M. J.; Riggin, R. M. Pharm.
`Res. 1991, 8, 427-436.
`3. Wang, Y. J.; Hanson, M. A. J. Parenter. Sci. Technol. 1988, 10,
`3-26.
`4. Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Aurell-Wistrom,
`C. Biochem. J. 1987, 242, 1-10.
`5. Carpenter, J. F.; Crowe, J. H. Cryobiology 1988, 25, 244-255.
`6. Gray, C. J. Biocatalysis 1988, 1, 187-196.
`7. Townsend, M. W.; DeLuca, P. P. J. Parenter. Sci. Technol. 1988,
`42, 190-199.
`8. Pikal, M. J.; Dellerman, K.; Roy, M. L. Dev. Biol. Stand. 1991,
`74, 21-38.
`9. Oliyai, C; Borchardt, R. T. In Formulation and Delivery of
`Proteins and Peptides; Cleland, J. L., Langer, R., Eds.; American
`Chemical Society: Washington, DC, 1994; pp 46-58.
`10. Oliyai, C.; Patel, J.; Carr, L.; Borchardt, R. T. Pharm. Res. 1994,
`11, 901-908.
`11. Eichner, K. In Water Relations of Foods; Duckworth, R. B., Ed.;
`Academic Press: New York, 1975; pp 417-434.
`12. Carpenter, J. F.; Crowe, L. M.; Crowe, J. H. Biochim. Biophys.
`Acta 1987, 923, 109-115.
`13. Finot, P. A.; Aeschbacher, H. U.; Hurrell, R. F.; Liardon, R. The
`Maillard Reaction in Food Processing, Human Nutrition and
`Physiology; Birkhauser: Basel, 1990.
`14. Cipolla, D. C.; Shire, S. J. In Techniques in Protein Chemistry
`II; Villafranca, J. J., Ed.; Academic Press, Inc.: New York, 1990;
`pp 543-555.
`15. Canova-Davis, E.; Baldonado, I. P.; Teshima, G. M. J. Chro-
`matogr. 1990, 508, 81-96.
`16. Stults, J. T.; Bourell, J. H.; Canova-Davis, E.; Ling, V. T.;
`Laramee, G. R.; Winslow, J. W.; Griffin, P. R.; Rinderknecht,
`E.; Vandlen, R. L. Biomed. Environ. Mass Spectrom. 1990, 19,
`655-664.
`17. Canova-Davis, E.; Kessler, T. J.; Lee, P. J.; Fei, D. T.; Griffin,
`P.; Stults, J. T.; Wade, J. D.; Rinderknecht, E. Biochemistry
`1991, 30, 6006-6013.
`18. Shire, S. J.; Holladay, L.; Rinderknecht, E. Biochemistry 1991,
`30, 7703-7711.
`19. Nursten, H. E. In Maillard Browning Reactions in Dried Foods
`in Concentration and Drying of Foodstuffs; Macarthy, D., Ed.;
`Elsevier Applied Science: London, 1986; pp 53-87.
`20. Ledl, F.; Schleicher, E. Angew. Chem., Int. Ed. Engl. 1990, 29,
`565-594.
`21. Nguyen, T. H.; Burnier, J.; Meng, W. Pharm. Res. 1993, 10,
`1563-1571.
`22. Li, S.; Nguyen, T. H.; Scho¨neich, C.; Borchardt, R. T. Biochem-
`istry 1995, 34, 5762-5772.
`23. Harris, J. I.; Cole, R. D.; Pon, N. G. Biochem. J. 1956, 62, 154-
`159.
`24. Strickley, R. G.; Brandl, M.; Chan, K. W.; Straub, K.; Gu, L.
`Pharm. Res. 1990, 7, 530-536.
`
`Acknowledgments
`S.L. was supported by fellowships from the PMA Foundation and
`the Syntex Corporation. Research support was provided by Genen-
`tech, Inc. The authors would like to acknowledge Mr. Long Truong
`(Analytical Chemistry, Genentech, Inc.) for LC/MS analysis and Dr.
`Richard Schowen (Department of Pharmaceutical Chemistry, The
`University of Kansas) for his helpful suggestions and discussions.
`JS950456S
`
`Journal of Pharmaceutical Sciences / 877
`Vol. 85, No. 8, August 1996
`
`R'CH-0
`I
`I
`OHfH,
`R-COO- + NH2-CH-COO-
`
`OH
`I
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`- - R-f=t( J:
`fH co,-
`
`OH
`
`R'CH-0
`I
`I
`OHfH,
`R-COO- + NH2-CH-COO-
`
`Scheme 1
`
`ditional reaction site is probably Lys-A(17) or Arg-B(13) on
`the T2-T7 fragment. Maillard reactions have been recognized
`as one important factor in determining long-term storage
`stability of lyophilized protein formulations containing sugars
`as excipients. This type of modification is a particular
`problem in the solid state as the initial aminocarbonyl
`condensation reaction toward the formation of the Schiff base
`is accelerated at low water activity.19,20
`One unexpected finding was the facilitated hydrolysis,
`induced by glucose, of the Trp28Ser29COOH peptide bond on
`the B-chain of Rlx. The resulting degradation product des-
`Ser Rlx was not observed when mannitol or trehalose was
`used as the excipient. Earlier work on Rlx indicated two types
`of chemical instabilities of the protein in solution: hydrolytic
`cleavage of the N-terminal Asp on the B-chain and the
`oxidation of Met residues.21,22 The lability of -XSerY-
`peptide bonds has been described previously in the litera-
`ture.23,24 Among five Ser residues in Rlx (Figure 1), the
`hydrolysis of the Trp28-Ser29 peptide bond is pronounced,
`possibly because of its higher flexibility on the C-terminal
`region of the protein. It was shown that the Trp28-Ser29
`peptide bond hydrolysis was much more significant in the solid
`state than in solution (Figure 6). This result seems to suggest
`that a high content of glucose is necessary for the reaction to
`occur. In the solid state, the effective concentration of glucose
`was high enough to initiate the reaction; therefore, there was
`not an obvious difference in the amount of des-Ser Rlx
`formation when glucose concentration varied from 1 to 2%.
`Moisture level could be varied depending on the lyophilization
`conditions (e.g., freezing temperature). It is one major vari-
`able that contributes to the amount of des-Ser Rlx formation
`in the storage of lyophilized protein.
`We propose that glucose might induce des-Ser formation
`through one of the intramolecular reactions or other similar
`processes shown in Scheme 1. Glucose could initially react
`with the Ser side chain and consequently form a cyclic
`intermediate (Scheme 1a), or alternatively, the Ser side chain
`could stabilize transition states by hydrogen bonding in a
`direct reaction with glucose at the amide functional group
`(Scheme 1b). It is known that glucose exists predominantly
`in a cyclic structure with a small amount of open-chain form.
`Although the open-chain compound is the reactive form, it can
`be replenished fairly rapidly. It cannot be concluded at this
`point that the modification of protein by glucose is specific to
`
`5 of 5
`
`Fresenius Kabi
`Exhibit 1037
`
`