`
`CSL EXHIBIT 1059
`
`Page 1 ofll
`
`CSL V. Shire
`
`Page 1 of 11
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`CSL EXHIBIT 1059
`CSL v. Shire
`
`

`

`PHARMACEUTICAL RESEARCH
`An Official Journal of the American Association of Pharmaceutical Scientists
`Pharmaceutical Rese,irch publishes inn ovative basic resea rch and techno logica l ad vances in the pharmaceutica l-bio med ical sciences. R esearch areas covered ·
`jo urn al include: pha rm aceutics and d rug de livery. pharmacok inetics and pharmacodynamics, d rug metabolism, pharmacology and toxicology, medicinal che
`natural products chemist ry, analyt ical chemistry. che mical kineti cs and drug stabi lit y. bio technology, pharmaceutica l technology. a nd cli nical investigations. •
`as a rt icles o n the social. econom ic. o r managemen t aspects of th e pharm aceutica l sciences.
`
`EDITOR-IN-CHIEF
`Vincent H. L. Lee, Depa rtme nt of Pharmaceuti cal Sciences. Universit y o f
`South ern Ca lifo rnia, Los Ange les. Califo rni a
`
`EDITOR-E UROP E
`Daan Crommelin, Utrecht U nive rsit y. Utrecht. The Netherl ands
`
`EDITOR-JAPA N
`Mitsuru Hashida, Kyoto Uni ve rsit y. Kyoto. Japan
`
`A SSOCIATE EDITORS
`Kinam Park, Purdue Unive rsity, West Lafayette. Indi ana
`David E. Smith, Uni versit y of Mi chigan, A nn Arbor, Michiga n
`
`ASSOCIAT E EDITORS- EU ROP E
`Meindert Danhof, Le iden Uni versit y. Leiden. The
`Josef J. Tukker, U trecht Universit y. U trecht. T he
`
`e th e rla nds
`eth erla nds
`
`EDITORIAL ADVISORY BOARD
`Maria Jose Alonso, University of Santiago de Compostela. Campus Sur, Spain
`Gordon L. Amidon, Universit y of Michigan. A nn Arbo r. Mi chigan
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`Per A rtursson, Uppsala University. Uppsala. Sweden
`Jessie L.-S. Au, Ohi o State U ni ve rsity, Columbus. Ohio
`You-Han Bae, Kwang-Ju Institute of Science & Techn ology. Kwang-Ju,
`South Ko rea
`Peter Bonate, ILEX Oncology. Sa n Anto nio , Texas
`Ronald T. Borchardt, Unive rsity o f Ka nsas, Lawre nce. Ka nsas
`Joke Bouwstra, Leiden- Amsterda m Cente r fo r Drug Research. The
`Ne the rlands
`Harry Brittain, Ce nte r for Pharmaceutica l Ph ysics. Milford.
`ew Jersey
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`North Carolina
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`ew Jersey
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`Gary Fujii, Mo lecular Express, Inc .. Los Angeles, Ca liforn ia
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`ew Yo rk
`Sven Frokj:.er, Ro ya l Danish School of Ph armacy. Copenh agen. De nmark
`Igor Gonda, Acruz. Melbourne , A ustralia
`Margareta Hammarlund-Udenaes, University of Uppsala, Uppsala. Sweden
`Ian Haworth, U ni versit y of So uthern Ca lifo rnia. Los A ngeles. Californi a
`Masahiro Hayashi, To kyo Uni ve rsity of Pharmacy & Life Sciences, Tokyo. Japan
`Wim E. He nnink, Utrecht lnsitute fo r Pha rm aceutica l Scie nces. Ut recht. T he
`Netherlands
`Susan Hershenson, A mge n Inc .. Tho usand Oaks. Ca liforn ia
`A nthony J. Hickey, U niversity of No rth Caroli na at C hapel Hi ll. C hapel Hill .
`North Carolina
`Sung-Joo Hwang, Chungnam Na tio nal Uni versit y, So uth Ko rea
`Tatsuji lga, U ni ve rsit y o f T okyo Hosp it al. Tokyo , Japa n
`Lisbeth lllum, West Ph armaceutica l Se rvice. No ttingham. Uni ted Kingdo m
`
`Kenichi lnui, Kyoto Universi ty Hospita l. Kyoto. Japan
`Myron K. Jacobson, Uni versity o f A rizona, T ucson. A rizona
`Rudy L. Juliano, U nive rsity of North Caroli na at Chapel Hill, Chapel
`No rth Carolin a
`Tetsuya Kamataki, Ho kkaido University, Japa n
`Mats 0 . Karlsson, Uppsala Uni ve rsity, Uppsala. Sweden
`Kwang-Jin Kim, Uni ve rsity of Southern Ca lifo rnia. Los A ngeles, Cali!
`Sung Wan Kim, Uni versity of Utah. Salt Lake City. U tah
`Toshikiro Kimura, Ok ayama Unive rsity. O kayama, Japan
`A.-N. Tony Kong, Rutge rs- The State Unive rsity o f New Jersey, Pisca
`New Jersey
`Jindrich Kopecek, Universit y of U tah. Salt La ke City. Utah
`Deana Kroetz, U niversity of Ca li fo rnia. Sa n Francisco. California
`Peter R. Langguth, Jo ha nnes G ute nberg- Unive rsi ty. Mainz. Germany
`Chi-Ho Lee, Pusan Natio nal Unive rsity, Pusan. So uth Korea
`Kang Choon Lee, Sung Kyunk wan University. Suwon City, South Korea
`Seung Jin Lee, Ehwa Wo mens U niversity. Seoul, So uth Korea
`Claus-Michael Lehr, Uni versity of Saa rland , Saarbruecken, Germany
`Thomas M. Ludden, G lo bo Max LLC. Hanover. Maryland
`Kristina Luthman, Goteborg Unive rsity. Swede n
`Panos Macheras, University of A the ns, A thens. G reece
`Randall J. Mrsny, Cardi ff Un iversit y, Card iff. United Kingdom
`Tadanori Mayumi, Osaka Unive rsi ty. Osaka. Japan
`Jerry R. Nedelman, Nova rtis Ph armaceuticals, East Hanover, New Je
`Derek T. O'Hagen, C hiron Corpora tio n. Eme ryville. Califo rnia
`Teruo Okano, Tokyo Wome n's Med ical College. Tokyo. Japan
`Michael J. Pikal, Uni ve rsit y of Connecticut. Sto rrs. Connecticut
`Mark Prausnitz, G eo rgia Instit ute of Technology. G eorgia
`Mary Reiling, St. Jude C hi ld re n's Research Hospita l. Me mphis, Tenn
`A braham Rubinstein, T he Hebrew Uni ve rsi ty o f Jerusalem, Jerusalem.
`Wolfgang Sadee, Ohio State U ni versity, Colum bus. O hio
`W. Mark Saltzman, Co rn ell Uni ve rsit y, Ith aca , New Yo rk
`Wei-Chiang Shen, Unive rsity of Southern Cali forn ia. Los Angeles,
`Steven J. Shire, Ge ne ntech. Inc .. S. Sa n Francisco. Ca lifo rnia
`Ke nneth B. Sloan, Uni versit y of Flo rida. Gainesville. Florida
`David E. Smith, Uni versit y of Michigan, An n Arbor. Michigan
`Valentino J. Stella, Uni versit y of Kansas. Lawre nce . Kansas
`Yuichi Sugiyama, Uni versit y of To kyo. Tokyo, Japan
`Scan Sullivan, Uni ve rsity o f Flo rida . Ga in esvi lle , Fl orida
`Yoshinobu Takakura, Kyoto Uni ve rsit y. Kyoto. Japan
`Kozo Takayama, Hoshi Uni ve rsity. To kyo . Japan
`Tetsuya Terasaki, Toho ku Unive rsity. Se nda i. Japan
`Bernard Testa, Un iversity of Lausanne. Lausanne. Switzerland
`Kenneth Thummel, Universi ty of Washingto n, Seattle , Washington
`A kira Tsuji, Kanazawa U ni versit y. Kanazawa. Japan
`A rio U rtti, Unive rsity of Kuo pi o, Kuo pio. Finl and
`Keiji Yamamato, C hi ba Uni versity. C hi ba. Japan
`
`BOOK REVIEW EDITOR
`Kinam Park, Purdue U ni ve rsity. School of Pharmacy, West Lafayene,
`47097
`
`EDITORI A L ASS ISTANTS
`Ruth Ellis-Ballard
`Elizabeth B. Gongora
`Tomomi Uchiyama
`
`PH A RMACEUTI CA L RESEA RCH ( ISSN: 0724-874 1) is published mo nthl y by Klu wer Academic/Plenum Publishers. P.O . Box 322. 331Xl AH Do rdrecht . The Netherland,_
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`Page 2 of 11
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`

`

`PHARMACEUTICAL RESEARCH
`Official Journal of the American Association of Pharmaceutical Scientists
`
`February 2002
`
`CONTENTS
`
`ce of the Structure of Drug Moieties on the in Vitro Efficacy of HPMA Copolymer-Geldanamycin
`Derivative Conjugates
`Yuji Kasuya, Zheng-Rong Lu, Pavla Kopeckova, S. Esmail Tabibi, and Jindfich Kopecek
`
`Determination of Binding of Cisplatin to DNA in the Presence of Biological Thiols: Implications
`of Dominant Platinum-Thiol Binding to Its Anticancer Action
`Erika Volckova, Lea P. Dudones, and Rathindra N. Bose
`
`Dependency of DL-Lactide/Glycolide Copolymer Particulates for Intra-Articular Delivery System
`on Phagocytosis in Rat Synovium
`Eijiro Horisawa, Katsuaki Kubota, Izumi Tuboi, Keiichi Sato, Hiromitsu Yamamoto,
`Hirofumi Takeuchi, and Yoshiaki Kawashima
`
`llular Processing of Poly(Ethylene Imine)/Ribozyme Complexes Can Be Observed m Living
`Cells Using Confocal Laser Scanning Microscopy and Inhibitor Experiments
`Thomas Merdan, Klaus Kunath, Dagmar Fischer, Jindrich Kopecek, and Thomas Kissel
`
`parative Inhibitory Effects of Different Compounds on Rat Oatpl (Slc2/ al)- and Oatp2
`(Slc2/a5)-Mediated Transport
`Yoshihisa Shitara, Daisuke Sugiyama, Hiroyuki Kusuhara, Yukio Kato, Takaaki Abe,
`Peter J. Meier, Tomoo ltoh, and Yuichi Sugiyama
`
`n Gradient-Dependent Transport of Valproic Acid in Human Placental Brush-Border Membrane
`Vesicles
`Hiroaki Nakamura, Fumihiko Ushigome, Noriko Koyabu, Shoji Satoh, Kiyomi Tsukimori,
`Hitoo Nakano, Hisakazu Ohtani, and Yasufumi Sawada
`
`eability Profiles of M-Alkoxysubstituted Pyrrolidinoethylesters of Phenylcarbamic Acid across
`Caco-2 Monolayers and Human Skin
`Lenka Gyiirdsiova, L eena Laitinen, Johanna Raiman, Jo zef Ciimarik, Eva Sedlarova, and
`Jouni Hirvonen
`
`Potential of Chitosan in Enhancing Peptide and Protein Absorption across the TR146 Cell
`Culture Model-An in Vitro Model of the Buccal Epithelium
`Ana Portero, Carmen Remufian-L6pez, and Hanne M¢rck Nielsen
`
`'de Acylation by Poly(a-Hydroxy Esters)
`Andrea Lucke, Josef Kiermaier, and Achim Gopferich
`
`115
`
`124
`
`132
`
`140
`
`147
`
`154
`
`162
`
`169
`
`175
`
`Page 3 of 11
`
`

`

`CONTENTS (Continued)
`
`Experimental and Computational Screening Models for Prediction of Aqueous Drug Solubility
`Christel A. S. Bergstrom, Ulf Norinder, Kristina Luthman, and Per Artursson
`
`Pharmaceutical Engineering
`
`Production and Characterization of a budesonide Nanosuspension for Pulmonary Administration
`Claudia Jacobs and Rainer Helmut Muller
`
`Thermophysical Properties of Pharmaceutically Compatible Buffers at Sub-Zero Temperatures: Implications
`for Freeze-Drying
`Evgenyi Y. Shalaev, Tiffany D. Johnson-Elton, Liuquan Chang, and Michael J. Pikal
`
`Pharmacokinetics/Pharmacodynamics
`
`Effect of Testosterone Suppression on the Pharmacokinetics of a Potent GnRH Receptor Antagonist
`Eugenia A. latsimirskaia, Margaret L. Gregory, Kenna L. Anderes, Rosemary Castillo,
`K. Eric Milgram, David R. Luthin, Ved P. Pathak, Lance C. Christie, Haresh Vazir,
`Mark B. Anderson, and John M. May
`
`Ignoring Pharmacokinetics May Lead to Isoboles Misinterpretation: Illustration with the Norfloxacin(cid:173)
`Theophylline Convulsant Interaction in Rats
`Miren Cadart, Sandrine Marchand, Claudine Pariat, Serge Bouquet, and William Couet
`
`AAPS ELECTRONIC SCIENTIST
`Debbie Werfel
`
`Page 4 of 11
`
`

`

`Pharmacewica/ Research, Vol. 19, No. 2, February 2002 (© 2002)
`
`Research Paper
`
`Thermophysical Properties of
`Pharmaceutically Compatible Buffers
`at Sub-Zero Temperatures:
`Implications for Freeze-Drying
`
`4
`Evgenyi Y. Shalaev1
`, Tiffany D. Johnson-Elton1
`•
`Liuquan Chang3, and Michael J. Pikal3
`
`2
`•
`
`,
`
`Received October 10, 2001; accepted November I, 2001
`
`Purpose. To evaluate crysta llization behavior and collapse tempera(cid:173)
`ture (Tg') of buffers in the froze n sta te, in view of its importance in
`the deve lopment of lyophilized fo rmul at ions.
`Methods. Sodium ta rtrate, sodi um ma late, potassium citrate, and so(cid:173)
`dium citrate buffers we re prepared with a pH range within the ir
`individual buffering capacities. Crysta llization a nd th e Tg' were de(cid:173)
`tected duri ng heating of the frozen solutions using standard DSC and
`modulated DSC.
`Rtsults. Citrate and mala te did not ex hibit crystallizatio n, while suc(cid:173)
`cinate and tartrate crystallized during heating of th e frozen solutions.
`The citrate buffer had a highe r Tg' than malate and tartrate buffers at
`the same pH. Tg' vs. pH graphs fo r citrate a nd malate buffers studied
`had a similar shape, with a maximum in T g' at pH ra ngi ng from 3 to
`4. The Tg' maximum was explained as a result of a compe tition
`between two opposi ng trends: an increase in the viscosity of th e amor(cid:173)
`phous phase beca use of an increase in e lectrostatic interacti on, and a
`decrease in the Tg' beca use of an increase in a water conce ntration of
`the freeze-concentrated solution.
`Conclusion. Citrate buffer was identified as th e pre fe rred buffer for
`lyophilized pharmace uti cals because of its higher Tg' and a lower
`crystallization tendency.
`
`IEY WORDS: lyop hili za ti on; freezin g; buffers; collapse; glass tra n(cid:173)
`sition; DSC.
`
`INTRODUCTION
`
`Many pharmaceuticals contain a buffer to control pH, to
`ensure optimal chemical and physical stability of a drug mol(cid:173)
`ecule. Buffering capacity and a possibility of a buffer-specific
`catalysis are the major buffer properties which are usually
`taken into consideration in development of liquid pharma(cid:173)
`ceutical formulations (1). For lyophilized formulations , there
`are two additional physical chemical parameters to consider,
`Le., buffer crystallization potential at sub-ambient tempera(cid:173)
`tures, and the collapse temperature. A buffer component may
`aystallize during freezing producing significant pH changes
`(2) that are usually undesirable and should be avoided. Crys(cid:173)
`tallization and pH changes of phosph ate buffer at sub-zero
`temperatures were studied in detail in the presence of differ(cid:173)
`ent metal ions and in a wide range of pH and concentration
`3-7). Systematic studies of equilibrium freezing behavior of
`
`Groton Laboratories, Pfizer Inc., Groton, Connecticut 06340.
`School of Pharmacy, U ni ve rsi ty of Minnesota, Minnesota.
`School of Pharmacy, U niversity of Connecticut, Storrs, Connecticut
`1Wi269.
`To whom correspondence shou ld b e add ressed. (e- m a il :
`evgenyi_y _shalaev@groto n. pfizer .com)
`
`phosphate buffer were performed by van den Berg et al. in
`1950-1960. In these studies, liquid (unfrozen) portions of a
`frozen solution was physically separated at sub-zero tempera(cid:173)
`tures, and the pH and composition of the liquid portion were
`measured at room temperature (2-4). Later, other methods
`were used such as measurements of pH at sub-zero tempera(cid:173)
`tures with a low-temperature electrode (5,6,8,9) and pH in(cid:173)
`dicators (10), X-ray diffraction measurements at sub-ambient
`temperatures (7), and DSC studies (11-13) . Significant pH
`changes were observed depending on a metal ion type and
`experiment setup (sample size, cooling rate). Based on these
`results, phosphate buffer is genera lly regarded to be undesir(cid:173)
`able for lyophilized formulations, at least if high buffer con(cid:173)
`centrations are required to maintain high buffer capacity (14).
`There are some studies of other buffers of pharmaceutical
`interest (citrate, glycine, succinate, carbonate) in very narrow
`ranges of solution pH and concentration (9,10,15).
`Another physical chemical parameter critical for lyophi(cid:173)
`lization is the collapse temperature (16). Freeze drying above
`the collapse temperature produces loss of the cake-like struc(cid:173)
`ture that one desires. Obviously, materials with a higher col(cid:173)
`lapse temperature can be freeze-dried at a higher tempera(cid:173)
`ture, hence providing a faster and more robust lyophilization
`cycle. If the collapse temperature of a fo rmulation is relatively
`low, it is more difficult and sometimes impossible to lyophilize
`such a formulation in a practical process. As a rule, presence
`of amorphous buffer in a formulation decreases the coll apse
`temperature resulting in recommendations to minimize buffer
`concentration in lyophilized formulations (17). The collapse
`temperature can be measured by different techniques, with
`freeze drying microscopy and DSC being the methods of
`choice in most cases. With DSC, a thermal transition denoted
`Tg' is measured as the temperature of an endothermic step
`which precedes the me lting endo th erm on DSC heating
`curves of frozen so lutions (16). It should be stressed that
`interpretation of the physical nature of the Tg' thermal event
`is still controversial. There are two alternative interpretation
`of the Tg'. The Tg' therm al event has been explained as either
`a glass transition of th e freeze-concentrated solution (18,19),
`or onset of ice melting marked as Ts (softening tempera ture)
`(20), or Tm (21). D espite of this controversy, there is a com(cid:173)
`mon agreement that the Tg' corresponds closely to the col(cid:173)
`lapse temperature, the collapse temperature normally being
`higher by 1-3°C (16). Frequently, one detects a second very
`weak apparent glass transition roughly 20°C lower than Tg'.
`This lower transition is denoted Tg", and does not appear to
`be related to the collapse phenomena. The Tg' of several
`acids and bases (ascorbic acid , citric acid , glycine, HEPES,
`TRIS) and buffers (citrate, TRIS, acetate, glycine, and histi(cid:173)
`dine) has been determined in (12,15,22-25). In majority of
`these studies, the Tg' was determined at a single pH value
`(with exceptions (25) for histidine and (15) for glycine). There
`is a lack of systematic data on collapse temperatures and
`crystallization behavior as a functi on of pH for buffers of
`pharmaceutical significance.
`In the present study, crystallization behavior and col(cid:173)
`lapse temperature of several buffers (citrate, succinate, ma(cid:173)
`late, tartrate) have been studi ed using DSC. Each buffer was
`prepared at different pH to cover the buffering range of the
`particular buffer. It should be emph asized that variation of
`
`195
`
`0724-874 1/02/0200-0195/0 © 2002 Plenum Publishing Corporation
`
`Page 5 of 11
`
`

`

`196
`
`Shalaev, Johnson-Elton, Chang, and Pikat
`
`pH is equiva lent to va ri ation of the mo lecular/ionic species in
`solution as pH variations alters the extent of ioniza tion of
`bu ffe r. Significant changes in the crystallization behavior, and
`th e Tg' were observed as a fun ction of solution pH. An un(cid:173)
`expected patte rn of Tg' changes with solution pH was ob(cid:173)
`served for all buffers studied with Tg' having a maximum
`around pH 4. In addition, it appea red th at a metal ion type
`(i.e., Na vs. K) has a signi fica nt impact on Tg' of a citrate
`bu ffer.
`
`MATERIALS AND METHODS.
`
`Materials
`
`Reagent grade succinic acid and DL-malic acid were pur(cid:173)
`chased fro m Fisher Scie ntific and Sigma, respectively. Citric
`acid of US P grade, L-( + )-tartaric acid of NF grade, and so(cid:173)
`dium hydroxide of NF grade were obtained from JT Baker. In
`additi on, DL-tartaric acid of reagent grade from EM Science
`was used. Deionized water was used to prepare all of the
`bu ffe r solutions that were studied. Citric acid, succinic acid,
`tartaric acid, malic acid were prepared as 0.25M solutions.
`The acids were titrated with 0.25M sodium hydroxide to the
`desired buffer pH . In addition, citric acid/potassium hydrox(cid:173)
`ide solutions were prepared by the sa me methods. Weight of
`the added base was measured. The pH range studied was
`chose n as to be within the pH range where the buffer system
`had significa nt bu ffe ring capacity (1).
`
`DSC Experiments
`
`DSC experiments were performed with a Perkin-Elmer
`Pyris 1 instrument and TA Instruments modul ated DSC 2920
`instrument equipped with Refrigerated Cooling System. Ap(cid:173)
`prox. 15 µI of solution were placed in aluminum pans, and
`empty aluminum pans were used as a reference with both
`instruments. Other details of the experiments performed with
`the Perkin-E lmer instrument are as fo llows. The instrument
`was ca libra ted using melting poin ts of indium at heating rate
`l0°C/min . The ca libration was checked using de-ionized wa(cid:173)
`ter. The uncertainty in th e temperature ca libration was esti(cid:173)
`mated to be within l.5°C. Samples were cooled to -60°C at
`10°C/min , then held at -60°C for 5 min , and then heated from
`-60°C to 25°C at 10°C/min. The Tg' and Tg" temperatures
`were determined as extrapolated onset temperatures using
`Pyris software. Experiments were perfo rmed with the TA
`instrument as fo llows. Calibrations were performed using in(cid:173)
`dium as standard at a heating rate 10°C/min and 1 °C/min for
`standard and modul ated method, respectively. The purge gas
`used was nitrogen with a fl ow rate at 50 ml/min. Samples were
`run in two diffe rent modes: (i) Standard DSC mode, which
`the sa mples were cooled to -60°C at l0°C/min , equilibrate at
`-60°C for 5 min , and then heated to 25°C at 10°C/min. (ii)
`Modulated DSC mode, which the samples were cooled to
`-60°C and then hea ted to 25°C at the same hea ting and cool(cid:173)
`ing ra te o f 1 °C/min. The run was modulated with an ampli(cid:173)
`tude ±0.5°C and a period 100 seconds. The Tg' and Tg" tem(cid:173)
`pera tures were determined as extrapolated onset tempera(cid:173)
`tu res using T A universa l analysis so ftware.
`
`RESULTS
`
`Typica l DSC curves o f sodium and potassium citra te
`buffe r are shown in Figs. l a and lb, respectively (ice me lting
`
`A
`
`pH3
`
`endo
`
`t
`
`, ,mw
`
`pH3.S
`~ pH4
`.s
`
`3:
`0
`u::::
`iii
`Q)
`I
`
`pH4.S
`
`HS
`pHS.S
`
`pH6
`
`-20
`
`-40
`
`endo
`
`I 1 mW
`
`Tg'
`
`pH2.S
`
`pH 3
`
`~ B
`.s
`3:
`0
`u::::
`iii
`Q)
`I
`
`pHS
`
`-40
`Temperature {0C)
`
`-20
`
`Fig. 1. Representati ve DSC heating curves of citric acid/NaOH (A)
`and citric acid/KO H (8 ) solutio ns. Magni fied low-te mperature por(cid:173)
`tio ns o f the DSC scans are shown . Numbe rs present solution pH.
`Scannin g ra tes: 10°C/min . The experime nts were run with Perkin(cid:173)
`Elme r Pyris- 1 DSC.
`
`end oth erm is not shown ). Two co nsecutive endothermic
`events, Tg" and Tg' , were observed in majority of cases which
`is typica l for frozen aqueous solutions (18-21). Th ere is a
`common agreement th at a higher temperature even t (Tg')
`corresponds to the collapse temperature.
`A "dip" in the baseline was observed on DSC heating
`curves of sodium citrate at pH 's from 5 to 7. Such "dip" on a
`DSC heating curve co uld be due to an exothermic event sue~
`as crystallization; if this is the case, assignment of the Tg
`event is uncertain. To determine if crystallization occu rred 10
`these samples, two types of DS C ex perime nts were per(cid:173)
`form ed. In the first experiment, a therm al cycling study was
`perform ed; therm al cycling allows one to separate reversible
`therm al transitions (such as glass tra nsition and melti ng) fron1
`irreversible tra nsitions (such as crystallization) and artifac_ts
`(such an event associated with a change in a sample sh ape 10
`th e DSC pan) (20,26). In this thermal cycling experiment, the
`frozen solution was first hea ted to - 44.5°C (which is the onset
`temperature of the thermal event under consideration) fol(cid:173)
`lowed by cooling to -65°C, and th en hea ted from -65 to 25°C.
`If th e therm al event under consideration is th e (ice) crystal(cid:173)
`lization exotherm , the second DSC heating curve should have
`a different appea rance because crystall ization wo ul d occur
`only during the fi rst run and thus would be irreversi ble. Jn
`particul ar, the temperature of the first endoth e rm ic steP
`wo uld be shifted to higher temperature (if ice crystallize) or
`to the lower temperature (if solute crystallizes). Results of the
`therm al cycling experiment are shown in Fig.2a. lt ca n be seen
`
`Page 6 of 11
`
`

`

`'fhermophysical Properties of Buffers at Sub-Zero Temperatures
`
`A
`
`Tg '
`
`"-..
`
`~
`.s
`
`3:
`0
`u::::
`iii
`a,
`J:
`
`Tg' "
`
`\ ~
`
`heating interrupted
`
`1mW
`
`I
`
`endo
`
`-30
`
`-40
`
`-so
`
`-60
`
`6
`'<--
`.... ::,
`Q)
`«i
`....
`ro
`C.
`E
`Q)
`I-
`
`197
`
`A
`
`•
`
`-60
`
`-50
`
`-40
`
`-30
`
`Temperature (°C)
`
`2
`
`3
`
`s
`4
`Solution pH
`
`6
`
`7
`
`B
`
`0.1 mW I
`
`endo
`
`-SO
`
`-40
`
`-30
`
`-20
`
`Temperature {°C)
`Ilg. 2. Thermal cycling (A) and mod ulated DSC (B) runs of ci tric
`acid/NaOH solution with pH 6. The da ta were obtained with TA
`bsc.
`
`that first and second hea ting curves are practically identical,
`i.e., the position of the first endothermic step did not change
`on the second scan. Hence, the res ults of the therm al cycling
`llperiment suggested that crystallization did not occur during
`ating of the sodi um citrate buffer solution.
`In addition , modul ated DSC experim ents were per(cid:173)
`rmed. Modulated DSC allows separation of irreversible
`uch as crystallization) and reversible (such as glass transi-
`. n) thermal events (19). Mod ulated DSC heating curves are
`own in Fig. 2b. The reversing heat flow curve shows two
`secutive endothermic events (Tg" and Tg'), similar to a
`gular DSC sca n. The nonreversing hea t flow curve shows
`t there is perh aps some crystallization, as evident from the
`ak exothermic peak centered at -45°C; however the mag(cid:173)
`'tude of the exotherm is very small, and the modulated DSC
`ta are not consistent with a significant amount of crystalli-
`tion occurring during heating the frozen solution. Tg' tem(cid:173)
`rature determined from the modul ated DSC run is slightly
`er than determined from the regular DSC sca n; however,
`difference is close to the estim ated experimental error.
`8otb thermal cycling and modul ated DSC experiments indi(cid:173)
`tlte that regular (non-modu lated) DSC sca ns can be used to
`asure Tg' and Tg" of the sodium citrate solutions at pH 5
`to 7. We did not attempt to investiga te the origin of the ap(cid:173)
`Parent "dip" which was observed prior to the Tg' event for
`IOdium citrate solutions at pH 5 to 7 in more detail. Figure 3
`lhows Tg' and Tg" as a function of pH for sodium citrate and
`Potassium citra te. There is good agreement between resu lts
`Obtained with the two diffe rent DSC instruments and wi th
`
`-30
`
`0 -40
`'<--
`a,
`.... ::,
`"§ -SO
`Cl! a.
`E
`a,
`I-
`
`-60
`
`I
`0
`
`3
`
`•
`
`0
`
`0
`
`6
`
`s
`4
`Solution pH
`
`B
`
`0
`
`7
`
`Fig. 3. Tg' and Tg" of citric acid/NaOH (A) and citric acid/KO H (B)
`solutions as a fu nction of pH. D: Tg' measured wi th TA DSC; • : Tg'
`measured with Perkin-Elmer DSC; D: Tg" measured with TA DSC;
`e : Tg" measured wit h Perkin-Elmer DSC; 6 : Tg' measured with TA
`MDSC; T: Tg" measured with TA DSC; 0 : Tg' measured wit h TA
`DSC in thermal cycl ing experiment; X: Tg" measured with TA DSC
`in thermal cycling experi ment. Each data point corresponds to a
`single DSC run. Lines are given as an visual aid.
`
`different sa mple preparations. For sodium citrate, both Tg'
`and Tg" passed through a maximum at pH - 4. Similarly, fo r
`potassium citra te, Tg' passed through maximum between pH
`3 and 4 .
`DSC curves of malic acid/NaOH solutions had a similar
`appearance to the ci trate buffer solutions with one or two
`endothermic steps (Tg" and Tg') fo llowed by ice melting peak
`(curves are not shown). Tg" and Tg' of malate buffer as a
`function of solution pH are shown in Fig. 4a. Again , Tg' goes
`through a maximum at pH 4.
`Succinic acid/NaOH and tartaric acid/NaOH so lutions
`demonstra ted a di fferent thermal behavior. Representative
`DSC heating curves of succinic acid/NaOH and tartaric acid/
`NaOH systems are shown in Figs. 5 and 6, respectively. Buffe r
`crystallization occurred in all three succi nic acid/NaO H mix(cid:173)
`tures studied (pH 4, 5, 6) as evidenced from observation of
`both the exothermic peak (D) and endothermic peaks (M)
`prior to ice melting. A wea k endothermic step immediately
`before the crystallization exotherm , which was observed at
`pH 5 and 6 in succinate buffer (Fig. 5, inset), may be assigned
`to either Tg' or Tg". We did not attempt to characterize this
`transition in more detail. Crystalliza tion was not detected in
`pure succinic acid. Lack of crystallization observed fo r the
`free acid indica tes that a sa lt (not the free acid) crysta llized
`during heating of frozen succin ic acid/NaOH solutions. In
`
`Page 7 of 11
`
`

`

`198
`
`-40
`
`A
`
`D
`I
`
`-50
`
`G
`~
`Q) ...
`::::,
`~
`Q)
`C.
`E
`Q)
`I-
`
`-60
`
`-30
`
`-35
`
`::::,
`
`-40
`
`G
`~
`Q) ...
`1\1 ... Q)
`C.
`E -45
`Q)
`I-
`
`3
`
`4
`Solution pH
`
`5
`
`6
`
`B
`
`~
`
`Shalaev, Johnson-Elton, Chang, and PikaJ
`
`M
`
`pH 4
`
`pH 5
`
`pH 6
`
`Tg"?
`
`11 mW
`
`-40
`
`-20
`
`endo
`
`l
`
`lsmw
`
`D
`
`pH6
`
`pH5
`
`pH4
`
`succinic acid
`
`~
`
`E -;:
`-ct!
`
`0
`u..
`
`(I)
`I
`
`-50
`
`3
`
`Solution pH
`
`4
`
`5
`
`Fig. 4. Tg' and Tg" of malic acid/NaOH (A) and tartaric acid/NaOH
`(B) solutions as a function of pH. All data point but open squares in
`Fig. 48 correspond to solutions prepared with L-( + )-tartaric acid, and
`open squares correspond to solutions prepared with DL-tartaric acid .
`See Fig. 3 for other symbols. Each data point corresponds to a single
`DSC run. Lines are given as a visual aid.
`
`agreement with this hypoth esis, both crystallization exo(cid:173)
`therms and melting endotherms were stronger in succinate
`solution with pH 6 (solution with a higher salt content). In
`addi tion, multiple melting peaks were observed at pH 4 and 5,
`indicating that several crystalline succinate salts were formed
`(i.e., perhaps mono- and di-sodium succinate and th eir hy(cid:173)
`drates).
`A Tg' event followed by a strong crystallization peak was
`observed in a solution of L-( + )-tartaric acid/NaOH at pH 3
`(Fig. 6a). In addition , a weaker exotherm was observed in
`solution of pH 4. We note that a solution of DL-tartaric acid/
`NaOH at pH 3 had a much weaker exothermic event (scan is
`not shown) indicating that the racemic reagent had a lower
`crystallization tendency. Fig. 4b shows Tg' and Tg" vs. pH for
`tartaric acid/NaOH system. Despite of the difference in crys(cid:173)
`tallization behavior, there was no significant difference in the
`Tg' between solutions prepared with either L-( + )-tartaric acid
`or DL-tartaric acid. Both Tg' and Tg" for tartaric acid/NaOH
`solutions were slightly higher at more acidic pH (pH 3) than
`at pH 5.
`
`DISCUSSION
`
`