.AM
`
`.hxE.AIn
`B.m
`MAIA V. BRACCO
`
`IPR PETITION
`
`
`
`
`MAIA Exhibit 1013
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`Pharmaceutical Formulation
`Development of Peptides
`and Proteins
`
`Edited by
`SVEN FROKJAER AND LARS HOVGAARD
`
`
`
`
`

`

`First published 2000 by Taylor & Francis
`I I New Feller Lane, London EC4P 4EE
`
`Simultaneously published in the USA and Canada
`by Taylor & f-rancis Inc
`325 Chestnut Street, 8'h r-loor, Philadelphia P /\ 19 106
`
`Ta_1•/o!-_i\Frw1l'is is a11 i11111ri11t o( the foy/or & Fra11cis Gro1111
`.·'
`(© 200(/lor & Francis Limited
`Typeset in Times by Graphieraft Limited, 1 long Kong
`Printed and bound in Great Britain by TJ International Ltd, l'adstow, Cornwall
`
`/\II rights reserved. No part of this book may be reprinted or
`reproduced or utilised in any form or by any electronic, mechanical,
`or other means, now known or hereafter invented, including
`photocopying and recording, or in any information storage or
`retrieval system, without permission in writing from the publishers.
`
`Every effort has been made to ensure tlwt the advice and
`information in this book is true and accurate at the time or going to
`press. I lowevcr, neither thr.: publisher nor the authors can accept
`:my legal responsibility or liability for any errors or omission:-; that
`may be mallc. In the casr.: or drug a<lministrntion, any mr.:dic,11
`procedure or the use or technical equipment mentioned within this
`book, you arc strongly advised to consult the manufoctun:rs'
`guidelines.
`
`British l,ihrC11y Cawlog11i11g in />11hlicatio11 /)ata
`/\ catalogue record for this book is available from the British Library
`
`/,ih,w:I' ii/" Co11grr1ss Catalogi11g ill /'11hlirntio11 Data
`/\ catalog record for this book has been requested
`
`ISBN 0-748-40745-6
`
`
`
`
`

`

`Contents
`
`List offig11rcs
`List of tahh·s
`Confrihutors
`Pre/i1ce
`
`Peptide Synthesis
`
`Bernard A. Moss
`
`Introduction
`1.1
`1.2 Chemical synthesis of peptides
`1.2.1 Solution and solid phase peptide synthesis
`1.2.2 Large-scale peptide synthesis
`1.3 Concluding remarks
`Relcrenccs and additional sources
`
`,-': .,•'
`
`2 Basics in Rccomhinant DNA T echnology
`
`/
`
`Nanni Din and Jan Enghcrg
`
`~ ----
`'
`- -1-
`..:.-1.
`~ _i '
`
`\ :
`'T
`
`2.1
`2.2
`
`2.3
`
`2.4
`
`Introduction
`General methods in gene technology
`2.2.1 DNA cloning tools
`2.2.2 Cloning of cDNA
`2.2.3 PCR cloning and DNA database mining
`Expression of recombinant proteins
`2.3. 1 Transcription. translation amt protein
`modi !ications
`2.3.2 Choice of expression system
`Protein design
`2.4. 1 Protein variants
`2.4.2 Protei n chimeras
`2.4.3 Epitopc display libraries
`
`This.mat@ria l w as.<opi@d
`at the NLM and m a y b-e
`Subject US-Copyright Lilw.s
`
`page xi
`XIII
`xv
`xvii
`
`I
`2
`4
`6
`10
`10
`
`12
`
`12
`13
`13
`15
`16
`18
`
`18
`19
`22
`22
`24
`24
`
`V
`
`
`
`
`

`

`Vl
`
`Co11te111.,·
`
`2.5 Recombinant protein therapeutics - status and future trends
`References
`
`3 Protein Purification
`Lars I lovgaard, Lars Skrivcr and Sven Prokjacr
`3.1
`Introduction
`3.2
`f-ractionation strategics
`3.2.1
`Initial fractionation step
`3.2.2
`Intermediate purification step
`3.2.3 Final polishing step
`3.2.4 The 11nished product
`3.3 Protein stability in downstream processing
`3.3.1 Protein conformation stability
`3.3.2 Protein instability
`3.3.3 Essential process-related parameters
`References
`
`4 Peptide and Protein Characterization
`Miroslav Baudys and Sung Wan Kim
`4.1
`Introduction
`4.2 Chromatogrnphy
`4.2.1 Reversed phase chromatography
`4.2.2
`I lydrophobic interaction chromatography
`4.2.3
`Ion-exchange chromatography
`4.2.4 Size-exclusion chromatography
`4.3 Electrophoresis
`4.3. I Gel electrophoresis
`4.3.2 Two-dimensional gel electrophoresis
`4.3.3 Capillary electrophoresis
`4.4 Structural characterization
`4.4.1 Primary structure
`4.4.2 Mass spectrometry
`4.5 Secondary and tertiary structure
`4.5.1 Absorption and fluorescence spectroscopy
`4.5.2 Circular dichroism spectroscopy
`4.5.3
`Infrared spectroscopy
`4.5.4 Other methods
`4.(> Conclusion
`References
`
`5 Chemical Pathways of Peptide and Protein Degradation
`Chimanlall Goolclrnrran, Mehrnaz Khossravi and
`Roarnlcl T. Borchardt
`5.1
`Introduction
`5.2 1 lydrolytic pathways
`5.2.1 Deamidation or Asn and Gin residues
`5.2.2 Degradation or Asp residues
`
`This materlsl wascoDied
`
`25
`27
`
`29
`
`29
`30
`30
`31
`32
`32
`33
`33
`34
`37
`38
`
`41
`
`41
`43
`43
`46
`47
`47
`48
`48
`50
`50
`51
`52
`53
`55
`56
`57
`58
`59
`60
`60
`
`70
`
`70
`71
`71
`75
`
`
`
`
`

`

`Confellfs
`
`5.2.3 Degradation of N-terminal sequences containing
`rcnultimate Pro residues via dikctopircrazine formation
`5.3 Oxidation pathways
`5.3.1 Autooxidation
`5.3.2 Metal-catalysed oxidation
`5.3.3 Photooxidation
`5.3.4 Strategies to prevent oxidation
`5.4 Other chemical pathways
`r3-Elimination reactions
`5.4.1
`5.4.2 Disulphide exchange reactions
`5.5 Conclusion
`References
`
`6 Physical Stability of Proteins
`.Jens Brangc
`
`I ntro<luction
`6.1
`(i.2 Protein structure
`(>.2.1 Stabilizing interactions
`6.2.2 Role of water in structure and stability
`6.3 Protein destabilization (denaturation)
`6.3.1 Unfolding intermediates (molten globule)
`6.3.2 Temperature-induced changes
`Influence or pl I
`6.3.3
`lnnuencc of pressure
`6.3.4
`6.4 Aggregation and precipitation
`6.4.1 Mechanisms of aggregation
`6.4.2 Precipitation and fibrillation phenomena
`6.4.3 Factors inllllCllcing nggregation and precipiWtion
`6.5 Surface adsorption
`6.6 Solid phase stabi lity
`6.6.1 Lyophil ization-induccd aggregation
`(1.7 Stabilization of protein drugs
`6.7. 1 Stabilization strategics
`References
`
`7 Peptides and Proteins as P;1renteral Suspensions: an Overview
`of Design, Development, mul Man11facl11ring Considerations
`
`Michael R. DeFclippis and Michael J. Akers
`
`7. I
`7.2
`7.3
`
`Introduction and scope
`Rationale for suspension development
`Types of suspensions and particle formation
`In sif11 particle formation
`7.3.1
`7.3.2 Combination of particles and vehicle
`Excipient selection
`7.4
`7 .5 General requirements for suspension products
`Testing and optimiz:1tion of chemical, physical, and
`7.6
`microbiological properties
`
`vii
`
`78
`79
`80
`80
`82
`83
`84
`84
`84
`85
`86
`
`89
`
`89
`90
`92
`93
`94
`95
`98
`98
`99
`99
`100
`102
`105
`106
`107
`107
`107
`108
`109
`
`113
`
`113
`114
`116
`11 (i
`122
`124
`126
`
`127
`
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`

`

`VIII
`
`Co11te11/s
`
`7.7
`7.8
`
`7.6.I Chemical properties
`7.6.2 Physical properties
`7.6.3 Microbiological properties
`Techniques !cir characterizing and optimizing suspensions
`Suspension manufacturn
`7.8. l Scale-up
`7.8.2 Manufacturing controls: special considerations for peptide
`and protein suspensions
`7.9 Other related systems
`7.10 Conclusions
`Acknowledgements
`References
`
`8 Pc1>tidcs and Proteins as Parenteral Solutions
`Michael .I. Akers and Michael R. DeFelippis
`8.1 Overview and introduction
`8.2 Optimizing hydrolytic stabi lity
`8.2.1 Uu fl'ers
`8.2.2
`Ionic strength
`8.3 Optimizing oxidative stability
`8.3.l Antioxidants
`8.3.2 Chelating agents
`8.3.3
`Inert gases
`8.3.4 Packaging and oxitlation
`8.3.5 Other chemical stabilizers
`8.4 Optimizing physical stability
`8.4. 1 Dcnaturation
`8.4.2 Protein aggregation
`8.4.3 Adsorption
`8.4.4 Precipitation
`8.4.5 Surfactnnts
`8.4.6 Cyclodcxtri ns
`8.4.7 Albumin
`8.4.8 Other physical complexing/stabilizing agents
`8.5 Optimizing microbiological activity: antimicrobial
`preservatives (APs)
`8.6 Osmolality (tonicity) agents
`8.7 Packaging
`8.8 Processing
`8.9 Conclusion
`References
`
`/
`
`9 Roles of Protein Conformation and Glassy St11tc in the Storni.:e
`Stability of Dried Protein Formulations
`.John F. Carpenter, Lotte Kreilgaard, S. Dean Allison and
`Thcodm·c W. Randolph
`9.1
`Introduction
`9.2
`Infra red spectroscopy to study protein secondary structure
`
`127
`129
`133
`133
`136
`136
`
`136
`138
`139
`139
`139
`
`145
`
`145
`147
`150
`151
`153
`154
`157
`157
`157
`158
`158
`159
`160
`162
`163
`163
`165
`166
`167
`
`167
`170
`170
`171
`171
`172
`
`178
`
`178
`180
`
`
`
`
`

`

`Co11te11ts
`
`9.3 Physical factors affecting storage stability of dried protein
`formulations
`9.4 Summary nm! conclusions
`Acknowledgements
`References
`
`IO Peptide and Protein Drug Delivery Systems for Non-parenteral
`Roules of Administralion
`Mette lngcmann, Sven Frokjaer, Lars llovgaard and
`llcllc Brnndsled
`
`I 0.3
`
`JO.I
`Introduction
`I 0.2 Non-parenteral routes of delivery for peptides and proteins
`10.2.1 Barriers to non-parenteral administration of peptides
`nnd protci ns
`I 0.2.2 General approaches to bypass enzymatic and
`absorption barriers
`formulatioti principles for peptides and proteins
`I 0.3.1 Entrapment and encapsulation
`I 0.3.2 Covalent binding
`Immobilized proteins intended for local effect in the GI tract
`- a case study
`I 0.4.1 0ml enzyme supplementation therapy - phenylalanine
`ammonia-lyasc
`I 0.5 Future rcrspcctives
`I 0.6 Summary
`References
`
`I 0.4
`
`11 Peptide and Protein Derivatives
`Gitte Juel Friis
`11.1
`In troduction
`I 1.2 4-Jmidazolidinonc prodrugs
`11.3 Prodrugs or TRI I
`11.4 Derivatives of desmoprcssin
`11.5 Derivatives of' insulin
`11.6 Cyclic prodrugs
`11.7 Conclusions
`References
`
`12 Chemical and Phannacculical Documcnlalion
`Karen Fich and Deirdre Mannion
`12.1
`Introduction
`12.2 Composition
`12.3 Method or numuf'acture
`12.4 Control of starting materials
`12.4.1 Active substances
`12.4.2 Excipic111s
`12.4.3 Packaging materials
`
`ix
`
`181
`186
`I 8(1
`18(1
`
`189
`
`189
`J 89
`
`191
`
`192
`194
`194
`198
`
`200
`
`200
`202
`203
`203
`
`206
`
`206
`207
`209
`210
`212
`213
`214
`2 15
`
`220
`
`220
`221
`222
`222
`222
`22(>
`226
`
`
`
`
`

`

`X
`
`lntcnncdiatc products
`12.5
`12.(1 Control tests on tile finished product
`12.7 Stability
`12.7.1 Expcrtrcport
`l?e/en:11ces
`
`illdex
`
`Co11te11ts
`
`227
`227
`228
`230
`23 1
`
`232
`
`
`
`
`

`

`8
`
`Peptides and Proteins as Parenteral
`Solutions
`
`MICHAEL J. AKERS AND MICHAEL R. DEFELIPPIS
`Lilly J.:vw,1rc/1 L.1i.J<H,1torie.~, Jndi,m,rf)oli~, lnc/i;in.1, USA
`
`8.1 Overview an<l inlroduclion
`8.2 Oplimizing hydrolylic stability
`8.2. 1 Buffers
`8.2.2
`Ionic slrenglh
`8.3 Optimizing oxidative stability
`8.3.1 Anlioxidants
`8.3.2 Chelating agents
`8.3.3
`Inert gases
`8.3.4 Packaging and oxidation
`8.3.5 Other chemical stabilizers
`8.4 Oplimizing physical stability
`8.4. 1 Denaluralion
`8.4.2 Protein aggregalion
`8.4.3 Adsorption
`8.4.4 Precipitation
`8.4.5 Surfactants
`8.4.6 Cyclodexlrins
`8.4. 7 Albumin
`8.4.8 Other physical complexing/stabilizing agents
`8.5 Optimizing microbiological activity: antimicrobi:il preservatives (A Ps)
`8.6 Osmolality (tonicity} ~•gents
`8.7 Packaging
`8.8 Processing
`8.9 Conclusion
`References
`
`11.1 Overview and introduction
`The purpose or this chapter is to provide praclical guidance to formulation scicntisls
`charged with the development of stable, manuf:.,cturahle. and elegant solution dosage
`forms or peptides and proteins. The chapler wi ll cover !he basics or chemical stabi liiation,
`physical stabilization. and microbiological qualily or proteins and peptides in solution.
`We will place more emphasis on the approaches used to solve protein /peptide solution
`
`145
`
`-145-
`
`
`
`
`

`

`146
`
`Akers and Dc:Felippis
`
`formulation rroblcms than on discussing the nature or degradation mechanisms which arc
`covered elsewhere in this text and in many other excellent publications. We also will
`have some coverage or packaging and mnnufocturing of protein solution dosage forms, in
`the spirit or emphasizing that scientists developing these dosage forms must be equally
`concerned with the formulation, the package, and the manufacturing rroecss.
`There nrc at least 22 protein products on the market, four or which arc stored as ready-to(cid:173)
`usl.! solutions, and the rest or wh ich arc stored as freeze-dried powders, then reconstituted
`into solutions by adding a diluent before adm inistration. Approximately 200 peptides and
`proteins are being studied in the clinic, most of which arc freeze-dried products. It is reason(cid:173)
`able to assume that nearly every one of these pcrtide and protein products, commercial or
`in clinical study, has had to overcome and control stability issues in solution. The type or
`stability issue and the degree of complexity of the degradation mechanism differ from protein
`to protein, but approaches to resolve instabi lity issues in solution arc relatively universal.
`Therl.! arc some basic guidelines lo consider in the development of parenteral solut ions
`of proteins and peptides. These arc summarized as fol lows.
`
`A thorough understanding of the physical anc..l chemical properties of the protein or
`peptide bulk drug substance is necessary. Well-documented analytical techniques arc
`now available for studying these properties in solution. Effects of temperature, pl I,
`shear, oxygen, buffer type and concentration, ionic strength, and protein /peptide con(cid:173)
`centration must be understood. From prcfonnulation studies, protei n/peptide chemical
`and physical degradation pathways will be better understood so that the final formu la(cid:173)
`tion, manufocturing process, and packaging system will be rationally dcvclorcd.
`
`2 The route of admi 11istrat ion must be known in order to select the final dosage form,
`vehicle, volume, and tonicity requirements for the product. For cxamrlc, if the prim(cid:173)
`ary route of' administration is intravenous, the vehicle has lo be water although
`some water-miscible co-solvents can be used. The volume can be limitless (unless an
`antimicrobial preservative is rart of the formulation, in which case the volume is
`limited to 15 ml), and the ton icily docs not necessarily have to be isotonic because the
`injected solution will be raridly diluted. I lowevcr, if the route or administration w ill
`be subcutaneous or intramuscular, then the vehicle can be aqueous or nonaqucous, the
`volumes arc limited (usually no more than 2 ml for subcutaneous, 3 ml for intramus(cid:173)
`cular), and the tonieity or the product needs to be more tightly controlled since the
`product is not quickly or readily diluted. The rntc of injection is also a factor to be
`considered in selection of final formulation ingredients in that some ingredients. includ(cid:173)
`ing the protein/peptide itself~ can be irritating and even cause local inflammatory
`reactions if injected too quickly and/or at too high a concentration.
`
`3 Careful screening and choice of solutes for solubilization, stabilization, preservation,
`and tonicity adjustment must take place. These aspects will be the thrust or this chapter.
`
`4 Potential effects of the manufocturing process on the stability of the protein/peptide in
`the final formulation must he understood. Proteins/peptides cannot withstand terminal
`sterilization techniques (heat, gas, radiat ion) and, thus, must be sterilized by aseptic
`filtr<1tion. The filter used must be qualified so that it does not bind the protein/peptide.
`The effect of !low rate during filtration and filling on solution stability must be
`studied. Also, the effect of shear (mechanical stress) that is cncoumered during manu(cid:173)
`facturing must be known. Time limitations must be established from the time the
`protein solution is compou11tkd unt il it is sterile-filtered in order to avoid any increase
`in cndotoxin levels from whatever the bioburden, however small. may be in the non(cid:173)
`sterile solution. llarwood C'f al. ( 1993) and Nail and Akers (2000) arc excellent
`
`-146-
`
`
`
`
`

`

`PeJJ!ides a11cl JJrvfei11s as parenteral solutiom
`
`147
`
`references that deal thoroughly with all aspects or the manufacturing of sterile protein
`and peptides dosage forms.
`5 Selection of the most compatible container/closure system is tremendously important.
`Formulation scientists must appreciate that the container and closure system is just
`as important as the final solution formulation in assuring long-term stability and
`maintenance of sterility and other quality parameters of the product. Proteins and
`peptiucs a1c well known to atlsorb to glass, so experiments must be designed to study
`this possibility and, if adsorption occurs significantly, additives such as albumin must
`be considered to reduce the adsorption. Glass leachates and particulates arc possible,
`and the formulator must be aware or this. Experiments must be conducted to assure
`elimination of this potential problem. The choice of rubber closure is particularly
`important because of known potential for the closure to leach some or its own ingredi(cid:173)
`ents into a solution, to adsorb components of the protein/peptide formulation, to core
`(rubber particulates) when penetrated by a needle, to generate particulates, and to leak
`due to problems with the fitmcnt on the glass vial or resealability of the elastomer
`after needle penetration. Studies on adsorption of the protein to plastic surfaces will
`be necessary irthe final product will be a plastic container. Even if plastic is not part
`of the primary container, protein-plastic compatibility studies should be done since
`plastic tubing such as silicone or polyvinyl chloride wil l be used in pharmaceutical
`process equipment (e.g. filling machines), and the final dosage fo1111 might be added
`to large-volume parenteral solutions contained in plastic bags.
`6 Studies must be conducted to understand the effects of distribution and storage on the
`stabi lily of the final product. Temperature excursions during shipping, mechanical
`stress, exposure to light, and other simulated shipping and storage conditions must be
`studied. From these studies, appropriate procedures for distribution and long-term
`storage or these relat ively unstable dosage forms can be developed.
`Table 8. 1 provides or summary or the key steps in the development of solution dosage
`forms of peptides and proteins.
`
`n.2 Optimizing hydrolytic stability
`
`Tile effect of solution pH on stability is a very important factor to study in early protein
`solution development. Figure 8.1 schematically depicts expected stability problems of
`proteins as a function or pH. Preformulation stability studies arc conducted very early in
`the product development cycle lo elucidate relative protein solubility and stability over an
`approprintc pl I range (normally pl I 3 to pl I I 0). The relationship of stability and solubil(cid:173)
`ity at various pl-I values usually follows a pattern of higher solubility, lower chemical
`stability; or lower solubility, lower physical stability. Protein solubility is minimum gen(cid:173)
`erally at its isoclectric point. Insulin, for example, has an isoclcctric point of 5.4, and at
`this pl-I it is quite insoluble in water (<0.1 mg/ml). Adjusting the solution pl I to less than
`4 or greater than 7 greatly increases insulin solubility (>30 mg/m l, depending on zinc
`concentration and species source or insulin), but also increases the rate of' dcamidation at
`the pll ranges ([3range <.!I al., 1992b). An example or the effect or pl I on denmidation aml
`polymcrizntion or insulin is shown in Figure 8.2 (Urangc and Langkjaer, 1993). In dosage
`l'onn development, the scientist must flrst determine what pl I range provides acceptable
`solubility of the protein for proper dosage, then determ ine whether this pH rnngc also
`provides acceptable stability. There is usually a give-and-take relationship between solu(cid:173)
`bility and stability, ~ml it is up to the scientist to idcntiry what pll is optimal for both.
`
`-147-
`
`
`
`
`

`

`148
`
`Akers and DeFcli11pis
`
`Table ll.1 Development str.1tegy for protein and peptide parenteral solution dosage forms
`
`Formulation and package development studies
`
`Process studies
`
`Final strategy/objc<.:tivcs
`Development of' final formulation
`
`•
`
`Justification of' choice of excipients, pH,
`specilications
`
`Selection of container/closure
`
`• Extractables
`
`• Container/closure integrity
`• Glass leachates, particulates
`
`Stability and compatibility studies
`•
`
`Effects of light, oxygen. high temperature,
`frce;:ing
`
`•
`
`Interaction of cxcipicnts wi th active
`components
`
`• Long-term stability studies of final
`container formulation in finnl container/
`closure system
`
`• Temperature/shipping excursions
`
`Microbiological characteristics
`
`• Antimicrobial properties
`• Preservative efficacy
`• Endotoxin control
`
`Optimization studies of' cxcipicnts. pl I. other
`possible variations
`Process development
`
`•
`
`Process control (e.g. time, temperature
`during each processing step
`
`• Filter selection/validation
`
`Microbial retention
`- Adsorption
`-
`Extractables
`
`• Effect or terminal stcril ization
`Justification of excess
`•
`• Process vnlidation
`Sterilization of components
`-
`
`/\septic process
`-
`- Cleaning
`-
`Filling
`
`Establishment of' critical process parameters
`Establishment of control strategy
`
`C-tcnninal
`deamidation
`
`Carbonyl-amine
`reactions
`
`Un-ionized protein
`amides
`
`Un-ionized terminal
`carboxylic acid
`
`Un-ionized side-chain
`carboxylic acids
`
`Selectively oxidized
`under acidic conditions
`
`Asp transpcptidation
`
`Ii-elimination
`
`Met oxidation
`
`Deamidation
`
`Peptide cleavage
`
`Various oxidations
`
`Base-catalysed
`
`Ionized cysteinc
`
`to reactive species
`
`Di.saccharidc browning
`
`Acid
`
`Neutral
`pll
`
`Disulphide
`shifting
`
`!3asc
`
`Figure H.1
`
`l'rotein reactions ;is a function of pH (courtesy of Dr Lc>e Kirsch).
`
`-148-
`
`
`
`
`

`

`fl<'plides ontl proteins us p<1rc11terul s11l11tio11s
`
`149
`
`A
`
`100 - . - - - - - - - - - - - - - - - -~
`
`80
`
`Q)
`
`g' 60 -r=
`
`(1)
`(.)
`ci> 40
`Q..
`
`(cid:127) Monodesamido
`~ Didesam ido
`D Spilt product
`
`20
`
`0
`3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
`pH
`
`B
`
`20 -.---A-- - -- -- - -- - --
`
`-
`
`-.
`
`15
`
`Q)
`Cl
`
`~ -r= 10
`
`(1)
`0
`'--(1)
`Q..
`
`II Dimer
`(cid:143) Trimer
`(cid:127) Tetramer
`
`5
`
`0
`3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
`pH
`
`I lit'( I ol pl I 011 clt'.1micl.11io11 ,rnd polynwri/,1lio11 ol in,ulin: t lwrnic ,11
`Figure U.2
`1r,1mlom1.ilion duling , to1 ,1g<• ll'i C, 12 111011111') ol rl10111holH•dr ,1 I hovi,w in~ulin ( ry,t,11,
`(ll. 7'Y., N.i( I, ll.2'Y., 11hvnol> ,1, .i fun< lion ol pl I. (/\) I rnm,1tio11 ol 1lw hyd1oly,b pwclull,
`111ont><i<•,,1mido .111<1 clidt•,,1111ido i11,uli11, .111d tlw in,u l in ,pl 11 product (!\II /\'l).
`(ll) I rn111<1tion ol < ov,1 lt•11l clinl('1~ ,llld o lig<>lllt'r,. Rt·p1 inl<·d with p1•1111i,,io11 fron1
`ll1 ,111g(• ,ll)d I ,111gkj,t•1 ( 11) 1) I); r.01'il'lllllll I'll'''·
`
`1 lydrnlysis or dcamidation occurs wi th peptides and protei11s containing susceptible
`J\sn anti ( il n amino acids. lhc only 1,,0 amino acid:-. thnt ;1rc primary amines. The sitk(cid:173)
`chain amide linkage in ;i (iln and J\sn residue may underg<1 deamidation to form f'rcc
`carho:xylic acid. Dca111idatio11 can be promoted by a vmic.:ty or factors includi11g high pl I.
`temperature and ionic strenglh (Manning. et ul .. 1989). The rak: of'dc,11nid:.11ion is alTcttcd
`
`-149-
`
`
`
`
`

`

`150
`
`Akers all(/ DeFelippis
`
`by amino acid sequence, particularly lhc amino acid immcdiatcly following the Asn or
`Gin amino acids. Oliyai and Borchardt ( I 994) studied the influence of primary amino
`acid sequence on the degradation of Asp residues under both acidic and alkaline condi(cid:173)
`tions. /\s expected, the rate of intramolccular formation of the cyclic imidc, the first step
`in the hydrolytic degradation pathway (Pntcl and Borchardt, 1990), was most affected by
`the size or the amino acid on the C-tcrminal side of the Asp residue. Dcamidation rates
`for peptides will be highest when Asn is immediately followed by a Gly amino acid since
`Gly has no side-chain, thus no opportunity lo hinder the hydrolysis reaction stcrically.
`C-termi nal substitution of Gly with increasingly more bulky residues (Ser, Val) inhibi ts
`the amount of cyclic imidc produced. I lowcver, with respect lo Asp amide bond hydrolysis
`with adjacent am ino acids either before or after Asp, such structural changes had little or
`no effect.
`For larger protein structures, the effects of adjacent amino acid sequences on the
`dcmnidation rates of Asn and Gi n arc more difficult to estimate simply due to the three(cid:173)
`dimensional complexities of these structures. However, it certainly is intuitive that
`adjacent amino acids and their size will have some effect on J\sn and Gin deamidation
`regardless of the size of the total protein. l'lydrolytic stability or peptides and proteins can
`be minimized, therefore, through one or more of the frillowi11g approaches.
`
`Optimization of ami no acid sequence, i.e. engineering protein structures to remove
`unstable amino acids or insc11 amino acids that sterically hinder J\sn or Gin dcamidation,
`ns long ,is this docs 1101 affect protein activity, potency, toxicity, or any other quality
`attribute.
`
`2 Formulate at optimal solution pl-I . For example, human epidermal growth factor 1- 48
`demonstrates some interesting pH-dependent stabil ity in that at pl! <6 succinimidc
`fo rmation at Asp11 is favoured, while at pH >6 deamidation of /\sn 1 is favoured
`(Scnderoff ct al., 1994 ). The optimal pl-I, therefore, is right a l pl I = 6.
`
`3 Store at low temperatures, alt hough this will always create difficulties during distribu(cid:173)
`tion and long-term storage of the product.
`
`4 Optimize the effects of ionic strength (to be discussed in section 8.2.2).
`
`ll.2. 1 Buffers
`
`Buffers arc used to prevent small changes in solution pH which can affect protein solubi l(cid:173)
`ity and stability. Buflcrs arc composed of sails of ionic compounds, the most common or
`which arc acetate, ci trate, and phosphate. 13uffcr systems acceptable for use in parenteral
`solutions arc listed in Table 8.2.
`The proper selection of buffer type and concentration is determined by performing
`solubility and stability studies as a function of' pl I and buffer species. Normally, the pl I
`or lllaximum solubility is not the pl l or maximulll stability. I Eowcvcr, a pH range that is
`a good compromise between solubi lity and stability can he selected and maintained with
`the proper selection of the appropriate buffer component.
`In the pll range of 7-12, buffer concentration can have a significant effect on the rate
`of deamidation indicating general acid- base catalysis. Generally, dcamidation is much
`slower at acidic pl I than al neutral or alka line pH. /\CTI I dernnidation in the pH range or
`7- 11 is catalysed by increasi ng buffer concentrations, whereas there is no buffer catalysis
`
`-150-
`
`
`
`
`

`

`Peptides a11d proteins as J>are11reral solutions
`
`151
`
`Table 11.2
`
`lluffcrs used in protein formulations
`
`Buffer system
`
`pKa
`
`pl I Range or use
`
`J\cctatc
`Carbonate
`Citrate
`Glutamate
`Glyeinatc
`llistidinc
`Lactate
`Malcate
`!'hosp hate
`Sueeinatc
`Tartratc
`Tris
`
`4.8
`6.4
`3.14. 4.X. 5.2
`2.2. 4.25, 9.()7
`2.4, 9.X
`1.8, 6.0, 9.2
`3.8
`1.92. (1.23
`7.2(pKa2)
`4.2, 5.64
`2.93, 4.23
`6.2 (pKb 7.8)
`
`2.5-6.5
`5.0- 11.0
`3.0- X.0
`X.2- 10.2
`6.5- 75
`(,.2- 7.8
`3.0- 6.0
`2.5- 5.0
`3.0- 8.0
`4.8- 6.3
`3.0- 5.0
`6.8- 7.7
`
`at pl I 5- (1.5 (Patel, I 993). 1 lowcvcr, insulin dcamidation at the A2 I position predomin(cid:173)
`ates at acidic pl I while deamidation at B3 predominates at neutral pll (11range el al.,
`1992b).
`Several potential problems arc associated with using buffers in parenteral solutions.
`For example, it should not be expected that in large-scale manufacturing the compounded
`solution containing the buffer will always result in the exact pH specified. Dilute solu(cid:173)
`tions of strong acids (hydrochloric acid) or bases (sodium hydroxide) arc usually required
`lo 'fine-tune' the final solution pl I. Excessive use or the pl I adjustment solutions may
`alter the buffer capacity and ionic strength or the buffered solution ( Niebergall, 1990).
`Increasing buffer capacity to control pH better could signilicantly increase ionic strength
`which, in turn, may cause increased potential of' pain upon injl:ction due to the increase in
`solution osmolali1y.
`General acid and/or general base buffer catalysis can accelerate 1he hydrolytic degrada(cid:173)
`tion or the protein. An example is shown in Figure 8.3 (Yoshioka ct al., 1993) where the
`inactivation rate of' ~-galactosidase increased with increasing concentrations or phosph(cid:173)
`ate buffer up to 0.5 M, then decreased, presumably related to higher buffer componenls
`causing a reduction in water mobility. Cleland <'f al. ( 1993) cite several examples whcr·c
`the rate of pro1cin dcamidation was markedly dependent on the buffer anion. Capasso
`ct al. ( 1991) compared the dcamidation rate or a small peptide using different hurlers and
`fi.n11HJ that the peptide was most unstable in a phosphate buffer and most stable in Tris
`buffer. Wang et al. ( 1996) found that buffer type and concentration affected aggregation
`or basic fibroblast growth foctor depending 011 pll. At pl I 5, aggregation increased as
`citrate buffer concentration increased. Citrnte buffer at pH 3.7 caused aggregation, whereas
`acetate buffer rit pH 3.8 did not. At pH 5.5- 5.7, phosphate, acetate and citrate buffers all
`showed similar aggregation rates.
`
`8.2.2
`
`Ionic strength
`
`Ionic strength is a measure of the intensity of the electrical field in a solution. It depends
`on the total concentration of' ions in solution and the valence or each ion. The ionic
`strength of a 0.1 M solution of sodium chloride is 0. 1. The ionic strcng1h of a 0.1 M
`
`-151-
`
`
`
`
`

`

`152
`
`Akers and DeFeliJJJJis
`
`en
`C:
`C:
`C'il
`E
`Q) ...
`
`>, -> -(.) <
`
`~ 0
`
`100
`
`50
`
`0
`
`0
`
`100
`Time (min)
`Figure II.]
`lnac:liv.ition of 11-g.ilactosicl.isc in pH 7.4 phosph,1te buffer solution al 50'(, JS
`a function of phosph.ite buffer conccntr;'llion: (6.) l 0, (0) 50, ((cid:143)} 100, ('v) 200, (& ) 500,
`(e ) 700, ((cid:127) ) 900 mM. The concentration of f1-g;ilactosiclase was 0.1 mg/ml. l~crxinted
`with permission from Yoshiok.i et al. (1 'J'JJ); [)Plenum Press.
`
`200
`
`2-----------------,
`--- KCI
`_._ NaCl
`
`1.5
`
`'
`
`\
`
`\
`
`\
`
`\
`
`\
`
`\
`
`£
`~ 1
`~
`~
`..:c
`
`0.5
`
`\
`
`• .... .... .... .... .... .... ..
`
`o-+-------,.---
`0.8
`0
`0.4
`[salt] (molarity)
`
`1.2
`
`Figure 8.4 Effect of salt concentr;ition on r/\/\T solution slaliilily. Reprinted with
`permission from Vcmuri et al. (l <J'fl); ©l'lcnu111 Press.
`
`solution or sodium sulphate is 0.3, because sulphate ions have a valence or 2 added to the
`valence of I for the sodium ions. Ionic strength may have an effect 011 protein stability
`in solution. The Dcbye-l luckcl theory predicts that increased ionic strength would be
`expected to decrease the rnte or degradation or oppositely dwrgcd reactants. and increase
`the rate or degradation or similarly charged reactants.
`Ionic strength will affect the stability of' a protein, but in which direction (increase
`or decrease) di ffcrs with di ffcre nt proteins. r:or example. increasing ionic strength wi II
`increase the stability of recombinant alpha 1 antitryrsin (Yemuri et al., 1993) (figure 8.4 ).
`
`-152-
`
`
`
`
`

`

`PeJ>tides (//Id pmteins (IS p({renteral solutions
`
`153
`
`100
`
`-
`E -Cl
`.s
`-(/) 10
`..c ...
`Ql :c
`::::,
`0 en
`
`\
`
`'\
`
`' ~
`
`/
`
`,L
`
`I
`,,.
`
`.
`~✓
`
`1 ...... -----,.---..------,.---
`4
`5
`7
`6
`pH
`Figure 8.S Effect of ioni<.; stn:.mgth ()11 rbSt solul)ility-pH profiles. Ionic strength ft
`inc:re,1secl with increasing NaCl: ((cid:127) ) p == 0.24; (0 ) p = 0 .12; (e ) p = 0.072; (0) fl= 0.024;
`( "') ~l == 0.0011. Reprinted with permission from D;ivio and Hageman ( 1 <)<J3); ©Plenum
`l'ress.
`
`8
`
`9
`
`10
`
`Conversely, increasing ionic strength will increase the rate or deamidation or human
`growth hormone (Pearl man and l3ewley, 1993) and bovine somatotropin (Davio and
`I Jageman, I 993) (Figure 8.5).
`
`11.3 Optimizing oxidative stability
`
`Proteins containing methionine, cysteine, cystine, histidine, tryptophan, and tyrosine may
`be sensitive to oxidative and/or photolytic degradation depending on the conformation of
`the protein and resultant exposure of these amino acids to the solvent and environmental
`conditions such as presence of oxygen, light, high temperature, metal ions, and variours
`free radical initiators. Oxidation of sulphydryl-containing amino acids (e.g. methionine
`and cystcinc) wi ll lead to disu lphide bond formation and loss or biological :1ctivity. The
`free thiol group that is present in a Cys residue of any nativt: biologically active protein
`not only may oxidize to produce an incorrect disulphide bridge, but also can resu lt in
`other degradation reactions such as alkylntion, addit ion lo double bonds and complcxation
`with heavy metals.
`I lu man growth hormont:, chymoslrypsin, lysosyme, parathyroid hormone, human
`grnnulocyle-colony stimulating factor, insulin-like growth factor I, acidic and basic
`libroblast growth foctors, relax in, the mo11oc