Parenteral Medications
`Third Edition
`Volume 1: Formulation
`and Packaging
`
`Eton Ex. 1033
`1 of 44
`
`

`

`Pharmaceutical Dosage
`Forms
`
`Eton Ex. 1033
`2 of 44
`
`

`

`Pharmaceutical Dosage
`Forms
`
`Parenteral Medications
`Third Edition
`
`Volume 1
`Formulation and Packaging
`
`Edited by
`
`Sandeep Nema
`Pfizer, Inc.
`Chesterfield, Missouri, U.S.A.
`
`John 0. Ludwig
`Pfizer, Inc.
`Chesterfield, Missouri, U.S.A.
`
`informa
`healthcare
`
`Eton Ex. 1033
`3 of 44
`
`

`

`First published in 1984 by Marcel Dekker, Inc., New York, New York_
`Th.is C'd..ition published in 2010 by Jnformn Het1Utic\lre, Telephone Hou.i,,c, 69-77 P.iul Street, London EC2A
`4LQ, UK.
`
`Siml.tltnnoously publishL-d in the USA by Inform., Healthcarc1 52 Vnnderbill Aven1..1~, '71h Floor, New York.
`NY 10017, USA
`lnforma Hc.-ilthcar~ is a tr.:tding division or lnfonna UK L.td. Registen.>d Office: 37-41 Mortimer Street,
`London WIT 3)H, UK. Registered in Engl.md and Walt"' number 1072954.
`
`4'2f)10 lnforma Het1lthc11rc, except as olherwisc indicated
`
`No claim to original U.S. Cove.rnment works
`
`Rcprmh,.od mnterfal is quoted with permission. Although every effort h.as been made to ensure: that all
`ownt>rs of copyright m.tterial have been cicknowledged in th.is publication, we wouJd be g lad to
`acknowledge in subsequent reprints or editions any omissions brought to our attention.
`
`All rights reserved. No part of this publication may be rl?produccd, stor<.-d in a retrieval system, or
`tri.msmilled, in any fotrn or by zmy me1ms, ck-ctronic, mec.hanial., photocopying, recording, or othenvise,
`unless with the prior writt~n p::nnission of the publisher or in accordi1nce with the provisions of the
`Copyright, Design! and Patents Act 1988 or under th.-: tenns of any lkc:ncc pen:n.itting limited copying
`issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London Wl P OLP, UK, or lhe
`Copyright Clearance Center, Inc., 222 Rosewood Drive, DanverS, MA 01923, USA (http://www.
`copyright.com/ or telephone 978-750-8400).
`
`Produc1· or corporate names may be ttademarks or registered trademarks, and are used only for
`identification and explanation without intent to infringe.
`
`This book contains information from reputable sources and although reasonable efforts have been made to
`publish accurate information, t.he publisher makes no warranties (either express or implied) as to the
`accuracy or fitness for ai p3rticular purpose of the lnformat'ion or advice contained hcn..""'in. The publisher
`wishes to make it clear th.M any views or opi.nton.s expressed in this book by Utdividual authors or
`contrib\1tors are their perso:nal views and opinions and do not necessarily reflect lhc views/opinions of
`the publisher. Any information or guidance contained in this book is intended for use solely by medic~1.l
`professlon;ils strictly as a .supplement to the medical professional's own judgcmCJ\l, knowledge of the
`patient's medlcaJ h.fstory, relevant manufacturer's instructions and the appropriate best practice
`guidelines. Because of the rapid advances in medical science, any information or advice on dosages.
`procedures, or diagnoses should be independently verifi<.-d. Thls book docs not indicate whether a
`pa.rricuJ;"tr m?atment ts appropriate or swtable £or a p.irlicular individual. Ultimately a is the sole
`responsibility of the medical professional to tnake his or her own professional judgements, so as
`appropriately t() advise and treat patients. Saw for death or personal injury caused by the publisher's
`negligence and to the fullest extent otherwise permitted by law, neither the publisher nor any person
`engaged or employed by the publisher shalJ be responsible or liable for any loss~ injury or damage caused
`to any person or property arising in auy way from the use of this book.
`
`A CIP record for this book is available from tl1e British Library.
`
`Library of Congress Cataloging-in-Publication Data available on appliation
`
`ISBN-13, 9781420086430
`ISBN- 13, 9781420086539 (three-volume set)
`
`Orders may be sent to: lnforma Healthcare. Sheepen Plar.e, Colehester, E.~sex C03 31.P, UK
`Telephone, +44 (0)20 7017 5540
`Email: CSDhealthcarebooks@informa com
`Website: http://informah~althcarebooks.com/
`
`Por corpo,r3te .s:iles p le.lse cont.'lct: CorpomteBookslHC@inform.'l.com
`For foreign right5 p lease cont.,ct RightsUiC@i.nforml11.com
`For reprint permissions please conbct: Pennf.ssJomlHC@informt\,com
`
`Typeset by MPS Limited, A Macmi\k,n Comp.,ny
`Printed and bound in Jndi.A
`
`Eton Ex. 1033
`4 of 44
`
`

`

`We dedicate this work to those who have inspired us.
`To my parents Walter and Ruth Ludwig and my wife Sue Ludwig
`To my parents llari and Pratibha Nema and my wife Tina Busch-Ncma
`
`Eton Ex. 1033
`5 of 44
`
`

`

`Foreword
`
`I was a faculty_ member at the University of Ten.nessee and a colleague of Dr. Kenneth Avis
`".'hen h~_conceived, orgamze?, and edited (along with H.A. Lieberman and L. Lachman) the
`first edition of this book series that was publ.ished in 1984. It was so well received by the
`pharmaceutical science community that an expanded three-volume second edition was
`publi~hed in 1992. Dr. Avis_did not survive long enough to oversee a third edition, and it wa,
`questionable whether a third edition would ever be published until two of his graduate
`students, prs. Nema and Ludwig, took it upon themselves to carry on Dr. Avis' tradition.
`Thetr oversight of this third edition is work that their mentor would be highly pleased
`and proud of. From 29 chapters _in the second edition to 43 chapters in this new edition'. this
`three-volume series comprehensively covers both the traditional subjects in parenteral s(cid:143) en~e
`and technology as well as new and expanded subjects. For example, separate chapter topics m
`this edition not found in previous editions include solubility and solubilization, depot delivery
`systems, biophysical and biochemical characterization of peptides and proteins, container(cid:173)
`closure integrity testing, water systems, endotoxin testing, focused chapters on different
`sterilization methods, risk assessment in aseptic processing, visual inspection, advances in
`injection devices, RNAi delivery, regulatory considerations for excipients, techniques to
`evaluate pain on injection, product specifications, extractables and leachables, process
`analytical technology, and quality by design.
`The editors have done an outstanding job of convincing so many top experts in their
`fields to author these 43 chapters. The excellent reputations of the auU1ors and editors of this
`book will guarantee superb content of eaci, chapter. There is no other book in the world that
`covers the breadth and depth of parenteral science and technology better 1lan this one. ln my
`opinion, the editors have achieved their primary objectives- publishing a book that conta.ins
`current and emerging sterile product development and manufacturing information, and
`maintaining the high standard of quality that readers would expect.
`
`Michael /. Akers
`Baxter BioPharma Solutions
`Bloomington, Indiana, U.S.A.
`
`Eton Ex. 1033
`6 of 44
`
`

`

`I Preface
`
`~hnmrnce11ticnl Dosnge Fom,s:. Pn~enfernl Medications was originally published in 1984 ~nd
`1IDmed1ately accepted as a definitive reference in academic institutions and the pharmaceutical
`indus~y. The second edition was published in 1993. The ensu.ing years have produced
`,ncrechble technological advancement. Classic small-molecule drugs are now complemented
`by complex molecules such as monoclonal antibod.ies, antibody fragments, aptamers,
`an~ense, ~Ai therape~tics, and DNA vaccines. There have been significant innovations in
`delivery ~evices, ana_l~tical techniques, in-silica modeling, and manufacturing and control
`technologies. In add.1tion, the global regulatory environment has shifted toward greater
`emphasis on science-based risk assessment as evidenced by the evolving cGMPs, quality by
`design (Q~~)'. p~ocess analy~cal technology (PAT), continuous processing, real time release,
`and other 1.n.ttiatives. The rapidly changing landscape in the parenteral field was the pnmary
`reason we undertook the challenging task of updating the three volumes. Our objectives were
`to (i) revise the text with current and emerging sterile product development and
`manufacturing science and (ir) maintain the high standard of quality the readers expect.
`The third edition not only reflects enhanced content in all the chapters, but also more
`than half of the chapters are new underscoring the rapidly advancing technology. We have
`divided the volumes into logical subunits-volume 1 addresses formulation and packaging
`aspects; volume 2, facility design, sterilization and processing; and volume 3, regulations,
`validation and future directions. The authors invited to contribute chapters are established
`leaders with proven track records in their specialty areas. Hence, the textbook is authoritative
`and contains much of the collective experience gained in the (bio)pharmaceutical industry over
`the last two decades. We are deeply grateful to all the authors who made tlris U10rk possible.
`Volume 1 begins with a historical perspective of injectable drug therapy and common
`routes of administration. Formulation of small molecules and large molecules is presented in
`depth, including ophthalmic dosage forms. Parenteral packaging options are discussed
`relative to glass and plastic containers, as well as elastomeric closures. A definitive chapter is
`provided on container closure integrity.
`Volume 2 presents chapters on facility design, cleanroom operations, and control of the
`envirumm:nt. A chapter diacussing pham1aceutical water ayalems b included. Key quality
`attributes of sterile dosage forms are discussed, including particulate matter, endotoxin, and
`sterility testing. The most widely used sterilization techniques as well as processing
`technologies are pre.cented. Volume 2 concludes with an in-depth chapter on lyophilization.
`Volume 3 focuses on regulatory requirements, risk-based process design, specifications,
`QbD, and extractables/leachables. In addition, we have included chapters on parenteral
`administration devices, siRNA delivery systems, injection site pain assessment, and control,
`PAT, and rapid microbiology test methods. Volume 3 concludes with a forward-looking
`chapter discussing the furure of parenteral product manufacturing.
`These three volumes differ from other textbooks in that they provide a learned review on
`developing parenteral dosage forms for both small molecules and biologics. Practical guidance
`is provided, in addition to theoretical aspects, for how to bring a drug candidate forward from
`discovery, through preclinical and clinical development, manufacturi.ng, validation, and
`eventual registration.
`The editors wish to thank Judy Clarkston and Lynn O'Toole-Bird (Pfizer, Inc.) for their
`invaluable assistance and organizational support during this project, and Sherri Niziolek and
`Bianca Turnbull (lnforma Healthcare) for patiently leading us through the publishing process.
`
`Eton Ex. 1033
`7 of 44
`
`

`

`We also acknowledge the assistance or Pfizer, L11c. colleagues Lin Chen a d
`n Mtn Ii¾\
`reviewing several of the ch1111ters.
`:a C~nivl!rlih.
`We would like to express sp«ial &':'titude to . U1e Late Kenneth E. Avl
`g rlll-
`Tennessee College of Pham1acy) for his dedication to teaching and
`lTing Pr -, Of
`knowledge In Lhe area of parenteral medications to so many studentss
`including us. Fi.11aUy, we acknowledge the co11LributionR of Dr Avis Leo over Ifie~
`11 laclutia_. 'vs,
`Herbert A. Lieberman who edited U1e earlier editions of this book seri·es'
`.,, •lld
`.
`
`5QlllktpN
`/0/111 o t,}"'4
`. ""(L>ig
`
`Eton Ex. 1033
`8 of 44
`
`

`

`I Contents
`
`Foreword Michael J. Akers
`Preface
`ix
`Contributors
`
`xiii
`
`vii
`
`1. Parenteral dosage forms: Introduction and historical perspective
`John D. Ludwig
`
`2. Parenteral drug administration: routes of administration and devices
`Hima11sJ1u Bhaltacharjee and Laura A. 11wma
`
`7
`
`30
`3. Biophannaceutfos of NCEs and NBEs
`Balaji Agoram, Kazuko Sagawa, Ravi M. Shanker, and Salis/, K. Si11gi,
`
`4. Preformulation
`N. Murli Vemuri
`
`57
`
`5. Formulation development of small and large volume injections
`Mad/rav Kamat a11d Patrick P. DeLuca
`
`76
`
`134
`6. Drug solubility and solubilization
`C/1i11g-Chiang Su, Lan Xiao, and Michael Hagema11
`
`158
`7, Formulation of depot delivery systems
`fames J. Cunni11gham, Marc]. Kirchmeier, and Sac/1i11 Mittal
`
`8. Biophysical an d biodtemical characterization of peptide and protein
`drug product
`194
`Ta11an K. Das and James A. Carroll
`
`9. Formulation of protein• and peptide-based parenteral products
`Gaozhong Zhu and Y. John Wang
`
`222
`
`254
`10. Development of ophthalmic formulations
`Paramila Bandyopadhyay, Martin /. Coffey, and Molrmmad 51,moer
`
`ll. Glass containers for parenteral products
`Robert Swift
`
`287
`
`12. Plastic packaging for parenteral drug delivery
`Vi11od D. Vilivalam and Frances L. DeGrazio
`
`305
`
`Eton Ex. 1033
`9 of 44
`
`

`

`IJ. Ll,OIOm~ri< clo~u:rn for pa.,,nle~I,
`RltwtJJ.n>tn
`
`31(cid:141)
`
`14.
`
`ra.,,nlt'flll pmduct <0nlaln,r clo,u.,, lnlegrily 1 .. Ung
`"'"" \1, ,,,,,., °""-"
`
`358
`
`Eton Ex. 1033
`10 of 44
`
`

`

`Contributors
`
`Balaji Agoram Pfizer, Inc., Sandwich, U.K.
`
`pa.ramita Bandyopadhyay Bausch & Lomb, Rochester, New York, U.S.A.
`
`Hiutanshu Bhattachatjee Department of Pharmaceutical Sciences, College of Pharmacy,
`University of Tennessee Health Science Center, Memphis, Tennessee, U.S.A.
`
`James A. Carroll BioTherapeutics Pharmaceutical Sciences, Pfizer, Inc., Chesterfield,
`Missouri, U.S.A.
`
`Martin J. Coffey Bauseh & Lomb, Rochester, New York, U.S.A.
`
`James J. Cunningham Pham,aceutical Research and Development Sciences, Merck Research
`Laboratories, West Point, Pennsylvania, U.S.A.
`
`Tapan K. Das Bio Therapeutics Pharmaceutical Sciences, Pfizer, Inc., Chesterfield, Missouri, U.S.A.
`
`Fra.nces L. DeGrazio West Pharmaceutical Services, Inc., Lionville, Pennsylvania, U.S.A.
`
`Patrick P. Deluca Pharmaceutical Sciences, University of Kentucky College of Pharmacy,
`Lexington, Kentucky, U.S.A.
`
`Dana Morton Guazzo RxPax, LLC, Bridgewater, New Jersey, U.S.A.
`
`Michael Hageman Bristol-Myers Squibb Research, Princeton, New Jersey, U.S.A.
`
`Renaud Janssen Helvoet Pharma, Alken, Belgium
`
`Madhav Kamat Biopharmaceutics R&D, Bristol-Myers Squibb Company, New Brunswick, New
`Jersey, U.S.A.
`
`Marc J, Kirchmeier Vaccine Formulation Deve.lopment, Variation Biotechnologies, Inc.,
`Cambridge, Massachusetts, U.S.A.
`
`John D. Ludwig BioTherapeutics Pharmaceutical Sciences, Pfizer, Inc., Chesterfield,
`Missouri, U.S.A.
`
`Sachin MIiia! Pharmaceutical Research and Development Sciences, Merck Research Laboratories,
`West Point, Pennsylvania, U.S.A.
`
`Kazuko Sagawa Pfizer Global R&D, Groton, Connecticut, U.S.A.
`
`Ravi M. Shanker Pfizer Global R&D, Groton, Connecticut, U.S.A.
`
`Mohannad Shawer Bausch & Lomb, Rochester, New York, U.S.A.
`
`Eton Ex. 1033
`11 of 44
`
`

`

`Salish K. Singh
`U.S.A.
`
`B,oTI,erapeulk'5 Pharmn~ulkal Sciences, Pfiter, In c., Chesterf 1
`
`C01tr~IB(n
`°'ls
`
`ie d, M'·
`
`Ching-Chiong Su BnShll-Myers Squibb Researd1, Pr lncclon, New Jersey, US.A.
`
`Robert Swlfl AlhgQn, 11,c., TI10us11nd Oaks, C~lllom la, U.S.A.
`
`Laun A. n,oma 1..X>p,1rtmcnt of l'hnm,nttutlrol Sciences, College ol Phannacy U
`l\lv•r-.ity or
`T~Ml'SS<!I! J lcallh Sdencc C(•n1er, McmphlB, Tennessee, U.S.A.
`'
`
`N. Murti Vl'murl Sanofi-Avel\llS. Brldgt'water, New Jersey, U.S.A
`
`Vlnod O. Vilivalam West l'harmaccutiml Services, Jnc., Lionvllle, Pen nsylvania, US.A.
`
`Y. John Wang Genentech, South San Francisco, California, U.S.A.
`
`Lan Xiao Bristol-Myers Squibb Research, Princeton, New ]e=y, U.S.A.
`
`Caozhong Zhu Shire Human Genetic Therapies, Jnc., Cambridge, Massachusetts, US-A.
`
`Eton Ex. 1033
`12 of 44
`
`

`

`Formulation of protein- and peptide-based
`parenteral products
`Gau.hong Zhu and Y. John Wang
`
`INTRODUCTION
`Sine., the early 1970s, scientific advances in molecular biology and genetic enginec .
`led to cnonnous sucCl'SS in protein- and pcptidc-baSt.>d therapeutics for the treatmcn~np have
`O many
`human diseases They cover almost all lhcrapcutic categories, mcluding cardio
`hemost.isis, antineoplastic, dfabetei: and cnd_ocrinology, anti-infccli_vc, neurophannaC::~hir
`enzyme replacement, wound healing, respiratory, and ~.nc _cartilage. Protein-based ~cal
`peutics are cmel'j\ing as a major class of new molecular cnlihes in the pharmaceutical ind hera.
`Over 200 biotechnology and plrnrmoceulical con,panics are developing protein-~
`therapeutics. More than 150 biologics are currently marketed, and over 400 are in adva
`stages of testing and clinical trials (I).
`Reed
`Unlike small molecules, which are typically synthesized through chemical pr0cesses
`proteins arc produced in living sys1ems. The main technology used to produce proic· '
`utilizes recombinant DNA lechniques, to pr~uce prot~ln _mol~es in a host cell. Several 1~
`of host cells have been employed, 111dud1ng Esc/1wc/11n colr, yeast, mammalian cells leg
`Chinese hamster ovary (CHO) cells and human fibr~blastsJ,_and plant-derived cells. Sev~,,;j
`other technologies are also used to produce therapeutic protems. Small proteins and peptides
`such as calcitonin, may be produced by chemical synthesis. Most human serum albumin ~
`sourced from human blood, urokinasc from urine, and streplokinase from fungi. Recombinant
`human antithrombin (ATryn®>, a new product approved by the FDA in 2009, is produced by
`h';msgenk a..1limals.
`
`CHARACTERISTICS OF PROTEINS AND PEPTIDES
`Compared with small-molecule drugs, protein-based pharmaceuticals are not only larger in
`molecular weight, but they also contain more complex compositions and higher order
`structures. Intrinsically, most proteins have poor stability and a very short half.life m vivo.
`Because of their poor oral bioavailability, most proteins require parenteral administration
`routes. In some cases, they require specific delivery systems targeting the specific site or action
`to achieve sufficient efficacy. Therefore, formulating these proteins as therapeutic agents with
`proper efficacy and safety profiles has been a challenging task. For successful product
`development, one needs to have a thorough understanding of the protein's phys1cochemical
`and biological characteristics, including stability, immunogenicity, and pharmacokinetic
`properties. The characterization of proteins is lherefore an important step in formulation
`development.
`
`Molecular Composition, Structuro, and Heterogeneity
`A protein, or polypeptide, is formed through the linkage of pephde bonds of ammo aods.
`Generally, prolein structures arc described at four levels: primary, secondary, tertiary, and
`quaternary. Details aboul these can be found in the preceding chapter of this volume.
`Because of their complex manufacturing process, from cell culture to downstream
`purification, protein products generally contain multiple species in tenns of molecular weight
`or size, which could be due to vanous modifications to the polypepbde side chains or glycans,
`reversible or irreversible formation of o\igomers by either noncovalent or covalent linkages,
`and formation of large soluble and/or insoluble aggregates. It is important to characteriz~and
`quantify llll species, as they may dhcctly affect product efficacy, safety, and immunogerudty,
`Depending on its size and the nature of its associations, several analytical techniques c:'n
`be used to characterize a protein's size Routinely, electrophorctic and chromatographic (with
`multiangle light-scattering detector) techniques have been used to estimate protein size up 10
`
`Eton Ex. 1033
`13 of 44
`
`

`

`FORMULATION OF PROTEIN- AND PEPTIOE·BASED PARENTERAL PRODUCTS
`
`223
`
`oligomers. ~y combining a den~turing electrophorelic technique (sodium dodecyl sulfa_te
`p0Jyacrylam1de gel electrophoresis [SOS-PAGE]) with size-exclusion high-performance liquid
`chrOITlatography (HPLC) or _a naliv_e electrophoretic technique (Native PAGE), the size of
`proteiJ\S and the nature of their associations (covalent vs. noncovalent) in Mtive and denatured
`states can also be estimated._ To '.nore accurately determine the size of proteins, mass
`spectrometry, such as matrix-assisted_ laser desorption/ionization time-of-flight ma~s
`spectrometry (MALDT-TOF MS) or hqu1d chromatography mass spectrometry (LC-MS), is
`often used. _However, because of_ the ma~ix effect and the high energy applied, the molecular
`weight or size deternu~ed by tlus t~cl~mque may not be the true size in solution.
`To me~sur~ tile s•~e of a pr~tein m solu tion up to 100 nm, several biophysical techniques
`may be feas~ble'. mcludmg ~nalyttcal ultracentrifugation (AUC), field flow fractionation (FFF),
`and d ynruruc l~ght scattenng. _It should be noted that the size distribution of proteins in
`solulio1'., e~peoally for reversible asso~iation, may be highly dependent on the soluti?n
`properties, U1dudmg pH, sal~ concentration, a nd protein concentration. Therefore, the mobile
`phase used _in these analy~es 1s pref~rably the same as the formulation vehicle, and the impact
`of the dilution factor dunng analysis should be assessed.
`Insolu~le aggregate~ or particles larger than 100 µm can be observed by visual inspection
`with the unaided eye. TJ:ietr_size can be estimated by microscopy. Subvisible insoluble aggregates
`betWeen 10 and 100 µrn m size can be quantified and sized either by a light obscuration test or by
`a microscopic particle count test per USP method <788>. It is still technically challenging to
`accurately quantify and size particles between 0.1 and 10 µrn. A technique using Micro-FlowTM
`iinaging (MFI) has been used for particles as small as 0.75 µm (2).
`
`1soelectric Point
`Proteins that contain both positively and negatively charged amino acids are amphoteric
`molecules. One property that characterizes a protein's charge profile is its isoelectric point, or
`pl. The pl of a protein is the pH at which it carries no net electrical charge. At a pH below its pl,
`a protein is positively charged; above its pl, it is negatively charged.
`The pl may be approximately calculated from the amino acid composition data, that is,
`pl = (pK1 + pK2 + pK3 . .. + pK.)/ri for n ionizable groups. However, because the dielectric
`constant in the immediate vicinity of an ionizable group depends on protein structure, and
`because hydrogen bonding may alter dissociation constants (K.), the true pl can differ
`significantly from the calculated one. Seve.ra I websites provide theoretical estimations of pl for
`proteins (e.g., http://www.scripps.edu/~cdputnam/protcalc.httru, http://www.expasy.ch/
`tools/pi_tool.htrnl, and http:/ /www.nihilnovus.com/Palabra.html).
`Some proteins have multiple species with different charge profiles, and each species has
`its own pl, so tl1ese proteins appear to have more than one pl. Some glycoproteins in particular
`exhibit complicated pl patterns because of the heterogeneity in their glycan composition. Also,
`some proteins comprise multiple deamidation species, which also results in complicated
`charge profiles that could be characterized by seve.ral teclutiques, including isoelectric focusing
`(IEF), ion exchange chromatography (IEC), and capillary electrophoresis (CE).
`Proteins show a broad range of pls, mostly in the range of 2 to 12. The pl of a protein may
`play an important role in solubility and stability. In general, protein solubility is at its
`minimum when the pH is near its pl. Also, because zero net charge at pl should presumably
`allow maximum interaction between salt bridges and exert the least interaction between
`protein molecules, it could be expected lo be the most stable condition for conformation.
`However, studies have shown that the optimal pH for conformational stability can be quite
`different from the pT and in many cases is found at a pH corresponding to a large net charge of
`the protein (3).
`
`Solubility
`The varieties of functional groups (charged, hydrophobic, etc.) on the side chain of amino acids
`and glycans (for glycoproteins) make protein solubili ty dependent on the pH, salt concen(cid:173)
`~ation, and polarity of the solvent. The over~ size of the protein_ does not necessarily
`influence solubility. For example, antibodies, which have molecular welghts of approX1Inately
`150 kDa, can often achieve aqueous solubility greater than 100 mg/mL.
`
`Eton Ex. 1033
`14 of 44
`
`

`

`VOLUME 1: FORMULATION AND PAC"•G
`""1//G
`
`224
`
`-8 not easy to determine because pepti-'
`.
`"es
`r proteu1 1
`d
`.d
`1
`form gels, or may ev~_op aggre~ates U!JOn
`The a ueous solubility of a p_epb e o
`_q
`t difficult. In addition, solubility "ilri
`h' h concentrations may
`and prot~ at u~g making solubility a~essm~ solubility determined by most methOcls es
`:fn~~:=~?de~nding on the conform~:~~it;of a protein as ~ hydr~colJoid is _difficult 1~
`g
`te a protein solution usmg a sem1permeabl
`t 1 b"l"ity because the true so
`apparen so u 1
`t·
`·
`h
`,
`.
`t concentrc1
`e
`d fine A common approach 1s o_
`1 'ghest protein concentra 10n 1s reac ed. Anothe
`.:emb~ane with centrifugation w1~1l th: u tide and then add water t? t~e point Wher;
`approach is to lyophilizc a protein o 0hrn a limited amount of J:'rotem i_s available, one
`undissolved material is barely_ ?re~!nt~I ethylene glycol (PEG) soluti~~ (typically 1-9%) and
`approach is to determine solub1hty
`PEdt determine aqueous solub1hty (4).
`then extrapolate the solubility to 0% P . , osolubility include its intrinsic properties and th
`•t·
`:ne a protem s
`f
`•
`e
`The factors that determu, .
`.
`.
`ro erties are the compos1 10n o ammo acids, the
`composition of the solvent. The mt:msi~ P ~position and structure of glycans. Generally a
`folded structure, and for glycop_rote•;~ d;oc~iobic ami.Jlo acids such as Phe'. :Yr, and Trp ~W
`protein made of a large proporhOn ° Y ~ cans increases water solubility. The solvent
`have low water solubility, and adding g rnd specific ligands, can also significantly affect
`properties, including pH, s~l_t concei:t:11
`• of pH is typically in the shape of a U or V, where
`the solubility. Protein solubility as a
`he on e exceptions. The solubility of a protein at low
`the minimum is at the P!· However,.~~~ a:alt concentration, which is called the salting-in
`ionic strength generally mcr_eas~s wit ses ~e additional counter-ions shield the ionic charge
`effect. As th~ salt concentratio_n m7~ili~- As salt concentration continues to increase, protein
`and t~~reby increase the prot~lll ~o u~ effect). At high salt concentration, the salts begin to
`solubility ~ecreas~ (the ~lht·ng of the protein for the solvation of the polar solvent, which
`compete with the 10111c mo1e 1es O
`th b' d
`t th
`t ·
`. •
`bili A 5 ecific ligand or stabilizer at m s o
`•
`.
`e pro em may also
`. P
`results m decrea~mg solu
`ty.
`I bility f fibroblast growth factor was observed
`O
`influence solubility. For example, increased so u
`..
`· or heparin-like substances (5). Also, alteplase solubility was
`in t e presence o
`.
`
`f h eparm
`h
`sed b th dd"t' n of arguun· · e (6). However, one needs to as~~~~ whether the hgand or
`·
`h fi 1 <
`u1
`d"
`• ·
`mcrea
`y e a 110
`excipient is acceptable for the intended clinical use before ad mg it mto t e na 1orm abon.
`
`Thermal Transition Midpoint
`Because native proteins exhibit folded structure in solution, they can undergo transition from
`native form to unfolded or denatured form with increasing temperature. The thermal
`transition midpoint (T ml, defined as the temperature at which equal amounts of native and
`denatured forms exist in equilibrium, is an important characteristic of proteins, measuring
`their thermal stability. Generally, a higher Tm value indicates better thermal stability.
`The most commonly used technique to determine Tm is differential scanning calorimetry
`(DSC), as this method not only provides an accurate measurement of Tm but also can assess
`reversibility of transition and estimate apparent enthalpy. Temperature-controlled spectrom(cid:173)
`etry, including circular dichroism (CD), fluorescence, and ultraviolet (UV) absorbance
`spectroscopy, is also sometimes used to differentiate the transitions by tertiary structure
`from those by secondary structure.
`Measurement of Tm has been widely used in preformulation and formulation develop(cid:173)
`ment. The profile of T.,, as a function of pH provides important information in selecting the
`optimal ~H_ for formulation. This method has also been used in screening different stabilizers,
`as an exc1p1en_t that el~vates Tm is expected to be a potential stabilizer (7). However, it should
`be noted that 111 choos111g the fom1ulation, one also needs to consider other information, as Tm
`alone is only indicative of thermal stability.
`. P~oteins in solid state also_e>dtib!t ~ennal transitions upon heating. These are typically
`detenruned by ~ - However, it is difficult to measure the true thermal transitions of solid
`prot~, because m most cases other components present in the solid dosage form aJso
`contr:bute to the overall thermal transition. Recently glass transition temperatures (T8) for
`• r al es
`1 ·
`• • '
`·
`tr
`Prote111s have been estimated by
`me~s~ed at t very fast scannmg rate in binary mixtures of protein and another glass form
`. ex apo atmg exap1ent concentration to zero using g v u
`
`exop1ent, sue as sucrose, ove.r a range of excipient concentrations (8).
`
`Eton Ex. 1033
`15 of 44
`
`

`

`FORMULATION OF PROTEIN- ANO PEPTIDE-BASED PAR
`ENTERAL PRODUCTS
`
`225
`
`instability: Key Degradation Pathways
`.
`th
`The structural complexi~ of proteins mak
`_em susceptible to processing and handling
`,.,.,ndition5 that can result m structural and fes
`f
`1
`d
`unctional odifi
`·
`__ ,.;ety o cova ent an noncovaJent rea "
`cations. A protein can undergo a
`m
`~v
`c .. ons or modif'
`.
`tw
`.
`• .., .
`.
`.
`o mam categories· Ci) ph
`classified mto
`•cations, wluch may be generally
`. 1
`ho
`'cal
`·
`ys1ca or non-<
`or covalent bond degrad r
`1
`and (i,1 chenu
`ova ent
`nd degradation pathways
`pathways include denaturation or unfoJdin a •~n pathways. Common physical degradation
`rorces- Chemical degradation pathways ~ 8
`1 s~rptlon, and aggregation due to noncovalent
`eXchange, deamidation, isomerization, race:~ u . e covalent-bonded aggregation, disulfide
`Maillard reaction, diketopiperazine formati J.Zahon, fragmentation, oxidation, ~-elimination,
`0
`~~r::.'d Tso on. Oftentimes, physical degradation
`fl,cilitates chemical degradation, and vice
`athways have been extensively described .
`· he funda1?'entals of these degradation
`~ brief description of each degradation p •~hseveral review articles and book chapters (9-14).
`some proteins, and remedies are presente: b wl ay, the factors responsible for degradation in
`eow.
`
`•
`
`Oenat11mtio11
`Denaturation is the process of altering prot ·
`,
`1
`ctures) from its native folded ta
`ems ru~ture (i.e., secondary, tertiary, or quaternary
`.5
`te. Denatu~ation may result in an unfolded state, which
`s~d further facilitate oth
`h
`~ uired for proteins t;r P . ~sic~ a_nd c~emical degradations. Because a specific structure·
`is req I ss of efficac
`d . exer Ph ys~ological and pharmacological activities, d enaturation
`causes O
`Y an
`incurs t e nsk of safety such as immunogenicity.
`Many times, _the denaturation process can be described as N .... I ... D. The folded native
`structure (N) unwinds ~d passes through a partially unfolded or intermediate state m to a
`denatured ~late (D). This .process may be reversible or irreversible, depending on conditions.
`For rev~rsible ~e_nat~ration, the unfolded protein will regain its native slate once the
`denatun.ng condition 1s removed.
`Many_ factors can cause_ denaturation, including heat, freezing, extreme pHs, organic
`solvents, high. salt con~~ntrati?n, lyophilization, surface adsorption, and mechanical stress.
`These denaturing conditions dis.rupt a protein's higher order structure, which is held together
`by intramolecular forces including hydrogen bonding, salt bridges or electrostatic forces,
`hydrophobic interactions, and van der Waals forces.
`Hydrogen bonds are critical in determining overall protein conformation, since they are
`the major forces that stabilize the secondary ct-helices and ~sheets, as well as the overall folded
`structure. Water, the nearly ubiquitous medium for proteins, contributes to this hydrogen
`bonding. Cosolvents such as ethanol and acetone and chaotropic agents such as urea and
`guanidine hydrochloride disrupt the hydrogen bonds and thus readily denature proteins.
`The ionic side