Pharmaceutics
`I’: lg S§Z{i:-i1\(1:»i 0:‘ 3
`DO5/\(J: : :0 RM D i -.5 I.m\
`
`Edited by
`M. E. Aulton
`
`/)CCCCCCCLL
`
`j_ LIVINGSTONE
`
` AMGEN INC.
`
`Exhibit 1014
`
`EX. 1014- Pae1 of 15
`
`Ex. 1014 - Page 1 of 15
`
`AMGEN INC.
`Exhibit 1014
`
`

`
`Edited by
`
`Michael E. ALlItOh BPharm PhD FAAPS MRPharmS
`Professor of Pharmaceutical Technology,
`School of Pharmacy,
`De Montfort University,
`Leicester, UT{
`
`SECOND EDITION
`
`))CHURCHILL
`
`LIVINGSTONE
`
`T E
`
`DINBURGH LONDON NEW YORK OXFORD PHILADELPHIA ST LOUIS SYDNEY TORONTO 2002
`
` EX. 1014- Pae2 of 15
`
`Ex. 1014 - Page 2 of 15
`
`

`
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`First published 1988
`Second Edition 2002
`Reprinted 2002 twice, 2003
`
`Standard edition ISBN 0 443 05517 3
`
`International Student Edition ISBN 0 443 05550 5
`Reprinted Z002 twice, 2003
`
`British Library Cataloguing in Publication Data
`A catalogue record for this book is available from the British
`Library
`
`Library of Congress Cataloging in Publication Data
`A catalog record for this book is available from the Library of
`Congress
`
`Note
`
`Medical knowledge is constantly changing. As new
`information becomes available, changes in treatment,
`procedures, equipment and the use of drugs become
`necessary. The editor, contributors and the publishers have
`taken care to ensure that the information given in this text is
`accurate and up to date. However, readers are strongly
`advised to confirm that the information, especially with
`regard to drug usage, complies with the latest legislation and
`standards of practice.
`
`your source for books,
`journals and rnulcirnedia
`in the lrecrltlr sciences
`www.e|sevierhealth.com
`
`The
`publisher's
`policy is to use
`paper manufactured
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`Printed in Spain
`
`Ex. 1014- Pae3of15
`
`Ex. 1014 - Page 3 of 15
`
`

`
`1 solvent in clotrimazole
`:0 widely used as cosol-
`although their main use
`ater-miscible ointment
`glycols available which,
`roducts for human use,
`acesses or as solvents in
`and horticultural solu-
`opylene glycol, diethyl-
`and their monoethyl
`hol possessing three
`le, is also widely used,
`:h water for oral use. At
`used in, for example,
`n.
`
`»und and is thought to
`gs
`through the skin.
`solvent for veterinary
`:r for idoxuridine, an
`I to human skin.
`
`for the extraction of
`
`f its own therapeutic
`reparation of formula-
`. however, used as a
`e collodions.
`
`al makes it unpleasant
`t
`is often used as 3
`
`:ion of drugs in emul-
`e light liquid paraffifl
`for Oily nasal drops.
`zause ofthe possibiiitl’
`a if they are inhaled
`use in Veterinary for-
`rarnple, anthelminthlc
`trachloride.
`
`,
`
`insecticides such as pyrethrum and
`for
`solvent
`piperonyl butoxide.
`Xylene is present in some ear drops for human use
`to dissolve ear wax, and glycofurol is a useful solvent
`for parenteral products.
`it may be possible to
`As with aqueous systems,
`improve the solubility of a drug in a particular vehicle
`by the addition of a cosolvent. For example, nitrocel-
`lulose is poorly soluble in both alcohol and ether but
`adequately soluble in a mixture of both. The formula-
`tion of Digoxin Injection, too, is best achieved by the
`inclusion of both ethyl alcohol and propylene glycol.
`
`TOTHERT FORMULATION Aooitn/Es
`
`Buffers
`
`These are materials which, when dissolved in a
`solvent, will enable the solution to resist any change
`in pH should an acid or an alkali be added. The
`choice of suitable buffer depends on the pH and
`buffering capacity required. It must be compatible
`with other excipients and have a low toxicity. Most
`pharmaceutically acceptable buffering systems are
`based on carbonates, citrates, gluconates, lactates,
`phosphates or tartrates. Borates can be used for
`external application, but not to mucous membranes
`or to abraded skin.
`Although solutions of drugs that are themselves
`weak electrolytes will act as buffers, their buffering
`Capacities are not usually sufficiently robust and
`Should be enhanced by one of the systems described
`‘above.
`As the pH of most body fluids is 7.4, products
`such as injections, eye drops and nasal drops should,
`in theory, be buffered at this value to avoid irritation.
`Many body fluids themselves, however, have a
`buffering Capacity and’ when formulating 10W_
`"volume intravenous injections or eye drops, a wider
`19H range can be tolerated. This is potentially useful
`‘lhould a compromise be necessary when choosing a
`that
`is physiologically acceptable for a drug
`M1036 optimum stability, solubility and/or bioavail—
`
`Careful control both of the density of such injections
`and ofthe position ofthe patient on the operating table
`will enable precise control of the area to be anaes-
`thetized. The terms used to describe the density of
`injections in relation to that of spinal fluid are isobaric,
`hypobaric and hyperbaric, meaning ofequal, lower and
`higher density, respectively. The most widely used
`material for density modification is dextrose.
`
`lsotonicity modifiers
`
`Solutions for injection, for application to mucous
`membranes, and large-volume solutions for oph-
`thalmic use must be made iso-osmotic with tissue
`fluid to avoid pain and irritation.
`The most widely used isotonicity modifiers are
`dextrose and sodium chloride.
`lsotonicity adjust-
`ments can only be made after the addition of all
`other ingredients, because each ingredient will con-
`tribute to the overall osmotic pressure of a solution.
`
`Viscosity enhancement
`
`It may be difficult for aqueous—based topical solu-
`tions to remain in place on the skin or in the eyes for
`any significant time because of their low viscosities.
`To counteract
`this effect,
`low concentrations of
`gelling agents can be used to increase the apparent
`viscosity of the product. Examples include povidone,
`hydroxyethylcellulose and carbomer.
`
`Preservatives
`
`When choosing a suitable preservative it must be
`ensured that:
`
`- adsorption of the preservative onto the container
`from the product does not occur, and
`-
`its efficiency is not impaired by the pH of the
`solution or by interactions with other ingredients.
`F01” €Xat1'1P1€: many Ofthe Wid€1Y used P31‘ahYd1'0XY'
`736112010 acid esters Can be adSOI‘b€d
`into the
`micelles of some non-ionic surfactants and,
`a1th011gh their Presence Can b6 detected by Chemical
`analysis,
`they are in fact unable to exert
`their
`
`317
`
`
`
`Ex. 1014- Pae4of15
`
`Ex. 1014 - Page 4 of 15
`
`

`
`
`
`The simple use of sweetening agents may not be
`sufficient to render palatable a product containing 3
`drug with a particularly unpleasant taste. In many
`cases, therefore, a flavouring agent can be included.
`This is particularly useful in paediatric formulation
`to ensure patient compliance. The inclusion of
`flavours has the additional advantage of enabling mé,
`easy identification of liquid products.
`*
`Flavouring and perfuming agents can be obtainegil
`from either natural or synthetic sources. Natur"l
`products include fruit juices, aromatic oils such ;r
`peppermint and lemon, herbs and spices, and d'_.
`tilled fractions of these.They are available as conce
`trated extracts, alcoholic or aqueous solutions, M
`or spirits, and are particularly widely used in
`manufacture of products for extemporaneous u 3».
`Artificial perfumes and flavours are of purely s‘
`thetic origin, often having no natural counte
`They tend to be cheaper, more readily available, 1
`variable in chemical composition and more s
`than natural products. They are usually available-*
`alcoholic or aqueous solutions or as powders.
`The choice of a suitable flavour can only be
`as a result of subjective assessment and, as cons aw
`preferences vary considerably, this is not easy. M
`guidance can, however, be given by reference
`Table 21.1, which shows that certain flavours-i’
`particularly useful for the masking of one or mo. "
`the basic taste sensations of saltiness, bitter n"
`ll“
`sweetness and sourness.These tastes are detect
`sensory receptors on various areas of the Vii"1
`
`
`
`
`Suitable masking flavour
`
`Taste of
`product
`Salty
`Bitter
`Sweet
`
`
`Apricot, butterscotch, liquorice, peach, Vanll
`Anise, chocolate, mint, passion fruit, wild if
`Vanilla, fruits, berries
`
`
`
`
`Sour
`
`Citrus fruits, liquorice, raspberry
`
`EX.1014-Pae5of15
`
`
`
`ing agents such as sodium metabisulphite, or anti-
`oxidants
`such as butylated hydroxyanisole or
`butylated hydroxytoluene. For unit-dose parenteral
`products, such as injections of nicotinamide and
`ascorbic acid, it is possible to use Water for Injections
`free from dissolved air and to replace the air in the
`headspace by nitrogen or another inert gas.
`
`-
`
`sweetening agents
`
`
`
`Low molecular weight carbohydrates, and in partic-
`ular sucrose, are traditionally the most widely used
`sweetening agents. Sucrose has the advantage of
`being colourless, very soluble in water, stable over a
`pH range of about 4-8 and, by increasing the viscos-
`ity of fluid preparations, will impart to them a pleas-
`ant texture in the mouth. It will mask the tastes of
`both salty and bitter drugs and has a soothing effect
`on the membranes of the throat. For this reason,
`despite its cariogenic properties, sucrose is particu-
`larly useful as a vehicle for antitussive preparations.
`Polyhydric alcohols such as sorbitol, mannitol and,
`to a lesser extent glycerol, also possess sweetening
`power and can be included in preparations for
`diabetic use, where sucrose is undesirable. Other
`less widely used bulk sweeteners include maltilol,
`lactilol, isomalt, fructose and xylitol. Treacle, honey
`and liquorice are now very rarely used, having only
`a minor application in some extemporaneously
`prepared formulations.
`Artificial sweeteners can be used in conjunction
`with sugars and alcohols to enhance the degree of
`sweetness, or on their own in formulations for
`patients who must restrict their sugar intake. They
`are also termed intense sweeteners because, weight
`for weight, they are hundreds and even thousands of
`times sweeter than sucrose and are therefore rarely
`required at a concentration greater than about
`0.2%.
`Only about six artificial sweeteners are permitted
`for oral use within the European Union, the most
`widely used being the sodium or calcium salts of sac-
`charin (E954). Both exhibit high water solubility and
`are chemically and physically stable over a wide pH
`
`
`
`
`
`318
`
`Ex. 1014 - Page 5 of 15
`
`

`
`
`
`Daan Cromme/in, Ewoud van Winden, Albert Mekking
`
`1
`
`i
`‘
`‘*
`
`
`lntrediictiohv
`.
`—
`*
`i
`“Protein structures '_544
`~ Sourcesof pharma'ceutica|
`.
`' ‘Specifi.e>cha|Ienges
`‘.545; :4
`
`"rotein_s'i“
`
`, y
`
`lINTaoDUcTIoN
`
`Protein structures
`
`
`
`C
`fl
`
`K
`tr
`
`‘ u‘
`to
`W]
`be
`“Cl
`d“
`‘of
`1 P“
`
`.
`
`'
`
`
`
`
`
`Pharmaceutical proteins are built up of amino acids
`chains (their primary structure). To be phar1naco~
`logically active, this amino acid sequence must form
`a well defined three-dimensional structure. Parts of
`the protein will fold in locally identifiable, discrete
`structures such as oz helices or [3 sheets (known as
`secondary structures). The overall (tertiary) struc-
`ture of the protein is established by the proper
`positioning of the different subunits relative to eacfi.
`other. In some cases individual protein moleculem
`form a quaternary structure, in which the individ ii
`protein molecules interact and build a larger, 11,
`defined structure (e.g. haemoglobin).
`‘.
`The formation and stability of the secondary, t ,
`tiary and quaternary structures is based on rel"
`tively Weak physical interactions (e.g. electrost 1?
`Z
`interactions, hydrogen bonding, van der
`forces and hydrophobic interactions) and not
`covalent chemical-binding principles. Rep
`.
`energy between apolar parts of the protein ..'
`Water are responsible for hydrophobic interacti
`The physical forces involved are relatively
`This means that protein structures can be
`easily changed, leading to a modification or 4..
`loss of their pharmacological characteristics.
`Amino acid chains can be modified by coval
`attaching non—amino acid sections, such as
`(glycoproteins), phosphate or sulphate gr011 L
`particular, the sugar part can make up a subs»
`part of the molecular Weight of the (g1YC0)P
`These groups may be essential for the P11 ‘
`logical effect of a therapeutic protein, not only"
`acting at its receptor sites, but also to PTO "'
`proper pharmacokinetic profile.
`1
`Pharmaceutical protein molecules are 1 '9“
`diffusional transport through epithelial barf.1
`
`
`
`'.:._é.J;i‘..2.-....-9.’'-.
`
`
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`pharniacehticél
`pa'renteraI .éd,mihistrati,on'. 3545,
`7‘ _Stab'ilityrlssu'es'I645"
`__
`‘ y_.,PhySical:inSlabi|ity''‘_'545;’
`_Ch[emlCi;lI;if)stabi|ity,—‘
`is _Excipients_f.used,
`
`
`1" Anatyfigau.tegtatsuss.ta1c;saa.;te;az.;;i,
`; proteins 549
`
`
`
`"
`
`'
`
`'
`
`be
`
`if
`
`7 A
`
`at 1
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`‘
`
`Administration _of'ptiarmaceutie Cproteinys,-V7550‘
`j3l?io:uté;fs..ofadministration 5502
`j Release colfitroli 552 ’
`‘
`T Concluding remarks7i»,553_l ~
`
`'
`I:
`
`i
`
`-V
`
`i
`
`‘_‘Bibliograpl_1y
`
`553’
`
`"
`
`
`
`
`
`
`Ex. 1014- Pae6of 15
`
`Ex. 1014 - Page 6 of 15
`
`

`
`>f amino acids
`
`ets (known as
`zrtiary) struc-
`
`be pharmaC°'
`1°Cmustform
`fture' Pfms Of
`lablei dlscrete
`‘Y the proper
`*1_3UVe to each
`am _mOIe.Cu1e:
`I-he mdmdua
`3 larger’ We“
`gcondary’ter‘
`ased on rel?‘
`- °1€°"°S"““°
`n der Waals
`and not On
`Prom .
`: interactions.
`atively weak.
`an be rather
`ition or even
`ristjcs,
`by covalently
`ugh as 81183‘
`36 groupS-
`a substantial
`§1YCo)protem-
`Le pharII13‘°'
`10: only WI“
`) provide ‘h’
`d
`ire large “ch
`
`1:
`
`barrief5 5“
`
`p
`
`ferred three—dimensional structure is
`readily irre—
`versiblydisturbed (e.g.throughheat,changesinpHor
`ionicstrength). Someanalyticalapproachestomonitor
`theprotein structure are discussedlater.
`The preferred shelf—life for pharmaceutical prod-
`rmulated as aqueous solutions, even
`whenkeptintherefrigerator.Therefore, theyhave to
`be stored in a dry form and be reconstituted before
`administration.These delicate structures are usually
`dried byfreeze—drying (see Chapter 26).The choice
`of the proper excipients (e g lyoprotectants) has
`proved to be extremely important.
`Sources of harmaceutical
`roteins
`p
`Nowadays most proteins used in therapy or under
`.;;:::::::“:.2;:§::d“::.:m“:.°I.::::::;:?:lA:1:
`b.
`gy
`.
`. gy
`iotech products). Examples are human insulin, ery—
`thropoietin, monoclonal antibodies, cytokines and
`lnterferons.They are all produced in cell cultures by
`prokaryotic or eukaryotic cells,
`ranging from
`Escherz'chz'a coli to mammalian cells such as Chinese
`hamster ovary cells, or transgenic animals From the
`examples listed one may conclude that many ofthe
`Pharmaceutical proteins are basically endogenous
`Pfoducts. However, a number of currently used
`biotech products are not exactly identical
`to the
`endogenous product For example, the glycosylation
`Patterns of the recombinant form may be repro—
`I dllcibly produced on a large scale, but not completely
`match the endogenous product. Extensive evaluation
`_ gfithese products in clinical trials has proved their
`‘ Cacy and safety
`.
`
`Specmc challenges
`It IS clear thatpharmaceutical proteins offer special
`challenges to the pharmaceutical formulator They
`are delicate, large molecules with many functional
`changed In Vivo this may directly affect the interac-
`tionwiththereceptor, change theirpharmacokinetic
`characteristics, e.g their clearance, or make them
`immunogenic. Moreover, theirepithelialpenetration
`capability is very low unless the proper transporter
`molecules are available Thus, as a rule, pharmaceu-
`tical proteins are administered parenterally
`In the sections that follow several issues will be
`dealt with in more detail.
`M
`.
`O
`i
`p
`- ,.
`,
`FORMULATION OF OHAOMAOEOTIOAL
`ROTEINS FOR PARENTERAL
`ADMINISTRATION
`Stabim issues
`In the introduction to this chapteritwas pointed out
`that pharmaceutical proteins are high molecular
`weight molecules with amino acid building blocks
`that are sensitive to degradation and with a specific
`three—dirnensional structure. Table 35.1 lists
`the
`pathways for degradation ofproteins.
`.
`.
`..
`Physical Instability
`Degradation rates depend on environmental
`conditions and the formulatorshould carefullyselect
`conditions for optimal
`stability. For example,
`
`EX. 1014- Pae7of 15
`
`545
`
`Ex. 1014 - Page 7 of 15
`
`

`
`
`
`,
`
`( I
`
`Pd
`C]
`1'1:
`
`I
`
`'
`
`
`
`H2N..... Asn.....COOH ——> H2N..... Asp.....COOH + —NH2
`
`‘
`
`'0'
`Oxidation
`H2N..... Met.....COOH —+ H2N..... Met.....COOH
`
`Disulphide scrambling
`r"—'"I
`
`In
`
`H2N..... Cys..... Cys..... Cys..... COOH +——+ H2N..... Cys..... Cys..... Cys..... COOH
`\
`/
`
`H2N.....Cys.....Cys.....Cys.....COOH
`1j____?1
`
`’
`‘
`Oligornerization
`‘
`n(H2N.....Cys.....Cys.....COOH)
`
`F>-
`
`7 "Aggregation
`
`*
`
`n(H2N
`
`COOH) —>
`
`(H2N
`
`COOH)n
`
`Cross-linking
`
`r\J
`
`n(H2N .
`
`.
`
`. .. COOH) —>
`
`H2N -
`
`-
`
`- -- COOH
`
`T
`n(H2N.....Cys.....Cys.....COOH)
`\
`n(H2N.....Cys.....Cys.....COOH)
`L
`
`
`
`;~Am’CUPY
`
`L
`
`H2N..l... cooH
`
`/\J
`
`\/\
`
`Denaturation
`
`
`
`NH2
`
`——>
`
`a(as
`
`re
`HI]
`re;
`an
`4
`te1'1
`3“x
`ion
`
`scrzI\
`lsor
`aha]
`
`
`
`
`
`Ex.1014—Pae8of15
`
`Ex. 1014 - Page 8 of 15
`
`

`
`)H
`
`Chemical instability
`
`full
`Because of the many amino acids involved,
`prevention of all chemical degradation reactions is
`difficult. The formulator should consider which
`chemical degradation pathways are relevant. Under
`neutral conditions the peptide bonds between amino
`acids are stable; only the asparagine—glycine and
`asparagine—proline bonds are relatively labile.
`Deamidation is a rather common degradation
`reaction in water. Asparagine and glutamine are the
`amino acids that can be deamidated. Deamidation
`reaction kinetics depend on pH and neighbouring
`amino acids.
`Oxidation is not limited to methionine and cys-
`teine (Table 35.1): histidine, tryptophan and tyro-
`sine are also sensitive to oxidation reactions.
`Oxidation is catalysed by traces of transition metal
`ions. An oxidative milieu may also cause free cysteine
`units to form disulphide bridges or disulphide bond
`scrambling.
`Naturally occurring amino acids are in the L form.
`Isomerization to the D form is possible and will
`change the structure of the protein.
`Improper choice of excipients may also cause
`degradation reactions. For example, sugars are often
`
`Table 35.2 lists the excipients used in protein-
`containing parenteral dosage forms. Not all of the
`ingredients listed are always needed, e.g. many phar-
`maceutical proteins are sufficiently soluble in water.
`This is in particular true for highly glycosylated mol-
`ecules. In this case no solubility—enhancing sub-
`stances
`are needed. However,
`if
`solubility
`enhancement
`is necessary,
`the selection of the
`proper pH conditions should first be considered.
`Protein solubility depends on its net charge. In
`general, as with low molecular weight drugs,
`uncharged protein molecules (at the pH of their iso-
`electric point,
`i.e.p.) have the lowest solubility in
`water. Therefore, choosing pH conditions ‘away’
`from the i.e.p. can solve the protein (and the
`problem). Some amino acids (e.g. arginine and
`lysine) increase protein solubility and reduce aggre-
`gation reactions by a not—well understood mecha-
`nism. Detergents such as polysorbate 20 and 80 or
`sodium dodecyl sulphate can also be used to prevent
`aggregation. These compounds prevent the adsorp-
`tion of proteins to interfaces (air/water and con-
`tainer/water) and thereby interface-induced protein
`unfolding. Human serum albumin has a strong ten-
`dency to adsorb to interfaces and may therefore be
`added to therapeutic protein formulations as an anti-
`aggregation agent.
`
`
`—; Table 35.21’ Excipients,’used iniiparenteral ‘dosage forms’and their function
`Excipient
`Function
`
`J
`
`Examples
`
`increase solubility of proteins
`Reduction of adsorption and
`aggregation prevention
`Stabilizing pH
`Growth inhibition in vials
`for multiple dosing
`Prevent oxidation
`
`Preservation of integrity while in dry form
`Ensure isotonicity
`
`Amino acids, detergents
`Albumin, detergents
`
`Phosphate, citrate
`
`Phenol, benzylalcohol, organic Hg-compounds
`
`Ascorbic acid, sulphites, cysteine
`Sugars
`
`Sugars, NaCl
`
`
`
`Solubility-enhancing substances
`‘ Antiadsorbent/aggregation
`blockers
`Buffer components
`Preservatives
`
`3 Antioxidants
`Stabilizers during storage
`(lvoprotectants)
`Osmotic compounds
`
`
`'
`
`Ex. 1014- Pae9 of15
`
`Ex. 1014 - Page 9 of 15
`
`

`
`
`
`_of the product in the secondary drying stage.
`
`Microbiological requirements
`
`Typically, pharmaceutical proteins are administered
`via the parenteral
`route. This implies that
`the
`product should be sterile. In addition, virus and
`pyrogen removal steps should be part of the
`purification and production protocol.
`Pharmaceutical proteins cannot be sterilized by
`autoclaving, gas sterilization or ionizing radiation,
`because these procedures damage the molecules.
`Therefore, sterilization of the end—product is not pos-
`sible. This leaves aseptic manufacturing as the only
`option.
`
`alcohol and phenol are often used for this purpose.
`Buffered aqueous protein solutions may be stable
`for 2 years under refrigerator conditions. Some mon-
`oclonal antibody formulations,
`for example, are
`available as aqueous solutions, but
`the more
`common situation is that the formulation has to be
`
`freeze—dried in the vials to avoid degradation and to
`ensure that the product can be readily reconstituted.
`During freeze-drying (Chapter 26) water
`is
`removed by sublimation.
`In the freeze—drying
`process three discrete phases can be discerned. The
`first is freezing of the solution to temperatures typi-
`cally around —35 to —40°C, followed by a sublima-
`tion phase with temperatures of around —35°C and
`low pressures to remove the frozen water (phase 2),
`and a final, secondary drying stage to remove most
`residual water. The pressure must remain low, but
`the temperature can rise up to about 20°C without
`collapse of the porous cake (see below). A lyoprotec-
`tant (e. g. sugar) is necessary to stabilize the product
`as the removal of water may irreversibly affect the
`protein structure. Moreover, sugar lyoprotectants
`also happen to form readily reconstitutable porous
`cakes.
`
`The freezing temperature should be low enough to
`convert the aqueous solution with the sugar and the
`protein into a glass. Glass formation in sugar solu-
`tions usually occurs around —30°C. Just below the
`glass transition temperature the sublimation process
`can begin during the lowering of the pressure in the
`chamber. The sublimated water is collected on a
`
`All utensils and components must be presterilized
`(by heat sterilization,
`ionizing radiation or mem-
`brane filtration) before assembling the final formula-
`tion to minimize the bioburden. Protein products are
`manufactured under aseptic conditions in class 100
`areas (fewer than 100 particles > 0.5 um per cubic
`foot). This low level contamination is reached by
`filtration of air through HEPA (high-efliciency par-
`ticulate air) filters. Finally, the product is filled into
`the containers through sterile filters with 0.22 pm
`pores before capping or freeze drying/capping.
`Pharmaceutical proteins are produced by livinl
`organisms. Viruses can be introduced into th3_
`product either by the use of contaminated cultuzfi
`media or via infected (mammalian) production c
`It is therefore important that purification and In
`condenser with a considerably lower temperature
`facturing protocols contain viral decontamina
`(typically —60°C). As sublimation extracts a large
`steps.Viral decontamination can be accomplishe
`amount of latent heat from the system, the tempera-
`virus removal and/or by viral
`inactivation.
`ture in the vials containing the frozen protein solu-
`problem faced when selecting inactivationltcv
`tion could fall even lower
`than the starting
`niques is
`that
`there is often a narrow "‘
`temperature, slowing down sublimation. The vials
`between successful viral inactivation and pres
`are therefore heated in a controlled way to keep them
`tion of the integrity of the pharmaceutical pr
`at temperatures low enough to preserve their glassy
`structure.
`_
`_
`
`
`texture, but high enough to let
`the sublimation
`Viruses can be removed by filtration, pr6C1P1
`
`
`process proceed at a sufficiently high speed.
`or chromatography. For virus inactivation, heat —'”
`
`
`The mechanism(s) of action of lyoprotectants
`ment (pasteurization), radiation or crosslinkiflg 3
`5‘ f
`
`
`(non-reducing sugars) are not fully understood. The
`(e.g.
`fl-propiolactone) can be used. AS no "
`
`
`process guarantees complete virus removal:
`following may play a role:
`
`548
`
`.
`
`
`
`EX. 1014-Pae 10 of15
`
`
`
`Ex. 1014 - Page 10 of 15
`
`

`
`in vivo tests, use of test animals
`in vitro tests (sensitive cells)
`immunological tests
`ELISA
`RIA
`
`Analytical approaches
`Spectroscopic
`UV spectroscopy
`fluorimetry
`CD spectroscopy
`infrared spectroscopy
`mass spectrometry
`
`Electrophoretic approaches
`SDS-PAGE
`iEF
`
`High-performance liquid chromatography (HPLC)
`GP (gel permeation)
`Hl (hydrophobic interaction)
`Affinity chromatography
`IEC (ion exchange)
`RP (reversed phase)
`
`CD, circular dichroism;
`ELISA, enzyme-linked immunosorbent assay;
`IEC, ion-exchange chromatography;
`IEF, isoeiectric focusing;
`HPLC, high-performance liquid chromatography;
`MALDI, matrix-assisted laser desorption ionization;
`MS, mass spectrometry;
`"
`FHA, radioimmunoassay;
` j SDS-PAGE, s
`odium dodecyl sulphate polyacrylamide gel electrophoresis;
`
`
`
`stage.
`
`ministered
`;
`that
`the
`virus and
`irt of the
`
`erilized by
`radiation,
`molecules.
`is not pos-
`1.8 the only
`
`resterilized
`or mem-
`
`al formula-
`roducts are
`1 class 100
`
`l per cubic
`‘eached by
`:iency par-
`filled into
`
`1 0.22 ,um
`ping.
`I by living
`into the
`ed culture
`ction cells-
`and manu-
`taminatiofl
`
`tplished by
`ition. The
`tion tech-
`V window
`_ preserva—
`:al protein
`
`'ecipitau'on
`heat Heat‘
`(mg agent5
`no Single
`)V31: Often
`
`pharmacopoeial criteria, and this can be done, for
`example, through anion-exchange chromatography.
`
`ANALYTICAL TECHNIQIUIES TO
`CHARACTERIZE PROTEINS
`
`It is clearly important to be able to guarantee the
`integrity of a protein. As mentioned earlier, a protein
`
`TableV3i5.3 Approaches toiconfirm protein structure,
`Approach
`
`provides unequivocal structural evidence.
`Therefore, a set ofpharmacological, immunological,
`spectroscopic, electrophoretic and chromatographic
`approaches is used to characterize the protein as
`closely as possible. Table 35.3 lists a number of regu-
`larly used analytical techniques and the information
`that is obtained.
`Quality assessment used to be based on func-
`tional
`tests in vivo (relevant animal models). An
`example is the pharmacopoeial test for insulin: the
`lowering of the blood glucose level in rabbits upon
`
`information obtained
`
`Pharmacological effect
`Functional test
`
`Interaction with one epitope on protein
`interaction with one epitope on protein
`
`Secondary/tertiary structure
`Secondary/tertiary structure
`Secondary/tertiary structure
`Secondary/tertiary structure
`Secondary/tertiary structure
`
`Molecular weight
`lsoelectric point
`
`Molecular weight/aggregates
`Hydrophobic interactions
`interaction with specific ligand
`Charge patterns
`
`EX. 1014- Pae11 0115
`
` r 1
`
`Ex. 1014 - Page 11 of 15
`
`

`
`que“
`becal
`Tk
`piasn
`dire?‘
`reiat“
`mlght
`Steps:
`may I
`Sh°,rt"
`net“: I
`presen
`may tr
`able’
`1
`exampl
`PEG?”
`Comp“
`
`
`
`-.
`
`be slow and incomplete. Oral vaccines containing
`antigenic protein material are an exception to the
`general rule that proteins should not be adminis-
`tered orally. With vaccines, even low uptake levels
`may still deliver sufficient material
`to lymphoid
`tissue just below the epithelium (in the so—called
`Peyer’s patches) to induce a strong (both local and
`systemic) immune response.
`When a protein is delivered intravenously clearance
`from the blood compartment can be fast, with a half-
`life of minutes, or slow, with a half—life of several days.
`An example of a rapidly cleared protein is tissue plas-
`minogen activiator (tPA), with a plasma half—life of a
`few minutes. On the other hand, human monoclonal
`antibodies have half-lives of the order of days.
`Protein drugs
`are often administered sub-
`cutaneously or intramuscularly. These routes of
`administration are considered to be more patient
`friendly and the injection process easier than with the
`intravenous route. Upon intramuscular
`(i.m.) or
`subcutaneous (s.c.) injection the protein is not instan-
`taneously drained to the blood compartment. Studies
`monitoring the fate of a protein upon s.c. injection
`demonstrate that passage of a protein through the
`endothelial barrier lining the local capillaries at the site
`
`
`
`
`
`Lymphrecovery(%ofdose)
`
`70’
`
`60-
`
`50-
`
`40 —
`
`30 _
`
`|FN-oc~2a
`
`Insulin
`
`Cytochrome C
`
`
`
`O
`
`F.UdFlIIIIIII|
`2
`4
`6 8101214161820
`Molecular weight (kDa)
`
`and RIA (radioimmunoassay) belong to the class of
`immunological tests. Here the interaction of a mon-
`oclonal antibody with one epitope region on the
`protein is determined. The rest of the molecule is
`not ‘probed’.
`Electrophoretic techniques such as SDS-PAGE
`(sodium dodecyl sulphate—polyacrylamide gel elec-
`trophoresis) and IEF (isoelectric focusing) are pow-
`erful tools to assess product purity and to provide
`molecular weight and isoelectric point (i.e.p.) infor-
`mation regarding the protein.
`Table 35.3 lists a number of chromatographic
`techniques that elucidate product characteristics. In
`particular, impurities and degradation products can
`be picked up at an early stage. Gel chromatography
`discriminates mainly on the basis of molecular size
`and is a powerful technique to monitor aggregate
`formation. Ion-exchange resins separate on the basis
`of subtle variations in protein charge patterns and
`are being used to detect oxidation (e.g. methionine)
`and
`deamidated
`(converted
`glutamine
`and
`asparagine) products. Modern mass spectroscopic
`techniques such as MALDI—TOF (matrix-assisted
`laser desorption ionization—time of flight) mass
`spectroscopic analysis, or a combination of HPLC
`(high-pressure liquid chromatography) with electro-
`spray ionization-induced mass spectrometry give
`detailed information on amino acid sequence and
`glycosylation patterns.
`In conclusion, to ensure pharmaceutical protein
`quality one must follow a strict protocol regarding
`the definition of the protein production cell lines
`used, the chosen culturing conditions and down-
`stream processing conditions, and the filling/
`(drying)/ finishing process. Analytical approaches
`to confirm the protein structure will always include
`a long list of approaches, ranging from in vivo tests
`in animals to information provided by highly
`sophisticated analytical technologies.
`None of these tests tells the whole story; together
`they tell more, but
`there is never the situation
`encountered with many low molecular weight
`molecules whereby a full description of the drug,
`including‘ a detailed impurity profile, is available.
`
`
`
`
`
`
`
`0 O
`
`Fig. 35.1 Correlation between the molecular weight 3"d "
`cumulative recovery of recombinant interferon (IFN <1-Zali
`-
`cytochrome C, inulin and 5—F|uoro-2’-deoxyuridine lF”dHl‘
`
`efferent lymph from the right popliteal lymphnode follo T‘
`administration into the lower part of the right hind I99 0'
`
`(from Crommelin and Sindelar, 1998)
`
`
`550
`
`EX. 1014—Pae 12 of15
`
`
`
`Ex. 1014 - Page 12 of 15
`
`

`
`
`
`10
`
`O
`
`I
`15
`
`I
`I
`I
`12
`9
`6
`Days after so administration
`PEG-IL-2 pharmacokinetics and pharmacodynamics
`Fig. 35.2
`(changes in blood lymphocyte count) after subcutaneous
`adminstration of 10 MIU/kg in rats. PEG = poly(ethylene glycol);
`(from Crommelin and Sindelar 1998)
`
`in the number of blood lymphocytes) is observed
`long afterwards (can be days) (Fig. 35.2).
`Finding alternatives for the parenteral route has
`been an area of interest for many years. Table 35.4
`lists different possible routes of delivery for proteins.
`With the exception of the pulmonary route, all
`other options have a low bioavailability. Some
`bioavailability data on the intratracheal administra-
`tion of proteins in rats are shown in Table 35.5. The
`extent of absorption depends strongly on the nature
`
`Route
`
`Nasal
`
`Pulmonary
`
`Rectal
`
`Buccal
`
`Transdermal
`
`Relative advantage
`
`Easily accessible, fast uptake, proven track record
`with a number of ‘conventional’ drugs, probably lower
`proteolytic activity than in the GI tract, avoidance of first-pass effect,
`spatial containment of absorption enhancers is possible
`Relatively easy to access, fast uptake, proven track record
`with ‘conventional’ drugs, substantial fractions of insulin
`are absorbed, lower proteolytic activity than in the GI tract,
`avoidance of hepatic first-pass effect, spatial containment of
`absorption enhancers (?)
`
`Easily accessible, partial avoidance of hepatic first-pass effect,
`probably lower proteolytic activity than in the upper parts
`of