! ?"c
`O.C y
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`1,'· I
`Hydro car bans
`• •
`\ Methanogens \
`CH4 + C02
`
`Polymers
`cellulose~
`Monomers
`xylans
`sugars ~
`proteins
`amino acids Products
`lipids
`nucleic acids
`fatty acids
`VF A's, acetate
`bases
`alcohols, C02
`H2, formate ~
`I Fermenting microbes I
`acetate
`I Syntrophs I
`H2 + C02
`
`I
`
`t
`
`CSL EXHIBIT 1044
`CSL v. Shire
`
`Page 1 of 10
`
`

`

`Current Opinion in Biotechnology
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`

`

`ELSEVIER
`
`__ ,,
`Available online at www.sciencedirect.com
`·.;~ ScienceDirect
`
`Volume 20, issue 6, December 2009
`
`CONTENTS
`
`Abstracted/indexed in: BIOSIS, CAB Abstracts International, CAB Health, Chemical Abstracts, EM BASE, Index Medicus,
`Medline. Also covered in the abstract and citation database SCOPUS®. Full text available on ScienceDirect®
`
`Reviews
`
`Chemical biotechnology
`Edited by Kazuya Watanabe and George Bennett
`
`607
`
`Kazuya Watanabe and George Bennett
`Editorial overview: Chemical biotechnology: an expanding
`discipline that contributes to sustainable development in the
`21st century
`
`610 Masanori Arita
`What can metabolomics learn from genomics and proteomics?
`
`616
`
`623
`
`633
`
`642
`
`651
`
`659
`
`Taku Uchiyama and Kentaro Miyazaki
`Functional metagenomics for enzyme discovery: challenges to
`efficient screening
`
`Michael J Mcinerney, Jessica R Sieber and Robert P Gunsalus
`Syntrophy in anaerobic global carbon cycles
`
`Kazuya Watanabe, Mike Manefield, Matthew Lee and
`Atsushi Kouzuma
`Electron shuttles in biotechnology
`
`Dayakar V Badri, Tiffany L Weir, Daniel van der Lelie and
`Jorge M Vivanco
`Rhizosphere chemical dialogues: plant-microbe interactions
`
`Guillermo Gosset
`Production of aromatic compounds in bacteria
`
`Shen-Long Tsai, Shailendra Singh and Wilfred Chen
`Arsenic metabolism by microbes in nature and the impact on
`arsenic remediation
`
`Pharmaceutical biotechnology
`Edited by William Strohl and David Knight
`
`668 William R Strohl and David M Knight
`Editorial overview: Discovery and development of
`biopharmaceuticals: current issues
`
`673
`
`678
`
`Richard Stebbings, Stephen Poole and Robin Thorpe
`Safety of biologics, lessons learnt from TGN1412
`
`Huijuan Li and Marc d'Anjou
`Pharmacological significance of glycosylation in therapeutic
`proteins
`
`685 William R Strohl
`Optimization of Fc-mediated effector functions of monoclonal
`antibodies
`
`692
`
`700
`
`708
`
`715
`
`722
`
`Chichi Huang
`Receptor-Fc fusion therapeutics, traps, and MIMETIBODY"'
`technology
`
`Yves Durocher and Michael Butler
`Expression systems for therapeutic glycoprotein production
`
`Steven J Shire
`Formulation and manufacturability of biologics
`
`Melody Sauerborn and Huub Schellekens
`B-1 cells and naturally occurring antibodies: influencing the
`immunogenicity of recombinant human therapeutic proteins?
`
`Patrick Y Muller, Mark Milton, Peter Lloyd, Jennifer Sims
`and Frank R Brennan
`The minimum anticipated biological effect level (MABEL) for
`selection of first human dose in clinical trials with monoclonal
`antibodies
`
`The Cover
`Chemical interactions in anaerobic environments that facilitate the
`decomposition of organic polymers, including plant and animal residues,
`petroleum, and anthropogenic pollutants. Diverse microbes are essential
`for metabolism of organic polymers.
`
`DOl 10.1 016/50958-1669(09)00160-8
`
`Page 3 of 10
`
`

`

`_,,
`Available online at www.sciencedirect.com
`·:;" ScienceDirect
`
`ELSEVIER
`Formulation and manufacturability of biologics
`Steven J Shire
`
`An important challenge in the pharmaceutical development of a
`biologic is the optimization of safety and efficacy while ensuring
`the ability to manufacture the drug while maintaining quality
`and stability. The manufacturing process consists of several
`operational steps referred to as 'unit operations' where the
`biologic is subjected to different stresses and conditions that
`may compromise quality and stability. Moreover, recently the
`requirement for the development of subcutaneous formulations
`for high dose drugs, such as monoclonal antibodies, at high
`protein concentrations has created additional challenges for
`many of the unit operations. These challenges can be mitigated
`by modification of the manufacturing process and/or
`development of formulations to prevent degradation. In
`particular, formulations have been designed to minimize
`protein aggregation and decrease viscosity, which has led to
`successful manufacture of the biologic.
`
`Addresses
`Genentech, Inc., 1 DNA Way, MS# 96A, S. San Francisco, CA 94080,
`United States
`
`Corresponding author: Shire, Steven J (shire.steve@gene.com)
`
`Current Opinion in Biotechnology 2009, 20:708-714
`
`This review comes from a themed issue on
`Pharmaceutical Biotechnology
`Edited by William Strohl and David Knight
`
`Available online 31st October 2009
`
`0958-1669/$ - see front matter
`© 2009 Elsevier Ltd. All rights reserved.
`
`Introduction
`Before the advent of recombinant DNA technology,
`biologics were produced from either animal tissue, for
`example pancreatic tissue for insulin, pituitary glands for
`human growth hormone or blood, for example blood
`coagulation proteins such as Factor VIII. However, with
`the commercialization of recombinant DNA technology,
`the shift has been to produce human versions of protein
`drugs from bacterial fermentation or mammalian cell
`culture. The pharmaceutical development of these bio(cid:173)
`logics requires the selection of a formulation that is based
`on optimizing safety and efficacy while ensuring the
`ability to manufacture the drug while maintaining quality
`and stability. In addition the formulation needs to be
`appropriately designed for its intended route of admin(cid:173)
`istration, as well as the 'marketability' of the final
`drug product (DP). The latter is often tied in with the
`
`challenges of a marketplace with a competitive environ(cid:173)
`ment, where products that more adequately address the
`needs of patients through ease of use, have an advantage.
`However, not to be overlooked is the industry's respon(cid:173)
`sibility to address the needs of patients even in noncom(cid:173)
`petitive environments. Thus, the development of a
`biologic candidate into a successful DP must address
`all these challenges leading to a set of requirements often
`referred to as the 'Target Product Profile' or TPP. In this
`paper we will focus on the challenges and issues that need
`to be addressed during the design, development, and
`scale-up of a formulation to enable consistent and robust
`performance in a manufacturing environment.
`
`The manufacturing of these biologics begins with the
`expression in biologic, cell-based systems of proteins that
`then requires recovery and purification, often by multiple
`chromatographic techniques. The general overall biopro(cid:173)
`duction process is executed in a series of steps often
`referred to as 'unit operations'. Herein lies one of the
`biggest differences between small molecule and biologic
`development. Instead of obtaining a drug candidate from
`chemical synthesis that leads to separate efforts to pre(cid:173)
`pare formulated drug, the manufacturing step for the
`formulation of a biologic is often the last step in the
`recovery and purification process. Thus the final recovery
`and purification step requires a technology that can effi(cid:173)
`ciently exchange the components of the chromatographic
`eluent into the components of the formulation for the
`bulk drug substance (OS). The OS is further subjected to
`additional unit operations to eventually obtain the final
`DP, each of which may subject the therapeutic protein to
`a variety of stresses. Thus, the success of these processing
`steps will be highly dependent on the appropriate design
`of the formulation as well as modifications of process and
`process equipment. The focus of this review will be on
`the interplay between formulation and process design,
`and the needs of key unit operations that occur from the
`initial OS manufacturing step to the DP in its final
`container/closure system.
`
`Drug substance manufacturing
`Solution exchange technologies
`After elution from the final chromatographic recovery/
`purification step a unit operation is required to exchange
`the components of the chromatography elution buffer
`with the chosen formulation components. This can be
`done using size exclusion chromatography or binding
`followed by elution from ion exchange resins. However,
`the use of buffer exchange or size exclusion may
`result in further dilution of protein and large volume
`handling requirements, whereas bind/elute_ ion exchange
`
`Current Opinion in Biotechnology 2009, 20:708-714
`
`www.sciencedirect.com
`
`'. \
`
`·I
`'i
`rl 'I
`.1 , I
`
`Page 4 of 10
`
`

`

`Formulation and manufacturability of biologics Shire 709
`
`Figure 1
`
`80
`70
`
`60
`
`(j)
`ell 50
`a..
`g 40
`.i':'
`.iii 30
`0
`0
`CIJ 20
`>
`10
`
`0 ..
`
`0
`
`•
`
`•
`
`• t•..
`50
`
`chromatography may be limited by ionic strength and
`pH requirements of the elution buffer necessitating
`additional exchange steps. The predominant technology
`has been used in the industry for buffer exchange and
`concentration is a form of ultrafiltration/diafiltration
`called tangential-flow filtration (TFF) [1-3]. In this tech(cid:173)
`nology, the protein solution is recirculated under pres(cid:173)
`sure, tangentially to an ultrafiltration membrane. A
`. pressure differential is maintained across the membrane,
`which allows for the selective passage of water and
`small solutes but not macromolecule of interest. This
`technology works well for formulated OS at low to mod(cid:173)
`erate concentrations of protein drug. However, the emer(cid:173)
`gence of monoclonal antibody therapies for home-use,
`subcutaneous (SC) administration, has necessitated the
`development and manufacture of high concentration
`formulations >100 mg/mL [4 •• ]. The attainment of
`high concentration formulations by TFF systems can
`be difficult because the required transmembrane flux
`may lead to a higher concentration gradient at the mem(cid:173)
`brane boundary. Depending on a protein's propensity to
`interact with and unfold at the surface, this could lead to
`decreased flux and eventual membrane fouling. Proteins
`are sensitive to generated air-water interfaces, resulting
`in denaturation [S-7] and the continuous circulation
`through tubing and pulsation of the pumps may also
`cause cavitation [8], which results in protein unfolding
`and precipitation. Thus, formulations that can stabilize
`the native protein structure when exposed to these stres(cid:173)
`ses may mitigate some of these problems. In addition to
`the stresses encountered by proteins during TFF, another
`potential limitation is the ability to concentrate highly
`viscous protein solutions. In particular, high viscosity may
`lead to high back-pressures, exceeding pump capacity
`during the TFF process. Moreover, as the viscosity
`increases during protein concentration, the consequent
`reduction in the diffusion coefficient can lead to a very
`high protein concentration at the membrane causing a
`further decrease in the transmembrane flux. The higher
`viscosity makes it increasingly difficult to remove the
`concentrated protein from the TFF unit, leading to low
`yields for the process that may be economically unaccep(cid:173)
`table. While some of these challenges can be overcome by
`redesign or modification of the TFF equipment, it can
`lead to a greater expense as well as significant delays in
`development timelines. Design of an appropriate formu(cid:173)
`lation to decrease solution viscosity may help address
`some of these manufacturing process limitations. Success(cid:173)
`ful implementation of a formulation design to decrease
`solution viscosity at high concentration is shown in the
`viscosity profile for an lgG1 monoclonal antibody (here(cid:173)
`after referred to as MAb1), where the addition of appro(cid:173)
`priate excipients lowered the viscosity dramatically
`(Figure 1) [9,10 •• ]. The large viscosity in this antibody
`is caused by reversible protein self-association that
`appears to be governed by electrostatic attractive inter(cid:173)
`actions at high concentration. Increase of the formulation
`
`Current Opinion in Biotechnology
`
`Viscosity versus concentration for an lgG1 monoclonal antibody (MAb1).
`Formulation without (e) and with (.A) added excipients that decrease
`viscosity.
`
`ionic strength or addition of particular compounds such as
`arginine can decrease these interactions and viscosity
`[11,12••]. In situations where greater decreases
`in
`viscosity are required, additional improvements in the
`TFF process can be achieved by running the process at
`higher temperature [13••,14]. At the higher temperatures
`where the viscosity is lower, greater membrane flux for
`MAb1 can be attained at a specific transmembrane pres(cid:173)
`sure (Figure 2). However, in such cases, protein stability
`may be compromised by prolonged exposure to higher
`processing
`temperatures. Formulation design
`thus
`becomes critical for not only lowering the viscosity but
`also controlling degradation at the higher temperatures.
`This can be a very challenging exercise since physical
`degradation and chemical degradation routes may have
`
`Figure 2
`
`100.0
`90.0
`80.0
`g 70.0
`"' ~ 60.0
`>< 50.0
`:::J
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`30.0
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`5.0
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`
`/
`
`I
`
`.....
`
`... _..
`
`---~---
`
`20.0
`15.0
`10.0
`Transmembrane Pressure (PSI)
`Current Opinion in Biotechnology
`
`25.0
`
`The tangential-flow filtration (TFF) flux (UM2/H) versus transmembrane
`pressure (PSI) at different temperatures for MAb1, initially at 30 mg/ml
`and at 23 (e), 40 <•l. and 46"C (6.).
`
`www.sciencedirect.com
`
`Current Opinion in Biotechnology 2009, 20:708-714
`
`••
`•
`•
`•
`•
`
`. ·
`
`& A A
`
`• .....
`100
`Cone. (mg/mL)
`
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`
`Page 5 of 10
`
`

`

`71 0 Pharmaceutical Biotechnology
`
`Figure 3
`
`(a)
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`
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`ca·
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`....
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`
`0.10
`
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`
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`0.07
`
`5.0 5.5 6.0 6.5 7.0 7.5
`pH
`
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`
`8.0
`
`0.010
`
`(b)
`
`180
`
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`. / -------,~'
`
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`'
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`
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`5
`
`/::,.
`
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`
`/::,.
`
`/::,.
`
`/::,.
`
`6
`
`I
`
`a
`
`9
`
`7
`pH
`Current Opinion in Biotechnology
`
`(A) The pseudo-first order rate constants for isomerization (f!) and deamidation (•) versus pH for MAb1. (B) The kinematic viscosity at 25°C versus pH
`for MAb1 at 130 mg/mL.
`
`very different dependencies on solution conditions. This
`is exemplified by the behavior ofMAb1 shown in Figures
`3A and B where the isomerization and deamidation
`degradation kinetics are optimized at a pH where the
`viscosity is at a maximum value. Ultimately it may not be
`possible to control degradation only by formulation
`design, necessitating evaluation, and optimization of
`the impact of the processing.
`
`Bulk OS storage
`Once formulated, the OS needs to be stored in order to
`manage product inventory, that is the results of a man(cid:173)
`ufacturing campaign may produce large quantities of OS
`that needs to be stored over a period of time. It is also
`often desirable to minimize manufacturing campaigns,
`and thus a robust formulation needs to be provided to
`ensure stability of OS that can be as long as five years. -
`Freezing of OS bulk solutions is an obvious strategy that
`has been widely used in the industry [15.]. This requires
`filtration and filling of large volumes of OS into storage
`vessels followed by freezing. The filtration using 0.2 f.Lm
`filters may be more difficult at high protein concen(cid:173)
`trations especially with high viscosity [16,17], and thus
`formulation to lower viscosity may be beneficial at this
`step. After sterile filtration the freezing step at large
`volumes can lead to cryoconcentration as a result of water
`freezing and exclusion of formulation solutes and protein
`[18,19]. If the solution/ice mixture is not mixed during
`thawing, the protein will be exposed to concentration and
`thermal gradients. In addition the generation of a matrix
`consisting of ice and protein can result in the alteration
`of protein conformation near the ice surface due to
`weakening of hydrophobic bonds and protein adsorption
`onto the ice surface [20,21]. The disruption of protein
`structure may result in the exposure of hydrophobic
`
`portions normally found in the interior of the protein,
`and coupled with the increased concentration of protein
`during freezing, can lead to increased protein aggregation.
`Thus freeze-thaw vessel design is very important and
`several systems have been created that ensure efficient
`heat transfer and mixing during the thawing step
`[15.,22,23]. Although the development of freeze-thaw
`systems can minimize the formation of gradients during
`thawing, the protein is still exposed to ice surfaces during
`the freezing process. Thus, formulation excipients such as
`sugars, which can function as cryoprotectants can help
`stabilize the protein conformation, and are often included
`[24.,25]. An important consideration of such excipients is
`that they are not prone to crystallization during freezing
`[26]. Mannitol and recently sorbitol, have proven to be
`problematic as protein stabilizers since they readily crys(cid:173)
`tallize resulting in a phase separation whereby the sugar is
`no longer able to interact and potentially protect the
`protein from damage during freezing [27-29]. Formu(cid:173)
`lation components that have been shown to be effective
`as cryoprotectants include disaccharides such as trehalose
`and sucrose [24•], although recent evidence suggests that
`trehalose may be prone to crystallization during freezing
`making it a less attractive choice than sucrose (T Patapoff,
`unpublished data). When considering sugars as cryopro(cid:173)
`tectants it is important to avoid reducing sugars, which
`can result in glycation adducts to primary amines [30,31].
`However, sucrose although not a reducing sugar, can
`hydrolyze at lower pH resulting in the formation of the
`reducing sugar glucose [32,33]. Sucrose formulations at
`pH 6 or greater are usually not an issue, but buffer salts
`that are prone to crystallization such as dibasic sodium
`phosphate can lead to large decreases in pH (from 7 to 4)
`and should be avoided [24•]. Another important consider(cid:173)
`ation in formulation design for OS frozen bulk storage is
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`the glass transmon temperature Tg' of the ice/water/
`solute frozen matrix. Storage of the frozen formulation
`at temperatures above the Tg' results in greater molecular
`mobility and can result in enhanced degradation, especi(cid:173)
`ally protein aggregate formation [34,35]. Typical Tg'
`values
`for carbohydrate-based
`formulations are at
`approximately -30°C [24•] and thus storage of large
`volumes at temperatures lower than -30°C is desirable,
`but often not practical or economical. Tonicifiers such as
`NaCl are typically added to formulations to create iso(cid:173)
`tonic solutions that prevent cell rupture, particularly in
`the case of red cells. However, the use of N aCl should be
`carefully evaluated because at -21 oc water and NaCl
`form a eutectic mixture where the NaCl is at 23.3% w/w,
`which could enhance the mobility at -zooc [36], a
`temperature typically used for large-scale bulk storage
`[15•]. In addition, the common stainless steel alloy 316L
`used in freezing/storage vessels is susceptible to corrosion
`induced by chloride [37]. The metal corrosion and leach(cid:173)
`ing of metal ions into the protein formulation can lead to
`,, metal-induced oxidation degradation of the protein drug
`
`[38].- Thus development of formulations may require a
`screening of other counterions, which do not promote
`metal corrosion. Development of alternate materials,
`especially plastic disposables for freeze-thaw storage
`systems are attractive but will require a thorough analysis
`of potential leachates from the plastic materials into the
`protein formulation [39,40]. Another consideration as to
`how formulation can benefit this particular unit operation
`is the development of higher concentration OS formu(cid:173)
`lations that can be frozen resulting in a decrease in the
`volumes used for storage, which then require less tank
`storage. Alternatively bulk freeze-drying or spray drying
`formulations could be developed. The literature is
`replete with examples on formulation development using
`such processes [41 •• ,42-50]. Although freeze-drying for
`bulk storage could be done in trays, spray drying would
`require appropriate collection of the powder and then
`aseptic filling into the storage containers.
`
`Formulation and manufacturability of drug
`product
`The manufacturing challenge for DP will be dependent
`on the final form chosen. For liquid products, especially at
`high concentration, addition of viscosity lowering exci(cid:173)
`pients may be required since the viscosity of the product
`can impact sterile filtration before fill, as well as the
`accuracy of the filling equipment. In particular, highly
`viscous liquids may result in excessively long times to
`filter DP as in the case of OS or result in 'hanging drops' at
`the tip of the filling tube resulting in carry over into the
`next vial. Generation of air-water interfaces and pump
`cavitation during filling can induce protein denaturation
`but can be mitigated by the addition of surface-active
`agents such as surfactants to the DP formulation. How(cid:173)
`ever, some of the common surfactants that are used such
`as polysorbate 20 and 80 are prone to degradation, which
`
`www.sciencedirect.com
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`
`Formulation and manufacturability of biologics Shire 711
`
`may impact the stability of the protein drug [51]. In
`particular, oxidation of amino acid residues such as meth(cid:173)
`ionine has been shown to occur as a result of peroxides in
`the surfactants [52].
`
`The choice of the final container/closure may also dictate
`choices for formulation components. Prefilled syringes
`tend to expose the protein to higher levels of silicone oil,
`which can lead to increased aggregation [53,54). Recent
`experience with Eprex illustrates the problems that may
`occur as a result of formulation changes and leachates
`from drug contact surfaces. In that particular example it is
`proposed that addition of the surfactant polysorbate 80
`may have increased the amount of leachates into the
`formulation that enhanced an anti-erythropoietin immu(cid:173)
`nogenic response leading to pure red-cell aplasia (PRCA)
`in patients [55]. Other potentialleachates, such as tung(cid:173)
`sten salts and oxides that result from the process used to
`make staked needle syringes have been implicated in
`increasing the propensity for aggregation [56•]. If this
`problem cannot be handled by formulation then the
`manufacturing process may need to be altered to mini(cid:173)
`mize the levels of tungsten.
`
`In the case of solid, freeze or spray dried dosage forms
`excipients that can act as cryoprotectants as well as drying
`protectants need to be added. Excipients such as sugars
`and surfactants have been shown to be effective in
`protecting the protein from the stresses encountered
`during typical drying techniques such as lyophilization
`or spray drying [19,24.]. A common degradation pathway
`that occurs during lyophilization is protein aggregation,
`which can be controlled by such stabilizers (Figure 4).
`The addition of sugars as proteccive agents during drying
`is believed to occur via one or both of two proposed
`mechanisms. One is the formation of a glassy amorphous
`state where molecular motions are decreased relative to
`
`Figure 4
`
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`2
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`2000
`1500
`1000
`500
`Sucrose:Protein (moVmol)
`
`2500
`
`Current Opinion in Biotechnology
`
`The pseudo-first order rate constants at 5ooc for aggregation of
`lyophilized MAb1 versus sucrose:MAb molar ratio.
`
`Current Opinion in Biotechnology 2009, 20:708-714
`
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`Page 7 of 10
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`712 Pharmaceutical Biotechnology
`
`proteins in solution and the other is through the ability of
`sugars to replace the lost hydrogen bonding from removal
`of water [19,57•]. Recent studies have further elucidated
`how sugars such as sucrose protect proteins during lyo(cid:173)
`philization [41 •• ]. Surfactants when added to solid, dried
`dosage forms are useful in preventing the formation of
`particulates during the reconstitution process. An essen(cid:173)
`tial property of these dosage forms is that the excipients
`remain in an amorphous state since crystallization results
`in a phase separation whereby the stabilizer no longer can
`interact with the protein [28]. Another aspect of solid
`dosage form development is the possibility of developing
`high dosage form