lon in bloicaruaoké'i-
`v. 20. m s (2009 Deal
`lIlMIIIIIIIlIIIlI
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`\ Monomers
`809973 \
`amino acids
`Law adds
`35“
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`Methanogens
`CH4 + CO2
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`Products
`vFA's, aoelate
`alcohols. co2
`H2. female \
`
`make: serge.
`
`Fermem‘ing microbes
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`Chemical biotechnology
`Edited berazuya Watanabe arid George Bennett
`' Pharmaceutical biotechnology
`Edited by William 51?th éfi‘d Da‘Qi’d’ Knight
`'
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`CSL EXHIBIT 1044
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`

`

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`‘ HEW"
`
`ELSEVIER
`
`Available online at www.5ciencedirect.com
`-"’
`"7 ScienceDirect
`
`Volume 20. issue 6, December 2009
`
`
`
`CONTENTS
`”f—__——_——————l
`Abstracted/indexed in: BIOSIS, CAB Abstracts lntemational, 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
`
`610
`
`615
`
`823
`
`633
`
`642
`
`651
`
`659
`
`Kazuya Watanabe and George Bennett
`Editorial overview: Chemical biotechnology: an expanding
`discipline that contributes to sustainable development in the
`21 st century
`
`Masanorl Arita
`What can metabolomics learn from genomics and proteomics?
`
`Taku Uchiyama and Kentaro Miyazaki
`Functional metagenomics for enzyme discovery: challenges to
`efficient screening
`
`Michael J Mclnerney, Jessica R Sieber and Robert P Gunsalus
`Syntrophy in anaerobic global carbon cycles
`
`Kezuya Watanabe. Mike Manefield. Matthew Lee and
`Atsushl Kouzuma
`Electron shuttles in biotechnology
`
`Dayakar V Badrl, Tiffany L Weir. Daniel van der Lelie and
`Jorge M Vivanco
`Rhizosphere chemical dialogues: plant-microbe interactions
`
`Guillermo Gusset
`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 VWlliam Strohl and David Knight
`868
`William R Strohl and David M Knight
`Editorial overview: Discovery and development of
`biophamtaceuticals: current issues
`
`673
`
`678
`
`685
`
`692
`
`700
`
`708
`
`715
`
`722
`
`Richard Stebblngs. Stephen Poole and Robin Thorpe
`Safety of biologics. lessons learnt from TGN1412
`
`Huiluan Li and Marc d‘Anlou
`Pharmacological significance of glycosylation in therapeutic
`proteins
`William R Strohl
`Optimization of Fc-mediated effector functions of monoclonal
`antibodies
`
`Chichl Huang
`Receptor-Fr: fusion therapeutics, traps. and MIMETIBODYT“
`technology
`
`Yves Durocher and Michael Butler
`Expression systems for therapeutic glycoprotein production
`Steven J Shire
`Formulation and manufacturability of biologics
`
`Melody Sauerbom 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 cfinical 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.
`
`DOI 10.1016/50958-1669(09)00160-8
`
`Page 3 of 10
`
`

`

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`,_,_.rs...-\myl~'u-v-faluv-I—’:—
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`
`
`
`
`
`Available online at www.5ciencedirect.com
`
`"3' ScienceDirec’t
`
`Formulation and manufacturability of biologics
`Steven J Shire
`
`
`Curggtp pinion'
`
`
`
`Blollét’éh.- 9.1.9.9 .
`
`
`r
`
`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, lnc.. 1 DNA Way. MSti 96A. S. San Francisco. CA 94080,
`United States
`
`Corresponding author: Shire, Steven J (shiresteveagenecom)
`
`Current Opinion in. laid-technologymzoog, 202708-714
`This review comes from a themed issue on
`1- Pharmaceutical Biotechnology
`- Edited by William Strohl and David Knight
`
`Available online 313t October 2009
`
`I 0958-16695 — see front matter
`‘- © 2009 Elsevier Ltd. All rights reserved.
`
`. DOI 10.1016/j.00pbio2009.10.006
`
`challenges of a marketplace with a competitive environ-
`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-
`sibility to address the needs of patients even in noncom~
`pctitive environments. Thus,
`the development of a
`biologic candidate into a successful DP mu5t address
`all these challenges leading to a set of requirements often
`referred to as the ‘Targct 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-
`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-
`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-
`ciently exchange the components of the chromatographic
`eluent into the components of the formulation for the
`bulk drug substance (DS). The DS 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 DS manufacturing step to the DP in its final
`container/closure system.
`
`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 prOtcins such as Factor VIII. However, with
`the commercialization of recombinant DNA technology,
`Drug substance manufacturing
`Solution exchange technologies
`the shift has been to produce human versions of protein
`drugs from bacterial fermentation or mammalian cell
`After elution from the final chromatographic recovery/
`culture. The pharmaceutical development of these bio~
`purification step a unit operation is required to exchange
`logics requires the selection of a formulation that is based
`the components of the chromatography elution buffer
`on optimizing safety and efficacy while ensuring the
`with the chosen formulation components. This can be
`ability to manufacture the drug while maintaining quality
`done using size exclusion chromatography or binding
`and stability. In addition the formulation needs to be
`followed by elution from ion exchange resins. However,
`appropriately designed for its intended route of admin-
`the use of buffer exchange or size exclusion may
`istration, as well as the ‘marketahility’ of the final
`result in further dilution of protein and large volume
`drug product (DP). The latter is often tied in with the
`handling requirements, whereas bind/clute ion exchange
`www.5ciencedirect.com
`Current Opinion In Biotechnology 2009. 202708-714
`
`Page 4 of 10
`
`

`

`
`
`Formulation and manufacturablllty of biologic: Shire 709
`
`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-
`nology, the protein solution is recirculated under pres-
`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 DS at low to mod-
`erate concentrations of protein drug. However, the emer-
`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 transrnembrane flux
`may lead to a higher concentration gradient at the mem-
`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 [5-7] 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-
`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 TF F process. Moreover, as the viscosity
`increases during protein concentration, the consequent
`reduction in the diffusion coefficient can lead to a very
`high prorein concentration at the membrane causing a
`further decrease in the transmembranc flux. The higher
`viscosity makes it increasingly difficult to remove the
`concentrated protein from the TF F unit, leading to low
`yields for the process that may be economically unaccep-
`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-
`lation to decrease solution viscosity may help address
`some of these manufacturing process limitations. Success-
`ful implementation of a formulation design to decrease
`solution viscosity at high concentration is shown in the
`viscosity profile for an IgGl monoclonal antibody (here-
`after referred to as MAbl), where the addition of appro-
`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-
`actions at high concentration. Increase of the formulation
`www.3ciencedirect.com
`
`Page 5 of 10
`
`Em
`5.
`.Z‘
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`>
`
`Current Opinion in Biotechnology
`
`100
`
`Cone. (mg/mL)
`
`150
`
`200
`
`mscosity versus concentration for an IgG‘i monoclonal antibody (MAb1).
`Formulation without (0) and with (A) added exclpients that decrease
`viscosity.
`
`ionic strength or addition of particular compounds such as
`argininc can decrease these interacrions and viscosity
`[1],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
`MAbl can be attained at a specific transmembrane pres-
`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
`
`
`
`20.0
`15.0
`10.0
`Transmernbrane Pressure (PSI)
`Current Opinion in Biotechnology
`
`‘ 5.0
`
`25.0
`
`The tangential-flow filtration (TFF) flux (L/M2/H) versus transmembrane
`pressure (PSI) at different temperatures for MAbi. inltially at 30 mg/mL
`and at 23 (o), 40 (I). and 46°C (A).
`
`Current Opinion in Biotechnology 2009, 20:708—714
`
`
`
`
`
`
`
`
`
`
`
`
`r
`
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`(v
`.0Sin
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`

`

`710 Pharmaceutical Biotechnology
`
`Figure 3
`
`oo
`
`PON
`
`
`
`constatnt(days-1)
`
`2EC
`
`.9u
`G
`.5L.
`
`E.L’.’
`
`uonapguieaa Viscosity
`
`(cSt)
`
`
`
`(psliep)tutetsuoo
`
`are;
`
`8
`
`ao'"o
`§§§
`
`o
`
`5.0
`
`5.5 6.0
`
`6.5
`
`1.0
`
`7.5
`
`3.0
`
`pH
`
`
`
`Current Opinlon h Biotechnciogy
`
`(A) The pseudo-first order rate constants for lsomen’zatlon (o) and deamldation (I) 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 of MAbl 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.
`
`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
`Bulk Ds storage
`the freezing process. Thus, formulation excipients such as
`Once formulated, the DS needs to be stored in order to
`sugars, which can function as cryoprotectants can help
`stabilize the protein conformation, and are often included
`manage product inventory, that is the results of a man-
`ufacturing campaign may produce large quantities of DS
`[24°,25]. An important consideration of such excipients is
`that they are not prone to crystallization during freezing
`that needs to be stored over a period of time. It is also
`[26]. Mannitol and recently sorbitol, have proven to be
`often desirable to minimize manufacturing campaigns,
`problematic as pr0tein Stabilizers since they readily crys-
`and thus a robus: formulation needs to be provided to
`ensure stability of US that can be as long as five years.‘
`tallize resulting in a phase separation whereby the sugar is
`Freezing of DS bulk solutions is an obvious strategy that
`no longer able to interact and potentially protect the
`protein from damage during freezing [27—29]. Formu-
`has been widely used in the industry [15']. This requires
`lation components that have been shown to be effective
`filtration and filling of large volumes of DS into storage
`as cryoprotectants include disaccharides such as trehalose
`vessels followed by freezing. The filtration using 0.2 pm
`filters may be more difficult at high protein concen-
`and sucrose [24°], although recent evidence suggests that
`trations especially with high viscosity [16,17], and thus
`trehalose may be prone to crystallization during freezing
`formulation to lower viscosity may be beneficial at this
`making it a less attractive choice than sucrose (T Patapoff,
`step. After sterile filtration the freezing step at
`large
`unpublished data). When considering sugars as cryopro-
`volumes can lead to cryoconcentration as a result of water
`tectants it is important to avoid reducing sugars, which
`freezing and exclusion of formulation solutes and protein
`can result in glycation adducts to primary amines [30,31].
`However, sucrose although not a reducing sugar, can
`[18,19]. If the solution/ice mixture is no: mixed during
`thawing, the protein will be exposed to concentration and
`hydrolyze at lower pH resulting in the formation of the
`thermal gradients. In addition the generation ofa matrix
`reducing sugar glucose [32,33]. Sucrose formulations at
`conSisting of ice and protein can result in the alteration
`pH 6 or greater are usually not an issue, but buffer salts
`that are prone to crystallization such as dibasic sodium
`of protein conformation near the ice surface due to
`weakening of hydrophobic bonds and protein adsorption
`phosphate can lead to large decreases in pH (from 7 to 4)
`onto the ice surface [20,21]. The disruption of protein
`and should be avoided [24']. Another important consider-
`structure may result
`in the exposure of hydrophobic
`ation in formulation design for DS frozen bulk storage is
`wwwsciencedireetcom
`Current Opinion In Biotechnology 2009, 20.708—714
`
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`Formulation and manufacturability of biologlcs Shire
`
`711
`
`may impact the stability of the protein drug [51]. In
`particular, oxidation of amino acid residues such as meth-
`ionine has been shown to occur as a result of peroxides in
`the surfacrants [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-
`nogenic response leading to pure red—cell aplasia (PRCA)
`in patients [55]. Other potential leachates, such as tung-
`sten salts and oxides that result from the prooess 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-
`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
`
`Current Opinion ‘n Biolechnoiogy
`
`500
`
`1000
`
`1500
`
`2000
`
`2500
`
`SucrosezProtein (moi/mo!)
`
`The pseudo—first order rate constants at 50°C for aggregation of
`lyophlllzed MAb1 versus sucrose:MAb molar ratio.
`
`Current Opinion in Biotechnology 2009. 20:708-714
`
`a»
`
`h
`
`M
`
`A'
`
`.‘in
`s7Q~—
`
`xv
`x
`
`the glass transition 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-
`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-
`tonic solutions that prevent cell rupture, particularly in
`the case of red cells. However, the use of NaCl should be
`carefully evaluated because at —21°C water and NaCl
`form a eutectic mixture where the NaCl is at 23.3% w/w,
`which could enhance the mobility at —20°C [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-
`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 DS formu-
`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-
`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 DS 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-
`ever, some of the common surfactants that are used such
`as polysorbate 20 and 80 are prone to degradation, which
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`Page 7 of 10
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`

`Figure 5
`
`Current Opinion in Bioiedinology
`
`100150200 250300 350400
`Time (days)
`
`Soluble aggregate kinetics for iyophilized MAb1. Lyophilized
`formulations for sucrosetMAb molar ratio after reconstitution of 250:1
`stored at 5°C (I) and 30°C (0) and sucrosetMAb molar ratio after
`reconstitution of 500.1 stored at 5°C (A) and 30°C (0).
`
`resulted in an isotonic solution after reconstitution with
`
`final proteinzsucrose molar ratio of 250:1 or one that was
`hypertonic at 500:1. Although isotonicity is not necess-
`arily required for SC administration, it may be desirable
`for minimizing injection pain or inflammation [64] or
`tissue damage [65]. Thus, in order to lower tonicity the
`amount of added protectants must be decreased. The
`challenge then is to add sufficient lyoprotectants to attain
`acceptable stability while assuring a final reconstituted
`DP solution that is isotonic. As shown in Figure 5, the
`formulation with a final reconstituted 500:1 sucrosezpro-
`tein molar ratio for MAbl was significantly more stable at
`controlled room temperature (30°C) than at the lower
`250:1 molar ratio. If stability were the only concern then
`this would have been the appropriate choice. However,
`this MAb was going to be used mainly in clinics and
`doctors offices in the US market where refrigeration was
`readily available. At 5°C storage there was little differ-
`ence in stability between the 250:1 and 500:1 molar ratio
`formulations, and thus the lower ratio was chosen to
`assure an isotonic formulation.
`
`Conclusion
`
`The manufacturing of therapeutic proteins requires sev-
`eral unit operations, which subject the protein to different
`environments and stresses. The change in conditions and
`stresses can lead to physical and chemical alterations of
`the protein drug, which may impact safety and efficacy. In
`addition, physical properties such as high viscosity may
`make it difficult to conduct the unit operation success-
`fully to meet its targeted purpo