. Dressman
`of Frankfurt
`'harmaceutical
`~y Fra nkfurt
`many
`
`,. Hughes
`, of Florida
`,f Pharmacy
`lie, Florida
`
`W. Polli
`mithKline
`rria ngle Park
`Carolina
`
`P. Skelly
`ia, Virginia
`
`·e, Indiana
`
`' Pharmacy
`,m
`
`rs
`1ird Edition,
`
`lation,
`
`'On Shargel
`
`Sanford Bolton
`
`Sterile Drug Products
`Formulation, Packaging,
`Manufacturing, and Quality
`
`...
`Michael J. Akers, Ph.D.
`Baxter BioPharma Solutions
`Bloomington, Indiana, U.S.A.
`
`informa
`
`healthcare
`
`New York London
`
`Mylan Ex 1042, Page 1
`
`

`
`First publisht'CI in 2010 by Informa Healthcare, Telephone House, 69-77 Paul Street, London EC2A 4LQ UK.
`
`Simultaneoush- published in the USA by lnforma Healthcare, 52 Vanderbilt Avenue, 7th Floor, New York,
`NY 10017, CSA.
`
`I.nforma Healthcare is a trading division of lnforma UK Ltd. Registered Office: 37-n Mortimer Street, London
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`
`Preface
`
`This book is based
`formulation, packa
`35 years. I have bas.
`any reader who has
`and tables.
`This book is ,
`sterile product field
`basics are presente,
`intended to be a he
`dosage forms, be it
`manufacturing, eng
`chain, purchasing, !
`products. This boo]
`pharmacy schools a
`remain relevant for
`of old technologies
`The advent of
`enteral route of adn
`With continued ad\
`development and 1
`respect to numbers <
`have expanded the
`and technology. He
`university educatio
`external courses.
`This book is ,
`biopharmaceu ti cal
`parenteral science a
`
`1. Product deveh
`(chap. 2-11)
`2. Manufacturing,
`preparing steril,
`3. Quality and reg
`tions, sterility a!
`4. Clinical aspects,
`clinical setting (
`
`Chapters on J
`ment of sterile solu
`traditionally used t,
`cific formulation co
`agents, cryo- and 1)
`and emulsifying ag
`delivery systems, es
`ter 11 focuses on ov1
`how to approach fo
`
`Mylan Ex 1042, Page 2
`
`

`
`10 Formulation of freeze-dried powders
`
`=:=iMULATION OF FREEZE-Of
`
`\Vith the advent of biotechnology medicines, freeze-drying formulation and process develop(cid:173)
`ment have embarked on new heights of importance in the parenteral industry. Roughly 40%
`of commercial biopharmaceutical products are freeze-dried; this percentage likely will keep
`increasing with time. Freeze-drying and lyophilization mean the same thing. Freeze-drying
`perhaps is more accurate because the process involves both freezing of a solution and then
`removing the solvent from that solution that involves drying procedures. Lyophilization means
`to "love the dry state," but the title does not emphasize the cooling/freezing segment. Freeze(cid:173)
`drying involves:
`
`2.
`
`1. Compounding, filtering, and filling drug formulations as solutions into vials historically
`although now more syringes are being used as primary container for lyophilized prod(cid:173)
`ucts. Most of the discussion in this chapter will focus on the vial being the primary
`package.
`Inserting a partially slotted rubber closure on the neck of the vial (Fig. 10-1) and transferring
`the containers into a freeze-drying chamber. If the vial, as well as syringes or cartridges, is
`to be part of a dual-chambered device (lyophilized powder in one compartment, diluent
`solution in the other compartment, separated by a rubber plunger), then no rubber closure
`is inserted prior to lyophilization.
`3. Cooling the product to a predetermined temperature that assures that the solution in all
`containers in the freeze-dryer become frozen.
`4. Adjusting the temperatur~ of the shelf/shelves of the freeze-dryer that is as high as pos(cid:173)
`sible without causing the temperature of any product container to be above its "critical
`temperature" (eutectic temperature, glass transition temperature, collapse temperature).
`5. Applying a predetermined vacuum that establishes the required pressure differential
`between the vapor pressure of the sublimation front of the product and the partial pres(cid:173)
`sure of gas in the freeze-drying chamber that allows the removal of frozen ice from all
`product containers-the process of sublimation.
`6. Increasing the shelf temperature once all the ice is sublimed in order to remove whatever
`remaining water is part of the solute composition to a residual moisture level predetermined
`to confer long-term stability of the drug product.
`7. Completely inserting the rubber closure into the container via hydraulic-powered lowering
`of the dryer shelves.
`8. Removing all freeze-dried containers, completing the sealing (or for syringes/cartridges
`adding the rubber septum), and carefully inspecting eacJ:l product unit (inspection criteria
`for lyophilized products covered in chap. 22, Table 22-4).
`
`This chapter will focus on the formulation of freeze-dried products, whereas chapter 20
`will focus on the process of freeze-drying.
`
`ADVANTAGES AND DISADVANTAGES OF FREEZE-DRYING
`Freeze-drying is required for active pharmaceutical ingredients that are insufficiently stable in
`the solution state. Insufficiently stable means that the drug will excessively degrade in solution
`·within a period of time not amendable to marketing the product as a ready-to-use solution.
`Many small and large molecules are labile in the presence of water and within several days
`to se\·eral weeks will degrade to a point that is unacceptable, usually more than 10% loss
`of activity or potency compared to the label claim amount of active ingredient. Were it not
`for freeze-drying technology, many important therapeutic agents would not be commercially
`available.
`
`_ e 10-1 Partially slott1
`
`Tables 10-1 and 1
`nts general inforn
`-::'le quantitative for
`.:.are at the time of tJ(cid:173)
`ied formulated
`Besides overcomi1
`_::c., also offers the ac
`- be produced by
`~e drying, or steril1
`_ ertain advantag1
`• .:t can be dried wil
`·eel and maintaine
`
`~~-drying process ca
`.:ntainer prior and ·
`:.=rceze-drying als<
`- r powder-produ
`~ring. Volatile con
`• uired and high
`: ·losure. The free:
`.... - that usually ca1
`'."~Auct has been p
`-. must be maint,
`:-<lrying process it:
`as validating the
`• g into the chambe
`
`·i:al freeze-dried p
`:~ or, and appeare
`·tability, suffici1
`.:: shelf-life, suffici
`,.~anisms (sterilit:
`_ - m solution, it m
`
`Mylan Ex 1042, Page 3
`
`

`
`,rs
`
`. and process develop(cid:173)
`ndustry. Roughly 40°0
`ntage likely will keep
`~ thing. Freeze-drying
`>f a solution and then
`Lyophilization means
`2ing segment. Freeze-
`
`into vials historically
`for lyophilized prod-
`11 being the primary
`
`10-1) and transferring
`inges or cartridges, is
`:ompartrnent, diluent
`1en no rubber closure
`
`tat the solution in all
`
`1at is as high as pos(cid:173)
`be above its "critical
`1pse temperature).
`pressure differential
`and the partial pres(cid:173)
`f frozen ice from all
`
`to remove whatever
`level predetermined
`
`c-powered lowering
`
`syringes/ cartridges
`t (inspection criteria
`
`whereas chapter 20
`
`;ufficiently stable in
`degrade in solution
`.dy-to-use solution.
`vithin several days
`10re than 10% loss
`edient. Were it not
`Jt be commercially
`
`~ ' !..AT/ON OF FREEZE-DRIED POWDERS
`
`139
`
`-
`
`re 10-1 Partially slotted stoppers in solution vials prior to loading into freeze-dryer.
`
`Tables 10-1 and 10-2 present two lists of commercial freeze-dried products. Table 10-1
`';"'?Sents general information about these products, whereas Table 10-2 focuses more on the
`-::;,ecilic quantitative formulations for each product. They are not exhaustive and will not be up
`J ate at the time of this publication, but provide excellent representative information about
`_eze-dried formulated products being successfully used to save and affect lives.
`Besides overcoming stability problems by converting a solution to a dry powder, freeze(cid:173)
`·::,ing also offers the advantages of processing the product in the liquid form. Sterile powders
`~ also be produced by other processes (not covered in this book) such as spray-drying, spray(cid:173)
`-e:eze drying, or sterile crystallization followed by powder filling. However, freeze-drying
`-'€ rs certain advantages over other powder production processes including the fact that the
`uct can be dried without the need for elevated temperatures, product sterility is more easily
`..:hieved and maintained, the contents of the dried material remain homogeneously dispersed,
`,
`the reconstitution times generally are faster. Also, for drugs that are oxygen sensitive,
`~
`e-drying is a better powder-producing alternative, because the environment during the
`7ceze-drying process can be an oxygen-free condition and an inert gas can fill the headspace of
`~~ container prior and during closing of the container.
`Freeze-drying also has certain limitations, perhaps the foremost being cost compared
`· other powder-producing processes and certainly more expensive than liquid filling and
`!oppering. Volatile compounds in the formulation could be removed if high vacuum levels
`~ required and high vacuum has been known to increase the extractable levels from the
`-:.ibber closure. The freezing and drying steps are known to cause stability problems with some
`;roteins that usually can be overcome using stabilizers called cryo- or lyoprotectants. Because
`· e product has been previously sterilized prior to loading into the freeze-drying chamber,
`~terility must be maintained during the loading and unloading process and also during the
`-eeze-drying process itself. The ability to maintain aseptic conditions during these processes
`.. · well as validating the sterilization of the freeze-dryer chamber and all connections and gases
`. .:ading into the chamber must be demonstrated.
`
`ATTRIBUTES AND REQUIREMENTS OF A FREEZE-DRIED PRODUCT
`The ideal freeze-dried product has a very pleasing aesthetic appearance (i.e., intact cake, uni(cid:173)
`:orm color, and appearance) (Fig. 10-2), sufficient strength of active ingredient, chemical and
`. hysical stability, sufficient dryness and other specifications that are maintained throughout the
`product shelf-life, sufficient porosity that permits rapid reconstitution times, and freedom from
`microorganisms (sterility}, pyrogens, and particulate matter after reconstitution. Also, after the
`d rug is in solution, it must remain within certain predetermined specifications (e.g., potency,
`(Text continues on page 154.)
`
`Mylan Ex 1042, Page 4
`
`

`
`154
`
`STERILE DRUG PRODUCTS: FORMULATION, PACKAGING, MANUFACTURING, AND DUALITY
`
`(A)
`
`(8)
`
`Figure 10·2 Examples of a pharmaceutically elegant freeze-dry cakes. Source: Courtesies of Eli Lilly and
`Company (A) and Dr. Gregory Sacha, Baxter BioPharma Solutions (8).
`
`pH, freedom from particulate matter) for a certain period of time prior to administration.
`The desired minimum time for solution stability after reconstitution is 24 hours at ambient
`temperature although many products, especially biopharmaceuticals, are insufficiently stable
`at ambient temperature and must be refrigerated even for these short periods of time. Also,
`European requirements that generally have been applied throughout the world require prod(cid:173)
`ucts without antimicrobial preservatives to be used (administered) "immediately," generally
`meaning within three hours after reconstitution. Freeze-dried products reconstituted with dilu(cid:173)
`ents containing antimicrobial preservatives can be stored for much longer times depending
`more on drug stability in solution than on potential microbial contamination concerns.
`Freeze-dried formulation requirements usually are different depending on whether the
`active ingredient is a small molecule or large molecule. Formulation of a freeze-dried product
`containing a small molecule often does not need any additives, depending on the amount of
`active ingredient per container. For example, many freeze-dried antibiotic products contain
`only the antibiotic. If the active constituent of the freeze-dried products is present in a small
`quantity (usually less than 100 mg) where, if freeze-dried alone, its presence would be hard
`to detect visually, then additives are used. This is true for many small-molecule freeze-dried
`products, for example, those containing anticancer agents, and practically always true for large(cid:173)
`molecule freeze-dried products. The solid content of the original product ideally should be
`between 5% and 30%. Therefore, excipients often are added to increase the amount of solids.
`Such excipients are called "bulking agents"; the most commonly used bulking agent in freeze(cid:173)
`dried formulations is mannitol. However, most freeze-dried formulations must contain other
`excipients because of the need to buffer the product and /,or to protect the active ingredient
`from the adverse effects of freezing and/or drying. Thus, buffering agents such as sodium or
`potassium phosphate, sodium acetate, and sodium citrate are commonly used in freeze-dried
`formulations. Sucrose, trehalose, dextran, and amino acids such as glycine are commonly used
`lyoprotectants. Other types of stabilizing excipients often required in freeze-dried formulations
`are surface-active agents or competitive binding agents. Other reasons for adding excipients
`freeze-dried compositions, although typically these are part of the diluent formulation rather
`than the freeze-dried formulation, are tonicity-adjusting agents and antimicrobial preservatives
`for multiple-dose applications.
`Each of these substances contribute to the appearance characteristic of the finished dry
`product (plug), such as whether the appearance of the finished product is dull and spongy
`or sparkling and crystalline, firm or friable, expanded or shrunken, or uniform or striated.
`Therefore, the formulation of a product to be freeze-dried must include consideration not only
`of the nature and stability characteristics required during the liquid state, both freshly prepared
`and when reconstituted before use, but also the characteristics desired in the dried product as
`it is released for commercial use and distributed to the ultimate user.
`
`:;:=/MULATION OF FREEZE-£
`
`A "rule-of-thurr
`:1t:crter" because most
`. : freeze-dried prod
`lecule is different,
`y be as much or n
`radation.
`
`- RMULATION COiii
`=-n:eze-dried drug me
`- ·table molecules. E
`.--: maintaining pH, .
`:: • ing process. Addi
`ili ty and, in somi
`· ent. Such additive
`_ nts, and complexi
`- antimicrobial pres
`• reconstitution dil·
`-:nulations.
`Freeze-dried for
`jpients because of
`container), or add
`powder, bufferin~
`ability of the drug
`. le at least for the
`Stabilizing large
`--< and challenge. Fri
`_ ~ following addit
`· .e.:ule freeze-dried
`: mL levels), contai
`e binders to minir
`...:mg, disposable mi
`·..: . Certain additive:
`...::...allv are avoided t
`·-·tic or glass transi
`~ ,. tion effects on p
`• to be exhaustive
`
`,
`
`Some protein rr
`-rocess of freezini
`-:. cy. Certain exci:
`~;ed.- by freezing a;
`::Zing are called c1
`
`Additive Cal
`
`ers
`gizers" (prevent co
`ize aggregation
`rotection
`_ _ rotection
`· ize surface adsor
`
`• = se temperature mo
`•• modifiers
`
`Mylan Ex 1042, Page 5
`
`

`
`1NUFACTURING, AND QUALITY
`
`FORMULATION OF FREEZE-DRIED POWDERS
`
`A "rule-of-thumb" for freeze-dried products containing small molecules is " the drier, ;:;1,,.
`better" because most stability problems with small molecules are moisture-related. How2 ·t?r
`for freeze-dried products containing large molecules, "drier is not necessarily better. " Eac·
`molecule is different, but in general for large molecules, the effects of freezing and dryin_,
`may be as much or more deleterious to the active constituent as the potential for hydrolytic
`degradation.
`
`FORMULATION COMPONENTS IN FREEZE-DRIED PRODUCTS
`Freeze-dried drug molecules, evidenced by the requirement to be freeze-dried, are relatively
`unstable molecules. Even in the dry state, freeze-dried formulations typically require additives
`for maintaining pH, isotonicity, or protection against adverse effects of the freezing and / or
`drying process. Additives may also be required, not for dry-state purposes, but to maintain
`stability and, in some cases, solubility of the drug in solution after adding a reconstitution
`diluent. Such additives to enhance solution stability and solubility include buffers, surface-active
`agents, and complexing agents. For drugs reconstituted to serve as multiple-dose products,
`an antimicrobial preservative system must be part of the freeze-dried formulation or part of
`the reconstitution diluent. Table 10-3 lists examples of formulation additives in freeze-dried
`formulations.
`Freeze-dried formulations containing small molecules either do not require any additive
`excipients because of the large quantity of drug to be freeze-dried, (typically more than 100 mg
`per container), or additives required are for relatively simple purposes such as adding bulk to
`the powder, buffering the formulation, providing isotonicity, or perhaps helping to maintain
`solubility of the drug. Formulation challenges for small molecule formulations are relatively
`simple at least for the experienced formulation scientist.
`Stabilizing large molecules during freeze-drying requires much more formulation exper(cid:173)
`tise and challenge. Freeze-dried formulations of large molecules typically contain one or more
`of the following additives: bulking agents, lyoprotectants, surfactants, and buffers. Some large(cid:173)
`molecule freeze-dried formulations, typically when the protein content is so dilute (low mg to
`ng/ mL levels), contain human serum albumin or some other component to serve as compet(cid:173)
`itive binders to minimize loss of protein due to adsorption to manufacturing surfaces (filters,
`tubing, disposable mixing bags, stainless steel) and primary container surfaces (glass and rub(cid:173)
`ber). Certain additives such as mannitol and sucrose also may serve as tonicity modifiers. Salts
`usually are avoided because they decrease the critical temperature of the formulation (lower
`eutectic or glass transition temperature) and are known to cause concentration-dependent desta(cid:173)
`bilization effects on proteins. Table 10-2 presents a listing of freeze-dried protein formulations,
`not to be exhaustive but to give the reader an idea of the qualitative composition of these
`formulations.
`Some protein molecules can be adversely affected by the freeze-drying process, that is,
`the process of freezing and / or drying can cause the protein to denature and aggregate and lose
`potency. Certain excipient stabilizers ha\·e been found to minimize or prevent the problems
`caused by freezing and / or drying. Excipients that stabilize the protein against the effects of
`freezing are called cryoprotectants. The primary theory, although not completely accepted,
`
`Table 10-3 Additive Categories and Examples for Freeze-Dried Formulations
`
`Category
`
`Example(s)
`
`Bulking agents
`Stabilizers
`"Ridigizers" (prevent collapse)
`Minimize aggregation
`Cryoprotection
`Lyoprotection
`Minimize surface adsorption
`Buffers
`Collapse temperature modifiers
`Tonicity modifiers
`
`Mannitol, lactose, glycine
`
`Mannitol, glycine
`Polysorbate 20 or 80; poloxamer 188
`Polyethylene glycol, some sugars
`Sucrose, trehalose
`Human serum albumin, polysorbates
`Acetate, citrate, phosphate, Tris, amino acids
`Dextran, polyethylene glycol, disaccharide sugars
`Mannitol, sodium chloride, glycerin
`
`:ourtesies of Eli Lilly and
`
`ior to administration.
`; 24 hours at ambient
`re insufficiently stable
`periods of time. Also,
`e world require prod(cid:173)
`tmediately," generally
`·constituted with dilu-
`1ger times depending
`tion concerns.
`1ding on whether the
`, freeze-dried product
`ing on the amount of
`otic products contain
`is present in a small
`sence would be hard
`nolecule freeze-dried
`always true for large-
`1ct ideally should be
`the amount of solids.
`l.l<ing agent in freeze(cid:173)
`s must contain other
`the active ingredient
`ts such as sodium or
`used in freeze-dried
`! are commonly used
`e-dried formulations
`or adding excipients
`tt formulation rather
`crobial preservatives
`
`: of the finished dry
`is dull and spongy
`uniform or striated.
`nsideration not only
`oth freshly prepared
`:he dried product as
`
`Mylan Ex 1042, Page 6
`
`

`
`::ORMULATION OF FREEZ
`
`:empera ture (T g) in
`_ e physical state o
`_ ult in collapse or
`irtrimental to the s
`.znportant quality p
`Gradual com·
`_ ecre is adequate m
`~ .a term used to de
`~ er to lower the l
`ility of the am
`- ~ us solid is stor,
`occur at tempe
`• _Q). Molecular n
`· resonance (21
`;:_ and 13C solid(cid:173)
`~uity in lyophili
`...\dditives in ,
`molecular WE
`recombinant
`~rature, theret
`, re thought to
`- sral.
`.\1pha1 -anti tr
`;et does no
`
`·
`
`156
`
`STERILE DRUG PRODUCTS: FORMULATION, PACKAGING, MANUFACTURING, AND QUALITY
`
`for explaining the cryoprotective effects of certain additives, is called the "excluded solute"
`or "preferential exclusion" theory (1-3). Some scientists have suggested that solutes that help
`protect the protein from dissociating during freezing do so because they are excluded from
`the surface of the protein, as can be demonstrated by dialysis experiments (where the protein
`and the excipient are not found together in the dialysate). When solutes are excluded from the
`protein surface, the chemical potential of both the protein and the solute increase. This presents
`a thermodynamically unfavorable environment for the denatured form of the protein as the
`denatured form is an unfolded form and yields a greater surface area to the solvent. The native
`form, with less surface area, is therefore thermodynamically favored.
`Another way of explaining the effects of cryoprotectants is the fact that they induce
`preferential hydration of the surface of the protein because by not binding at the protein surface,
`this favors water molecules to bind preferentially and this helps to stabilize the native protein
`state.
`Sugars (sucrose, lactose, glucose, trehalose), polyols (glycerol, mannitol, sorbitol), amino
`acids (glycine, alanine, lysine), and polymers (polyethylene glycol, dextran, polyvinylpyrroli(cid:173)
`done) all serve as potential cryoprotectants. The best or, at least, most preferred cryoprotectants
`appear to be polyethylene glycol (PEG) (molecular weight 3350 Daltons), sucrose, and trehalose.
`For proteins requiring both cryo- and lyoprotection, it may be judicious to employ both
`an agent such as PEG along with a sugar. An example of a marketed therapeutic with this
`combination is Venoglobulin-S, which contains PEG and sorbitol. A potential caveat to using
`PEG in lyophilized formulations is the possibility of a liquid-liquid phase separation induced
`by freeze-concentration, an event implicated in protein unfolding (4).
`Proteins may not denature or experience any loss of potency during freezing or in the
`frozen state, but may experience adverse effects when the sublimation process occurs and when
`stored in the dry state. Such proteins need stabilizers called lyoprotectants. Lyoprotectants
`appear to stabilize proteins from the effects of drying and the dry state by what is referred
`to as the "water-substitute" hypothesis or the "vitrification" hypothesis. Sugars are excellent
`lyoprotectants. They provide a glassy matrix that retards molecular motions and reduces the
`rates of deleterious reactions (5,6). They also decrease protein-protein contacts and inhibit
`deleterious reactions depending on such contacts (e.g., aggregation) (7-9). Sugars serve as water(cid:173)
`replacement substrates that form hydrogen bonds to proteins in the dried state (4). The water(cid:173)
`replacement or substitute hypothesis is supported by solid-state studies exploring techniques
`such as Fourier-transform infrared (10), water sorption (11,12), and dissolution calorimetry (13).
`It is likely sugars have all these possible mechanistic roles in their ability to stabilize proteins.
`Often the same excipient can provide both cryo- and / or lyoprotection. An example of
`cryoprotection is the stabilization effect of sucrose, trehalose, sorbitol, and gelatin on a recombi(cid:173)
`nant adenoviral preparation (14). An example of lyoprotection is the stabilizing effect of lactose
`and other sugars on recombinant human growth hormone (rhGH) (15). However, lactose, a
`reducing sugar, is not preferred because of its potential adduct formation.
`In both dry state theories, it is important that the excipient stabilizer, the lyoprotectant.
`exist in the amorphous state, hence the name "vitrification" (glass formation). Protein stabili~
`in the dry state results from the protein existing with an amorphous solute in an inert, rigi •
`amorphic matrix where the water content in the matrix also helps to stabilize the protein.
`Obviously, too much excess water and the protein will degrade by chemical processes (e.g.
`deamidation), but proteins need a certain amount of water to maintain secondary and higher
`structure. Thus, excipients that remain amorphous during the freeze-dry process molecular!~
`interact with the amorphous protein and together the matrix confers stability on the protein fo(cid:173)
`long-term stability in the dry state. It has been shown that, for optimal stabilization, the sugar
`excipient should remain in the same amorphous phase containing the peptide or protein (ah
`of the above mechanisms are consistent with this observation). For example, crystallization ;
`mannitol has been implicated to explain incomplete stabilization of lyophilized rhGH (16) an::
`the structure of bovine serum albumin, ovalbumin, f3-lactoglobulin, and lactate dehydrogenax
`(LDH) upon freeze-drying (17). In addition to crystallization, separation of amorphous phases
`can also occur, particularly in the frozen state.
`Once excipients crystallize, they no longer molecularly interact with the protein and cann •
`protect it. Amorphous excipients, combined with the protein, have a unique glass transitio;:-
`
`Mylan Ex 1042, Page 7
`
`

`
`1ANLJFACTURING, AND QUALITY
`
`d the "excluded solute"
`ed that solutes that help
`they are excluded from
`1ents (where the protein
`~s are excluded from the
`:e increase. This presents
`:m of the protein as the
`J the solvent. The native
`
`e fact that they induce
`1g at the protein surface,
`bilize the native protein
`
`mnitol, sorbitol), amino
`<tran, polyvinylpyrroli(cid:173)
`·eferred cryoprotectants
`, sucrose, and trehalose.
`dicious to employ both
`:l therapeutic with this
`Jtential caveat to using
`ase separation induced
`
`ring freezing or in the
`'Ocess occurs and when
`!Ctants. Lyoprotectants
`te by what is referred
`s. Sugars are excellent
`>tions and reduces the
`1 contacts and inhibit
`Sugars serve as water(cid:173)
`·d state (4). The water-
`, exploring techniques
`ution calorimetry (13).
`to stabilize proteins.
`:ction. An example of
`l gelatin on a recombi(cid:173)
`lizing effect of lactose
`. However, lactose, a
`
`:er, the lyoprotectant,
`ion). Protein stability
`lute in an inert, rigid
`stabilize the protein.
`nical processes (e.g.,
`econdary and higher
`process molecularly
`ity on the protein for
`1bilization, the sugar
`iptide or protein (all
`)le, crystallization of
`Llized rhGH (16) and
`:tate dehydrogenase
`f amorphous phases
`
`e protein and cannot
`que glass transition
`
`=; - \1LJLATION OF FREEZE-DR/ED POWDERS
`
`perature (Tg) in the dry state. If storage temperature exceeds the glass transition tempera:- _
`physical state of the dried matrix changes from a glassy solid to a rubbery solid tha -.... -
`..:ult in collapse or partial collapse of the freeze-dried cake. Product collapse is not necessa.r_:,·
`:,;,rrimental to the stability of some proteins, although pharmaceutical elegance still remains ar.
`-,portant quality parameter of freeze-dried products.
`Gradual conversion of excipients from the amorphous to the crystalline state occurs i,·hen
`_ -"re is adequate molecular mobility for nucleation and crystal growth (12). Molecular mobility
`_ a term used to describe the movement of molecules in a formulation. Water will act as a plas(cid:173)
`::izer to lower the glass transition temperature of amorphous solids and increase the molecular
`bility of the amorphous system (18). Molecular mobility typically occurs when the amor(cid:173)
`:: ous solid is stored at a temperature greater than its glass transition temperature, but can
`occur at temperatures below the glass transition temperature of certain amorphous solids
`~ -,20). Molecular mobility of protein molecules can be measured by solid-state 1 H nuclear mag(cid:173)
`·tic resonance (21), nuclear magnetic resonance relaxation based critical mobility temperature
`-2), and 13C solid-state nuclear magnetic resonance (23). All these techniques measure water
`- .ability in lyophilized formulations and this can be correlated to protein stability.
`Additives in a formulation can prevent crystallization of carbohydrates. Examples include
`'gh molecular weight polymers (e.g., dextran and polyvinylpyrrolidone, (24) and proteins
`., recombinant bovine somatotropin (BST) (12). Polymers can increase the glass transition
`perature, thereby decreasing the mobility of the amorphous solute, whereas proteins such as
`-:: 5r are thought to interfere with either nucleation rates or number