-_°=aoSNo=xudate)N=°°oNoN4a@#<x2a4OozOo2Ooz>0z4udNLea
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`BONITO
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`John A.
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`TTe]
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`BONITO
`
`John A.
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`TTe]
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`
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`onacid-free
`
`Ma |attheaddressbelow.
`
`This bookis printed
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`
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`r
`
`4
`
`Preformulation
`Development of
`
`Parenteral Biopharmaceuticals
`
`JOHN A. BONTEMPO
`
`
`
`
`Biopharmaceutioal Product Development, East Brunswick, New Jersey
`
`I.INTRODUCTION
`92
`A.Considerations of Domestic and International
`Distribution of the Product
`93
`
`Points to Consider for Constituted Versus
`B.
`
`Lyophilized Fonnulations
`93
`c.Unit Dose or Muleidose
`94
`
`D.Physicochemical Factors to Be Considered for
`Protein Drug Formulations
`
`94
`
`II, INITIAL PREFORMULATION STUDIES:
`
`PARAMETERS AND VARIABLES TO BE TESTED
`95
`95
`A.Initial Variables to Be Tested
`B.Preliminary Analytical Development
`96
`
`C.Experimental Conditions for the Initial
`
`
`Preformulation Studies
`
`97
`
`Ill. MECHANICAL AND PHYSICAL STRESSES
`
`99
`A.Shaking Effect on Protein Solution at the
`
`Preformulation Level
`99
`
`Freeze-Thaw Experiments
`B.
`
`100
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`C. Aggregation
`D, Denaturation
`E. Precipitation
`F. Adsorption
`
`VI.
`
`SUMMARY
`
`REFERENCES
`
`1.
`
`INTRODUCTION
`
`104
`105
`105
`106
`
`106
`
`107
`
`Preformulation research studiesof protein therapeutics encompass biophar-
`maceutical, physicochemical, and analytical investigations in support of
`subsequent stable formulations for preclinical, clinical, and market usage.
`In this highly competitive protein therapeutics field, it is very impor-
`(ant to obtain significant, measurable progress with preformulationsstudies
`in a timely manner. How extensive these studies are will depend on the
`availability of the crude, active drug substance and the intended route
`of administration. Most often, these studies begin with extremely small
`amounts of crude bulk active substance and, as more material becomes
`available with greater purity, more studiesareinitiated,
`From an industrial point of view, the preformulation studies are de-
`signed to cover a wide range of properties in a short time to learn as much
`as possible, but not in great depth. The pharmaceutical formulation scien-
`tist is very much interested in identifying potential problems early enough
`to evaluate potential alternatives to stabilize future formulation(s).
`
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`Manyglobaljoint ventures and partnerships today in the biopharmaceuti-
`cal industry dictate various pharmaceutical, clinical, and marketing strate-
`gies, The regulatory requirements and acceptance of formulation excipi-
`ents, packaging components, unit dose versus multidose product, and
`stability conditions vary from continent to continent. Constituted and/or
`lyophilized dosage forms must also be considered. The developmentoffor-
`mulation considerations should be on a worldwide acceptancebasis,
`
`B, Points to Consider for Constituted Versus Lyophilized
`Formulations
`
`Someofthe key points to be considered for a constituted formulation are:
`* Aconstituted formulation maybe less stable than a lyophilized one
`* Effect of agitation during manufacturing and shipping
`*
`Interaction of the liquid with the inner wall of the glass vial and
`with the elastomeric closure
`* Aggregation problems
`* Head space within the vial
`* Preservative effectiveness
`
`Someof the key points to be considered for a lyophilized formulation
`
`are:
`
`* Better stability than a constituted product
`* Determination of an optimal lyophilization cycle
`* Effects of residual moisture on the activity and stability of the product
`* Ease of reconstitutability. Clinicians, nurses and trained home us-
`ers, prefer reconstitutability of the product within two minutes.
`
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`Cc. Unit Dose or Multidose
`
`The decision to select unit dose versus multidase should be based upon
`inputfrom clinical investigators, focus groups, marketing surveys, and com-
`petitors’ products. A multidose formulation will require significantly more
`time for development,
`The multidose will require the screening and incorporation of com-
`patible preservative(s) with the protein formulation. This formulation will
`be tested to determine ifit is efficacious enough to meet the United States
`Pharmacopeia (USP) requirements. Meeting these requirements, it can
`qualify as a “multidose” for the U.S. market. However,if the formulation
`is also designated for international market, there are three additional fac-
`tors that must be taken into account. Thefirst is that for the “antimicrobial
`effectiveness test,” a particular country may or may not accept the preserv-
`ative selected. Secondly, the concentrations of the preservative present in
`the formulation maybe different from the USP requirements. Thirdly, the
`time periods required for the inhibition of the bacteria and fungi strains
`tested may also differ, Consequently, I strongly suggest that the interna-
`tional regulatory requirements for compliance should be well researched
`and understood by the scientific and management staff. Other excipients
`should also be thoroughly reviewed for international acceptance.
`
`D. Physicochemical Factors to Be Considered for
`Protein Drug Formulations
`
`Someof the most important physicochemical properties of protein drugs
`required for the development of parenteral preformulations and formula-
`tions are found in Table 1.
`
`
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`pH Surface denaturation
`
`Since this may be an early stage of process development, some of the
`properties listed in Table 1 may not be available initially, simply because
`there was not enough time or personnel to perform the work.
`
`il.
`
`INITIAL PREFORMULATION STUDIES: PARAMETERS
`AND VARIABLES TO BE TESTED
`
`The pharmaceuticalformulationscientist will consider several factors in the
`preformulation designs. The data received from the Process/Purification
`section are reviewedfor structure, pH and purity of the substance, prelimi-
`nary bioassay, and an immune assay used in terms of semiquantitative
`measurements,
`Other important information that may or may not be available are
`product solubility, preliminary stability, potential degradation routes. From
`personal experience, there is only minimal crude bulk active substance at
`this early stage.
`
`A.
`
`Initial Variables to Be Tested
`
`Perhaps 10 or more initial preformulation combinations should be consid-
`ered. Theinitial variables to be tested with various protein concentrations
`are the effects of buffer species, ionic strength, pH range, temperature,
`
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`two different methods that are “stability indicators,” most often fully vali-
`dated, Dr. Sharma, in Chapter6, will cover the bioanalytical development.
`
`TABLE 2 Bioanalytical Methodsto EvaluateInitial Preformulation Development
`Method
`Function
`
`Bioassay
`
`Immunoassay
`
`pH
`SDS-PAGE (Reduced &
`nonreduced)
`RP-HPLC
`
`|EF
`
`SE-HPLC
`
`N-terminal sequencing
`UV
`
`CD (circular dichroism)
`in the UV region
`
`Measure of activity throughout shelf life of a
`formulation
`
`Purity assessment and measures concentra-
`tion of a particular molecular species
`Chemical stability
`Separation by molecular weight, characteriza-
`tion of proteins and purity
`Estimation of purity, identity, and stability of
`proteins. Separation and analysis af pro-
`tein digests,
`Determinesthe isoelectric point af the protein
`and detects modifications of the protein
`Method of separating molecules according
`to their molecular size and purity
`determination
`Elucidation of the C-terminus,identity
`Detection of individual component, concentra-
`tion, and aggregation
`Detects secondary and tertiary conformation
`and quantitates various structures
`
`
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`pH Range
`
`Initially, a range of pHs should be selected, for example, 3, 5, 7 and 9. Spe-
`cific pH units will be determined during the formulation studies. The pH
`changes may have varying impacts on thesolubility and stability of the for-
`mulation. pH control in pharmaceutical dosage formsis very critical (1).
`The proper pH selection is one of the key factors in developing a stable
`product.
`
`Buffers
`
`The buffer(s) selection should be made from the USP physiological buffers
`list and should be selected based upon their optimal pH range. Some of
`these buffers are acetate pH 3.8-5.8, succinate pH 3.2-6.6, citrate pH 2.1—
`6.2, phosphate pH 6,2-8.2, and triethanolamine pH 7.0-9.0, These pH
`ranges will differ from protein to protein.
`Buffer concentrations should be in the range of 0.01 to 0, molar con-
`centration, As buffer concentration goes up, so does the pain upon injec-
`tion. In selecting the proper buffer, phosphate should be the last in one’s
`choice. Phosphate buffer reacts with calcium from the glass vial and zinc
`from the rubber stopper to cause glass laminates and eventually haziness
`of the solution during stability periods.
`
`Other Excipients to Be Considered
`
`As it was stated previously, the objective of a preformulation study is to
`select potentially compatible excipients in orderto hasten the development
`of stable formulations. Based upon protein chemical and physical instabil-
`
`
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`Antioxidants
`
`Since oxidationis one of the majorfactors in protein degradation,it is highly
`recommended,should the use of a specific antioxidant be required, to in-
`clude into the preformulation an antioxidant such as ascorbic acid, sodium
`disulfide, monothio-glycerol, or alpha tocopherol. The role of an antioxi-
`dant is to deplete or block a specific chain reaction. Antioxidants will be the
`preferential target and eventually be depleted, or may block a specific chain
`reaction. Argon and/or nitrogen gascan also be usedto flood the head space
`of a vial or ampule duringsterile filling to prevent or retard oxidation. A
`recommended antioxidant dose would be about 0.05 to 0.1%.
`Preservatives
`
`If a multidose formulationis required, an antimicrobial agent,called pre-
`servative,is required to be incorporatedinto the formulation, The preserv-
`ative effectiveness must comply with the USP requirementsto be qualified
`as multidose. The most often used preservatives and respective concentra-
`tions are phenol (0.3 to 0.5%), chlorobutanol (0.3 to 0.5%) and benzyl al-
`cohol(1.0 to 3.0%). Additional details are provided in Chapter5.
`Surfactants
`
`Judicious selection of surfactants can result in the prevention of aggrega-
`tion and stabilization of proteins (2). Polysorbate 80, poloxamer 188, and
`pluronic 68 have been used in injectable formulation. The purity of the
`surfactant may have an impact on the chemicalstability of the preformula-
`tion. Peroxide residues in the surfactant have been implicated in oxidations
`of protein.
`
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`ceiving. Simulation of some of the functions described above need to be
`
`Membranefiltration is the most often used techniqueto sterilize protein
`solutions. The chemical nature of the filter and the pH ofthe protein solu-
`tion are the two mostimportant factors affecting the protein adsorption(3).
`However, there are other issues that require consideration, The formula-
`tion scientist must be aware ofparticles or fibers released duringthefiltra-
`tion, the potential extractables that may occur, the potential toxicity of the
`filter media and the product compatibility with the membrane. Of al] the
`filters tested (unpublished data) polyvinylidene difluoride, polycarbonate,
`polysulfone, and regenerated cellulose were found to be the most compat-
`ible with various proteins and with minimal amountsof protein binding and
`deactivation.
`
`to achieve greater compatibility, flexibility, low levels of particulates, and
`machinability. In addition, adsorption, absorption, and permeation through
`the stopper are essentially eliminated. Extensive details may be found in
`Chapter8.
`Membrane Filter Selection
`
`lil. MECHANICAL AND PHYSICAL STRESSES
`
`A. Shaking Effect on Protein Solution at the
`Preformulation Level
`
`Someof the various physical modesofvialed protein solution can undergo
`begin with the bulk active formulation,filling of formulated solution into
`vials or ampules, visual inspection, labeling, packaging, shipping, and re-
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`These experiments will also be describedin later chapters andwill be part
`of Chapter 5. These experiments require a fair amountof active drug sub-
`stance as well as a fair amount of work.At this point of developmentthere
`may not be enough active drug substanceavailable,
`
`C. Filling Systems
`
`Ofall the filling types employed to dispenseliquid, such as time-pressure,
`piston, and rotary pump,the rolling diaphragm metering pumpis the one
`ofchoice for filling biopharmaceuticalsolutions. The internalparts ofthe
`pump do not come in contact with one another where the liquid solution
`flows. This is the “TL Systems Rolling Diaphragm Liquid Metering Pump”
`(4). One of the most important features of this pumpis that it eliminates
`the principal cause of particulate generation which is most often induced
`by parts coming togethercreating shedding of microscopic particles.
`There are three other important parameters to control while dispens-
`ing protein solutions. (1) The speed at whichliquidis filled into the vials.
`With protein solutions the maximum speed is between 25 to 30 vials per
`minute, delivering 0.5 to 2.0 mL volume per 5- or 10-mL vial per single
`filling head. If a large numberofvials need to befilled,this filling system
`can accommodate variable numbers offilling heads, thus allowingit to fill
`a large numberofvials, Filling at a faster rate will result in protein precipi-
`tation and aggregation. (2) The inner diameterofthefilling cannula should
`not be so very small as to induce shearing and aggregation of the protein
`solution, (3) Thetip of the cannula for the filling head should be bent at
`such an angle as to deliver the fluid against the inner wall of the vial and
`not perpendicular to the bottom ofthevial, This will result in a gentle flaw
`
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`Continueif stable
`
`TABLE 3 Guideline for Preformulation Stability Studies
`Temperatures Timepaint
`Frozen controls (-80 and -20°C)
`Reference control sample as needed
`Refrigerated (2-8°C)
`T = 0, 6, 12, 24 & 48 weeks
`Continue if stable
`T=0, 4, 8, 12, 18, 24 weeks
`Continue if stable
`T=0,1, 2, 4,8, 12 weeks
`
`fers refrigerated temperature storage between 2 and 8°C.
`The presentstorage conditionsset up by the USP on storage require-
`ments are as follows:
`
`* Cold storage. Any temperature between 2 and 8°C
`* Cool. Any temperature between 8 and 15°C
`* Room temperature. Temperature prevailing in a working area
`* Controlled room temperature. Temperature controlled thermo-
`statically between 15 and 30°C
`« Excessive heat. Temperature exceeding 40°C
`
`Table 3 summarizes the initial guideline time points and tempera-
`tures that preformulation solutions should be exposed to. The results from
`the preformulationswill allow the review team to determine directions to
`manipulate the excipients to obtain better stability,
`
`
`
`Intermediate (20, 30, 37°C)
`
`High temperature (40, 45, 50°C)
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`to understand how proteins degrade, how theyare affected by the compo-
`sition of the formulation, and the effects ofstability conditions. The major
`pathways of protein degradation are chemical and physical. Under chemi-
`cal degradation, changes and modifications occur due to bond formation
`or cleavage, yielding new chemicalentities. One or moreofthe following
`can occur: oxidation, deamidation, hydrolysis, racemization, isomerization,
`beta elimination, and disulfide exchange. Physical instability can occur in
`the form of denaturation, aggregation, precipitation, and adsorption with-
`out covalent changes.
`
`A. Oxidation
`
`Oxidation of protein is perhaps one of the most common degradation
`mechanisms that can take place during various stages of the processing,
`such as fermentation, purification, filling, packaging, and storage of the
`biopharmaceuticals, Under oxidative stress and in the presence of trace
`metals, amino acids such as methionine (Met) can be oxidized to methion-
`ine sulfoxide, cysteine (Cys) to cysteine disulfide, as well as tryptophane
`(Try) and histidine (His) via other modifications.
`Oxidation can be controlled or minimized by (1) the addition of an-
`tioxidants, (2) havingstrict controls on the processing operations, (3) using
`nitrogen gas to flood head space of the container.
`Oxidized human growth hormone (hGH) retains only 25 percent the
`activity of the native molecule, recombinant interferon-beta loses consid-
`erable antiviral activity due to oxidation (5). Oxidation can be detected by
`reversed phase HPLC (RP-HPLC), high-performanceisoelectric chroma-
`tography (HP-IEC), peptide mapping, amino acids analysis, and mass spec-
`
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`Hydrolysis is another mostlikely cause of degradation ofproteins. It in-
`volves a peptide (amide) bondin the protein backbone (5). The most influ-
`ential factor affecting the hydrolytic rate is the solution pH.
`
`D. Racemization
`
`Proteins may also degrade via other modifications (10) such as racemiza-
`tion. This mechanisminvolves the removal of the alpha proton from an
`amino acid in a peptideto yield a negatively charged planar carbanion. The
`proton can then be replaced into this optically inactive intermediate, thus
`producing a mixture of D and L enantiomers (2). Racemization can yield
`enantiomers in both acidic and alkaline conditions.
`
`E.
`
`Isomerization
`
`Protein degradation is also induced by isomerization. Hydrolysis of cyclic
`amidesof asparagine, glutamine, and aspartic acid will result in isomeriza-
`tion. Low pH accelerates hydrolysis of asparagine and glutamine. However,
`high pH accelerates hydrolysis of aspartic acid and glutamic acid (2,11,12).
`
`F. Disulfide Exchange
`
`Disulfide exchange may result from a degradation other than covalent
`modification. These reactions may include the disulfide exchange ofcys-
`teine. This reaction is base, catalyzed and promoted bythiol antioxidants
`(13).
`
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`A. Covalent Aggregation
`This pathway involves modification of the chemical structures resulting in
`new chemical structures and may include reactions, such as oxidation, de-
`amidation, proteolysis, disulfide interchanges, racemization, and others.
`
`B. Noncovalent
`
`This instability may be induced byagitation, shear, precipitation, and ad-
`sorption to surfaces.
`
`C. Aggregation
`Protein aggregation derived from either physical or chemicalinactivation,
`is presently a major biopharmaceutical problem (17-21). Aggregation can
`be either covalent or noncovalent, occurring during any phase of product
`development from purification to formulation, An early detection of aggre-
`gation via biochemical or spectrophotometric methods,or both, can be of
`significant guidance to formulationscientists in selecting compatible excipi-
`ents to minimize and/or prevent its formation in the experimental formu-
`lation.
`Formation of aggregation can begin by the formationofinitial parti-
`cles from protein molecules via the Brownian movement. Thisis followed
`bycollision of these molecules and aggregatesofvarying sizes can be formed.
`These aggregates can be generated by shearorcollisional forces (22).
`Detection and measurementsof aggregations can be performed by a
`numberof techniques. Visual observations,light scattering, polyacrylamide
`gel electrophoresis, UV, spectrophotometry,laserlight diffraction particu-
`
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`Denaturationofproteins can be the result of several processes and reported
`by several investigators (27).
`Factors which induce denaturation are heat or cold, extreme pHs,
`organic solvents, hydrophilic surfaces, shear, agitation, mixing,filtering, shak-
`ing, freeze-thaw cycles, ionic strength, and others. Thermal inactivation
`processes will induce conformationalside reactions and destruction of amino
`acids (28). The loss of biological function may well be attributed to the
`effect of the temperature on the higher-ordered structure of the protein.
`Thermal denaturationofproteinsis of great interest to the formulation
`scientist. Thermal probesoffer tools to study protein structure andstability
`that ultimately can beofsignificant use to stabilize protein drug formula-
`tions. Modifications of protein thermaleffects have been reviewed (8).
`Theability of the protein to refold from a denatured state, a reversible
`heat denaturation, is also of considerable interest for the stability of a pro-
`tein formulation. These processes of renaturation are very complex (29),
`and each protein does have its own unique renaturation mechanisms.
`Since filtrations and volume reductions occur from the fermentation
`to process purification,thereis very likely inactivation of the protein attrib-
`utable to shearing effect.
`
`E. Precipitation
`
`Precipitation in formulations can occur by a variety of mechanismssuch as
`shaking, heating,filtration, pH, and chemical interactions, Aggregation is
`the initial onset of precipitation. The protein molecules form aggregations
`of varying sizes first, and later when the aggregates reacha critical mass,
`precipitate out of solution and are clearly visible.
`
`
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`air during mixing (process),filtration (process), and air in the process steps,
`a significant surface area has been encounteredto yield interphases.
`During the actual final manufacturing of vials. ampules, syringes,
`catheters, pumps, and their respective storage conditions, the proteins
`could be adsorbedat the interphase and removed from the solution.
`Several researchers (22,33,34,35,36) have investigated these bio-
`chemical mechanism problems. Since proteins have surfactant charac-
`teristics, they have a high affinity to adsorptionat the air—liquid and solid-
`liquid interphase. Hydrophobic and hydrophilic interactions which are con-
`centration dependent, determine the extent and the rate of adsorption. The
`adsorption effect on the protein is the unfolding of the protein. When this
`occurs at an interphase, it can lead to (1) inactivation of the protein solu-
`tion, (2) insoluble protein aggregates being formed at the adsorbedsite, (3)
`additional conformational changes occurring, and (4) chemical degrada-
`tion of the protein continuing duringstability periods.
`
`Vi. SUMMARY
`
`Theinitial critical parameters of preformulations have been addressed in
`this chapter, The formulation team, at this point of development,will re-
`view and evaluateall the results obtained from the preformulation studies.
`The pharmaceutical formulator will design several approachesfor the next
`stage of formulation development taking into accountall the parameters
`that may achieve one or more stable marketable formulations. In the for-
`mulation studies ahead, a numberofstabilizing ingredients should be con-
`sidered to achieve acceptable industrial stability.
`
`
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`
`. Fischer G, et al. Biochem Biophys Acta 1984; 87:791.
`. Bachinger HP, J Biol Chem 1987; 262:17144.
`. Kenney J, et al. Lymph Res 1986; 5:23.
`. Constantino RH,et al. Pharm Res 1994; 71:21.
`. Kenney WC,et al. Lymph Res 1986; 5:23.
`. Shihong L, et al. Pharm News 1995; 2:12.
`. Klibanov AM. Adv Appl Microbiol 1983; 29:1.
`. Manning MC,Patel K, Borchardt RT. Pharm Res 1989, 6:903.
`. Weiss M. Genetic Engineering News 1994, Jan.
`. Creighton TE.Proteins: Structures and Molecular Properties. New York: WH
`Freeman, 1984.
`. Scopes RK. Protein Purification Principles and Practices. 2d ed. Berlin: Sprin-
`ger-Verlag, 1987.
`. Glatz EC. Chemical and physical pathways of protein degradation. In: Ahern
`JT, Manning M,eds.Stability of Protein Pharmaceuticals, Part A. Plenum Press,
`1992.
`. Dingledine M,et al, Pharm Res Abstracts 1993; 10:5-82.
`. Watson E, Kenney WC. J Chromatogr 1988; 436:289.
`. Quinn R, Andrade JD. J Pharm Sci 1983; 72:1472.
`. Cantor CR, Timasheff SM. The Proteins. Vol. 5, 3d ed. New York; Academic
`Press, 1982:145,
`. Shirley AB. Chemical and physical pathways of protein degradation. In: Ahern
`JT, Manning M,eds, Stability of Protein Pharmacsuticals, Part A. Plenum Press,
`1992.
`. Volkin BD, Middaugh ER. Chemical and physical pathways of protein degra-
`dation. In: Ahern JT, Manning M, eds. Stability of Protein Pharmaceuticals,
`Part A. Plenum Press, 1992.
`. Jaenicke R. Prog Biophys Molec Biol 1987; 49:117.
`. Mizutani T. J Pharm Sci 1980; 69:279.
`
`
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`Biopharmaceutical Product Development, East Brunswick, New Jersey
`
`I.
`
`FORMULATION REQUIREMENTS
`A. Characterization, Homogeneity, and
`Reproducibility of the Bulk Active Drug
`B, pH Effect on a Formulation
`C. Stabilizers Used in Protein Formulations
`D. Surfactants
`E. Buffer Selection
`F. Polyols
`G. Antioxidants
`H. Antimicrobials (Preservatives)
`lL Tonicity
`
`fl. CONTAINER-CLOSURE INTERACTIONS
`A. Glass Vials
`B. Leakage Tests
`C. Plastic Vials
`D. Sorption of Preservatives by Plastic
`E.
`Siliconization of Elastomeric Closures
`F.
`Siliconization of Vials
`
`110
`
`110
`111
`11)
`111
`114
`114
`114
`116
`117
`
`117
`117
`117
`118
`119
`120
`120
`
`ang
`
`
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1026, p. 21 of 54
`
`
`
`Investigational New Drug (IND) Requirements
`D.
`E. Formulation Development Scale-Up Considerations
`F, Summary
`
`REFERENCES
`
`129
`140
`14]
`
`141
`
`i. FORMULATION REQUIREMENTS
`
`Preformulation Evaluation From the preformulation studies, there should
`be some key parameters that can be ofsignificant ‘aid in the designs of
`experimental formulations. These key parameters are (1) Initial compati-
`bility testing of the active drug substance with some excipients, (2) Effect
`ofstability factors such as temperature,light, packaging components, (3)
`Initial degradation products in the preformulation, and (4) the perform-
`anceof stability assays for the preformulation,
`The following are some of the major considerations to be taken into
`the experimental formulation designs:
`
`A. Characterization, Homogeneity, and Reproducibility
`of the Bulk Active Drug
`
`Characterization, homogeneity, and lot-to-lot reproducibility of the bulk
`active drug substance is of paramount importance. At this stage of dosage
`form development, a great deal of characterization of the bulk has been
`obtained, Regulatory compliance demandsthat the process in place yields
`reproducibility of the active drug substance, as well as whatever impurities
`
`
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1026, p. 22 of 54
`
`
`
`however, oxidation of methionine, cysteine, and tryptophan can occur, as
`well as other degradative mechanisms (2), The optimal pHis essential for
`better stability.
`Achangeof one pH unit will change the reaction one way oranother.
`The solution pH maybe one ofthe mosteffective ways ta stabilize a liquid
`formulation (3).
`
`C. Stabilizers Used in Proteln Formulations
`
`Degradation ofproteins can be a major biopharmaceutical problem during
`purification, characterization, preformulation, formulation development,
`and possibly during storage. Selective excipients are incorporated into the
`formulation in order to improve the physical and chemicalstability of the
`protein drug substance.
`A variety of molecules have been used as stabilizers, such as surfac-
`tants, amino acids, polyhydric alcohols, fatty acids, proteins, antioxidants,
`reducing agents, and metal ions. Someof the most often used excipients are
`stabilizers, and an explanation for their modeof action has been reported
`in the literature andlisted in Table 1 (4-19).
`
`D. Surfactants
`
`Protein surfactant interactions have also been investigated by other re-
`searchers (20-22). Most recently, the interaction of Tween 20, Tween 40,
`Tween 80, Brij 52, and Brij 92 were studied with recombinant human growth
`hormone and recombinant human interferon gamma for surfactant:protein
`binding stoichiometry.
`
`
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1026, p. 23 of 54
`
`
`
`Surfactants
`Polysorbate 20 & 80
`Poloxamer 407
`
`Fatty Acids
`Phosphotidy! choline
`Ethanolamine
`Acathyliryptophanate
`Polymers
`Polyathylens glycol (PEG)
`Polyvinylpyrrolidone (PVP) 10, 24, 40
`Polyhydric alcohol
`Sorbitol
`
`Mannitol
`Glycerin
`Sucrose
`Glucose
`Propylene glycal
`Ethylene glycol
`Lactose
`Trehalose
`Antioxidants
`Ascorbic acid
`Cysteine HCI
`Thioglycerol
`Thioglycalic acid
`Thiasorbitol
`Glutathione
`
`Retard aggregation
`Prevent denaturation
`Stabilize cloudiness
`
`Stabilizer
`
`Stabilizer
`
`Pravent aggregation
`
`Pravent denaturation
`Aggregation
`Cryoprotectant
`May act as antioxidant
`
`Strengthen conformational
`Prevent aggregation
`
`Retard oxidation
`
`
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1026, p. 24 of 54
`
`
`
`This stoichiometric relationship can be applied to protein formula-
`tions to determine stability. Poloxamer 407 (Pluronic F-127) was also tested
`with interleukin-2 and urease resulting in increased stability when the for-
`mulation was subjected to strong agitation (23). Recombinant urokinase
`losses were reduced by the addition of human serum albumin (HSA),
`Tween 80, and Pluronic F-68 (24).
`Interleukin-2 and ribonuclease A, when reconstituted with a variety
`of surfactants, amino acids, sugars and other substances, reduced aggrega-
`tion significantly (25).
`The formation of particulates with a monoclonal antibody wasinhib-
`ited by Tween 80 and recorded by visual and laser light diffraction particu-
`late analysis methods (26).
`Proteins will adsorb at interphases such asliquid/air or liquid/solid.
`When protein molecules are adsorbed they undergo physicochemical
`changes. [Insoluble particles begin to form,eventually resulting in aggrega-
`tion and precipitation and this, in turn, may lead to partial orfull loss of
`bioactivity.
`The addition of surfactants poloxamer 188 (Pluronic 68), or polysor-
`bate to a liquid formulation can prevent or reduce denaturation ofthe pro-
`tein at a liquid/air or liquid/solid interface of the protein in solution (27),
`The most recent literature concerning the use of nonionic surfactants,
`indicate that during bulk storage and usage, hydroperoxides may be formed
`and can degrade manyproteins (28).
`It is for this reason that, when these surfactants are purchased,a client
`must ask the vendorfora certificate of analysis specifying all the tests per-
`formed, including hydroperoxides.
`
`
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1026, p. 25 of 54
`
`
`
`alcohols and carbohydrates. These include mannitol, sorbitol, and glycerol.
`These have been found to stabilize proteins in solution in varying concen-
`tration from 1.0 to 10%. Although the modeof action ofprotein stabiliza-
`tion is not yet clear,it is suggested that the sugar exerts pressure to reduce
`the surface contact between the protein and the solvent (29,30).
`
`G. Antioxidants
`
`Oxidation is one of the major factors in protein degradation. A protein
`solution, from purificatioin to final product for an end user, goes through
`various equipment made of metal, glass, or plastic. At some points during
`the process, the protein solution comes in contact with catalyzing metals
`such as copper, iron, calcium, and manganese, thus inducing the potential
`loss of protein activity. A probablesolutionto this problem will be the in-
`corporation of a compatible antioxidant in the formulation. Some of the
`most often used antioxidants for parenteral preparations are ascorbic acid,
`sodium bisulfite, sodium metabiosulfite, monothio-glycerol, alpha toco-
`pherol, and others. The most frequently used concentrations are in the
`0.1% range and higher. The optimal concentrations are determined by the
`data the formulator obtains from experimental results on a case by case
`evaluation. Nitrogen and argon gasis also used to retard or prevent oxida-
`tive reactions and the gasis used by flooding the head spaceofa vial or
`ampule during sterile filling.
`Antioxidantsfall into one or more of the following categories (31):
`1. Chelating agents, Oxidative reactions catalyzed by metal ions.
`Chelating agents such as EDTA and citric acid de
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