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`4 P
`
`reformulation Development of
`Parenteral Biopharmaceuticals
`
`JOHN A. BONTEMPO
`
`Biopharmacautieal Product Development. East Brunswick. New Jersey
`
`INTRODUCFION
`A. Considerations of Domestic and lntemational
`Distribution of the Product
`8. Points to Consider for Constituted Versus
`
`Lyophilized Formulations
`C. Unit Dose or Multidose
`
`D. Physicoehemieal Factors to Be Considered for
`Protein Drug Formulations
`
`11.
`
`INITIAL PREFORMULA’I‘ION STUDIES:
`PARAMETERS AND VARIABLES TO BE TESTED
`A.
`Initial Variables to Be Tested
`
`B. Preliminary Analytical Development
`C. Experimental Conditions for the Initial
`Preformulation Studies
`
`III.
`
`MECHANICAL AND PHYSICAL STRESSES
`
`A. Shaking Effect on Protein Solution at the
`Preformulation Level
`
`B. Freeze—Thaw Experiments
`
`92
`
`93
`
`93
`94
`
`94
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`95
`95
`96
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`97
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`99
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`100
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`92
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`Bantampo
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`C. FillingSystems
`D. Stability Evaluation
`
`W. DEGRADATION MECHANISMS
`A. Oxidation
`B. Deamidation
`
`C. Hydrolysis
`D. Racemiaation
`E.
`Isomerization
`
`F. Disulfide Exchange
`G. Beta-Elimination
`
`V.
`
`PHYSICALDEGRADATIONS
`
`A. Covalent Aggregation
`B. Noncovaient
`
`C. Aggregation
`D. Denaturation
`
`E. Precipitation
`F. Adsorption
`
`VI.
`
`SUMMARY
`
`REFERENCES
`
`1.
`
`INTRODUCTION
`
`100
`10]
`
`102
`102
`103
`
`103
`103
`103
`
`103
`104
`
`104
`
`104
`104
`
`104
`105
`
`105
`106
`
`106
`
`107
`
`Preformulation research studies of 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‘
`tant to obtain significant, measurable progress with preformuiations studies
`in a timely manners 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 studies are initiated.
`From an industrial point of view, the preforrnulation 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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`Pretormulation Davampmont
`
`93
`
`As previously stated. there must be a strong interdisciplinary collabo-
`ration team to review. identify. and maximize the most productive leads
`toward formulation development. Preformulation studies are short in do—
`ration, two to three months. and some of these are performed undervarying
`stress conditions which will be described later in this chapter. It is important
`to remember that no two proteins are alike and studies designs will vaxy
`case by case.
`Prior to the onset of preforrnulations. the pharmaceutical team must
`review some very important factors which will have an impact on the pre-
`formulation and formulation development.
`
`A. Considerations of Domestic and International
`Distribution ot‘ the Product
`
`Many global joint ventures and partnerships today in the biopharrnaceuti-
`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 andlor
`Iyophilized dosage forms must also be considered. The development of for-
`mulation considerations should be on a worldwide acceptance basis.
`
`B. Points to Consider for Constituted Versus Lyophilized
`Formulations
`
`Some of the key points to be considered for a constituted formulation are:
`
`- A constituted formulation may be 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
`
`Some of the key points to be considered for a lyophilized formulation
`
`are:
`
`- Better stability than a constituted product
`‘- Determination of an optimal iyophilization qrclc
`- 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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`94
`
`Bontempo
`
`' Stability of the reconstituted product
`- Preservative effectiveness (if this is a multidose product)
`- Cost effectiveness. Lyophilization technology is expensive along
`with cost of utilities
`
`At the onset of preformulations studies. it is difficult to predict with
`certainty which of the two types of formulations will have a marketable
`advantage for an extended shelf life. At this early stage of devel0pment,
`there are usually very small amounts of the bulk active drug substance avail-
`able. The formulator must make very efficient use of the active drug sub-
`stance. Nevertheless, both formulations should be considered and started
`
`at the same time. Stability results should be the deciding factor as to which
`form will be selected for further deveIOpment.
`
`G. Unit Dose or Multidose
`
`The decision to select unit dose versus multidose should be based upon
`input from 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 if it is efficacious enough to meet the United States
`Pharmacopeia (USP) requirements. Meeting these requirements. it can
`qualify as a “multidose” {or the US market. However, if the formulation
`is also designated for international market. there are three additional fac-
`tors that must be taken into account. The first 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 [emulation may be 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 escipients
`should also be thoroughly reviewed for international acceptance.
`
`0. Physlcochemlcal Factors to Be Considered for
`Proteln Drug Formulations
`
`Some of the most important physieochemical properties of protein drugs
`required for the development of parenteral preformulations and formula-
`tions are found in Table l.
`
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`Preformulatlon Development
`
`95
`
`TABLE 1 Physicochemlcal Factors to Be Considered for Protein Drug
`Formulations
`
`Structure of the protein drug
`lsoelectrlc point
`Molecular weight
`
`Amino acid composition
`
`Disuilide bonds
`
`Spectral propenies
`Agents effecting solubility:
`Detergent
`Salts
`Metal ions
`
`pH
`
`Agents affecting stability
`pH
`Temperature
`Light
`Oxygen
`Metal ions
`Freeze—thaw
`Mechanical stress
`
`Polymorphism
`Stereoisomers
`Filtration media compatibility
`Shear
`
`Surface denaturatEOn
`
`Since this may be an early stage of process development, some of the
`properties listed in Table i may not be available initially, simply because
`there was not enough time or personnel to perform the work.
`
`II.
`
`INITIAL PREFOHMULATION STUDIES: PARAMETERS
`AND VARIABLES TO BE TESTED
`
`The pharmaceutical formulation scientist will consider several factors in the
`preformMation designs. The data received from the ProcesslPurification
`section are reviewed for 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 Tasted
`
`Perhaps 10 or more initial prefonnulation combinations should be consid-
`ered. The initial variables to be tested with various protein concentrations
`are the effects of buffer species. ionic strength, pH range, temperature,
`
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`96
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`Bantempo
`
`initial shear, surface denaturation, agitation. and aggregation. Since it has
`been well documented that protein solutions are unstable, some selective
`excipients from various classes of stabilizers should also be included in or-
`der to evaluate stability requirements. Stabilizers will be discussed later in
`this volume.
`
`B. Preliminary Analytical Development
`
`In order to determine the initial stability results. it is necessary to have
`developed, or to have under development, analytical methods to measure
`the potency of the specific formulation under various experimental condi-
`tions. Ultimately some of these analytical methods will be needed to moni-
`tor stability to detect physical and chemical degradation. Regulatory com—
`pliance for the beginning of Phase I Clinical Studies may require at least
`two different methods that are “stability indicators." most often fully vali-
`dated. Dr. Sharma. in Chapter I5, will cover the bioanalytical development.
`
`TABLE 2 Bioanatytlcal Methods to Evaluate initial Pretormulation Development
`
`Method
`
`Bioassay
`
`immunoassay
`
`pH
`SOS-PAGE (Reduced 8t
`nonreduced)
`RP-HPLC
`
`IEF
`
`SE-HPLC
`
`Function
`
`Measure of activity throughout shell lite ot a
`formulation
`
`Purity assessment and measures concentra-
`tion of a panicalar molecular species
`Chemical stability
`Separation by molecular weight. characterizes
`tion of proteins and purity
`Estimation of purity, identity. and stability of
`proteins. Separation and analysis oi pro
`tein digests.
`Determines the isoelectric point of the protein
`and detects modifications at the protein
`Method of separating molecules according
`to their molecular size and purity
`determination
`
`N-terminal sequencing
`UV
`
`Elucidation of the C-ten'ntnus. identity
`Detection at individual component. concentra«
`tion. and aggregation
`Detects secondary and tertiary conformation
`CD (circular dichroism)
`
`in the UV region and quantitatas various structures
`
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`Preformulalion Development
`
`97
`
`However. some of the following bioanalytical methods listed in Table 2 can
`be applied to begin initial evaluation of the preformulations degradation
`(if any) under test.
`
`C. Experimental Conditions for the initial
`Pretormulatlon Studies
`
`Protein Concentration
`
`Protein drugs are extremely potent: therefore, very low concentrations are
`required for their respective therapeutic levels. Dosage forms development
`need to be tested at varying ranges of activity. The respective concentra-
`tions may range from nanograms to micrograms to milligrams and the con-
`centration will vary from protein to protein.
`
`pH Flange
`
`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 the solubility and stability of the for-
`mulation. pH control in pharmaceutical dosage forms is very critical (1).
`The proper pH selection is one of the key factors in deveIOping 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 bu ffers 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 0.1 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 Exciplents to Be Considered
`
`As it was stated previously. the objective of a prefonnulation study is to
`select potentially compatible excipients in order to hasten the development
`of stable formulations. Based upon protein chemical and physical instabil-
`
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`93
`
`Bontempo
`
`ity, it is highly probable that some excipients may be included in the prefor-
`mulation. In so doing, the designs of formulations to follow can be more
`specific in selecting the proper excipient(s) to control specific degradation
`pathways.
`
`Cholating Agents
`
`The crude bulk protein drug during fermentation and purification steps has
`passed through and contacted surfaces such as metal, plastic, and glass. If
`metal ions are present in the liquid bulk active. it is highly recommended
`to use a chelating agent such as cthyienediamine tetraacidic acid (EDTA)
`to effectively bind trace metals such as copper, iron, calcium. manganese
`and others. A recommended dose of (EDTA) would be about 0.01 to
`0.05%.
`
`Antioxidants
`
`Since oxidation is one of the major facrors 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 andior nitrogen gas can also be used to flood the head space
`of a vial or ampule during sterile filling to prevent or retard oxidation. A
`recommended antioxidant dose would be about 0.05 to 0.1%.
`Preservatives
`
`If a multidose formulation is required, an antimicrobial agent, called pre-
`servative, is required to be incorporated into the formulation. "the preserv-
`ative effectiveness must comply with the USP requirements to 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»
`coho! (1.0 to 3.0%). Additional details are provided in Chapter 5.
`
`Surfactants
`
`J udicious selection of surfactants can result in the prevention of aggrega-
`tion and stabilization of proteins (2). Polysorbate 80, poloxarner 188, and
`pluronic 68 have been used in injectable formulation. The purity of the
`surfactant may have an impact on the chemicai stabiiity of the prefonnula-
`tion. Peroxide residues in the surfacrant have been implicated in oxidations
`
`of protein.
`
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`Preformulation Development
`
`99
`
`Glass Vial Selection
`
`Type 1 glass. as classified in the USP. should be used. The selection of a
`glass vial must also be taken into consideration when dealing with adsorp-
`tive properties of the respective protein. Adsorption of proteins will be
`treated later in this volume.
`
`Rubber Stopper Selection
`
`In studying both the liquid and reconstituted protein drugs. the selection of
`a rubber stepper is also of major concern considering the potential reactiv—
`ity of a protein solution with a rubber stopper. as well as the reactivity of
`the reconstituted lyophilized solution during storage conditions prior to
`use. For parenteral formulations, the biopharrnaceutical industry has been
`using rubber stoppers with a veryr thin film of various inert polymers in order
`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
`Chapter 8.
`
`Membrane Filter Selection
`
`Membrane filtration is the most often used technique to sterilize protein
`solutions. The chemical nature of the filter and the pH of the protein solu~
`tion are the two most important factors affecting the protein adsorption (3).
`However. there are other issues that require consideration. The female-
`tion scientist must be aware of particles or fibers released during the filtra-
`tion, the potential extractables that may about, the potential toxicity of the
`filter media and the product compatibility with the membrane. Of all 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 amoants of protein binding and
`deactivation,
`
`Ill. MECHANICAL AND PHYSICAL STRESSES
`
`A. Shaking Ettect on Protein Solution at the
`Pretormulation Level
`
`Some of the various physical modes of vialed 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-
`ceiving. Simulation of some of the functions described above need to be
`
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`mo
`
`Bantempo
`
`performed by doing some shaking experiments to determine their affect on
`aggregation induction.
`Some of these preformulation experiments should also contain vary-
`ing concentrations of surfactant(s) with appropriate controls. These short
`and inexpensive experiments can be set up on reciprocal shakers for periods
`of time from 1 to 6 to 24 hours. shaking from 10, 30, and 60 reciprocal
`strokes per minute. Reciprocal strokes disrupt and break up the flow of the
`liquid, while rotary strokes move the liquid circularly without breakup.
`These studies are intended to determine precipitation and aggregation ef-
`fects. Detailed aggregation experiments and results will be described later
`in these chapters.
`
`B. Freeze—Thaw Experiments
`
`These experiments will also be described in later chapters and will be part
`of Chapter 5. These experiments require a fair amount of active drug sub-
`stance as well as a fair amount of work. At this point of development there
`may not be enough active drug substance available.
`
`C. Filling Systems
`
`or all the filling types employed to dispense liquid, such as time«pressure.
`piston, and rotary pump, the rolling diaphragm metering pump is the one
`of choice for filling biOphannaceutical solutions. The internal pans of the
`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 pump is that it eliminates
`the principal cause of particulate generation which is most often induced
`by parts coming together creating shedding of microscopic particles.
`There are three other important parameters to control while dispens-
`ing protein solutions. (1) The speed at which liquid is 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 Ill-ml. vial per single
`filling head. If a large number of vials need to be filled. this filling system
`can accommodate variable numbers of filling heads. thus allowing it to fill
`a large number of vials. Filling at a faster rate will result in protein precipi-
`tation and aggregation. (2) The inner diameter of the filling cannula should
`not be so very small as to induce shearing and aggregation of the protein
`solution. (3) The tip 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 of the vial. This will result in a gentle flow
`
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`

`Prelormulatlon Development
`
`tat
`
`touching the inner wall of the vial when the cannula enters the vial and
`delivers the required amount of fluid. The proper bend on the tip of the
`cannula may also eliminate aggregation andlor shearing of the protein
`solution.
`
`0. Stability Evaluation
`
`The development of acceptable analytical methods while isolation, charac-
`terization, and purification of a bulk active drug substance are going an is
`very important. It can be an aid in generating semiquantitative and quanti-
`tative measurements of the active bulk drug at various stages of the process.
`Significant marketing advantages in this competitive pharmaceutical
`market wOuld be to achieve a longer shelf life of the product and storage
`temperature at room temperature. Today the lyophilized protein drug of-
`fers refrigerated temperature storage between 2 and 8°C.
`The present storage conditions set 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
`0 Excessive heat. Temperature exceeding 40°C
`
`Table 3 summarizes the initial guideline time points and tempera-
`tures that preforrnulation solutions should be exposed to. The results from
`the prefonnulations will allow the review team to determine directions to
`manipulate the excipients to obtain better stability.
`
`TABLE 3 Guideline tor Pretormulation Stability Studies
`
`Temperatures
`
`Timepolnt
`
`Frozen controls (~80 and —20'C}
`Refrigerated (24"0)
`
`Intermediate (20. 30. 37°C}
`
`High temperature {40. 45. 50°C)
`
`Reference control sample as needed
`T = O. B. 12. 24 8. 48 weeks
`Continue it stable
`
`T = 0. 4, 8, 12. 18. 24 weeks
`Continue it stable
`
`T = 0. 1. 2. 4. 8, 12 weeks
`Continue it stable
`
`
`
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`

`102
`
`Bantampo
`
`W. DEGRADATION MECHANISMS
`
`To predict degradation pathways of new biophannaceuticals is very diff-
`cuit. Depending on the stress conditions, each protein may react differently
`than another protein. As stated previously, the objectives of preformulation
`are to evaluate stress conditions such as pH, temperatures, and buffers and
`begin evaluation of some initial breakdown products. At this particular
`stage of development, it is necessary to have some analytical method(s) with
`some reliability to detect initial degradation. it is difficult to begin evalu-
`ation of degradation products without the reliability of these preliminary
`assay methods.
`The purpose of initial preformuiation studies is to begin understand-
`ing of protein instability via chemical and physical stress conditions (5). in
`order to stabilize potential useful pharmaceutical products, it is important
`to understand how proteins degrade. how they are affected by the compo-
`sition of the formulation. and the effects of stability 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 chemical entities. One or more of the 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
`biophannaceuticals. Under oxidative stress and in the presence of trace
`metals, amino acids sach as methionine (Met) can be oxidized to methion—
`ine sulfoxide, cysteine (Qrs) to cysteine disultide. as well as tryptophane
`(Try) and histidine (His) via other modifications.
`Oxidation can be controlled or minimized by (l) the addition of an-
`tioxidants. (2) having strict 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-performance isoelectric chroma-
`tography (HP-l EC). peptide mapping. amino acids analysis. and mass spec-
`
`MYLAN INST. EXHIBIT 1026 PAGE 14
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`

`Prelormulatlon Development
`
`103
`
`trometry (MS) (6). in terms of total protein concentration. ultraviolet Spec-
`trophotometry is the method most often used (1').
`
`B. Deamldation
`
`Deamidation is another more frequent degradation mechanism affecting
`pharmaceutical protein stability. Dcarnidation is the hydrolysis of the side
`chains amide on asparagine (Asp) and glutamine (Gln) to form Asp audior
`Gln residues. Extensive reports have elucidated mechanisms of deamida-
`tion reactions (8).
`Deamidation can be detected by isoelectric focusing, ion exchange
`chromatography. tryptic mapping and HPLC (9).
`
`C. Hydrolysis
`
`Hydrolysis is another most likely cause of degradation of proteins. It in-
`volves a peptide (amide) bond in the protein backbone (5). The most influ-
`ential factor affecting the hydrolytic rate is the solution pH.
`
`D. Racemlzation
`
`Proteins may also degrade via other modifications (10) such as racemiza-
`tion. This mechanism involves the removal of the alpha proton from an
`amino acid in a peptide to yield a negatively charged planar carbonion. 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.
`
`lsomerizatloa
`
`Protein degradation is also induced by isomerization. Hydrolysis of cyclic
`amides of 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. Disuh‘ide Exchange
`
`Disulfide exchange may result from a degradation other than covalent
`modification. These reactions may include the disulfide exchange of cys-
`teine. This reaction is base, catalyzed and promoted by thin] antioxidants
`(13).
`
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`194
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`Bootempo
`
`DiSulfide exchange can occur in misfolded conformers due to incor-
`rect intramolecular disulfide bonds (14).
`
`G. Beta-Elimination
`
`Another degradation residue can be the beta-elimination of ser, thr, cys, lys
`and phe residues. These reactions are accelerated by basic pH. tempera
`ture, and the presence of metal ions (16).
`
`V. PHYSICAL DEGRADATIONS
`
`Aggregation
`
`Protein aggregation can be of a covalent or noncovalem nature (11.18).
`
`A. Covalent Aggregation
`
`This pathway involves modification of the chemical structures resulting in
`newr chemical structures and may include reactions. such as oxidation. de-
`amidation, proteolysis. disulfide interchanges. racemization, and others.
`
`a. Noncova lent
`
`This instability may be induced by agitation. Shea r. precipitation. and ad-
`sorption to surfaces.
`
`C. Aggregation
`
`Protein aggregation derived from either physical or chemical inactivation,
`is presently a major biopharmaceutical problem (Ii—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 speetrophotometric methods, or both. can be of
`significant guidance to formulation scientists in selecting compatible excipi—
`ems to minimize andtor prevent its formation in the experimental formu—
`lation.
`Formation of aggregation can begin by the formation of initial parti-
`cles from protein molecules via the Brewnian movement. This is followed
`by collision of these molecules and aggregates ofvarying sizes can be formed.
`These aggregates can be generated by shear or collisional forces (22).
`Detection and measurements of aggregations can be performed by a
`number oftechniques. Visual observations, light scattering. polyacrylamide
`gel electrophoresis. UV, spectrophotometry. laser light diffraction particu—
`
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`Pretormulation Development
`
`105
`
`late analysis. fluorescence spectra and differential scanning colorimetry
`(DSC), RP—HPLC. and SE-HPLC (7.14). Conformational changes can also
`lead to aggregations and can be measured by DSC (23).
`A formulation scientist should focus on some important observations
`that need to be made to answer some potential problems on aggregation.
`
`0 Determination of initial approximate number of aggregates
`- Determination of approximate size and distribution of aggregates
`. Do the aggregates increase in size and number over time?
`- Do the aggregates affect the efficacy of the proteins?
`- What is the effect of aggregation on the long—term storage of the
`potential marketable product?
`
`D. Denaturation
`
`Denaturation of proteins can be the result ofseveral 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 conformational side 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.
`Then-ital denaturation of proteins is of great interest to the formulation
`scientist. Thermal probes offer tools to study protein structure and stability
`that ultimately can be of significant use to stabilize protein drug formula-
`tions. Modifications of protein thermal effects have been reviewed (8).
`The ability of the protein to retold 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. there is very likely inactivation of the protein attribn
`utable IO shearing effect.
`
`E. Precipitation
`
`Precipitation in formulations can occur by a variety of mechanisms such 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 reach a critical mass.
`precipitate out of solution and are clearly visible.
`
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`

`toe
`
`Boniempo
`
`From a biopharrnaceutical formulation point of view. precipitation
`can occur in membrane filters, filtration equipment. pumps. and tubing and
`loss of activity is very often recorded.
`Eventually. as the aggregation mechanisms are controlled and pre-
`vented. precipitation is essentially reduced or avoided. Details on the func-
`tions of stabilizers are discussed later in this volume.
`
`F. Adsorption
`
`Some of the most prevalent. ubiquitous factors of deactivation (30,31.
`32) that the protein biochemists and formulation scientists face. are the
`surface areas interactions from the purification. formulations. and stability
`stages.
`
`Essentially. at each point that the protein solution has encountered
`air during mixing (process). filtration (process). and air in the process steps.
`a significant surface area has been encountered to yield interphases.
`During the actual final manufacturing of vials. ampules. syringes.
`catheters. pumps. and their respective storage conditions. the proteins
`could be adsorbed at 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-
`ten’sties. they have a high affinity to adsorption at the air—liquid and solid—
`liquid interphase. Hydrophobic and hydrophilic interactions which are con-
`centration dependent. determine the extent a nd the rate ofadsorption. The
`adsorption effect on the protein is the unfol