PSTT Vol. 3, No. 4 April 2000
`
`research focus
`
`reviews
`
`The formulation of biopharmaceutical
`products
`Dave A. Parkins and Ulla T. Lashmar
`
`Biopharmaceutical products represent a diverse group of products
`
`that includes proteins, peptides, nucleic acids, whole cells, viral par-
`
`gation, precipitation and adsorption5 can also
`cause loss of activity.
`
`ticles and vaccines. The conformation of the macromolecule or cell
`
`must be maintained to retain biological activity, and animal models
`
`for biological activity and characterization assays are often devel-
`
`oped in tandem with initial formulation studies. This presents the
`
`formulation scientist with a unique set of challenges when compared
`
`to those for small molecules. This review focuses on approaches to
`
`the formulation of macromolecules into biopharmaceutical products,
`
`and provides examples of studies that have been undertaken within
`
`the authors’ laboratories.
`
`Dave A. Parkins
`Head of BioPharmaceutical
`Formulation Sciences
`GlaxoWellcome Research and
`Development
`Ware
`UK SG12 ODP
`tel: 144 (0)1920 883791
`fax: 144 (0)1920 883873
`e-mail:
`DAP1964@GlaxoWellcome.co.
`uk
`Ulla T. Lashmar
`BioPharmaceutical Formulation
`Sciences
`GlaxoWellcome Research and
`Development
`Beckenham
`UK BR3 3BS
`tel: 144 (0)208 6396578
`fax: 144 (0)208 6396332
`
`t For effective product development the formu-
`lation scientist must have a good understanding
`of the mechanism of degradation of the macro-
`molecule of interest, and its potential impact on
`such areas as its biological activity, metabolic
`half-life and immunogenicity.These mechanisms
`can, however, be complicated; encompassing
`many
`interrelated chemical and physical
`processes1,2. As degradation may occur during
`the production, isolation, purification, formu-
`lation, storage and delivery of the macromole-
`cule, it is necessary for the formulation scientist
`to work closely with those developing the bio-
`processing and analytical methodologies.
`Protein and peptide degradation can be
`broadly divided into those caused by chemical
`and physical mechanisms3. There are many
`chemical mechanisms of degradation; these may
`include bond formation or cleavage, whereas
`physical mechanisms involve changes in confor-
`mation, adsorption, aggregation and precipi-
`tation4. Nucleic acids generally require a chemi-
`cal modification to produce irreversible loss of
`biological activity, although shear stress, aggre-
`
`Chemical degradation
`The mechanisms by which proteins and peptides
`degrade include deamidation, racemination, hy-
`drolysis, oxidation, and disulphide exchange4,
`with deamidation and oxidation being the most
`common degradation pathways6. For nucleic
`acids the primary mechanism of degradation is
`the two-step process of depurination and
`b-elimination5,7.
`
`Primary structure and stability
`Examining the primary structure of a protein or
`peptide can provide an insight into how the mol-
`ecule may degrade. However, this will probably
`only be of real use for peptides and macromol-
`ecules when the amino acid sequence is exposed
`on the outer surface of the macromolecule, and
`therefore able to undergo chemical degradation.
`Disulphide bonds contribute to the formation
`of tertiary structures and the stabilization of sec-
`ondary structures8; disulphide bond interchange
`is possible at cysteine–cysteine links, leading to
`aggregation of the macromolecule. Deamidation
`is the hydrolysis of the side chain amide on glu-
`tamine and asparagine, which may be reduced
`by formulating at a pharmaceutically relevant pH
`below neutral pH (Refs 1 and 9). However, iso-
`merization is greatest at low pH for asparagine
`and glutamine. The side chains of tryptophan,
`methionine and cysteine and, to a lesser extent,
`tyrosine and histidine are potential oxidation
`sites; oxidation of these sites are most significant
`at neutral and alkaline pH. Methionine in
`particular is prone to oxidation from atmos-
`pheric oxygen4,9. Tryptophan is one of the most
`photosensitive amino acid residues in proteins10,
`whereas a lysine–threonine bond indicates sus-
`ceptibility to copper-induced cleavage. Hydrolysis
`
`1461-5347/00/$ – see front matter ©2000 Elsevier Science Ltd. All rights reserved. PII: S1461-5347(00)00248-0
`
`129
`
`Petitioner
`Ex. 1009, p. 129
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`

`
`reviews
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`research focus
`
`PSTT Vol. 3, No. 4 April 2000
`
`itol hexaphosphate and ethylenediaminebis(o-hydroxypheny-
`lacetic acid) for iron chelation15,16.
`
`Techniques to examine chemical degradation
`Factors such as pH, ionic strength, buffer composition, other
`excipients, light, atmospheric oxygen, metal ions and tempera-
`ture may all affect the mechanism of degradation6,17, and
`should be evaluated. The formulation scientist should avoid
`being dependent on any single analytical technique as different
`techniques can often produce apparently contradictory data. A
`range of complimentary analytical techniques should therefore
`be employed to ensure that a broad understanding of the
`degradation process is obtained.
`Bioassays are rarely used in early formulation screening stud-
`ies of proteins and peptides. This is because they are labour-
`intensive, relatively insensitive and can be variable.To allow for
`screening of a large number of samples and to measure changes
`of only a few percent in any given population, gel elec-
`trophoresis, size exclusion chromatography (SEC) looking for
`chain breaks, and aggregation and other high pressure liquid
`chromatography (HPLC) methods often provide the basis for
`stability analysis18–20. Light catalysed degradation and aggre-
`gation is routinely assessed in our laboratory using a suntest
`light cabinet as the light source and SEC–HPLC and iso-electric
`focusing (IEF) for the analysis. Figure 1 illustrates the difference
`in light-induced degradation for three monoclonal antibodies.
`Factors such as pH, ionic strength and buffer type can also be
`evaluated using SEC–HPLC; Figure 2 illustrates the use of the
`
`100
`
`95
`
`90
`
`85
`
`80
`
`75
`
`Monomer (%)
`
`of the peptide bond is possible at asparagine–proline and
`asparagine–tyrosine linkages, suggesting that these sequences
`are acid labile11. A summary of amino acids and amino acid
`sequences, potential reactions and formulation strategies is
`given in Table 1.
`Nucleic acids differ from proteins and peptides by virtue of
`the fact that they are all chains composed of the same limited
`number of nucleotides. Even though deamidation is a possible
`degradation path for deoxyribonucleic acid (DNA), it is prob-
`ably the chemical integrity of the phosphodiester backbone
`that is more important5. Depurination and b-elimination lead-
`ing to cleavage of the phosphodiester backbone7 is acid catal-
`ysed12, and at alkali pH this reaction occurs at a rate 20-fold
`lower than at neutral pH (Ref. 2). Long-term stability of a prod-
`uct may therefore be improved by formulating the final prod-
`uct to a pH above seven. If depurination and b-elimination are
`the only degradative mechanisms occurring, formulation above
`neutral pH may improve stability5. Free radical oxidation is an-
`other important source of strand damage5, with trace metal
`ions being able to catalyse many of these oxidative processes13.
`One of the most effective ways of reducing free radical oxida-
`tion is by inclusion of antioxidants such as ascorbic acid or glu-
`tathione14. Another potential method is to include chelating
`agents to remove trace metal ions. Ethylenediaminetetraacetic
`acid (EDTA) has been recommended for chelation of copper
`and Desferal, diethylenetriaminepentaacetic acid (DTPA), inos-
`
`Table 1. Amino acids or sequences susceptible to chemical
`degradation, together with formulation strategies to reduce
`degradation.
`
`Amino acid or
`sequence
`
`Mechanism of
`degradation
`
`Formulation
`strategy
`
`Cysteine–cysteine
`
`Aggregation
`
`Glutamine, asparagine
`
`Deamidation
`
`Tryptophan, methionine, Oxidation
`cysteine, tyrosine,
`histidine
`
`Addition of
`surfactants,
`polyalcohols and
`other excipients
`
`pH 3–5
`
`pH ,7
`
`Oxidation
`
`Protect from oxygen
`
`0
`
`1
`
`2
`Time (h)
`
`3
`
`4
`
`Pharmaceutical Science & Technology Today
`
`Photo decomposition Protect from light
`
`Copper induced
`cleavage
`
`Chelating agents
`
`Hydrolysis
`
`pH .7
`
`Figure 1. Light-induced degradation for three monoclonal antibodies
`with time in a Suntest light cabinet. The percentage of monomer left
`following exposure was measured by SEC–HPLC, illustrating the
`sensitivity of the method.
`
`Methionine
`
`Tryptophan
`
`Lysine–threonine
`
`Asparagine–proline,
`asparagine–tyrosine
`
`130
`
`Petitioner
`Ex. 1009, p. 130
`
`

`
`PSTT Vol. 3, No. 4 April 2000
`
`research focus
`
`reviews
`
`uble and insoluble aggregates can exist. Exposure to hydro-
`phobic surfaces, heating, lyophilization, reconstitution of
`lyophilized material, exposure to light, organic solvents, heavy
`metals, moisture in solid state and shaking during manufac-
`ture, shipping and handling may all induce aggregation26,27,
`and this may lead to reduced bioactivity28 and immunogenic
`reactions29. Insoluble aggregates may also lead to unacceptable
`physical product characteristics such as opalescence and pre-
`cipitates2. While aggregation of most proteins leads to loss of
`potency, this is not universally true; insulin, for instance, nor-
`mally exists as dimers or hexamers20.
`
`Techniques to determine aggregation and precipitation
`SEC–HPLC is commonly used to determine soluble covalent
`protein aggregates30. Because the formation of insoluble aggre-
`gates involves a change in the number and mass of particles,
`various techniques, such as light-scattering analysis31, spec-
`troscopy20,32 and ultracentrifugation33, have been used to
`assess the extent of insoluble aggregation.
`Fluorescence is a very sensitive technique that will detect in-
`soluble aggregates from protein solutions below 0.01 mg mL21;
`this technique is therefore useful as a screening tool for the evalu-
`ation of stress-induced aggregation. In our laboratory, fluorescence
`is used to evaluate the concentration of an excipient required to
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`15
`
`30
`
`800
`
`700
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`*
`
`0
`
`Intensity
`
`Zn
`4˚C 37˚C EDTA Fe, Ni
`Additive
`
`Cu Mn Mg Ca
`
`Pharmaceutical Science & Technology Today
`
`30
`
`25
`
`20
`
`15
`
`10
`
`5 0
`
`Low molecular weight fragments (%)
`
`Figure 2. Effect of the presence of metal ions and
`ethylenediaminetetraacetic acid on the appearance of low molecular
`weight fragments of a monoclonal antibody as measured by SEC–HPLC
`following storage at 378C for five weeks.
`
`technique to evaluate the protective effect of a chelating agent.
`Circular dichroism (CD) and high sensitivity differential scan-
`ning calorimetry (HSDSC) are useful complementary tools for
`confirming observations made by SEC–HPLC and IEF19,21. DNA
`formulations are routinely screened using ion-exchange–HPLC,
`capillary electrophoresis (CE) or agarose gel chromatography.
`CD and HSDSC can also be used to monitor changes5,22–25.
`
`Physical degradation
`Physical instability of proteins involves changes in their
`secondary, tertiary or quaternary structure and includes de-
`naturation, aggregation, precipitation and surface adsorption.
`Changes in their confirmation also cause loss of potency; it is
`therefore vital for both the stability and the potency of pro-
`teins that conformation is maintained. In contrast, the sec-
`ondary helix structure for DNA can change without an associ-
`ated change in potency26. The most common tertiary structure
`in DNA is supercoiled. A change in the degree of supercoiling
`or loss of supercoiling to an open circle-form or linear form
`may not necessarily cause total loss of activity, but may make
`the nucleic acid more susceptible to aggregation.Therefore ide-
`ally, DNA should be formulated in such a manner that its con-
`formation remains relatively unchanged with time5.
`
`Aggregation and precipitation
`For proteins and peptides aggregation may involve the inter-
`change of covalent bonds, such as disulphide bridges or non-
`covalent forces such as hydrophobic interactions, and both sol-
`
`60
`
`45
`Time (min)
`Pharmaceutical Science & Technology Today
`
`75
`
`90
`
`Figure 3. Fluorescence intensity values for 0.1 mg mL21 antibody in
`surfactant concentrations from 0% to 0.1%, stirred for up to 90 min at
`208C. The intensity from antibody solutions containing 0.0005%
`surfactant and above remained constant with stirring time, suggesting
`that these surfactant concentrations prevent shear-induced
`aggregation. 0% surfactant (black square); 0.0001% surfactant (star);
`0.0005% surfactant (black triangle); 0.001% surfactant (white square);
`0.01% surfactant (white circle); 0.1% surfactant (black circle).
`
`131
`
`Petitioner
`Ex. 1009, p. 131
`
`

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`reviews
`
`research focus
`
`PSTT Vol. 3, No. 4 April 2000
`
`10
`
`9
`
`8
`
`6
`
`5
`Fill volume (mL)
`
`6
`
`8
`
`Flow rate
`
`10
`
`4
`
`12 3
`
`99
`
`74
`49
`23
`–2
`
`4
`
`Amount of supercoiled remaining (%)
`
`Pharmaceutical Science & Technology Today
`
`Figure 4. The impact of operating conditions on the degradation of a
`DNA–lipid complex delivered by an airjet nebulizer.
`
`SEC–HPLC and gel electrophoresis. The study of heat-induced
`conversion of supercoiled DNA to the open circle and linear
`forms are typically monitored using agarose gel electropho-
`resis, capillary electrophoresis and various chromatographic
`techniques. HSDSC, UV and CD can also be used for screening
`purposes39.
`
`Formulation strategies for macromolecules
`Many macromolecules have a relatively short shelf life in aque-
`ous medium, which can lead to both poor bioavailability and
`poor stability in the final product. To improve bioavailability
`the macromolecule may be formulated as an oily injection,
`sustained release product, particulate delivery system or
`implant; these types of products often have the advantage of
`improved stability over an aqueous formulation. Where the
`formulation does not have adequate stability, then stability
`might be improved through the exclusion of water from the
`product or the addition of stabilizing excipients.
`When developing a biopharmaceutical product a wide range
`of formulation components have to be considered; these include
`buffer type and strength, use of ionic compounds, sugars, poly-
`ols and certain amino acids, the incorporation of surfactants, in-
`clusion of antioxidants, chelating agents and other substances.
`Figure 7 illustrates how a design expert system and denaturation
`temperature can be used to assist in the selection of several
`optimum parameters for a liquid formulation of an antibody.
`
`prevent agitation-induced antibody aggregation. Figure 3 illus-
`trates that a surfactant at 0.0005% was sufficient to prevent ag-
`itation-induced aggregation in a 0.1 mg mL21 antibody solu-
`tion. Electron paramagnetic resonance (EPR) has also been
`suggested as a tool for the determination of the amount of sur-
`factant required to prevent aggregation32.
`
`Adsorption
`Exposure of aqueous solutions of biopharmaceuticals to solid
`surfaces can lead to loss by adsorption onto liquid–solid inter-
`faces and loss by adsorption. The adsorption of drug to con-
`tainer surfaces or in filters requires particular attention as it can
`present a significant problem for biopharmaceuticals34,35.
`Different types of containers and filters have substantially dif-
`ferent affinities for macromolecules34. Adsorption is extremely
`rapid and can be prevented by various additives36.
`
`Techniques to determine adsorption
`Loss on contact with a material may be determined by solution
`depletion using ultraviolet (UV) absorption34, radiolabelling,
`or other suitable analytical techniques. Desorption and/or
`exchange techniques have also been used36.
`
`Denaturation
`Denaturation refers to an alteration in the secondary, tertiary or
`quaternary structure of the macromolecule, and can be caused
`by heating, cooling, freezing, denaturants, pH, organic solvents
`and shear4. Denaturation may be either reversible or irre-
`versible and is a useful tool to assess the comparative stability
`of a macromolecule in various formulations37. If a macromol-
`ecule is to be delivered to a patient using a delivery device,
`then the impact of the device must be fully understood. Figure 4
`illustrates how the operating conditions of an airjet nebulizer
`can significantly alter the degradation due to the shear experi-
`enced by a DNA–lipid complex.
`
`Techniques to determine denaturation
`Macromolecules often undergo thermal degradation by com-
`plex mechanisms.This means that often meaningful prediction
`of stability cannot be derived from short-term storage at el-
`evated temperatures. However, a useful insight into a formula-
`tion may be obtained by following thermal degradation with
`the use of calorimetry and determining the impact of formula-
`tion variables such as pH, buffer type, ionic concentration and
`the presence of stabilizing additives37,38. Figure 5 illustrates the
`variation in onset of denaturation for a monoclonal antibody
`with varying pH using HSDSC. These events can also be fol-
`lowed by CD and intrinsic fluorescence, as illustrated in Fig. 6.
`Other techniques of value in the detection of thermal de-
`naturation include UV, nuclear magnetic resonance (NMR),
`
`132
`
`Petitioner
`Ex. 1009, p. 132
`
`

`
`PSTT Vol. 3, No. 4 April 2000
`
`research focus
`
`reviews
`
`Figure 5. Denaturation profile for a monoclonal
`antibody at different pH values. Data generated
`using high sensitivity differential scanning
`calorimetry.
`
`pH7.1
`pH6.0
`pH5.0
`
`pH4.0
`
`pH3.0
`
`0.002
`
`0.000
`
`–0.002
`
`–0.004
`
`–0.006
`
`Cp (calc ˚C)
`
`20
`
`30
`
`40
`
`50
`60
`Temperature (˚C)
`
`70
`
`80
`
`90
`
`Pharmaceutical Science & Technology Today
`
`Exclusion of water
`The stability of many macromolecules can be enhanced by the
`exclusion of water from the product. Freeze-drying is most often
`employed for this purpose38, but spray drying40,41, drying by
`means of supercritical fluid processing42 and precipitation have
`also been investigated. Freeze-drying does not always
`result in a more stable product compared with a solution43.
`Macromolecules can denature during freezing because of
`changes in the microenvironment; for instance, from increasing
`salt concentration or changing pH in the unfrozen liquid26,44.
`Aggregation and denaturation can also occur during the sec-
`ondary drying process as a result of the loss of residual water45.
`The rate of freezing has also been found to affect the formation
`of aggregates46. Denaturation during spray drying has been re-
`ported as a result of the high air–product interfaces and high
`
`temperatures during drying47. The supercritical fluid technique
`may involve high pressure, high air–product interfaces and poss-
`ibly solvents, all of which can cause denaturation48. Residual
`moisture in the product can cause both chemical and physical
`degradation, which, apart from reduced bioactivity, can also
`cause reduced reconstitution and diffusional properties49.
`
`Additives
`A variety of excipients can be introduced into a formulation in
`an effort to increase the stability of the macromolecule during
`the manufacture, handling and delivery of the product.
`
`Sugars
`Sugars have been shown to protect macromolecules against de-
`naturation50 and are particularly used as cryoprotectants51. The
`
`0.0004
`0.0000
`–0.0004
`–0.0008
`–0.0012
`20
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`60
`40
`Temperature (˚C)
`
`80
`
`Figure 6. Use of circular dichroism (CD) and
`fluorescence to follow structural changes. Insert
`shows high sensitivity differential scanning
`calorimetry (HSDSC) profile. HSDSC reveals two
`exothermic transitions. CD and intrinsic
`fluorescence can also follow these events. Data
`generated using a monoclonal antibody. Molar
`ellipticity at 295 nm (red); molar ellipticity at
`225 nm (green); intrinsic fluorescence intensity
`at 350 nm (blue).
`
`30
`
`40
`
`50
`
`Temperature (˚C)
`
`60
`
`Pharmaceutical Science & Technology Today
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`133
`
`Petitioner
`Ex. 1009, p. 133
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`

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`reviews
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`PSTT Vol. 3, No. 4 April 2000
`
`Box 1. Excipients commonly used to stabilize
`macromolecules
`
`Excipient type
`
`Examples
`
`Amino acids
`
`Sugars
`
`Surfactants
`
`Salts
`
`Polyols
`
`Antioxidants
`Polymers
`
`Chelating agents
`
`Glycine, arginine, alanine, proline,
`aspartic acid, glutamic acid, lysine
`Trehalose, sucrose, maltose, fructose,
`raffinose, lactose, glucose
`Poloxamer 407, Poloxamer 188,
`polysorbate 80, polysorbate 20,
`octoxynol-9, polyoxyethylene-(23)
`lauryl alcohol, polyoxyethylene-(20)
`oleyl alcohol, sodium lauryl sulphate
`Sodium sulphate, ammonium sulphate,
`magnesium sulphate, sodium acetate,
`sodium lactate, sodium succinate,
`sodium proprionate, potassium
`phosphate
`Cyclodextrins, mannitol, sorbitol,
`glycerol, xylitol, inositol
`Ascorbic acid, glutathione
`Polyethylene glycol, dextran,
`polyvinylpyrrolidone
`EDTA, tris(hydroxymethyl)aminomethane
`(TRIS), diethylenetriaminepentaacetic
`acid, inositol, hexaphosphate,
`ethylenediaminebis
`(O-hydroxyphenylacetic acid), desferal
`
`the protein to exclude the more hydrophobic additive. At
`higher solvent concentration, the polyols may denature the
`protein. Cyclodextrins may prevent aggregation by molecular
`encapsulation57. Box 1 lists some useful polyols.
`
`Surfactants
`Surfactants are often added to stabilize macromolecules, and
`both nonionic20,55 and anionic58 surfactants have been used.
`The addition of surfactants may prevent adsorption of proteins
`to surfaces59, reduce agitation-induced aggregation20,60, and
`reduce denaturation58,61. Reports have suggested that surfac-
`tants are less effective in protecting against thermally induced
`denaturation52,55.The addition of surfactants has also been used
`to prevent freezing or freeze thaw-induced aggregation38 as
`well as delivery device-induced aggregation. Some studies in-
`vestigating the effect of surfactant concentration on protein
`aggregation suggested that the protective action coincides with
`the critical micelle concentration (CMC) for the surfactant,
`supporting the theory of the formation of a surfactant mono-
`layer at the air–water interface being their mechanism of
`action61. For other surfactants the maximum protective effect
`
`15.0
`
`12.8
`
`10.5
`
`8.3
`
`Concentration (%)
`
`82.6
`81.3
`79.9
`78.6
`77.3
`
`HSDSC peak maximum
`
`7.0
`
`6.4
`
`5.9
`
`pH
`
`5.3
`
`4.8 6.0
`
`Pharmaceutical Science & Technology Today
`
`Figure 7. Optimization of the stability of an antibody with respect to pH
`and concentration of the active using the design expert system.
`
`mechanism for stabilization is similar in both liquid and frozen
`systems and has been described by several groups19. The pres-
`ence of a sugar creates a thermodynamically unfavourable con-
`dition, because the chemical potential – the partial molar free
`energy for both the macromolecule and the sugar – is in-
`creased. Preferential exclusion of the sugar from the surface of
`the macromolecule minimizes thermodynamic activity, which
`in turn preserves the preferred conformation52,53. Sugars may,
`however, not be universally suitable for all conditions.
`Temperature-dependent hydrophobic interactions between a
`macromolecule and certain sugars may cause preferential bind-
`ing at higher temperatures, thus decreasing the activity of a
`macromolecule19. For optimum long-term stability, disaccha-
`rides such as trehalose and sucrose are recommended19.
`Reducing sugars can convert amide groups to ketoamine
`groups, resulting in a brown colour of the product. Non-
`reducing sugars should, therefore, always be used for tonicity
`adjustments and as stabilizers. A selection of stabilizers from
`this group are given in Box 1.
`
`Polyols
`Polyols include polyhydric alcohols and carbohydrates. They
`are used to stabilize macromolecules both in solution and on
`freeze-drying, and are particularly useful in preventing denatu-
`ration38,45,54,55. Some of the materials in this group, such as
`glycerol56, stabilize proteins through selective solvation of the
`protein, causing water molecules to pack more closely around
`
`134
`
`Petitioner
`Ex. 1009, p. 134
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`

`
`PSTT Vol. 3, No. 4 April 2000
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`research focus
`
`reviews
`
`concentrations greater than 1% have been shown to enhance
`antibody aggregation60. Dextran is commonly used in freeze-
`dried products as a cryostabilizer when it is necessary to raise
`the collapse temperature of a formulation38. Other excipients
`from this group are listed in Box 1.
`
`Ionic compounds
`Ionic compounds such as salts and buffers interact with macro-
`molecules via non-specific or specific binding66. Depending
`on the type of interaction, a salt may increase thermal stabil-
`ity52, increase the solubility and reduce the extent of aggre-
`gation67.The importance in considering the selection of buffer
`type is illustrated in Fig. 8. The Figure shows that a 200 mg
`mL21 antibody solution required the inclusion of an additional
`solubilizer if a phosphate buffer was used, whereas the selec-
`tion of an acetate buffer obviated this need. However, care is
`required, as proteins do tend to denature in high salt concen-
`trations68. Box 1 lists some ionic compounds.
`
`Chelating agents antioxidants and others
`Chelating agents and antioxidants are mainly used to prevent
`chemical degradation and their use as such has already been
`described in this review5,14. Lipids and fatty acids or their de-
`rivatives are used to stabilize proteins and nucleic acids, which
`is not surprising as many proteins in circulation are known to
`bind to lipids. Stabilization by fatty acids or their derivatives, in
`particular phospholipids, results from the association of their
`polar and non-polar portions with reacting groups on the
`protein or nucleic acid4,69.
`
`Conclusions
`In the past, the formulation of biopharmaceutical products has
`often been empirical, because products were considered too
`complex for adequate characterization. Developments in the
`biotechnology industry have meant that highly purified prod-
`ucts, that can be characterized, are increasingly being pro-
`duced. This is a trend that has been recognized by regulatory
`bodies such as Center for Biologics Evaluation and Research
`(CBER) with the introduction of the concept of a ‘well charac-
`terized’ product. A consequence of this change is that it is be-
`coming increasingly important that the formulation scientist
`has a good understanding of the macromolecule being formu-
`lated, in order to produce products that meet patient, commer-
`cial and regulatory requirements.
`
`Acknowledgements
`The authors would like to acknowledge the contribution of
`their colleagues in Biopharmaceutical Development at
`GlaxoWellcome Research and Development, and also Andrew
`Heron (Department of Chemistry, University of Glasgow, UK).
`
`135
`
`*
`
`*
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`Solubility mg ml–1
`
`Buffer type
`
`Pharmaceutical Science & Technology Today
`
`Figure 8. Solubility of an antibody in acetate, citrate and phosphate
`buffer. The pH of all the buffer solutions was the same and the
`solubility was determined from precipitation on concentration.
`*Indicates that the solution remained clear following storage.
`Acetate (black); citrate (white); phosphate (grey).
`
`occurred at concentrations much higher than the CMC, indi-
`cating that other factors are also involved20. Higher concen-
`trations of surfactants may destabilize a macromolecule6,60.The
`concentration required to protect a macromolecule should,
`therefore, be evaluated for each possible encountered stress.
`Surfactants commonly used to prevent adsorption, aggre-
`gation, precipitation, denaturation and freeze-thaw stresses are
`given in Box 1.
`
`Amino acids
`Amino acids have been added to biopharmaceuticals for a vari-
`ety of reasons; dicarboxylic amino acids such as aspartic and
`glutamic acid have been used to reduce aggregation19,62, and
`glycine, arginine and lysine have also been reported to prevent
`aggregation63. In addition, amino acids have been found to be
`useful as chelating agents64, and may reduce surface adsorption.
`They have also been reported to increase the thermal stability of
`proteins65. Box 1 lists some amino acids that have been used.
`
`Polymers
`Low concentrations of poly(vinylpyrollidone) (PVP) have been
`successfully used to inhibit antibody aggregation, whereas PVP
`
`Petitioner
`Ex. 1009, p. 135
`
`

`
`reviews
`
`research focus
`
`PSTT Vol. 3, No. 4 April 2000
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`3
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`7
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`8
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`9
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