16
`
`DEVELOPMENT
`OF FORMULATIONS
`FOR THERAPEUTIC
`MONOCLONAL ANTIBODIES
`AND Fc FUSION PROTEINS
`
`aaaaasSampathkumar Krishnan, Monica M. Pallitto, and
`Margaret S. Ricci
`
`16.1. INTRODUCTION
`
`Monoclonal antibody-based therapies have become a huge area for biopharmaceutical
`development, with 18 monoclonal antibodies (Table 16.1) on the market and nearly
`200 antibody molecules in clinical facilities [1–4]. Monoclonal antibodies for ther-
`apeutic and prophylactic indications over the years have moved from fully murine
`and humanized murine forms to completely human forms. There has also been a
`breakthroughs since the 1980s regarding purification [5], analytical methods including
`biological assays [6], and manufacturing aspects leading to the ability to prepare purer
`lots of monoclonal antibodies economically at large scales. The majority of mono-
`clonal antibodies that are currently approved or in clinical development are focused
`on meeting therapeutic needs in the areas of oncology, autoimmune, and inflammatory
`diseases [1,4].
`(typically
`Antibodies and Fc fusion proteins are large macromolecules
`>150 kDa), an order of magnitude larger than many other protein therapeutics such
`as cytokines, and are multidomain as well as typically glycosylated in nature, (if pro-
`duced by mammalian cell culture [7]. Domains of antibodies have naturally evolved
`to associate with a variety of targets such as antigens and FcRn receptors with high
`
`Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals,
`edited by Jameel and Hershenson
`Copyright © 2010 John Wiley & Sons, Inc.
`
`Ex. 2008-0001
`
`

`

`80
`
`IgG2a,anti-CD3
`
`Biotech
`
`Na,Kphosphate;pH7NaCl,polysorbate
`
`1
`
`IV/liquid
`
`Muromonab-CD3;murine
`
`J&J-Ortho
`
`dextran40
`
`NaCl,sucrose,
`
`polysorbate80
`
`Mannitol,
`
`benzylalcohol
`polysorbate20,
`
`Naphosphate
`
`Citrate;pH5.2
`Naphosphate,Na
`
`immunotoxin
`anti-CD33,
`humanizedIgG4,
`
`4(5mg)
`
`IV/lyophilized
`
`Gemtuzumabozogamicin;
`
`50
`
`SC/liquid
`
`IgG1,anti-TNFα
`
`Adalimumab;human
`
`IgG1,anti-HER2
`
`Trehalose,
`
`Histidine;pH6
`
`21(440mg)
`
`IV/lyophilized
`
`Trastuzumab;humanized
`
`IV/liquid
`
`IgG1,anti-EGFR
`Cetuximab;chimeric
`
`Wyeth
`
`Abbott
`
`Genentech
`Systems
`
`Imclone
`
`OKT3®
`Othroclone
`
`Mylotarg®
`Humira®
`
`Herceptin®
`Erbitux®
`
`Bexxar®
`Avasatin®
`Product
`Antibody
`
`Naphosphate;pH7.2NaCl
`
`polysorbate80
`NaEDTA,
`Na,K-phosphate,pH7NaCl,KCl,
`
`Naphosphate;pH7.2NaCl,maltose
`
`polysorbate20
`
`Naphosphate;pH6.4Trehalose,
`
`2
`
`30
`
`14
`
`25
`
`IgG1,anti-CD52
`
`ILEX
`
`IV/liquid
`
`Alemtuzumab;humanized
`
`radiolabeled(131I)
`anti-CD20,
`murineIgG2a,
`
`Campath®-1HMillennium-
`
`IV/liquid
`
`Corixam-GSKTositumomab-I131;
`IgG1,anti-VEGF
`
`IV/liquid
`
`Bevacizumab;humanized
`
`Genentech
`
`Excipients
`
`Components;pH
`
`Buffer
`
`DosageFormConcentration,amg/mL
`DeliveryRoute/
`
`FinalFormulation
`
`Description
`
`GenericName;
`
`Company
`
`Manufacturing
`
`TABLE16.1.DetailedListofAntibodyProductsApprovedandMarketedintheUnitedStates
`
`384
`
`Ex. 2008-0002
`
`

`

`NaCl
`80
`
`Naacetate
`
`Naphosphate;pH6.1NaCl;polysorbate
`
`NaCl;glycine
`
`polysorbate80
`glycine;
`mannitol,
`
`Histidine
`
`20
`
`20
`
`100
`
`80
`
`NaCl,polysorbate
`
`Nacitrate;pH6.5
`
`80
`
`Naphosphate;pH7.2NaCl,polysorbate
`polysorbate80
`
`10
`
`2
`
`NaCl,sucrose,
`
`Na,Kphosphate
`
`4(20mg)
`
`IV/lyophilized
`
`Basilixamab;chimeric
`
`Novartis
`
`Simulect
`
`Naphosphate;pH7.2Sucrose,
`
`10(100mg)
`
`IV/lyophilized
`
`polysorbate20
`
`Sucrose,
`
`Histidine;pH6.2
`
`100(150mg)
`
`SC/lyophilized
`
`Efalizumab;humanized
`
`Genentech
`
`IV/liquid
`
`IgG1,anti-CD20
`Idec-GenentechRituximab;chimeric
`
`Fab
`IgG1,anti-GPIIb/IIIa,
`
`IV/liquid
`
`Centocor-LillyAbciximab;chimeric
`
`IgG1,anti-TNFα
`Infliximab;chimeric
`IgG1,anti-CD11a
`
`Centocorb
`
`Rituxan®
`
`ReoPro®
`Remicade®
`Raptiva®
`
`aFromtotallyophilizedproductifapplicable.
`
`385
`
`albumin
`
`NaCl,human
`
`80
`
`pH7
`Na,K-phosphate;
`
`Naacetate,
`
`Naphosphate;pH6.9NaCl,polysorbate
`polysorbate20
`
`111In)
`radiolabeled(90Yor
`anti-CD20;
`murineIgG1,
`
`1.6
`
`5
`
`IV/liquid
`
`Ibritumomabtiuxetan;
`
`BiogenIdec
`
`IgG1,anti-CD25
`
`IV/liquid
`
`Daclizumab;humanized
`
`Roche
`
`IgG1,anti-IgE
`
`Sucrose,
`
`Histidine
`
`125(202.5mg)
`
`SC/lyophilized
`
`Omalizumab;humanized
`
`Genentech
`
`IV/liquid
`
`IV/liquid
`
`IgG2,anti-EGFR
`
`Panitumumab;human
`
`IgG4,anti-4α-integrin
`Natalizumab;humanized
`
`IgG1,anti-RSV
`
`Amgen
`
`BiogenIdec
`
`Tysabri
`
`IM/liquid
`
`Palivizumab;humanized
`
`MedImmune
`
`Synagis®
`
`IgG,anti-CD25
`
`Zevalin®
`Zenapax®
`Xolair®
`Vectibix
`
`Ex. 2008-0003
`
`

`

`386
`
`DEVELOPMENT OF FORMULATIONS FOR THERAPEUTIC MONOCLONAL ANTIBODIES
`
`affinity, which makes them useful protein therapeutics. In the body, antibodies typi-
`cally have a half-life of 30 days. On the other hand, they are expected to be stable
`in storage for more than 2 years. The goal of a formulation program for therapeutic
`antibodies and Fc fusion proteins, as well as other protein therapeutics, is generally to
`develop a stable, robust formulation that minimizes physical and covalent degradation,
`ensures long-term storage stability, and prevents any adverse in vivo effects such as
`injection site, immunogenic or anaphylactic reactions. Additionally, instability of the
`antibody molecules can alter the pharmacology of the drug product as it affects both
`the pharmacokinetics in the serum and drug clearance from the body.
`Antibodies and Fc fusion proteins, like other proteins, can be degraded under
`conditions where they are exposed to extremes of heat, freezing, light, pH, agita-
`tion, shear stress, metals, and substances such as silicone oil from prefilled syringes.
`Exposed surface residues of each antibody are unique and require specific formulation
`excipients to provide maximal stability against the aforementioned stresses. Assess-
`ment of the physicochemical and thermodynamic instability of antibodies using novel
`analytical technologies has led to the identification of several events that are more
`specific to the unique nature of this particular class of proteins such as variations in
`Fc glycosylation, Fc methionine oxidation, hinge region cleavage, and glycation of
`Lys residues [8]. An optimal formulation should minimize all such antibody degra-
`dation reactions in solution, or at minimum, mitigate those degradation reactions that
`impact critical quality attributes.
`Biotechnology companies and contract research organizations are using improved
`analytical methodologies to monitor the degradation of the protein therapeutics dur-
`ing stability testing. Forced denaturation, agitation, and freeze–thaw studies are used
`to simulate the conventional stresses that a protein can undergo during production,
`shipping, storage, and administration. The effectiveness of forced degradation and sta-
`bility studies done on a small scale to predict long-term, large-scale product stability
`depends on a number of factors: (1) the temperature dependence of the protein has
`to be understood; (2) accurate predictions of the shelf storage require that the protein
`system follow Arrhenius degradation kinetics over the temperature range that is used
`for the accelerated stability studies; and (3) the stability studies have to be conducted
`on multiple manufacturing lots that are representative of the commercial process.
`One of the main challenges facing the manufacturers of biologicals in terms of
`formulation is demonstrating biocomparability in terms of product stability and clin-
`ical bioequivalence. The production of biological products is a complex process that
`undergoes continual development and refinement before commercialization and may
`continue post-launch. In most cases, any alterations in the process used to manu-
`facture the antibody molecules can result in wide differences in the structural and
`functional properties of the molecules. These changes can alter the stability, clinical
`efficacy, and/or safety of the recombinant antibody therapeutic. Therefore, there is a
`need to perform formulation development studies on manufacturing lots that are rep-
`resentative of the commercial process for the approved and marketed drug product.
`Another issue is the complexity of Escherichia coli production processes for cytokines
`versus mammalian-cell-derived processes for antibody production, which results in
`heterogeneity in glycosylation patterns. Antibodies are often heterogeneous as a result
`
`Ex. 2008-0004
`
`

`

`MECHANISMS OF DEGRADATION
`
`387
`
`of charge variants, glycosylation differences, and disulfide chemistry. Formulation
`screening must be initiated early in development even before knowing the commer-
`cial drug dose and before the commercial process is set. The purity of the excipients
`used in the formulation may present an additional challenge. Vendor or lot differences
`in the purity of the excipients can jeopardize the consistency of the drug product. With
`all of these considerations, formulation development can be a considerable challenge.
`In addition, the formulation screens must be efficient to accommodate limited amounts
`of protein available for early formulation studies.
`Regulatory agencies require rigorous testing procedures to determine the stability
`of a pharmaceutical formulation over time. Regulatory perspectives of the charac-
`terization and stability testing procedures have changed with advances in analytical
`technologies, especially in the fields of mass spectrometry and chromatography, and
`there is an increased understanding of the biology of recombinant proteins, as well as
`preclinical and clinical experience with many approved products. Also, there is a reg-
`ulatory requirement to demonstrate that material or process changes during antibody
`production generate bioequivalent drug product.
`
`16.2. MECHANISMS OF DEGRADATION
`
`16.2.1. Physical Instability
`16.2.1.1. Aggregation and Particle Formation. Aggregation and related
`particle formation is a dominant degradation pathway of antibodies and can occur
`during all stages of protein therapeutic processing and storage [3,9,10]. Aggregation
`of light-chain antibody fragments and their deposition into amorphous precipitates or
`insoluble fibrils has also been linked to amyloid diseases such as systemic amyloidosis
`[11–13]. Knowledge of the mechanisms underlying the protein aggregation processes
`is essential to develop rational in vitro preventive strategies. The aggregation phenom-
`ena can be stipulated by protein structural changes or by colloidal effects affecting
`protein–protein interactions [14]. Such events for proteins in general could occur via
`a simple diffusion-limited mechanism [15] or involve nucleation as the primary stage
`for further growth and propagation of aggregates [16,17]. From earlier studies it has
`become evident that proteins with dominating β-sheet content are prone to aggregation
`[18,19] and can self-assemble into either amorphous precipitates [15] or well-defined
`fibrils [17]. The aggregation process is also sensitive to a wide range of factors such
`as protein concentration, hydrophobicity, and charge as well as solution pH, ionic
`strength, and temperature [3,9,10,20]. Particle formation due to aggregation is a major
`issue, and control of particle levels for parenteral administration is necessary to pre-
`vent potential adverse reactions, as well as potential clogging of intravenous lines and
`filters. When high therapeutic doses are required, the need for high volumes may be
`countered by increasing the concentration of the antibody (sometimes several orders
`of magnitude higher than conventional protein therapeutics). This, in turn, may result
`in increased problems relating to aggregation and particulation.
`Antibody aggregation is complex and can proceed through covalent or
`noncovalent association that is highly dependent on the solution conditions, including
`
`Ex. 2008-0005
`
`

`

`388
`
`DEVELOPMENT OF FORMULATIONS FOR THERAPEUTIC MONOCLONAL ANTIBODIES
`
`pH, ionic strength, and excipients [21,22]. This association can be due to disulfide
`or nondisulfide covalent bonds, while the noncovalent associations can be due
`to hydrophobic or electrostatic interactions. Adding to the complexity, a given
`antibody can undergo multiple mechanisms of aggregation. Antibodies can undergo
`domain swapping, which can lead to altered structure and aggregation. Increasing
`the temperature and pH of the formulation often results in covalent crosslinking due
`to disulfide shuffling, while protein concentration, salt content, and other factors can
`promote non-covalent association. Antibodies have multiple intradomain as well as
`interdomain linkages through the disulfides [7], and these linkages have been found to
`undergo shuffling during processing leading to product heterogeneity and aggregation.
`Antibodies are also susceptible to photo-oxidation, which can lead to aggregation.
`In most cases aggregation of protein molecules proceeds through a partially and
`reversibly unfolded conformational state that results from partial unfolding. This con-
`formational state can be populated through the effects of solution conditions and
`the internal conformational stability of the molecule on the transition from native
`to unfolded states. A protein aggregate is formed between two or more molecules
`because of this higher-order structural disruption and exposure of hydrophobic regions
`leading to intermolecular interactions. This can eventually lead to aggregation and/or
`particulation [10,14].
`After storage in solution under physiological conditions for a sufficiently long
`period, dimers may represent the main component of total aggregates [approximately
`10%–30% (w/w) at a protein concentration of ∼160 mg/mL] [23]. More recent studies
`have examined kinetic and thermodynamic aspects of the dimerization of IgG1s in
`solution [21,22]. Using a recombinant human monoclonal antibody that recognizes
`vascular endothelial growth factor (rhMAb-VEGF) as a model [21], it was found that
`aggregation rates were greater in slightly alkaline (pH 7.5–8.5) compared to slightly
`acidic (pH 6.5–7.5) conditions. A high-salt environment (1 M NaCl) also enhanced
`dimerization. The nature of the IgG1 dimers was found to be highly complex, resulting
`from different associations of the antibody domains [22]. In our laboratory, we have
`studied the structure, stability, and conformational dynamics of the Fab, Fab
`, and
`Fc fragments of an IgG1 molecule [24]. Structural studies of the intact antibody
`and fragments showed that the structure of the Fc fragment is most susceptible to
`pH changes. Thermal, guanidine HCl–induced and urea unfolding studies at pH 7.4
`and 5.0 showed differences in conformational stability of the various fragments at
`these two pH levels. Incubation studies performed with the intact protein and the
`◦
`◦
`fragments at 37
`C and 50
`C showed that the Fc fragment aggregated faster than did
`the Fab and the intact antibody. We proposed that Fc–Fc and possibly Fab–Fc are
`responsible for the aggregation and particle formation of the IgG1 antibody molecule
`as a result of temperature-induced stress. McAuley et al. [25] showed that disulfide
`bond formation in the human CH3 domain plays an important role in antibody stability
`and dimerization. This domain contains a single buried, highly conserved disulfide
`bond. The authors showed that this disulfide bond significantly affects the stability and
`monomer–dimer equilibrium of the human CH3 domain, which may have implications
`for the stability of the intact antibody.
`Another study of empirical phase diagrams of monoclonal antibody solutions pro-
`duced from spectroscopic data suggested (1) the existence of similar structural states
`
`(cid:3)2
`
`Ex. 2008-0006
`
`

`

`MECHANISMS OF DEGRADATION
`
`389
`
`at low temperatures independent of concentration and (2) a decrease in the temperature
`at which phase changes were observed with increasing concentration. The decrease in
`structural stability observed in these studies is probably the result of aggregation or
`self-association of the recombinant MAbs on heating in crowded solutions, and not a
`decrease in the intrinsic structural stability of the MAbs [26]. A related investigation
`[27] found that at a given concentration, the phase separation temperature for proteins
`in general strongly increases with the molecular weight of the oligomers. These
`findings imply that for phase separation, the detailed changes of the surface properties
`of the proteins are less important than the purely steric effects of oligomerization.
`During manufacturing or shipment, proteins endure high mechanical or shear
`stress through filtration, mixing, and agitation and are exposed to various interfaces.
`Partially denatured molecules expose hydrophobic regions within the molecule, which
`can then result in interaction, protein aggregation, and particle formation [9,28]. Anti-
`bodies, like other proteins, can interact with air–water interfaces and surfaces such
`as metals and other hydrophobic components. Prior to delivery to the patient, protein
`pharmaceuticals often come in contact with a variety of surfaces (e.g., syringes and
`stoppers), which are treated to facilitate processing or to inhibit protein binding. One
`such coating, silicone oil, has previously been implicated in the induction of protein
`aggregation [29].
`
`16.2.2. Factors Affecting Physical Instability
`16.2.2.1. Solution Conditions. Vermeer and Norde have noted that pH has
`a strong influence on the antibody aggregation rate [30]. Table 16.2 outlines typical
`antibody-related degradation reactions and their mediation through use of appropriate
`solution conditions such as pH. Proteins in general are often stable against aggre-
`gation over narrow pH ranges, and may aggregate rapidly in solutions at pH values
`outside these ranges (Fig. 16.1). Examples include low-molecular-weight urokinase
`[31], relaxin [32], recombinant human granulocyte colony-stimulating factor (rhGCSF)
`[33], and insulin [34]. Both pH and salt can play a very important role for antibodies
`in solution, as they control physical properties such as conformational and colloidal
`stability as well as the chemical stability of the protein. In our studies, we found
`that different domains of the antibody have different pH sensitivities. It has also been
`shown that low pH (pH 4–6) and appropriate salt concentration reduce aggregation
`of antibodies in solution by affecting the noncovalent interactions between the anti-
`body molecules. The Fab fragment is most sensitive to heat treatment, whereas the
`Fc fragment is most sensitive to decreasing pH. The structural transitions observed by
`DSC and CD studies in the whole IgG is the sum effect of those determined for the
`isolated Fab and Fc fragments [30,35].
`The total charge on the protein is affected by the solution pH and electrostatic
`interactions within the antibody molecules and with the ions. Electrostatic interac-
`tions can affect protein stability in different ways. The amino acids in the antibody
`can be charged with increasing acidity or basicity of the solution. This can happen
`at a pH away from the isoelectric point (pI) of a protein [36]. The increasing charge
`repulsion between these charged groups of the antibody in such a solution can desta-
`bilize the folded or native state because of the high charge density. Thus, pH-induced
`
`Ex. 2008-0007
`
`

`

`390
`
`DEVELOPMENT OF FORMULATIONS FOR THERAPEUTIC MONOCLONAL ANTIBODIES
`
`TABLE 16.2. Typical Antibody-Related Degradation Reactions in Therapeutic Formulations
`
`Degradation
`
`Causes
`
`Possible Solutions
`
`Covalent aggregation
`Isomerization
`Deamidation
`Clip formation
`Oxidation
`
`Noncovalent aggregation Structural changes, colloidal
`stability, heat and other
`physical stress, sorbitol
`crystallization during freezing
`Disulfide rearrangement
`pH ∼ 5
`pH<5 and pH>6
`Proteases, metals, impurity
`Free radicals, reactive oxygen,
`metals, impurities, hydrogen
`peroxide
`Low antibody concentration,
`binding to surfaces,
`hydrophobicity
`
`Surface denaturation
`
`pH, buffer salt, ionic additives,
`protein concentration,
`improving raw-material purity
`
`pH, prevent association
`pH, magnesium chloride
`pH
`pH, chelation of metals, purity
`pH, free-radical and reactive
`oxygen scavengers, metal
`chelation
`Surfactants, protein
`concentration, pH
`
`pH
`
`2
`
`3
`
`Aggregation and particle formation [45, 139, 167]
`7
`6
`5
`4
`
`8
`
`9
`
`Acid-unfolding [30, 35]
`
`Reversible dimerization
`[21, 23]
`
`pI precipitation [36]
`
`Clip-mediated aggregation
`[143]
`
`Disulfide-linked aggregation
`[8]
`
`pH
`
`2
`
`3
`
`4
`
`Covalent instability [156, 157]
`5
`7
`6
`
`8
`
`9
`
`Deamidation [41, 78]
`
`Iso asp /
`Cyclic Imide
`[79, 83, 166]
`
`Deamidation [41, 78]
`
`Oxidation due to active oxygen species, metals etc [80]
`
`Acid related
`Hydrolysis [82, 83]
`
`Proteolysis,
`Other mechanisms [82, 83]
`
`Figure 16.1. Different physical instabilities observed for antibodies at different pH levels
`along with some relevant references.
`
`Ex. 2008-0008
`
`

`

`MECHANISMS OF DEGRADATION
`
`391
`
`conformational unfolding can lead to a state of lower electrostatic free energy [14]
`Also, specific charge interactions, such as salt bridges, can affect antibody confor-
`mational stability. These salt bridges can stabilize the folded state and in some cases
`cause self-interaction [36,37]. When proteins possess both positively and negatively
`charged groups, the differential charge distribution on the surface of the antibody
`can cause protein–protein interactions, making assembly processes such as antibody
`aggregation energetically favorable [38].
`
`16.2.2.2. Ligands and Cosolutes. Ligands and cosolutes are used in formu-
`lations to increase the physical stability of antibodies similar to other proteins. The
`Wyman linkage function applied by Timasheff [39] is commonly used to explain the
`effects of ligands and co-solutes in the formulation such as sucrose and salts. Through
`the Wyman linkage function, differential binding of ligand in two-state equilibrium
`will shift the equilibrium toward the state with the greatest binding. Binding of Zn2+
`to human growth hormone or insulin is a very common example in which the free
`energy of unfolding for these proteins is increased and the native state of the protein
`is favored [40].
`The Wyman linkage function can also be used to explain the effect of weakly
`interacting ligands (i.e., cosolutes), especially protein stabilizers such as sucrose and
`glycerol that are preferentially excluded from the surface of a protein molecule. In
`this case the degree of exclusion is proportional to the solvent-exposed surface area of
`the protein [39]. These cosolutes are excluded in the domain of the protein, and water
`takes its place in that domain, resulting in preferential hydration. Preferential exclusion
`can thus be interpreted as negative binding. During unfolding, protein surface area
`increases, leading to a greater degree of preferential exclusion. The net effect of greater
`negative binding to the unfolded state is to favor the native state.
`Ligands and cosolutes that alter protein conformational stability also influence
`protein aggregate formation. For example, in the presence of polyanions, aggregation
`of acidic fibroblast growth factor [41] and native recombinant keratinocyte growth
`factor [42] are greatly inhibited. It has also been shown that the addition of weakly
`interacting, preferentially excluded solutes such as sucrose can inhibit aggregation of
`immunoglobulin light chains [17] and rhGCSF [15].
`
`16.2.2.3. Salt Type and Concentration. Salts have complex effects on pro-
`tein physical stability by modifying conformational stability and colloidal solubility,
`and may have different effects according to the surface charge of the protein or anti-
`body. Salts bind to proteins, and destabilization of the protein can occur if the ions bind
`more strongly to the nonnative or unfolded state compared to the native state [43]. For
`example, the rate of aggregation of recombinant factor VIII SQ [44] was decreased
`in the presence of NaCl, while salt increased the aggregation rate for rhGCSF [33].
`Moore et al. [21] found that salt increased dimer formation for IgG1 antibody.
`Salts also modulate the strength of electrostatic interactions between the
`charged groups, at both intra and intermolecular levels. Thus, whereas intramolecular
`charge–charge interactions affect conformational stability, intermolecular electrostatic
`
`Ex. 2008-0009
`
`

`

`392
`
`DEVELOPMENT OF FORMULATIONS FOR THERAPEUTIC MONOCLONAL ANTIBODIES
`
`interactions affect degradation rates. The overall effect of salt on protein stability is
`a fine balance of multiple mechanisms by which salt interacts with protein molecules
`and affects protein–protein interactions. Because pH determines the type,
`total,
`and distribution of charges in a protein, salt binding effects may be strongly pH-
`dependent. These results suggest that protein stability can be increased by improving
`the coulombic interactions among charged groups on the protein surface [37].
`
`16.2.2.4. Preservatives. Antimicrobial preservatives, such as benzyl alcohol,
`are often utilized in liquid protein and antibody formulations to prevent bacterial
`growth during storage. In particular, multidose formulations of proteins require effec-
`tive preservatives to prevent microbial growth after opening and administration of the
`first dose. Preservatives are also required for certain drug delivery systems. However,
`preservatives can interact with proteins and often induce aggregation of protein in
`aqueous solution. For example, preservatives (e.g., phenol, m-cresol, benzyl alcohol)
`have been shown to induce aggregation of recombinant interleukin-1 receptor [45]
`and recombinant human interferon gamma (rhIFNγ) [46].
`Preservatives can bind to the nonnative or unfolded states and make the molecule
`prone to aggregation. For example, it was observed that addition of benzyl alcohol
`perturbed the tertiary structure of rhINFγ without affecting its secondary structure,
`and the rate of rhINFγ aggregation increased as the molar ratio of benzyl alcohol to
`protein increased [46]. Also, preservatives reduced the apparent melting temperature
`of recombinant interleukin-1 receptor [45].
`
`16.2.2.5. Surfactants. Nonionic surfactants are often utilized in protein and
`antibody formulations to prevent aggregation, surface denaturation, and adsorption
`during purification, filtration, transportation, freeze drying, spray-drying, and storage.
`Surfactants (surface-active agents) are amphiphilic molecules that tend to orient such
`that the exposure of the hydrophobic portion to the aqueous solution is minimized.
`For example, surfactants adsorb at air–water interfaces, forming a surface layer of
`surfactant molecules oriented so that only their hydrophilic ends are exposed to water.
`Such orientation and surface adsorption can also occur at solid–water interfaces such
`as those found in vials, syringes, tubing, and other containers. Protein molecules are
`also surface-active and adsorb at interfaces. Surface tension forces at interfaces can
`perturb protein structure, often resulting in aggregation. Surfactants inhibit interface-
`induced aggregation by limiting the extent of protein adsorption [47].
`As in other cosolutes, differential binding of surfactants to native and unfolded
`states of protein influences the protein’s conformational stability. In some cases sur-
`factants still can kinetically inhibit protein aggregation at interfaces despite causing a
`reduction in the thermodynamic stability of the protein conformation. This often helps
`prevent adsorption of antibodies formulated at low concentrations to interfaces such
`as IV lines, bags, and storage containers. In addition, surfactants have been shown to
`act as chemical chaperones, increasing rates of protein refolding and thus reducing
`aggregation [48,49].
`
`Ex. 2008-0010
`
`

`

`MECHANISMS OF DEGRADATION
`
`393
`
`16.2.2.6. Freeze–Thaw–Related Damage. Freeze–thawing is a common
`stress to which a therapeutic protein can be exposed to during manufacturing, ship-
`ping, and storage. Therapeutic proteins are purposely frozen for storage of bulk drug
`substance or for storage of analytical samples. The final commercial product also may
`be frozen accidentally because of mishandling. This process may happen once or mul-
`tiple times, with additional damage to the protein potentially occurring during each
`subsequent freeze–thaw cycle.
`Protein aggregation during freeze–thawing has been attributed to partial unfolding
`of protein molecules caused by the perturbing conditions arising during the pro-
`cess [50]. Perturbation of the protein conformation can be caused by low temperature
`[51], freeze concentration of solutes [52], pH changes due to buffer crystallization
`[53], exposure of protein molecules to the ice–liquid interface, and/or adsorption to
`the container surface [54,55]. Additionally, freezing-induced increases in salt concen-
`tration can reduce intermolecular repulsion (i.e., colloidal stability) between protein
`molecules via charge shielding, resulting in more favorable intermolecular interactions
`that lead to aggregation [14].
`During freezing, there is also an increase in the concentration of protein molecules
`[56] when ice crystallizes and phase separates from the remaining amorphous material.
`Additional excipients (e.g., salt, mannitol) may also crystallize. In aqueous solution,
`increased protein concentration typically corresponds to an increase in the rate of
`aggregation. Although freeze concentration of a protein would therefore be expected
`to promote aggregation, it has often been observed that increasing the initial concen-
`tration of a protein will actually reduce the percentage of aggregation occurring during
`freeze–thawing [50]. It has been suggested that increasing the initial protein concen-
`tration reduces the fraction of protein molecules that is exposed to the ice–liquid
`interface, resulting in reduced aggregation. Thus, the effect of changing the initial
`protein concentration on damage during freeze–thawing can be difficult to predict.
`Numerous factors can affect
`the magnitude and nature of freezing-induced
`stresses, as well as the protein’s responses to them. Among the most critical are the
`pH and ionic strength of the solution, because these factors, in general, modulate
`both the conformational and colloidal stability of protein [14] as well as a protein’s
`response to physical stresses. In addition, the warming and cooling rates used during
`freeze–thaw can alter the degree of macroscopic freeze concentration, surface area
`of the ice–liquid interface, and duration of exposure of the protein to these potential
`stresses. The container material, geometry, and volume can also affect protein damage
`during freeze–thawing by modulating the effects of adsorption of protein molecules
`at the liquid–container interface, and by altering cooling and warming rates.
`In some cases additional changes in physical state have been observed during
`frozen storage. Piedmonte et al. [57] observed protein aggregation during storage
`of sorbitol-containing formulations at −30
`◦
`C. The aggregation correlated with DSC
`melts that are characteristic of crystalline substances and suggest that the sorbitol
`crystallizes over time in the formulation. During freezing, the excipient must remain
`in the same phase as the protein to provide protein stability. By crystallizing, the
`sorbitol is phase-separated from the protein, which leads to protein aggregation.
`
`Ex. 2008-0011
`
`

`

`394
`
`DEVELOPMENT OF FORMULATIONS FOR THERAPEUTIC MONOCLONAL ANTIBODIES
`
`16.2.2.7. Lyophilization-Induced Stresses. Typically, it is desirable to for-
`mulate therapeutic proteins in liquid formulations for ease of administration and lower
`cost of production. However, proteins in liquid formulations are generally at a greater
`risk of and physicochemical degradation. Liquid formulations are also less robust with
`respect to stresses experienced during shipping and handling. If the desired shelf life
`cannot be achieved in a liquid formulation, lyophilization is often the alternative.
`Lyophilized proteins are typically less susceptible to physicochemical degradation
`because of the scarcity of water and the greatly reduced mobility of molecules in
`the dried state [9,58]. Spray drying is also a potential technology for producing fine
`protein powders for inhalation drug delivery [59,60].
`Although biopharmaceuticals are generally more stable in the dried state, it is
`well known that lyophilization and spray-drying processes themselves can be greatly
`damaging to proteins. Lyophilization involves two major steps; freezing of a pro-
`tein solution and drying of the frozen matrix under vacuum. The freezing step can
`potentially destabilize or denature proteins by a variety of mechanisms, including
`cold denaturation, concentration and pH effects, and ice–liquid interfacial effects.
`The drying step can potentially damage proteins by disruption and/or removal of the
`hydrogen-bonding network of water molecules. These dehydration-induced stresses
`are also present during spray drying. Moreover, gas–liquid interface and exposure to
`high temperatures used during spray dry