B i o P r o c e s s Technical
`
`Stability Considerations for
`Biopharmaceuticals, Part 1
`Overview of Protein and Peptide Degradation Pathways
`
`Jalpa Patel, Ruchi Kothari, Rashbehari Tunga, Nadine M. Ritter, and Binita S. Tunga
`
`T o ensure product safety and
`
`efficacy, protein therapeutics
`must meet defined quality
`characteristics immediately after
`manufacture as well at the end of their
`designated shelf lives. Many physical
`and chemical factors can affect the
`quality and stability of
`biopharmaceutical products,
`particularly after long-term storage in a
`container–closure system likely to be
`subject to variations in temperature,
`light, and agitation with shipping and
`handling. Compared with traditional
`chemical pharmaceuticals, proteins are
`considerably larger molecular entities
`with inherent physiochemical
`complexities, from their primary amino
`acid sequences through higher-order
`secondary and tertiary structures —
`and in some cases, quaternary elements
`such as subunit associations (1).
`Many proteins are glycosylated, and
`some have other posttranslational
`modifications such as
`phosphorylation, which also affects
`
`Product Focus: PePtides and Proteins
`
`Process Focus: Formulation/stability
`
`Who should read: analytical and
`Formulation develoPment, Process
`develoPment, Qa/Qc and regulatory
`aFFairs
`
`KeyWords: Forced degradation,
`Protein degradation, oxidation,
`disulFide scrambling, deamidation,
`aggregation, hydrolysis
`
`level: intermediate
`
`20 BioProcess International
`
`January 2011
`
`their potential degradation pathways
`as well as the kinetics of their
`degradation. Proteins are typically
`sensitive to slight changes in solution
`chemistry. They remain
`compositionally and conformationally
`stable only within a relatively narrow
`range of pH and osmolarity, and many
`require additionally supportive
`formulation components to remain in
`solution, particularly over time (2).
`Even lyophilized protein products
`experience degradation (3, 4).
`Advances in analytical chemistry
`have identified many degradation
`pathways that can occur in
`recombinant protein therapeutics over
`time. These pathways generate either
`chemical or physical instability.
`Chemical instability refers to the
`formation or destruction of covalent
`bonds within a polypeptide or protein
`structures. Chemical modifications of
`protein include oxidation, deamidation,
`reduction, and hydrolysis (5).
`Unfolding, dissociation, denaturation,
`aggregation, and precipitation are
`known as conformational or physical
`instabilities (5). In some cases, protein
`degradation pathways are synergistic:
`A chemical event may trigger a
`physical event, such as when oxidation
`is followed by aggregation.
`Here, we present several protein
`degradation events: oxidation,
`photodegradation, disulfide
`scrambling, deamidation, aggregation,
`precipitation, dissociation, and
`fragmentation. We illustrate the
`biochemistry of each, showing
`potential means of induction and
`
`www.photos.com
`
`suggesting formulation considerations
`for prevention. In an upcoming issue,
`Part 2 will conclude with methods of
`detection and strategies for validation
`of stability-indicating methods. Our
`objective is to provide an introduction
`(or refresher) to the major degradation
`pathways of protein products, with
`references for each. Readers are
`encouraged to consult those references
`for expanded details on the basic
`biochemistry of each pathway, case
`studies describing experiments with
`specific proteins, and further
`information on formulation
`development strategies.
`oxidation, Photodegradation,
`and disulFide scramBling
`Proteins and peptides are susceptible
`to oxidative damage through reaction
`of certain amino acids with oxygen
`radicals present in their environment.
`Methionine, cysteine, histidine,
`tryptophan, and tyrosine are most
`susceptible to oxidation: Met and Cys
`because of their sulfur atoms and His,
`
`Nexus Ex. 1031
`Page 1 of 10
`
`

`

`Figure 1: oxidation of methionine (top) and cysteine (bottom) *
`
`COOH
`
`H3N+
`
`C
`
`H
`
`CH2
`
`CH2
`
`S
`
`Oxidant
`
`COOH
`
`H3N+
`
`C
`
`H
`
`CH2
`
`CH2
`
`S O
`
`COOH
`
`H3N+
`
`C
`
`H
`
`CH2
`
`CH2
`O S O
`
`Oxidant
`
`CH3
`Methionine
`
`CH3
`Methionine Sulfoxide
`
`CH3
`Methionine Sulfone
`
`Oxidant
`
`H
`
`COOH
`H3N+ C
`CH2
`CH2
`S O
`OH
`Sul(cid:30)nic Acid
`pKa ~ 1.9
`
`H
`
`COOH
`H3N+ C
`CH2
`CH2
`O S O
`OH
`Cysteic Acid
`pKa ~ (cid:31)5.7
`
`COOH
`H3N+ C
`CH2
`S
`
`H
`
`Cystine
`
`COOH
`H3N+ C
`CH2
`S
`
`H
`
`n t
`
`a
`
`x i d
`
`O
`
`Oxidant
`
`H3N+
`
`COOH
`
`H
`
`C
`CH2
`
`Oxidant
`
`S O
`
`H
`Sulfenic Acid
`pKa ~ 5.7
`
`COOH
`
`H3N+
`
`H
`
`C
`CH2
`SH
`Cysteine
`pKa ~ 8.5
`
`* Griffiths SW. Oxidation of the Sulfur-containing amino acids in Recombinant human α1-antitrypsin (thesis).
`Massachusetts Institute of Technology: Cambridge, MA, 2002.
`
`environment, cysteine oxidation
`involves nucleophilic attack of thiolate
`ions on disulfide bonds, generating
`new disulfide bonds and different
`thiolate ions. The new thiolate can
`then react with another disulfide bond
`to form cysteine.
`Such intermolecular disulfide links
`formed by protein degradation
`accumulate mispaired disulfide bonds
`and scrambled disulfide bridges, which
`can alter protein conformation and
`subunit associations (6). Cysteine
`residues may also undergo spontaneous
`oxidation to form molecular byproducts
`— sulfinic acid and cysteic acid — in
`the presence of metal ions or nearby
`thiol groups (11). For example, human
`fibroblast growth factor (FGF-1)
`exhibits copper-catalyzed oxidation
`that can create homodimers (12).
`Spatial orientation of thiol groups in
`proteins plays an important role in
`cysteine oxidation. The rate of
`oxidation is inversely proportional to
`the distance between those thiol groups
`(13). This can eventually lead to
`formation of large oligomers or
`nonfunctional monomers, as with basic
`fibroblast growth factor (bFGF), which
`contains three cysteines that are easily
`oxidized and form intermolecular or
`intramolecular disulfide bonds (13).
`
`This oxidation often induces
`conformational modifications of the
`protein because cysteine disulfide
`increases side-chain volume in the
`protein’s interior and leads to
`unfavorable van der Waals interactions
`that maintain the original structure (13).
`Histidine residues are highly
`sensitive to oxidation through reaction
`with their imidazole rings, which can
`subsequently generate additional
`hydroxyl species (6). Oxidized
`histidine can yield asparagine/
`aspartate and 2-oxo-histidine (2-O-
`His) as degradation products during
`light and/or metal oxidation (6, 14). It
`may be a transient moiety because it
`can trigger protein aggregation and
`precipitation, which can obscure
`isolation of 2-O-His as an individual
`degradant (15). Oxidation of tyrosine
`may result in covalent aggregation
`through formation of bityrosine (16).
`Spatial factors may also affect tyrosine
`and histidine oxidation. Adjacent
`negatively charged amino acids
`accelerate tyrosine oxidation because
`they have high affinity to metal ions,
`whereas positively charged amino-acid
`residues disfavor the reaction (17, 18). If
`an adjacent amino acid is bulky, it may
`mask oxidation of neighboring amino
`acids and prevent them from getting
`
`Trp, and Tyr because of their aromatic
`rings (6). Oxidation can alter a
`protein’s physiochemical
`characteristics (e.g., folding and
`subunit association) and lead to
`aggregation or fragmentation. It can
`also induce potential negative effects
`on potency and immunogenicity
`depending on the position of oxidized
`amino acids in a protein relative to its
`functional or epitope-like domain(s).
`For example, parathyroid hormone
`biological activity was differentially
`affected by a single oxidation of either
`Met-8 or Met-18 and double oxidation
`(Met-8 with Met-18) when each
`specie was isolated and testing using
`in vitro bioassays (7, 8). Similarly,
`oxidation of Met-36 and Met-48 in
`human stem cell factor (huSCF)
`derived from Escherichia coli decreased
`its potency 40% and 60% (respectively)
`while increasing the dissociation rate
`constant of SCF dimer by two- to
`threefold, which suggests an effect on
`subunit binding and tertiary structure
`(9). In other cases, oxidation had no
`measurable impact on protein potency
`even when substantial structural
`changes were seen. For example,
`oxidized Met-111 in interferon α-2b
`affected the molecule’s primary,
`secondary, and tertiary structure and
`prevented site-specific epitope
`recognition by a monoclonal antibody
`(MAb) without altering in vitro
`biological activity (10).
`Mechanism and Factors Involved:
`Figure 1 shows biochemical pathways
`for oxidation of methionine and
`cysteine residues. Methionine is
`oxidized by atmospheric oxygen and
`oxygen radicals in solution to form
`methionine sulfoxide and methionine
`sulfone. Both species are larger and
`more polar than nonoxidized
`methionine, which can alter protein
`folding and structural stability (11).
`The rate of methionine oxidation in
`recombinant human parathyroid
`hormone (rHu-PTH) by hydrogen
`peroxide is enhanced at alkaline pH (8).
`Cysteine oxidation is also more
`prevalent at alkaline pH, which
`deprotonates thiol groups. Oxidation
`of cysteine induces disulfide bond
`breakage in a reducing environment
`(Figure 1, bottom). In such an
`
`22 BioProcess International
`
`January 2011
`
`Nexus Ex. 1031
`Page 2 of 10
`
`

`

`Figure 3: hydrolysis of a peptide bond
`
`N H
`
`H2O
`
`O
`
`H
`
`OH
`
`N H
`
`O
`
`presence of denaturing/unfolding
`reagents in solution can increase the
`extent of protein oxidation. Excipients
`such as polyols and sugars involved in
`stabilizing protein structure can
`decrease the rate of oxidation (6).
`Oxidative modification depends on
`intrinsic structural features such as
`buried and exposed amino acids. In the
`case of human growth hormone, Met-
`14 and Met-125 are readily oxidized by
`H2O2 because they are exposed to the
`surface of the protein, whereas Met-170
`in its buried position can be oxidized
`only when the molecule is unfolded
`(21). Also, atmospheric oxygen can
`cause protein oxidation over time.
`Headspace oxygen contributed to the
`loss of 50% potency by four months in
`multidose vials of tuberculin purified
`protein (TPP) (25).
`Oxidation can be induced during
`protein processing and storage by
`peroxide contamination resulting from
`polysorbates and polyethylene glycols
`(PEGs), both commonly used as
`pharmaceutical excipients. A
`correlation has been observed between
`the peroxide content in Tween-80 and
`the degree of oxidation in rhG-CSF,
`and peroxide-induced oxidation
`appeared more severe than that from
`atmospheric oxygen (26). Peroxide can
`also leach from plastic or elastomeric
`materials used in primary packaging
`container–closure systems, including
`prefilled syringes (27, 28).
`Preventive Measures: One
`molecular engineering strategy for
`minimizing oxidative degradation is to
`replace oxygen-labile amino acids with
`oxygen-resistant ones if a protein’s
`nature permits. In therapeutic
`Interferon beta (IFN-β), cysteine at
`position 17 was replaced by serine,
`because the former loses antiviral
`activity during storage to oxidation
`and disulfide scrambling (29).
`Substitution of methionine of
`epidermal growth factor (EGF) with a
`nonnaturally occurring norleucine also
`prevented oxidative degradation (30).
`
`Ala
`
`O C
`
`CH2
`
`Figure 2: Deamidation pathway of asparagine *
`O
`C
`
`Val Tyr Pro
`
`CH2
`CH
`
`NH2
`NH
`
`?
`
`C
`O
`L-Asn Peptide
`
`O
`C
`
`CH2
`CH
`
`O
`NH
`
`O
`C
`
`CH2
`
`C
`O
`L-Normal Peptide
`?
`D-Normal Peptide
`
`O
`C
`
`C
`
`CH2
`CH
`
`O
`
`C
`
`N
`
`CH2
`
`O
`L-Imide Peptide
`
`D-Imide Peptide
`
`O
`C
`
`CH2
`CH
`
`O
`C
`
`NH
`
`CH2
`
`O
`
`C
`O
`L-Iso Peptide
`
`?
`D-Iso Peptide
`
`* Deamidation, Isomerization, and Racemization at Asparaginyl and Aspartyl Residues in Peptides:
`Succinimide-Linked Reactions that Contribute to Protein Degradation. J. Biolog. chem. 262(2) 1987: 785–794.
`
`oxidized. It has been observed that
`histidine present in a sequence
`markedly increases both the peptide
`oxidation rate and methionine
`sulfoxide production. The strong
`metal binding affinity of the
`imidazole ring on the histidine side
`chain brings oxidizing species close to
`the substrate methionine (6).
`Light Degradation: Photooxidation
`can change the primary, secondary,
`and tertiary structures of proteins and
`lead to differences in long-term
`stability, bioactivity, or
`immunogenicity (19). Exposure to
`light can trigger a chain of
`biochemical events that continue to
`affect a protein even after the light
`source is turned off. These effects
`depend on the amount of energy
`imparted to a protein and the presence
`of environmental oxygen.
`Photooxidation is initiated when a
`compound absorbs a certain
`wavelength of light, which provides
`energy to raise the molecule to an
`excited state. The excited molecule
`can then transfer that energy to
`molecular oxygen, converting it to
`reactive singlet oxygen atoms. This is
`how tryptophan, histidine, and
`tyrosine can be modified under light
`in the presence of O2 (6). Tyrosine
`photooxidation can produce mono-,
`di-, tri-, and tetrahydroxyl tyrosine as
`byproducts (18). Aggregation is
`observed in some proteins due to
`
`cross-linking between oxidized
`tyrosine residues (20). Photooxidation
`reaction is predominately site specific
`(21). For example, in human growth
`hormone treated with intense light,
`oxidation is carried out predominantly
`at histidine-21 (22). In addition, the
`peptide backbone is also a
`photodegradation target (23).
`Alternatively, the energized protein
`itself can react directly with another
`protein molecule in a photosensitized
`manner, typically via methionine and
`tryptophan residues at low pH (6).
`Excipients and leachables can
`synergistically affect the oxidation
`(and hence, degradation) of a protein.
`Formulation components influence the
`rate of photooxidation in some
`instances: e.g., phosphate buffer
`accelerates the rate of methionine
`degradation more than other buffer
`systems do (22). Metal-ion–catalyzed
`oxidation depends on concentration of
`metal ions in the environment. The
`presence of 0.15-ppm chloride salts of
`Fe3+, Ca2+, Cu2+, Mg2+, or Zn2+ does
`not affect the rate of oxidation for
`human insulin-like growth factor-1,
`but when the metal concentration
`increased to 1 ppm, a significant
`increase in oxidation was observed
`(23). Oxidation can be exacerbated in
`the presence of a reducing agent such
`as ascorbate. Ascorbic acid increased
`oxidation of human ciliary
`neurotrophic factor (24). Also, the
`
`24 BioProcess International
`
`January 2011
`
`Nexus Ex. 1031
`Page 3 of 10
`
`

`

`
`
`
`
`Removal of headspace oxygen by
`degassing may be effective for
`preventing oxidation in some cases.
`Filling steps are carried out under
`nitrogen pressure, and vial headspace
`oxygen is replaced with an inert gas
`such as nitrogen to prevent oxidation
`(21, 25). With some oxidation-sensitive
`proteins, processing is carried out in
`the presence of an inert gas such as
`nitrogen or argon. For multidose drug
`preparations, use of cartridges with
`negligible headspace overcomes
`oxidation and related consequences (25).
`Care must be exercised when
`container–closure changes are
`considered. Many such changes for
`protein therapeutics (from vials to
`prefilled syringes or prefilled syringes
`to pen devices, for example) are
`considered to enhance patient
`convenience and ease of use. But
`historical experience with container–
`closure systems based only on chemical
`pharmaceuticals should be reevaluated
`when the same materials are used with
`protein-based products because of
`potential for unexpected, unique
`impacts on protein degradation.
`Controlling or enhancing factors
`such as pH, temperature, light
`exposure, and buffer composition can
`also mitigate the effects of oxidation
`by affecting a protein’s environment.
`Cysteine oxidation often can be
`controlled by maintaining the correct
`redox potential of a protein
`formulation, such as with addition of
`thioredoxin and glutathione.
`Antioxidants and metal chelating
`agents also can be used to prevent
`oxidation in protein formulations.
`Antioxidants are chemical “sacrificial
`targets” with a strong tendency to
`oxidize, consuming chemical species
`that promote oxidation. Scavengers
`such as l-methionine and ascorbic
`acid are used for this purpose in
`biotherapeutic formulations (31). In
`the absence of metal ions, cysteine as a
`free amino acid may act as an effective
`antioxidant. As chelating agents,
`EDTA and citrate might form
`complexes with transition metal ions
`and inhibit metal-catalyzed, site-
`specific oxidation (6). Addition of
`sugars and polyols may also prevent
`metal-catalyzed oxidation because of
`
`their complexion with the metal ions.
`Protective effects of glucose, mannitol,
`glycerol, and ethylene glycol against
`metal-catalyzed oxidation has been
`observed with human relaxin (32).
`Physical protection from UV/white
`light exposure with either a primary or
`secondary packaging system may be
`necessary to protect light-labile
`proteins from photooxidation.
`deamidation
`With many recombinant proteins,
`changes in peptide and protein
`structure are observed through the
`nonenzymatic deamidation of
`glutamine and asparagine residues.
`This can have varying effects on their
`physiochemical and functional stability
`(33, 34). It has been observed that
`deamidation of hGH alters proteolytic
`cleavage of the human growth
`hormone (33). And it was reported that
`deamidation of IFN-beta increased its
`biological activity (35). It has been
`determined that deamidation of
`peptide growth-hormone–releasing
`factor leading to aspartyl and iso-
`aspartyl forms reduces the bioactivity
`by 25- and 500-fold, respectively, as
`compared with the native peptide (36).
`Deamidation at an Asn-Gly site in
`hemoglobin changes its affinity to
`oxygen (37). Asparagine deamidation
`perturbs antigen presentation on Class
`II major histocompatibility complex
`molecules (38). It was reported that
`isomerization of Asp 11 in human
`epidermal growth factor led to a
`fivefold reduction in its mitogenic
`activity (39). And deamidation at two
`Asn-Gly sequences in triose-phosphate
`isomerase resulted in subunit
`dissociation (40).
`Mechanism and Factors Involved:
`Deamidation is a chemical reaction in
`which an amide functional group is
`removed from an amino acid.
`Consequences include isomerization,
`racemization, and truncation of
`proteins. Figure 2 shows the
`mechanism of asparagine degradation
`by deamidation.
`Isomerization: Isomerization of
`aspartate to isoaspartate residues in a
`protein solution is the most commonly
`observed outcome of nonenzymatic
`deamidation (41, 42).
`
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`
`BPI0101-QP-Pall.indd 1
`
`12/13/10 12:53:03 PM
`
`Nexus Ex. 1031
`Page 4 of 10
`
`

`

`Racemization: Succinimide
`intermediates formed during
`asparagine deamidation are highly
`prone to racemization and convert to
`d-asparagine residues (41, 42).
`Racemization of other amino acids,
`except glycine, is observed at alkaline
`pH (42).
`Truncation: At low pH levels,
`peptides and proteins experience
`deprotonation of the amide group on
`their asparagine side chain, followed
`by nucleophilic attack by the nitrogen
`atom of the amide anion on the
`peptide carbonyl carbon of the
`asparagine residue (43). This generates
`peptide chain cleavage by forming a
`succinimide peptide fragment.
`Subsequent hydrolysis of the
`succinimide ring can yield asparaginyl
`and β-asparaginyl peptides.
`Mechanisms for the aspartate–
`isoaspartate deamidation and
`isomerization reactions are similar
`because they both proceed through an
`intramolecular cyclic imide
`intermediate (44). Deamidation rates
`for individual amide residues depends
`on their primary sequence and three-
`dimensional (3D) structure as well as
`solution properties such as pH,
`temperature, ionic strength, and buffer
`ions (45). The deamidation rate for
`glutamine residues is usually less than
`that of asparagine residues (46, 47).
`If pH is >5.0, deamidation occurs
`through very unstable cyclic imide
`intermediate formation, which
`spontaneously undergoes hydrolysis.
`Under strong acidic conditions (pH
`1–2), direct hydrolysis of the amide side
`chain becomes more favorable than
`formation of cyclic imide (48). Peptide
`bond cleavage occurs to a greater extent
`in direct amide hydrolysis. At neutral
`pH, deamidation can lead to structural
`isomerization.
`The rate of deamidation is also
`influenced by protein secondary
`structure. Increasing helical structure
`decreases the rate of deamidation in
`some proteins (50). The rate of
`deamidation in several growth-
`hormone–releasing factor analogs was
`examined as a function of methanol-
`induced α-helical structure. Addition
`of methanol increased the level of
`α-helicity and decreased the rate of
`
`26 BioProcess International
`
`January 2011
`
`deamidation (51). In its native
`structure, RNAase resists
`deamidation possibly because of the
`relatively rigid backbone in the loop
`stabilized by a disulfide between
`Cys-8 and Cys-12 and by the β-turn
`at residues 66–68, which could
`hinder the formation of the cyclic
`imide (52). But if it is reduced and
`denatured, then refolded, aspartic
`and isoaspartic forms are generated,
`demonstrating different enzymatic
`activities. Replacement of Asp-67
`with Iso-Asp-67 showed that the
`isoaspartic form refolds at half the
`rate of the fully amidated form (51).
`Storage temperatures can affect a
`protein’s deamidation rate in the
`presence of certain biological buffers.
`Because amine buffers (e.g., Tris and
`histidine) have high temperature
`coefficients, storage at temperatures
`that are different from the temperature
`of preparation could shift formulation
`pH. Deamidation and isomerization
`reactions are pH-sensitive processes, so
`those shifts in formulation pH could
`alter the rate of deamidation. Another
`indirect effect of temperature is the
`dissociation constant of water: The
`hydroxyl ion concentration of water can
`vary as a function of temperature and
`thereby affect deamidation rates (39).
`Preventive Measures: Solution pH
`can substantially affect deamidation
`(44). Formulations at pH 3–5 can
`minimize peptide deamidation (48).
`AsnA-21 and AsnB-3 of insulin forms
`isoaspartate or aspartate, depending
`on the pH of solution (52). Insulin
`deamidates rapidly at Asn A-21 in low
`pH solutions (57). Steric hindrance
`also can affect deamidation rate:
`Bulky residues following asparagine
`may inhibit the formation of
`succinimide intermediate in the
`deamidation reaction (42).
`Replacement of a glycine residue with
`more bulky leucine or proline residues
`resulted in a 30- to 50-fold decrease in
`the rate (42). In lyophilized
`formulations, the deamidation rate is
`typically reduced, probably due to
`limited availability of free water in
`which the reaction can occur.
`Formulations that incorporate
`organic cosolvents can decrease their
`deamidation rates because addition of
`
`organic solvents decreases dielectric
`constants of a solution (44). Decreasing
`solvent dielectric strength — by
`addition of cosolutes such as glycerol,
`sucrose, and ethanol to a protein
`solution — leads to significantly lower
`rates of isomerization and deamidation
`(39, 44). Lowering dielectric strength
`of the medium from 80 (water) to 35
`(PVP/glycerol/water formulations) led
`to about a sixfold decrease in peptide
`deamidation rates (54). The lower rate
`of deamidation was attributed to less
`stabilized ionic intermediates formed
`during cyclization in the asparagine
`deamidation pathway. Insulin
`prepared in neutral solutions
`containing phenol showed reduced
`deamidation probably because of its
`stabilizing effect on the tertiary
`structure (α-helix formation) around
`the deamidating residue, which
`lowered the probability for formation
`of intermediate imides (53).
`aggregation and PreciPitation
`Aggregated proteins are a significant
`concern for biopharmaceutical
`products because they may be
`associated with decreased bioactivity
`and increased immunogenicity.
`Macromolecular protein complexes
`can trigger a patient’s immune system
`to recognize the protein as “nonself”
`and mount an antigenic response (55).
`Large macromolecular aggregates also
`can affect fluid dynamics in organ
`systems such as eyes (56).
`Aggregation is a common problem
`encountered during manufacture and
`storage of proteins (16). The potential
`for aggregated forms is often enhanced
`by exposure of a protein to liquid–air,
`liquid–solid, and even liquid–liquid
`interfaces (57). Mechanical stresses of
`agitation (shaking, stirring, pipetting
`or pumping through tubes) can cause
`protein aggregation. Freezing and
`thawing can promote it as well.
`Solution conditions such as
`temperature, protein concentration,
`pH, and ionic strength can affect the
`rate and amount of aggregates
`observed. Formulation in sucrose can
`increase aggregation over time because
`of protein glycation when sucrose is
`hydrolyzed (58). The presence of
`certain ligands — including certain
`
`Nexus Ex. 1031
`Page 5 of 10
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`

`

`
`
`
`
`ions — may enhance aggregation.
`Interactions with metal surfaces can
`lead to epitaxic denaturation, which
`triggers aggregate formation. Foreign
`particles from the environment,
`manufacturing process, or container–
`closure system (e.g., silicone oil) can
`also induce aggregation (21, 59, 60).
`Even handling protein products at
`compounding pharmacies can induce
`aggregation 10-fold above initially
`observed amounts (58).
`The impact of aggregation on
`product potency varies based on the
`physiochemical attributes of each
`protein relative to its functional
`domains and the nature of the activity
`being measured. Enzymes such as
`urease and catalase can lose up to 50%
`of their potency after shaking,
`fibrinogen clotting activity is
`decreased after shear stress, and
`recombinant IL-2 and recombinant
`interferon activity is substantially
`affected by aggregation from shaking
`and shearing (2). Aggregation also
`affects the mass balance of protein
`solutions, decreasing the concentration
`of the target protein. Microaggregated
`subvisible particles generated
`anywhere in a manufacturing process
`can develop into larger particles over
`time as a product is stored (62).
`Bevacizumab drug product lost 50% of
`active IgG after manipulations at the
`pharmacy triggered significant growth
`of micron-sized particles in
`repackaged solutions (58).
`Aggregates can be soluble or
`insoluble, reversible or irreversible,
`covalent or noncovalent (16). Soluble
`aggregates are usually reversible: e.g.,
`by altered solution conditions such as
`changing temperature or osmotic
`strength or by mild physical disruption
`such as swirling or filtration. Insoluble
`aggregates are typically irreversible.
`Under vigorous physical disruption
`(e.g., agitation or freezing and thawing)
`or over time in storage, they can grow
`into particles that may eventually
`precipitate. Covalent aggregates form
`when monomeric proteins become
`chemically crosslinked, e.g., though
`disulfide bonds. Although covalent
`linkages are necessary to stabilize the
`native tertiary structure of most
`polypeptide proteins, those that form
`
`by degradation can produce undesired
`crosslinks between protein moieties,
`which can lead to irreversible
`aggregation. Noncovalent aggregates
`are formed when proteins associate and
`bind based on structural regions of
`charge or polarity. Because such
`associates are weak (relative to covalent
`linkages), they are sensitive to solution
`conditions and usually reversible.
`Mechanism and Factors Involved:
`Because of the many physical and
`chemical manipulations required in
`upstream production and downstream
`processing, followed by formulation
`and filling operations, aggregation of
`protein biopharmaceuticals can be
`induced during nearly every step of the
`process including at hold points,
`shipping, and long-term storage (16, 21).
`Agitation (e.g., shaking, stirring, and
`shearing) of protein solutions can
`promote aggregation at the air–liquid
`interfaces, where protein molecules may
`align and unfold, exposing their
`hydrophobic regions for charge-based
`association (2). Agitation-induced
`aggregation has been seen in numerous
`protein products, including
`recombinant factor XIII, human
`growth hormone, hemoglobin, and
`insulin (2). Minimizing foaming caused
`by agitation during manufacture (as
`well as during product use) may be
`critical to preventing significant loss of
`protein activity or generation of visible
`particulate matter (62).
`Protein concentration also can
`promote aggregation, with or without
`agitation events. Results obtained
`from two PEGylated proteins and one
`Fc fusion protein demonstrated a
`direct correlation between protein
`concentration and aggregation under
`nonagitated (quiescent) conditions, but
`researchers found an inverse
`correlation between protein
`concentration and aggregation under
`conditions of shaking, vortexing, and
`simulated shipping (63).
`Antimicrobial preservatives used in
`multidose formulations also can
`induce protein aggregation. For
`example, benzyl alcohol accelerates the
`aggregation of rhGCSF because it
`favors partially unfolded
`conformations of the protein (64).
`Increasing antimicrobial preservative
`
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`12/13/10 12:53:03 PM12/13/10 12:53:03 PM
`
`Nexus Ex. 1031
`Page 6 of 10
`
`

`

`levels may increase the hydrophobicity
`of a formulation and could affect a
`protein’s aqueous solubility (62).
`Phenol and m-cresol can considerably
`destabilize a protein: Phenol promotes
`formation of both soluble and
`insoluble aggregates, whereas m-cresol
`can precipitate protein (65).
`Freezing and thawing — which can
`occur multiple times throughout
`production and use of protein
`therapeutics — can dramatically affect
`protein aggregation. Generation of
`water-ice crystals at a container’s
`periphery (where heat transfer is
`greatest) can produce a “salting out”
`effect, whereby the protein and
`excipients become increasingly
`concentrated at the slower-freezing
`center of a container (21). High-salt
`and/or high-protein concentrations
`can result in precipitation and
`aggregation during freezing, which is
`not completely reversible upon
`thawing. The effect can be seen with
`thyroid-stimulating hormone: When
`stored at –80 °C, 4 °C, or 24 °C for up
`to 90 days, it remained stable, but
`when frozen to –20 °C it lost >40%
`potency in that period, which was
`attributed to subunit dissociation (66).
`Multiple freezing and thawing cycles
`can exacerbate that effect and lead to a
`cumulative impact on the generation
`and growth of subvisible and visible
`particulates. A change in pH can
`come from crystallization of buffer
`components during freezing. In one
`study, potassium phosphate buffers
`demonstrated a much smaller pH
`change on freezing than did sodium
`phosphate buffers (21).
`Compendia currently limit the
`number of particles ≥10 μm and ≥25
`μm in size that may be present in
`injectible pharmaceutical prepar