`
`Effects of Surfaces and Leachables on the Stability
`of Biopharmaceuticals
`
`IARED S. BEE,‘ THEODORE W. RANDOLPH,2 IOHN F. CARPENTER,3 STEVEN M. BISHOP,1 MARIANA N. DIMITROVA‘
`
`lFormulation Sciences Department, Medlmmune, Gaithersburg, Maryland 20878
`
`2Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309
`
`3Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Aurora, Colorado 80045
`
`Received 19 October 2010; revised 11 January 2011; accepted 12 April 2011
`
`Published online 26 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002 /jps.22597
`
`ABSTRACT: Therapeutic proteins are exposed to various potential contact surfaces, particles,
`and Ieachables durin g manufacturing, shipping, storage, and delivery. In this review, we present
`published examples of interfacial- or leachable-induced aggregation or particle formation, and
`discuss the mitigation strategies that were successfully utilized. Adsorption to interfaces or
`interactions with leachables and/or particles in some cases has been reported to cause protein
`aggregation or particle formation. Identification of the cause(s) of particle formation involving
`minute amounts ofprotein over extended periods oftime can be challenging. Various formulation
`strategies such as addition of a nonionic surfactant (e.g., polysorbate) have been demonstrated
`to effectively mitigate adsorption-induced protein aggregation. However, not all stability prob-
`lems associated with interfaces or leachables are best resolved by formulation optimization.
`Detectable leachables do not necessarily have any adverse impact on the protein but control
`of the leachable source is preferred when there is a concern. In other cases, preventing pro—
`tein aggregation and particle formation may require manufacturing process and/or equipment
`changes, use of compatible materials at contact interfaces, and so on. This review summarizes
`approaches that have been used to minimize protein aggregation and particle formation dur—
`ing manufacturing and fill—finish operations, product storage and transportation, and delivery
`of protein therapeutics. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J
`Pharm Sci 100:4158—4170, 2011
`Keywords: protein aggregation; formulation; stability; agitation; air—water interface; adsorp—
`tion; particles; leachables; surface; biopharmaceuticals characterization
`
`INTRODUCTION
`
`Therapeutic proteins are used to treat a wide range
`of serious medical conditions, providing substantial
`benefits to patients. Proteins are complex molecules,
`subject to both intrinsic variation (eg, glycosylation
`pattern and charge isoforms) and a variety of chemi—
`cal (e. g., deamidation and oxidation) and physical (for—
`mation of soluble aggregates, particle formation, and
`reversible association) degradation pathways. Most
`common intrinsic degradation pathways for protein
`therapeutics include aggregation and often particle
`
`
`Abbreviations used: mAb, monoclonal antibody
`Correspondence to: Jared S. Bee (Telephone: +301—398-5912;
`Fax: +303—492—4341; E—mail: Bee-J@medimmune.com, jarcdsbec
`@gmailcoml
`Journal of Pharmaceutical Sciences, Vol. 100, 4158—4170 (2011)
`© 2011 Wiley—Liss, Inc. and the American Pharmacists Association
`
`formation, with the resulting degradation products
`normally making up a very small mass fraction of the
`therapeutic protein product. Not all molecular vari-
`ants or degradation products necessarily result in a
`loss of efficacy or a decrease in safety. Some types of
`protein aggregates may elicit immune responses in
`patients},2 However, the mechanisms for immuno—
`genicity of therapeutic proteins in patients are still
`not well understood and a link between immunogenic—
`ity and aggregates or particles in products remains
`unclear in many cases.3’4
`technology, biotechnology
`Using state—of—the—art
`companies use formulation and process control strate-
`gies to obtain high purity and stability in order to
`meet a typical goal of a 2-year shelf life.5 For the
`general case of bulk protein aggregation as described
`by Chi et al.,‘5 either partial unfolding or aggre—
`gate assembly can be the rate-determining step for
`
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`aggregation of proteins. The conformational and col-
`loidal stability of the protein can be optimized through
`the appropriate use of formulation buffer type, pH,
`and excipients.5‘10 Similarly, formulation conditions
`can also be used to maximize chemical stability of
`proteins5‘10 High-concentration monoclonal antibody
`(mAb) products present their own unique challenges
`such as self-association, viscosity, opalescence, and
`protein particle formation. 11,12
`Protein stability in bulk solution is only one of
`the key issues. During manufacturing, final fill—fin-
`ish, storage, and delivery, proteins may adsorb to sur-
`faces or react/bind with leachables. In some cases,
`this has resulted in aggregation, particle formation,
`or adsorption losses?!10 Figure 1 depicts some of the
`processes of how solid and liquid contact surfaces and
`leachables have caused instabilities in protein prod-
`ucts. Adsorption of proteins to surfaces is a complex
`process that is important in many fields.13~14 Protein
`
`surface adsorption can be driven by a combination
`of electrostatic forces, hydrophobic binding interac—
`tions, and entropy changes due to contributions from
`both water and protein”?15 These surface adsorption
`processes may be reversible or irreversible and may
`lead to either unfolding or partial unfolding of the ad-
`sorbed protein or only minimal perturbations to the
`protein structure. Depending on these factors, the ad-
`sorption of protein may be minimal and not cause any
`additional aggregation or particle formation. Simple
`adsorption can result in a reduction in the bulk pro-
`tein concentration that can be more of a concern for
`
`low concentration formulations. In other cases, pro-
`tein adsorption could nucleate further aggregation
`and particle formation. If adsorption is reversible,
`it is possible that the desorbed proteins may be re-
`leased in a structurally perturbed form that could
`lead to further aggregation or particle formation in
`the bulk.16 However, the detailed mechanism(s) has
`
`PATHWAY
`
`AGGREGATE FORMS
`
`000
`00
`
`No adverse impact of surfaces
`or leachables
`
`0o stopper
`
`00
`
`000
`CO
`
`No aggregates formed
`by surfaces or leachables
`
`%
`Protein particles
`
`Nucleation of protein particles
`1 at the air-water interface
`
`I
`
`Q};
`Nucleation of aggregates on
`heterogenous particles or surfaces
`
`" do
`
`Air—water
`interface
`
`0
`
`o 0
`Proteln O
`Vial surface 0
`
`0
`
`(Potential) particle
`or silicone droplet
`.
`
`O
`O
`(potential)
`Leachable ' .'
`O
`O
`#
`
`0 ’
`
`Physical or chemical instability
`
`caused by leachables
`O
`. O
`r6§q900
`o 852%
`Agglomeration of protein-coated
`particles or silicone droplets
`
`
`
`Coagulation with leachables
`
`Insoluble aggregates
`
`->
`
`006
`Soluble aggregates
`
`O
`_
`.
`.
`’ Modlfled or damaged protern
`
`o
`.0
`
`4‘9a"
`
`Heterogenous particles
`
`WA
`
`dsorption losses
`
`WA
`
`dsorption to solid surfaces
`
`Possible physical degradation pathways of proteins caused by interfaces, foreign
`Figure 1.
`particulates, and leachables described in this review. The processes in the figure correspond to
`specific examples that have been published and are discussed in the text. Although the figure
`shows a Vial as one example, these processes may also occur in other upstream operations and
`in other containers or delivery devices. These examples are also described and reviewed in this
`work.
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`not been fully determined for many published reports
`of adverse protein interactions with surfaces. Figure 1
`illustrates alternative mechanisms of particle forma—
`tion. These include agglomeration of protein—coated
`particles or silicone oil droplets and coagulation of
`proteins with leachables. This might occur when a
`few subvisible particles (SVPs) that were initially col-
`loidally stable (due to a high negative surface charge
`in the case of glass and silicone oil at common formu—
`lation pH conditions) become less stable when the sur—
`face charge is reduced by adsorption of protein. These
`protein-coated particles may then simply agglomerate
`together to form larger, more easily detectable parti—
`cles. It is also possible that foreign particles could ag—
`glomerate even if there is little or no protein adsorp-
`tion to the particles. A similar process of binding of
`leachables to proteins can lead to particle formation
`through colloidal destabilization of the protein, fol—
`lowed by precipitation of particles. Other leachables
`may also cause protein damage by directly reacting
`with the protein, potentially creating an aggregation—
`competent protein species. The last process for ag—
`gregation and particle formation we discuss is expo-
`sure to the air—water interface. Air—water interface
`
`exposure is one of the more common causes of par-
`ticle formation and aggregation described in the lit—
`erature. As with other interfaces, the details of the
`mechanism(s) of air~water interface—induced aggre—
`gation are not well described for many proteins. In
`this review, we present examples of the published ev-
`idence for these aggregation and particle formation
`processes and discuss rational mitigation strategies.
`Many of these examples of interface- and leachable-
`induced aggregation and particle formation processes
`are specific to certain products or conditions. We also
`note that detectable leachables may have no adverse
`impact of product safety, efficacy, quality, or protein
`stability. In this review, we have included many dif—
`ferent examples (even if they are less common) so that
`the lessons learned may be used to help in the practi—
`cal resolution of other similar issues in the future.
`
`MANUFACTURING AND FILL—FINISH
`OPERATIONS
`
`Manufacturing of therapeutic proteins is a complex
`process, which begins with production of the protein
`in cells cultured in a bioreactor wherein the protein
`is exposed to a multitude of solution species in the
`growth medium. The protein is then separated from
`the cell culture media by filtration or centrifugation.
`Recovery from inclusion bodies and refolding are per—
`formed if necessary. In downstream protein purifica-
`tion, viral inactivation and removal steps are often
`performed (e.g.,
`low pH incubation, nanofiltration,
`and solvent—detergent addition). Multiple chromatog—
`raphy (e.g., affinity, ion exchange, and hydrophobic
`
`interaction) and filtration steps are used to purify
`the protein further. The protein may be concentrated
`and formulated using diafiltration. The formulated
`bulk may then be frozen or held before the final
`sterile filtration and fill—finish operations. Following
`the final sterile filtration step, the product is filled
`into vials, syringes, or cartridges. Each of these steps
`may expose the protein to interfaces (i.e., solid—liq—
`uid and air—liquid) under a variety of solution condi-
`tions. In this review, we focus on downstream exam—
`ples of interfacial protein instabilities, although many
`of the aggregation and particle formation processes to
`which proteins are exposed during downstream unit
`operations could also be relevant to the cell culture
`environment.
`
`Diafiltration
`
`Air bubble entrainment and/or microcavitation have
`
`been cited as a cause of aggregation during diafil—
`tration operations.”“19 Adsorption to solid surfaces,
`contamination by particulates, and increased rate of
`aggregate assembly due to mixing could also be causes
`of aggregation.19 Simple adsorption losses and fouling
`of the protein onto the membrane can also occur. For
`instance, deactivation of aminoacylase was directly
`caused by adsorption losses to an ultrafiltration mem-
`brane surface.20 The type and brand offiltration mem—
`brane have been shown to result in different levels of
`
`protein adsorption.21
`Process controls may be used to minimize aggrega—
`tion during diafiltration by optimization of the oper-
`ation parameters such as the transmembrane pres—
`sure and cross-flow rate.22 It has been suggested
`that reducing turnover of the air—water interface and
`bubble entrainment would also reduce the formation
`
`of particles in biotherapeutics during diafiltration
`operationsm'19 It is possible that some formulation
`excipients can provide additional protection during
`diafiltration. This of course depends upon whether ex-
`cipients are added during the diafiltration operation
`or afterward by addition of a concentrated stock of
`the excipients. Although addition of a surfactant can
`suppress the formation of aggregates at the air—water
`interface when the protein is also exposed to shear,23
`this strategy may not be practical for diafiltration op—
`erations. Surfactants are normally added after the
`diafiltration operation because of the difficulties in
`controlling and predicting the final surfactant level
`in the retentate.24
`
`Freezing and Thawing
`
`Freezing is a common unit operation during the pro—
`duction of therapeutic protein products. Bulk inter-
`mediates are often frozen to increase their stability
`during production hold steps and freezing is the first
`step in lyophilization. Freezing and thawing can trig—
`ger aggregation and particle formation in proteins by
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`various different mechanisms.25 Storage temperature
`and freezing rate are important parameters for frozen
`stability. One other factor that is the focus of this re—
`view contributing to the overall destabilization of the
`protein is the choice of container material. For ex-
`ample, polytetrafluoroethylene and other commercial
`freezing containers fostered more aggregation than
`polypropylene during freeze—thawing of an IgGg.26
`Cryoprotectants, such as sucrose or trehalose, are of—
`ten added to protein formulations to protect against
`freezing and thawing damage.25
`The ice—solution interface itself can be destabi-
`
`lizing to proteins: increased intermolecular [l—sheet
`content was measured by infrared spectroscopy for
`two different proteins adsorbed to the ice surface.27
`Polysorbate addition has been shown to reduce for—
`mation of nonnative intermolecular D-sheet levels in
`proteins adsorbed to ice interfaces.27 In this case,
`the ability of polysorbate to reduce such structural
`changes in ice‘adsorbed protein molecules was pro—
`tein specific.27 Polysorbate 20 protected Factor XIII
`during freeze—thawing by competing with the par—
`tially unfolded protein for interfaces.28 Additionally,
`polysorbate 80 protected hemoglobin from damage
`at interfaces during freezing.29 The 40% or greater
`loss of interleukin-11 (IL—11) activity caused by ad—
`sorption to glass lyophilization vials was prevented
`by polysorbate 20, although for complete protection
`during lyophilization, trehalose and human albumin
`were also necessary.30
`The rate of cooling and the degree of supercooling
`affect the number and sizes of ice crystals and the
`time the protein is exposed to the ice interface. Each
`of these variables could potentially influence the ex—
`tent of freeze—thawing-induced protein aggregation.
`Because there are multiple variables in freeze—thaw
`stress, experimental studies to test the sensitivity of
`the specific protein formulation to realistic and worst—
`case freeze—thaw stresses can be used to determine
`
`appropriate mitigation strategies.
`
`Sterile Filtration and Fill—Finish
`
`Sterile filtration and fill—finish operations may exert
`adverse effects on stability by exposing the protein to
`production equipment surfaces (e.g., those presented
`by membranes, tubing, and pumps). In an engineering
`approach, choice of equipment to minimize air—water
`interface exposure and turnover, particle shedding,
`leachables, and cavitation can be employed to elimi—
`nate or minimize suspected causes of aggregation}9
`This type of optimization should be performed while
`also maintaining product homogeneity (i.e., ensuring
`mixing is adequate) and sterility, and overall robust-
`ness and quality. These same strategies could also
`be useful to minimize aggregation or particle forma—
`tion in other upstream unit operations. Formulation
`approaches can also be very effective at reducing ad—
`
`verse interactions with interfaces. For instance, the
`aggregation leading to membrane fouling during ster—
`ile filtration of human growth hormone was found to
`be caused by adsorption to hydrophobic interfaces and
`could be mitigated by addition of surfactant.“ Differ-
`ences in the magnitude of protein adsorption has been
`observed between different types and brands of ster-
`ilizing filters (e.g., polyvinylidene fluoride (PVDF),
`polyethersulfone (PES), cellulose acetate (CA), and
`Nylon).21 Various filters were found to adsorb polysor—
`bate 80, requiring appropriate setup of the prefiush
`step to avoid decreasing the levels of surfactant below
`the intended value for the final protein formulation.21
`Interestingly, it has been found that cellulose could
`preferentially adsorb soluble aggregates of a mAb
`from solution, although this did not have any adverse
`effect on the protein stability in bulk solution.32
`Stainless steel is ubiquitous in protein produc—
`tion equipment and has been reported to be a cause
`of protein aggregation or fragmentation in several
`cases: submicron steel particles shed from a pump in
`the laboratory environment caused “agglomeration of
`protein-coated particles” (see Fig. 1) and/or nucleated
`formation of larger aggregates of a mAb33; Fe ions
`caused hinge-region fragmentation of a mAb34; expo-
`sure to the stainless steel surface combined with ad—
`
`ditional shear stress resulted in aggregation of a mAb
`that exhibited a first—order dependence on protein
`concentration35; Fe ions leached from steel caused
`oxidation and aggregation36; Fe ions directly bound
`to a protein resulting in conformational destabiliza—
`tion followed by aggregation,37 and surface—induced
`soluble aggregation of a mAb had a second—order de—
`pendence on steel surface area and a zero—order de—
`pendence on bulk protein concentration that could
`be completely suppressed by polysorbate.38 Stainless
`steel surfaces typically are “passivated” or “electropol—
`ished” to create a more corrosion-resistant chromium
`
`oxide—rich surface layer. Factors that may impact the
`protein in solution include the following: the grade of
`steel alloy, the frequency of passivation, and chemical
`exposures of the steel. The impact of the formulation
`may play a particularly large role in the potential ad-
`verse interactions; for instance, exposure of steel to
`chloride ions at low pH has been shown to result in
`corrosion and release ofFe ions that subsequently cat—
`alyzed the oxidation of methionine residues.36 Stain—
`less steel exposure is an example of where there may
`be multiple distinct causes of aggregation or par—
`ticle formation: the steel surface itself, steel parti-
`cles shed from equipment, and the Fe ions leached
`from steel equipment. These examples would corre—
`spond to the scenarios of “physical or chemical in-
`stability caused by leachables” (Fe ions), “nucleation
`of aggregates on heterogenous particles or surfaces”
`(steel surface), and “agglomeration of protein—coated
`particles” (steel particles) shown in Figure 1. Here,
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`correct identification of the cause of the aggregation,
`fragmentation, or particle formation is crucial for cre—
`ating an effective mitigation strategy. Addition of a
`surfactant would be expected to reduce aggregation
`induced by surface adsorption, yet may not be effec-
`tive in eliminating oxidation, fragmentation, or con—
`formational destabilization caused by Fe ions. Rather,
`direct reduction of Fe ion levels by frequent passiva—
`tion of equipment and avoiding exposures of steel to
`extreme low pH in the presence of chloride or other
`corrosive ions might be a better strategy to eliminate
`negative effects of Fe ions on protein stability.36 Ad-
`dition of metal chelators has also been shown to be
`
`effective in eliminating the multiple adverse effects
`of Fe ions on protein stability, although care must be
`taken in the choice and level of the chelator.37 Nu—
`
`cleation of larger visible particles from smaller steel
`particles shed from pumps may not be completely sup—
`pressed by surfactant.26 In this scenario, a change in
`the process equipment has been shown to be effective.
`For instance, protein particle formation during fill—
`ing of an IgG was eliminated by replacement of a ra—
`dial piston pump with a rolling diaphragm pump.39 In
`some cases, there may be synergistic or compounded
`effects that may make identification of the problem
`and correct mitigation more difficult. A good example
`is where buffer—dependent conformational changes in
`a mAb increased the exposure of a site sensitive to
`Fez—catalyzed fragmentation.34
`Stainless steel is not the only important in-process
`surface to consider.
`In recent years, use of dis—
`posable containers has become a common practice
`in various steps of the manufacturing of protein
`therapeutics. Disposable containers pose potential
`challenges associated with leachables and possible
`shedding of particles, and are usually subjected to ex—
`tensive evaluation by biopharmaceutical companies
`before implementation.40
`
`CONTAINER CLOSURE
`
`Glass vials with rubber stoppers made of various poly—
`mers and coatings are commonly used primary con-
`tainers for protein therapeutics. Most recently, vials
`or syringes made ofcyclic polyolefin (clear plastic) are
`being evaluated as options for container—closure ma—
`terials for some biopharmaceuticals.41 Container clo—
`sures can be exposed to various solvents to extract and
`identify compounds that are then monitored as leach—
`ables under realistic product contact conditions.40
`This can result in an identification of a large number
`of extractables that are often not actually detectable
`in the formulation upon extended product contact. Di-
`rect health-based risk assessments can then be per—
`formed based on the extractables—leachables data for
`
`a given product configuration.40 Indirect effects of
`leachables could potentially include aggregation or
`
`particle formation.40 Detectable leachables may not
`necessarily have any adverse effects on product safety,
`efficacy, or quality.
`Rapid growth in the applications of targeted
`biotechnology products is driving the development of
`alternative delivery systems including prefilled sy—
`ringes (PFSs), autoinjectors (AIS), and infusion de-
`vices. Multiple commercial products are currently of—
`fered as PFSs and AI devices, and the number is
`expected to rapidly grow. The development of PFSs,
`AI, and infusion devices are associated with potential
`for component compatibility challenges. These poten-
`tial challenges include sensitivity of proteins to the
`silicone oil often used to enhance the gliding perfor—
`mance of the syringe/device, sensitivity to trace lev-
`els of metals such as tungsten, which may be used in
`the manufacturing of glass syringes with staked nee—
`dles, and potential leachables from the glass, silicone,
`rubber, and adhesive contact surfaces. These possible
`adverse interactions are addressed during compati—
`bility and stability studies during development. In
`addition, various types of syringes are being devel—
`oped currently by multiple vendors including silicone
`oil- and tungsten-free syringes, enabling a greater se—
`lection of container-closure systems to be potentially
`chosen from and/or evaluated during development.
`
`Glass
`
`Borosilicate glass is the most commonly used primary
`container material for biopharmaceuticals.41 During
`development, each product formulation is generally
`assessed and optimized for stability in glass vials
`(with stopper). Glass vials surface properties can vary
`between manufacturers and may change due to in—
`teractions with the solution or sterilization proce—
`dures, which could potentially result in pitting or
`delamination.41‘44 Glass has been successfully used
`for many commercial protein products without caus—
`ing aggregation or particle formation. Although re—
`ports of glass delamination are extremely rare for
`biotechnology products, the recent voluntary recall of
`a commercial protein therapeutic because some lots
`“. . .may contain extremely thin glass flakes (lamellae)
`that are barely visible in most cases” shows that de—
`lamination is still an important quality factor to be
`considered.45 We note that the voluntary recall also
`states that “To date, there have been no complaints
`or adverse events reported which can be directly at-
`tributed to the presence of glass lamellae.”45
`Excipients can also potentially interact with leach—
`ables from glass. Depending upon the exact supplier,
`glass can potentially leach ions such as barium or
`aluminum forming insoluble visible particles of bar-
`ium sulfate or aluminum phosphate when exposed
`to formulation excipients (sulfate and phosphate).46
`Proteins can adsorb to glass surfaces. In one case,
`the adsorption of protein to glass was minimized by
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`use of a siloxane coating and addition of surfactant.47
`Although they do not have identical surface chem—
`istry, silica nanoparticles have been used as a surro—
`gate surface for glass and were found to nucleate the
`growth of aggregates of recombinant human platelet-
`activating factor acetylhydrolase.48 Adsorption of the
`protein to glass and resulting aggregation could be
`reduced by choosing a solution pH at which both the
`protein and the glass had a net negative charge.48
`Also, in a more recent study with a mAb, it was found
`that there were differences in adsorption affinity as
`a function of pH and a surprising tendency for pref-
`erential adsorption of aggregates when the mAb was
`formulated with 150 mM NaCl in 10 mM sodium ac—
`
`etate at pH 5.0 and 5.5, and 150 mM NaCl in sodium
`phosphate at pH 6.0 and 6.5.49 It has been shown that
`for several mAbs, the structural changes on adsorp-
`tion to spiked glass particles were minimal, however,
`the protein—coated glass particles were less colloidally
`stabile, resulting in formation of larger agglomerated
`clusters of the spiked glass particles.”50 This spe—
`cific example corresponds to the general phenomenon
`of “agglomeration of protein—coated particles” illus—
`trated in Figure 1. The interactions between glass
`and protein are clearly both protein specific and for-
`mulation dependent. Simple adsorption losses at low
`concentrations may be reduced by use of coatings or
`surfactants.
`
`Stoppers
`
`Vial stoppers are often made from butyl and halobutyl
`rubber that may also contain other additives or poten—
`tial leachables depending upon the proprietary com—
`pound formulation.41 Not all detectable leachables
`will necessarily have any negative impact on product
`quality. In one case, vial stoppers released metal ions
`that in turn activated a metalloprotease, which then
`resulted in protein product damage.1 Polytetrafiuo—
`rethylene (PTFE) or other proprietary polymers have
`been used to coat vial stoppers to reduce the leaching
`of rubber components into the bulk formulation. The
`pure red cell aplasia (PRCA) experienced by Eprex®
`(Johnson & Johnson, New Brunswick, NJ) patients
`has been attributed to leachables released from un—
`
`coated rubber stoppers in the presence of polysorbate
`used to replace human albumin as a stabilizer51‘53 In
`other work, extractables from stoppers were not able
`to elicit “danger signal” responses in dendritic cells
`that would indicate their immunogenic potential.54
`Association of protein with polysorbate micelles has
`also been suggested as a possible cause of the im—
`mune responses,55 although this particular hypothe—
`sis has been questioned after additional studies by
`other workers suggested that association with mi—
`celles does not occur.53’56 The fundamental details and
`cause(s) of the observed PRCA cases remain the sub—
`
`ject of ongoing research and debate. Ultimately, the
`incidence of PRCA decreased when a FluroTec® (West
`Pharmaceutical Services, Inc., Lionville, PA) stopper
`configuration replaced the uncoated stopper and the
`route of administration was changed from subcuta-
`neous to intravenous.""*52
`Container-closure changes are considered high
`risk by regulatory agencies for parenteral biotech
`products.1 Optimization of
`the formulation and
`container—closure configuration is performed together.
`Although use of a PTFE stopper apparently improved
`the stability of Eprex®, PTFE was found to cause the
`aggregation of insulin in accelerated studies.57 For—
`mulation is important: Factor VIII adsorption to hy—
`drophobic surfaces, with concomitant unfolding and
`loss of activity of the adsorbed protein, could be re—
`duced by polysorbate 80.58 Interestingly, adsorption
`of Factor VIII to hydrophilic surfaces was not reduced
`by polysorbate, although no associated unfolding or
`loss of activity of the adsorbed protein was observed
`as was seen for hydrophobic surfaces.58 Stoppers are
`also often coated with silicone oil, and the amount of
`silicone coated is one variable that can be controlled.
`
`Vials are normally shipped and stored upright so the
`exposure to the stopper might be minimal for an ac-
`tual product. But for accelerated degradation studies,
`incubation in Vials that are inverted or laid side—on
`
`can also be used to assess the impact of the stopper
`on the stability of the product.
`
`Tungsten and Needle Glue
`
`Contamination of glass syringes with tungsten com—
`pounds released from the tungsten pin used to form
`the needle-mounting hole was identified as a cause of
`protein particle formation,59 and has been reported
`to have resulted in the formation of visible parti—
`cles in at least two therapeutic protein products.1
`In a recent study, it was demonstrated that tung—
`sten metal particles dissolved into negatively charged
`polyanions that electrostatically aggregated with a
`mAb, resulting in rapid visible particle formation.60
`Aggregation of the mAb by tungsten species was
`only observed below about pH 6.0 and above tung—
`sten levels of about 3—9 ppm, consistent with the
`fact that tungsten polyanions are the major soluble
`species formed under these conditions.60 The small
`number of tungsten metal particles that need to dis—
`solve to achieve this level of soluble polyanions high-
`lights their potential potency in causing aggrega—
`tion at lower pHs.60 Tungsten polyanion aggregation
`with proteins was largely reversible in phosphate—
`buffered saline.60 The mechanism of this aggrega—
`tion of protein with tungsten polyanions can be
`described as “coagulation:” coagulation is a colloidal
`science term for when a colloid (in this case the pro-
`tein) is electrostatically bridged and cross—linked by
`
`DO! 10.1002/jps
`
`JOLRNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO, 10, OCTOBER 201 1
`
`NOVITC(US)OO314257
`
`Regeneron Exhibit 1068.006
`
`
`
`4164
`
`BEE ET AL.
`
`charged species in solution (in this case tungsten
`polyanions), leading to particles and precipitate for—
`mation. Results for Fc fusion and cr—helical proteins
`spiked with extracts from actual tungsten pins used
`in syringe—forming operations were also consistent
`with polyanion-induced coagulation.61 Particle forma-
`tion was greatest at lower pHs and higher soluble
`tungsten levels, but in all cases, particles were only
`formed at tungsten levels 10 times higher than those
`actually measured in commercial PFS.61 These ex—
`amples correspond to “coagulation with leachables”
`as depicted in Figure 1. In response to the potential
`tungsten-induced protein degradation, syringe man—
`ufacturers have developed proprietary manufactur-
`ing processes and engineering changes that control
`or effectively eliminate residual tungsten contamina-
`tion in glass—staked needle syringes. Here we have
`referenced work showing different tungsten sensitiv—
`ities of a mAb, an Fc fusion protein, and an oc—helical
`protein.”61 These sensitivities varied based on mul—
`tiple factors such as the protein-to-tungsten ratio, the
`protein properties, protein concentration, and the for—
`mulation conditionsso’61 Therefore,