REVIEWS
`
`Effects of Surfaces and Leachables on the Stability
`of Biopharmaceuticals
`
`JARED S. BEE,1 THEODORE W. RANDOLPH,2 JOHN F. CARPENTER,3 STEVEN M. BISHOP,1 MARIANA N. DIMITROVA1
`
`1Formulation Sciences Department, MedImmune, 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 leachables during 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 of protein over extended periods of time 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 (e.g., 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: BeeJ@medimmune.com, jaredsbee
`@gmail.com)
`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.1,2 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
`Using state-of-the-art technology, biotechnology
`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.,6 either partial unfolding or aggre-
`gate assembly can be the rate-determining step for
`
`4158
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
`
`Novartis Exhibit 2040.001
`Regeneron v. Novartis, IPR2020-01317
`
`

`

`SURFACES AND LEACHABLES EFFECTS ON BIOPHARMACEUTICALS
`
`4159
`
`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
`proteins.5–10 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.5,10 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.14,15 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
`
`A
`
`–
`
`P
`
`V
`
`P
`
`L
`
`N
`
`N
`
`P
`
`A
`
`C
`
`A
`
`P
`
`I
`
`S
`
`M
`
`H
`
`A
`
`Figure 1. Possible physical degradation pathways of proteins caused by interfaces, foreign
`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.
`
`DOI 10.1002/jps
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
`
`Novartis Exhibit 2040.002
`Regeneron v. Novartis, IPR2020-01317
`
`

`

`4160
`
`BEE ET AL.
`
`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.17–19 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 of filtration 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
`operations.18,19 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
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
`
`DOI 10.1002/jps
`
`Novartis Exhibit 2040.003
`Regeneron v. Novartis, IPR2020-01317
`
`

`

`SURFACES AND LEACHABLES EFFECTS ON BIOPHARMACEUTICALS
`
`4161
`
`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 IgG2.26
`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 $-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 $-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.19
`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.31 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 preflush
`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 of Fe 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,
`
`DOI 10.1002/jps
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
`
`Novartis Exhibit 2040.004
`Regeneron v. Novartis, IPR2020-01317
`
`

`

`4162
`
`BEE ET AL.
`
`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
`Fe-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 of cyclic 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
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
`
`DOI 10.1002/jps
`
`Novartis Exhibit 2040.005
`Regeneron v. Novartis, IPR2020-01317
`
`

`

`SURFACES AND LEACHABLES EFFECTS ON BIOPHARMACEUTICALS
`
`4163
`
`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.32,50 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 Polytetrafluo-
`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 R(cid:1)
`(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 stabilizer.51–53 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 R(cid:1) (West
`Pharmaceutical Services, Inc., Lionville, PA) stopper
`configuration replaced the uncoated stopper and the
`route of administration was changed from subcuta-
`neous to intravenous.40,52
`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 R(cid:1), 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
`
`DOI 10.1002/jps
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
`
`Novartis Exhibit 2040.006
`Regeneron v. Novartis, IPR2020-01317
`
`

`

`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 "-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 "-helical
`protein.60,61 These sensitivities varied based on mul-
`tiple factors such as the protein-to-tungsten ratio, the
`protein properties, protein concentration, and the for-
`mulation conditions.60,61 Therefore, sensitivity of dif-
`ferent proteins and different formulations to tungsten
`leached from forming pins should be evaluated on a
`case-by-case basis because there may, or may not, be
`an impact at the actual potential worse case expected
`tungsten levels in PFSs.60,61 It is likely that the re-
`ductions of tungsten level in syringes, combined with
`knowledge of the pH effects on potential coagulation
`with protein, can now be used to effectively mitigate
`this specific issue for proteins which are sensitive to
`tungsten.
`The US Food and Drug Administration (FDA) re-
`ported in one case that if the specific glue used to
`attach the needle to the syringe tip was not allowed
`to fully dry, it could leach solvents, resulting in pro-
`tein oxidation.46 They reported that in this specific
`case, the issue could