504
`
`Biotechnol. Prog. 2008, 24, 504- 514
`
`Current Perspectives on Stability of Protein Drug Products during Formulation,
`Fill and Finish Operations
`
`Nitin Rathore*,† and Rahul S. Rajan‡
`
`Drug Product & Device Development and Process & Product Development, Amgen, Inc., Thousand Oaks, California 91320
`
`Commercialization of protein-based therapeutics is a challenging task in part due to the difficulties
`in maintaining protein solutions safe and efficacious throughout the drug product development
`process, storage, transportation and patient administration. Bulk drug substance goes through a
`series of formulation, fill and finish operations to provide the final dosage form in the desired
`formulation and container or delivery device. Different process parameters during each of these
`operations can affect the purity, activity and efficacy of the final product. Common protein
`degradation pathways and the various physical and chemical factors that can induce such reactions
`have been extensively studied for years. This review presents an overview of the various
`formulation-fill-finish operations with a focus on processing steps and conditions that can impact
`product quality. Various manufacturing operations including bulk freeze-thaw, formulation,
`filtration, filling, lyophilization, inspection, labeling, packaging, storage, transport and delivery
`have been reviewed. The article highlights our present day understanding of protein instability
`issues during biopharmaceutical manufacturing and provides guidance on process considerations
`that can help alleviate these concerns.
`
`Introduction
`
`The term “formulation, fill and finish” refers to the series of
`processing steps that are needed to turn a purified drug substance
`into the final dosage form, the finished product, for the market
`(1). The formulation step involves taking the purified protein
`at the desired concentration and dispensing it with the correct
`excipients that can ensure product quality and integrity during
`the subsequent fill/finish steps including filtration, filling,
`lyophilization, packaging, storage, transport and delivery. A
`robust formulation would need to keep the biopharmaceuticals
`stable not only during shelf storage but also during these
`manufacturing steps. At the same time, key operating and
`process parameters should be optimized to obtain a robust
`manufacturing process. The problems for protein therapeutics
`could be very different from the traditional small-molecule
`pharmaceutical processing and may require special handling and
`storage conditions to ensure product quality (2, 3). For instance,
`protein thermal instability is one of the main reasons why protein
`drugs need to be maintained under cold temperatures during
`storage and transport to achieve longer shelf life. Similarly, other
`stresses such as photo exposure and mechanical agitation could
`also impact the stability of protein products.
`Proteins are large macromolecules made up of a sequence of
`amino acids and characterized by a unique three-dimensional
`structure corresponding to their biologically active state. The
`native structure of a protein molecule is the result of a fine
`balance among various interactions including covalent linkages,
`hydrophobic interactions, electrostatic interactions, hydrogen
`bonding and van der Waals forces. Intraprotein and protein-
`solvent interactions both play an important role in maintaining
`
`* To whom correspondence should be addressed. Ph: 805-313-6393.
`Email: nrathore@amgen.com.
`† Drug Product and Device Development.
`‡ Process and Product Development.
`
`the protein structure and its stability. The free energy of
`unfolding has been generally reported to be quite small, in the
`range of 21-63 kJ/mol (2). Since the folded state of protein is
`only marginally more stable than the unfolded state, any change
`in the protein environment may trigger protein degradation or
`inactivation.
`The degradation pathways for protein therapeutics are many
`and often complex. Simplistically speaking, these pathways can
`be divided into two categories: physical degradations, which
`do not
`involve covalent bond modifications, and covalent
`modifications (for reviews, see refs 1-5). Physical degradations
`are most commonly manifested by protein aggregation. This
`type of degradation involves assembly of monomeric units of
`proteins, and dimerization is a common occurrence within these
`set of events (6). Higher order protein oligomers are often
`referred to as “high molecular weight species” or protein
`aggregates (4-8). These protein aggregates can be either soluble
`or insoluble. Recent evidence has suggested that protein
`aggregation occurs by a specific association of partially
`denatured polypeptide chains, as opposed to nonspecific co-
`aggregation (7, 8). Protein aggregation can be assessed by a
`variety of techniques, such as size exclusion chromatography,
`field flow fractionation and analytical ultracentrifugation for
`soluble aggregates (9) and light obscuration/scattering techniques
`for insoluble aggregates (10). A second type of protein degrada-
`tion is a change in the secondary, tertiary or quaternary structure
`of the protein, which does not involve protein-protein interac-
`tions. Biophysical techniques, such as circular dichroism, FT-
`IR and fluorescence are usually employed to assess such
`structural changes. These two degradation pathways can be
`intimately linked: a change in protein structure often precedes
`protein aggregation phenomena (4). Native aggregates are those
`in which there is only an assembly of protein monomers without
`a change in structure, whereas there is a change in protein
`
`10.1021/bp070462h CCC: $40.75
`
`© 2008 American Chemical Society and American Institute of Chemical Engineers
`Published on Web 05/17/2008
`
`Novartis Exhibit 2184.001
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Biotechnol. Prog., 2008, Vol. 24, No. 3
`
`505
`
`Formulation-fill-finish
`Unit Operations
`
`Freeze/Thaw
`Bulk DS
`
`Factors affecting product quality
`
`Ice-liquid interface
`Cryoconcentration
`Excipient crystallization, pH shifts
`Cold denaturation
`Container surface interactions
`Leachables and extractables from container
`
`Formulation (Dilution/
`Cone. addition)
`
`Excipient impurities initiating degradation reactions
`Shear during mixing
`Improper mixing of excipients
`Exposure and adsorption to various surfaces
`
`Filtration and /or
`UF/DF
`
`Protein loss due to adsorption
`Shear during filtration
`Protein unfolding due to membrane interactions
`
`DP Filling
`vials/syringes
`
`Lyophilization
`(if needed)
`
`Inspection
`
`labeling &
`Packaging
`
`DP Storage
`
`Transport
`
`Delivery Device
`
`Filling shear
`Foaming, dripping during filling
`Interactions with DP container
`Foreign particles introduction
`Light exposure
`Leachables and extractables
`
`Freezing/Drying related stresses
`pH and excipient control
`Residual moisture level
`Aggregate level after lyophilization
`
`Exposure to light and shear
`Micro-bubbles formation
`
`Temperature excursions
`Light exposure
`
`Long Term DP stability
`Interactions with Primary container
`Silicone oil interactions
`
`Shock and drop effects
`Change in pressures
`Vibration stresses
`Particle, bubbles and aggregate formation
`
`Protein-device surface interactions
`Interactions with silicone oil
`Shear during drug delivery
`
`Figure 1. Overview of the several formulation, fill and finish processes and the various factors that can affect product quality during these processing
`steps
`
`structure (secondary and/or tertiary) in the formation of non-
`native protein aggregates.
`There are several possible covalent modifications in proteins.
`One common modification is protein fragmentation, which
`involves the cleavage of a peptide bond. Residue specific
`modifications include but are not limited to aspartate isomer-
`ization, protein oxidation, deamidation, pyroglutamic acid
`formation and disulfide bond shuffling (5). Many times, proteins
`undergo post-translational modifications, such as glycosylation.
`Changes in glycosylation patterns under storage and processing
`conditions are also known to occur (11). Further, formulation
`excipients sometimes have the potential to interact with protein
`side chains, such as the glycation reaction between reducing
`sugars and side-chain or N-terminal amino groups (12). Covalent
`degradations that lead to changes in net charge of the protein
`can be captured by ion exchange chromatography (13, 14) and
`capillary isoelectric focusing (cIEF) (15). Weak cation exchange
`chromatography is commonly used to monitor antibody stability.
`Techniques combining reverse phase and mass spectrometry,
`
`such as peptide mapping, are more comprehensive and have
`the potential to detect most, if not all, covalent modifications
`(13, 16), but these are more time-consuming and resource
`intensive.
`This review article presents an overview of various formula-
`tion, fill and finish operations. The key aspects of processing
`steps that can affect stability and integrity of a product are
`discussed. Figure 1 presents the series of operations and various
`factors that can impact product quality. In the sections that
`follow, each unit operation is described in detail. The impact
`of some of the key operating input parameters on protein
`stability is included, and guidance is provided on scale-down
`studies needed to evaluate such destabilizing factors. For many
`fill and finish operations, such as freeze-thaw, mixing, and
`filtration, the main concern would be physical stability of the
`protein. However, exposure to light and various manufacturing
`equipment surfaces could trigger covalent modifications as well.
`Therefore, orthogonal assays to determine the physical and
`covalent stability of the molecule should be carried out to
`
`Novartis Exhibit 2184.002
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`506
`
`Biotechnol. Prog., 2008, Vol. 24, No. 3
`
`Heat
`transfer
`
`•• .... .
`• Frozen
`••
`•
`-• ♦ surface
`f
`•
`••• •• • -•
`• •
`• ....
`
`Ice- liauid interface
`
`♦ Protein
`• Excipients
`Figure 2. Slow freezing can result in cryoconcentration of proteins
`and excipients, which can further cause protein aggregation or
`precipitation. If the freeze front moves slowly, solutes are excluded
`from the solid-liquid interface, resulting in higher concentration in
`the regions that freeze later.
`
`determine the overall stability of the molecule to various
`bioprocessing stresses. In addition to formulation and other unit
`operations, protein instability issues may arise from interactions
`of drug product with packaging and components of delivery
`device. Current understanding of these challenges is included.
`Photodegradation of light-sensitive products is also discussed
`in a later section.
`
`Formulation and Fill-Finish Operations
`Bulk Freeze-Thaw: Advantages. Bulk freeze-thaw is
`commonly employed during biopharmaceutical manufacturing
`to gain operational flexibility while maintaining product quality.
`A frozen drug substance provides several advantages over liquid
`storage, including increased product stability, reduced possibility
`of microbial growth and alleviation of foaming issues during
`transportation, thereby eliminating the need to perform transport
`validation. Lowering the temperature to achieve a frozen bulk
`reduces the rates of degradation reactions and also immobilizes
`the protein molecule in a frozen matrix, thereby minimizing
`diffusive collisions that lead to aggregation. Lack of availability
`of free water also prevents several degradation reactions that
`are assisted by water, such as peptide bond hydrolysis and
`aspartic acid isomerization phenomena, further increasing the
`stability of frozen bulk in comparison to aqueous formulations.
`This greater assurance in product quality provides flexibility to
`schedule formulation-fill-finish operations based on the needs
`of the manufacturing facility. In this scenario, bulk drug
`substance is stored frozen and when needed is transported to
`the fill site where it is thawed, initiating a series of formulation
`and fill/finish steps. The application of freeze-thaw is not
`limited to storage of drug substance but is also used for the
`storage of pharmaceutical intermediates and formulated drug
`products.
`Protein Freezing: Stability Challenges. While bulk freeze-
`thaw offers numerous operational and product quality benefits,
`it may also prove detrimental to protein stability. Cryoconcen-
`tration is one of the common mechanisms through which protein
`destabilization could occur during freezing (17-19). As the
`freeze-front moves during the freezing process (Figure 2), the
`excipients as well as the proteins get excluded from the ice-
`liquid interface. As a result the concentration of the liquid bulk
`(yet to be frozen) close to the ice crystals increases progressively
`with freezing. Such concentration build up of excipients may
`result in changes in protein structure. Freezing of buffer solution
`can also cause change in pH due to selective precipitation of
`
`+ive
`
`Cl
`C:
`'o
`E
`
`C:
`:::,
`(!)
`<I
`
`- ive
`
`Temperature
`Td
`Tc
`Figure 3. Thermodynamic justification of cold denaturation of protein.
`The parabolic shape of Gibbs free energy implies that protein unfolding
`becomes favorable not only at elevated temperatures (T > Td) but also
`at very cold temperatures (T < Tc)
`
`buffer components, which can also result in protein destabiliza-
`tion (20). At the same time, increase in protein concentration
`also increases the possibility of molecular collisions and may
`result in protein aggregation or precipitation. The extent of
`cryoconcentration is maximized if the rate of freezing is slow.
`As a result, uncontrolled freeze-thaw processes, where the
`freeze front velocities are lower, are impacted to a greater extent
`by the destabilizing effects of cryoconcentration. One way of
`minimizing such freeze concentration effects is to reduce the
`freezing times by increasing the heat transfer from the container.
`Dendritic ice growth is also preferred in order to minimize
`cosolute exclusion during freezing. This can be achieved by
`establishing directional heat flow and avoiding mixing during
`freezing. Mixing could be detrimental as it would suppress
`dendritic ice growth, making the ice-liquid interface more flat,
`and therefore result in increased cryoconcentration.
`Proteins could also be susceptible to spontaneous unfolding
`at cold temperatures, referred to as “cold denaturation” (21).
`This effect is primarily attributed to the weakening of the
`hydrophobic effect with decrease in temperature. The thermo-
`dynamic justification of the cold denaturation temperature can
`be explained by the parabolic shape of the Gibbs free energy
`function as shown in Figure 3. A favorable negative free energy
`of unfolding favors thermal denaturation at higher temperature
`(Td). At lower temperatures, it is possible that ¢Gunfolding may
`become negative again below a certain critical temperature (Tc),
`resulting in protein unfolding. Such cold-induced denaturation
`phenomena, though rare, have been reported for certain proteins
`(22, 23). Further review of this phenomenon can be found in
`the literature (21).
`Very fast freezing rates can also prove to be detrimental to
`proteins (24). During freezing, protein molecules can concentrate
`and get unfolded (25, 26) on the ice-water interface, implying
`loss in protein activity. When freezing rates are very fast (e.g.,
`submerging container in liquid nitrogen (17)), smaller ice
`crystals are formed and result in a large ice-liquid interfacial
`area (25, 26). Increased protein aggregation and decreased
`activity have been reported for liquid-nitrogen-based freezing
`systems (27, 28). Fast freezing can also trap air that would be
`released during thawing and may cause protein denaturation on
`air-liquid interfaces (29, 30).
`Thawing Frozen Protein Solutions: Stability Challenges.
`Frozen bulk needs to be thawed before it can be formulated
`and processed. Thawing can cause further stress and damage
`to the protein. Slow thawing rates can result in ice recrystalli-
`zation with small ice crystals growing into larger ones. Proteins
`may get denatured at ice-liquid interfaces and lose their activity
`(25, 26). Cryoconcentration created during freezing can further
`harm the protein during thawing. Like freezing, faster thawing
`rates are usually preferred for protein stability. While mixing
`
`Novartis Exhibit 2184.003
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Biotechnol. Prog., 2008, Vol. 24, No. 3
`
`Table 1. Comparison of Freeze-Thaw Parameters for Carboy and
`Celsius Pak
`
`carboy (10 L)
`17.1 ( 0.9
`7.3 ( 0.4
`150 ( 15c
`static
`non-homogeneous
`
`process parameters
`freezing timea (h)
`FFVb (mm/h)
`thaw time (h)
`thaw type
`solution homogeneity
`after thaw
`a Freeze time refers to time taken by the solution to go from +3 (cid:176)C to
`-5 (cid:176)C.
`b Freeze front velocity c Thaw time reported in the table refers to
`the time needed to thaw 8.5 kg of protein solution in a 10 L carboy at
`2-8 (cid:176)C.
`
`Celsius-Pak (16.6 L)
`1.6 ( 0.2
`25.5 ( 2.5
`2.5 ( 0.5
`dynamic
`homogeneous
`
`during freezing may be detrimental to proteins, appropriate
`mixing during thaw is the key to minimize recrystallization and
`cryoconcentration related effects. Very slow mixing would
`contribute to longer thaw times and also would not be able to
`homogenize the solution (concentration gradients from the freeze
`step would continue to exist and may further increase). On the
`other hand, very high mixing rates would result in protein
`shearing, excessive foaming and plausible protein denaturation
`on the air-liquid interface. The mixing parameters should
`therefore be optimized to enhance thawing without affecting
`product quality.
`Freeze-Thaw Technologies And Process Scale-Down. The
`challenges faced during the freeze-thaw process would be
`dependent on the technology employed for the large-scale
`process. Most of the stability issues discussed above occur when
`very slow freeze-thaw rates are applied, which is usually the
`case for uncontrolled rate technologies (17). For example,
`polycarbonate carboys (10 L and 20 L) are commonly used to
`freeze and transport bulk drug substance. Freezing is conducted
`by placing these carboys in walk-in or upright freezers at -30
`or -80 (cid:176)C. Since the path lengths are large and the heat flux is
`slow, the process times for the freeze-thaw operations could
`be very long as shown in Table 1. As a result cryoconcentration
`becomes an important factor governing product quality in these
`containers. Controlled rate technologies such as Celsius Paks
`or Cryovessels, on the other hand, can achieve faster freezing
`and thawing rates by using a combination of small path length
`and increased flux for heat transfer (18). Table 1 shows a
`comparison of these process times for the uncontrolled rate
`(carboys) and controlled rate (Celsius Pak) technologies. It has
`also been shown in literature that the extent of cryoconcentration
`is minimal for Celsius Paks (18).
`The effects of bulk freeze-thaw on the product are protein-
`specific. It may not affect product quality for some protein
`solutions but may have negative effects on others. As a result,
`prior to large-scale processing, each product should be evaluated
`for the impact of multiple freeze-thaw operations on product
`quality. For early-stage products where product availability may
`be limited, scaled-down studies can be performed to mimic
`large-scale freeze-thaw process. For uncontrolled freeze-thaw
`processes, usually a smaller bottle with (a) surface area to
`volume ratio similar to that of the large scale and (b) material
`of construction identical to the large-scale container can be used.
`Freeze-thaw profiles from large-scale processes can also be
`mimicked on small scale using a controlled rate freezer.
`However, certain phenomena, such as cryoconcentration, could
`be process-scale-dependent and difficult to mimic in a small
`container. It is usually feasible to mimic the impact of protein-
`container interactions during freeze-thaw in smaller-scale
`experiments.
`For controlled rate technologies, the freeze-thaw process is
`often scalable in terms of freeze-thaw times and heat transfer
`
`30
`
`20
`
`10
`
`507
`
`l -
`
`-
`-
`
`Lab Scale (30 ml}J
`Pilot Scale (8.3 L}
`Full Scale (16.6 L)
`
`0
`~ -10
`f!
`~ -20
`..
`.. ...
`
`-30
`
`-40
`
`-50
`
`-60
`
`a.
`E
`
`0
`
`3
`2
`Time (hours)
`Figure 4. Scalability of controlled rate freeze-thaw as observed for
`Celsius Pak technology at different scales: 30 mL, 8.3L and 16.6 L.
`
`4
`
`5
`
`path length. For example, the path length for 30 mL, 100 mL,
`8.3 L and 16.6 L Celsius bags is identical (42 mm) and helps
`make the process scalable. The heat transfer fluid temperature
`profile over time can be programmed to achieve the same
`freeze-thaw profiles at all scales. Figure 4 shows how the
`thermal control unit of the Celsius technology can be used to
`obtain similar freeze-thaw profiles for lab-, pilot- and full-
`scale systems. This provides the flexibility to conduct stability
`characterization studies at lab scale with very limited material.
`While such disposable bag technology offer numerous advan-
`tages with the freeze-thaw process, the final impact on the
`product quality is also governed by the impact of the container.
`Issues such as increase in protein concentration associated with
`the loss of water vapor from plastic bags have been reported
`for prolonged storage at room temperature (31). The product-
`packaging interaction, robustness of the bag’s mechanical
`properties, permeability of the bags and the level of leachables
`and extractables should be characterized in detail to ensure that
`no impact on product quality over a period of storage time is
`observed.
`Formulation Step. The first step after thawing the bulk is
`to formulate it with the right buffer and to the target concentra-
`tion. The formulation step involves adding the desired excipients
`at target concentration and adjusting pH, conductivity and
`protein concentration (32). The final dosage form for the drug
`product could be different from that of the bulk drug substance.
`Sometimes it is operationally more favorable to store a drug
`substance at a higher concentration than the drug product, and
`therefore a dilution step would be needed during formulation.
`In other cases, a buffer exchange may be required between drug
`substance and drug product. To perform buffer exchange, a UF/
`DF step may then be required. There can be logistical challenges
`with the implementation of this step, such as whether to perform
`it at the bulk manufacturing site or at the drug product fill and
`finish site. One of the main challenges during the UF/DF process
`is arriving at the target bulk pH at the end of operation. Recent
`work by Stoner et al. have provided the groundwork for this
`phenomena and have provided a mathematical tool to calculate
`how much pH adjustment to make prior to the step to hit the
`target pH at the end (33). Similarly, a concentration step may
`also be needed if the drug product is formulated at a concentra-
`tion higher than that of drug substance. High product concentra-
`tion and viscosity could pose further challenges to membrane
`filtration during the concentration step. Impact of filtration on
`product is further discussed in the next section.
`The purity of the excipients could be another key factor
`affecting product quality at this step (34). Certain impurities in
`the raw materials can trigger degradation reactions. Using
`animal-derived excipients may carry a risk of causing TSE
`(Transmissible Spongiform Encephalopathies), and this would
`
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`
`

`

`508
`
`need to be carefully evaluated. Exposure to different surfaces
`including tubing and tanks could also affect protein stability.
`Surfactants, such as polysorbate 20, added to the formulation
`buffer as stabilizers, could get adsorbed on these surfaces and
`lower the surfactant concentration, resulting in protein desta-
`bilization. Leachables and extractables (especially if disposable
`bags are being used) from the various contact surfaces also have
`the potential to affect product integrity.
`Insufficient mixing during the addition of excipients could
`alter product quality due to solution inhomogeneity and also
`result in the final drug product not being able to meet its
`specifications. Excessive mixing, on the other hand, could create
`large shear stress that can denature proteins. Physical instability
`of proteins arising from mechanical stresses such as stirring and
`shaking in presence of various contact surfaces has been widely
`reported in literature (35-39). Air-liquid interfaces created
`during the mixing and pumping processes are another source
`of protein denaturation. Pumping can also result in addition of
`foreign particles in the final solution that can further trigger
`protein aggregation. The order in which excipients are added
`is also important in determining product quality. For example,
`addition of polysorbate is often performed after any UF/DF step
`in order to minimize loss due to membrane interaction.
`Small-scale characterization studies should be conducted to
`evaluate protein stability under formulation conditions. Buffers
`should be characterized to establish appropriate tolerances
`around excipient concentrations, pH, conductivity and osmo-
`lality. Hold time studies using the worst case scenarios for
`surface area exposed per unit volume should be designed to
`study the impact of different contact materials on protein
`stability. Temperature excursions should also be evaluated
`through hold time studies at various temperatures over prolonged
`duration of time. The characterization of the mixing process
`should include both product homogeneity testing as well as
`impact of mixing shear on product quality. Based on the tank
`and impeller geometry and product properties (viscosity and
`density), bulk and impeller tip shear can be computed for the
`manufacturing conditions. In the absence of appropriate scaled-
`down mixing systems, rheometers can be utilized in the lab to
`expose the product to the maximum applicable shear over the
`recommended duration of the mixing process. Final samples
`can be analyzed to assess the impact on product quality
`attributes. These findings can then be verified with fewer runs
`on the commercial scale to determine the impact during large-
`scale processing.
`Filtration. After the bulk drug substance has been formulated,
`it goes through sterile filtration. Sterile filtration is usually
`performed with a 0.22 (cid:237)m filter to make sure that the bulk is
`free from viable micro-organisms. An additional in-line filtration
`step might be incorporated just before filling. Dual filtration
`prior to filling may also be employed for risk mitigation in the
`scenario of a filter failure. The protein solution as a result could
`see multiple filtration steps before being filled as a final dosage
`form in the drug product container. It is therefore important to
`evaluate the impact of these filtrations on product quality. Sterile
`filtration at high trans-membrane pressure could stress the
`protein while pushing it through the filter pores.
`The protein can also selectively bind to the membrane
`resulting in either misfolding on the membrane surface or protein
`loss. It is therefore important to study the compatibility of the
`product to the membrane material. Figure 5 shows the binding
`of a protein drug product (at 1 mg/mL) to PVDF membrane. It
`is seen that in this case, up to 37.5 (cid:237)g of protein is adsorbed
`per unit cm2 of the filter area. The loss could be appreciable
`
`Biotechnol. Prog., 2008, Vol. 24, No. 3
`
`40
`
`N' 35
`E
`.!:!
`Cl
`~
`30
`
`C:
`
`"' "' .5!
`~ 0.
`
`25
`
`20
`
`0
`
`20
`
`60
`40
`Mass filtered (g)
`Figure 5. Protein adsorption on PVDF membrane as measured during
`sterile filtration through 0.22 (cid:237)m filter
`
`80
`
`for low concentration products if the batch size is relatively
`smaller or if the bulk is not being pooled after filtration and
`before filling.
`Similarly other formulation components, e.g., surfactants that
`are added as stabilizers, can get adsorbed on the membrane
`surface. This will cause the surfactant concentration in the
`solution to go below the target, which might result in product
`destabilization. Recent study by Mahler et al. (40) reported
`minimal loss of polysorbate 20 due to adsorption on filter
`membrane and also suggested that protein in formulation could
`influence surfactant concentration during dialysis process. While
`such losses may not be significant, it is advisable to test
`polysorbate concentrations under the final scale process condi-
`tions. A larger filter area will reduce filtration time, but at the
`same time it will maximize the protein and excipient losses
`associated with membrane adsorption. As a result, scale-down
`studies should be conducted prior to large-scale processing to
`assess the impact of filtration on product quality and to
`recommend the optimum filter size and the membrane type for
`the manufacturing process. Other filtration process parameters
`such as the trans-membrane pressure across the membrane, the
`temperature of the bulk and the liquid flow during filtration
`should also be evaluated for their impact on protein stability.
`Drug Product Filling. Once the drug substance has been
`formulated and sterile filtered, it is filled into the primary drug
`product containers, which are usually vials, or syringes for
`prefilled injectables. During this step, the drug product not only
`comes in contact with the primary container but also the various
`components such as stoppers, plungers, etc. All of these
`components are subjected to sterilization processes separately
`and brought together under aseptic processing conditions (41).
`Since there is no further sterilization step, it is critical to maintain
`the sterility of the drug product during this step. The environ-
`ment during the filling process could also contribute to foreign
`contaminants in the final drug product. The container closure
`systems and the environment of the fill chamber are qualified
`to be of the highest standards (class 100 room) needed to ensure
`product quality. Air flow patterns, HEPA filtration, humidity
`and operation design are used to minimize sources of foreign
`contaminants such as airborne dust, depyrogenation particles
`and fibers from operator garments, mobile machine parts and
`components. In addition to creating additional solid-liquid
`interfaces that may deactivate proteins, foreign particles pose a
`significant risk of causing immunogenicity (42, 43).
`Interactions with container surface and components, which
`come in direct contact with the drug product, can also affect
`protein stability. Siliconized stoppers can contribute to protein
`aggregation (44) and particulate formation in vials. Leachables
`and extractables from the container/component surface can
`further impact the physical and chemical stability of the drug
`
`Novartis Exhibit 2184.005
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Biotechnol. Prog., 2008, Vol. 24, No. 3
`
`product. Such protein stability issues are also common to
`primary container and delivery devices and are discussed in
`detail in the later section “Drug Product Storage, Transport and
`Delivery”.
`During filling the drug product can be subjected to high shear
`that can cause protein unfolding (45). Biddlecombe et al. (38).
`have reported significant levels of protein aggregation and
`precipitation in therapeutic antibodies due to shear in the
`presence of solid-liquid interfaces. Filing procedure can also
`affect protein stability. For products sensitive to oxidation and
`deamidation, fill can be performed under nitrogen to minimize
`degradation losses (46). The shape and height of the filling
`nozzle, as well as the fill speed, could contribute to foaming in
`vials that can possibly result in exposing the hydrophobic
`residues of the protein to air and initiate protein aggregation at
`the air/water interface. Fill parameters including the filler speed
`and nozzle design should be optimized to avoid other issues
`such as dripping and liquid spurting. For frozen formulations,
`it is important to have the fill volume in the vials below a
`threshold level to avoid any vial breakage issues. For lyophilized
`products, mannitol crystallization is attributed to be the cause
`of vial breakage during freeze-drying (47). Here too, it is
`recommended to keep the fill volume within 30% of the
`volumetric capacity of the vial. The filling speed also governs
`the process time for the fill step and hence the exposure to
`ambient
`temperature. Since filling is often done at room
`temperature, the stability data of the product should support the
`total exposure time for each processing step. Exposure to
`temperatures and durations outside the recommended window
`could affect product stability. Light exposure during the filling
`and post-filling processe