Wendy Saffell-Clemmer, BPS, is research director, R&D and innovation, at Baxter Healthcare.

Wendy Saffell-Clemmer

Aluminum compounds have been used as adjuvants in human vaccines for more than 75 years (1), and they are the most widely used adjuvants in both human and veterinary vaccines. Billions of doses of aluminum salts containing vaccines have been administered safely to a diverse population of patients globally (2). Despite their widespread usage, there is little regulatory guidance or published literature regarding release and stability testing of these products. Though there have been great gains in knowledge of therapeutic protein chemical and physical stability, there are few published examples of the applications of bioanalytical stability methods applied to protein once adsorbed on to the aluminum adjuvant. This article provides an introduction to aluminum adsorbed vaccines, reviews studies of antigen stability, and proposes a panel of test methods for the analysis of aluminum vaccine release and stability analysis.

	Early vaccines were prepared as alum precipitates, which could be heterogeneous. Preformed gels of aluminum hydroxide and, later, aluminum phosphate replaced alum precipitation and have resulted in standardized formulations of aluminum-adsorbed vaccines. Aluminum-based adjuvants continue to be used in new vaccine preparations as recombinantly expressed protein antigens are identified, which can serve as safer replacements for inactivated or attenuated pathogens.  Additionally, vaccines containing combination adjuvants linking aluminum with synthetic and natural ligands have been approved (2). The addition of adjuvants is often needed to induce a robust immune response and effective immunization to protein antigens, which may not possess strong immunogenicity. The physiological mode of action of aluminum adjuvants, however, is not completely understood (3). Potential mechanisms include that the aluminum forms a depot at the injection site from which the antigen is released slowly; that the particulate form of the adsorbed vaccine results in uptake by macrophages, neutrophils, and dendritic cells; and the immune system is stimulated directly. Additionally, it has been proposed that adsorption of the protein on the aluminum gel could result in structural destabilization increasing the protein’s immunogenicity (4).

	There are two common aluminum salt preparations used in human vaccines: aluminum hydroxide (Alhydrogel) and aluminum phosphate (Adju-Phos). The pH at which the net surface charge of the aluminum adjuvant is zero is called the point of zero charge (PZC), which is similar to the isoelectric point (pI) of a protein. The adjuvant surface is positively charged when the solution pH is below the PZC and negatively charged when the solution pH is greater than the PZC (5). Aluminum hydroxide has a PZC of approximately 11 and aluminum phosphate has a PZC of 4–5.5 (4), though consideration should be given to the buffer used in vaccine formulation as substitution of ions at the surface may lower the PZC and impact the adsorption of the antigen (5). For many proteins, antigen adsorption is best in pH intervals between pI of the protein antigen and PZC of the aluminum adjuvant because it is in this pH range that the protein antigen and the adjuvant will have opposite electric charges (1). For this reason, lysozyme, which has a pI of 11.35, has been used as a model antigen in studies of aluminum phosphate. Bovine serum albumin (pI of 4.7) and ovalbumin (pI of 4.5) bind more efficiently to aluminum hydroxide (4). Generally, Alhydrogel has a higher adsorption capacity than Adju-Phos, and the adsorption capacity of Alhydrogel decreases as its particle size increases and as the molecular weight of the proteins increase (6).

	The adsorption mechanism of proteins to aluminum adjuvants is not completely understood (6) and is likely a combination of physical phenomena including “electrostatic attraction, hydrogen bonding, apolar interactions, ligand exchange, and van der Waals’ forces” (4).  Studies of model proteins found that electrostatic interactions were the major mechanism for adsorption of lysozyme, human growth hormone, diphtheria toxoid, a monoclonal antibody, and PEGylated growth hormone, but that for one protein, ovalbumin, adsorption involved ligand exchange (6). The predominant mechanism for adsorption of HBsAg by aluminum hydroxide was determined to be ligand exchange between the phospholipids in HBsAg and the surface hydroxyls in aluminum hydroxide adjuvant (7). HBsAG was found to bind more tightly to an aluminum hydroxyphosphate sulfate adjuvant when the concentration of phosphate buffer was increased (8). The correlation of strength of adsorption to vaccine effectiveness is related to the adsorption mechanism. Phosphorylated proteins adsorbed via ligand exchange have an inverse relationship between high adsorption strength and poor antigen presentation and immune response (9). Studies of a trivalent Pneumococcal  protein vaccine demonstrated differences in immunogenicity resulting from selection of adjuvant impacted immunogenicity and found that lower microenvironment pH at the antigen surface and decreased strength of adsorption improved antigen stability (10).

	A significant challenge in vaccine formulation development is the understanding of the antigen stability following adsorption. While aluminum-precipitated and aluminum-adsorbed vaccines have been in use for 75 years, it has only been in the past 15 years that studies investigating the impact of adsorption on protein structure and stability have been reported, presumably as new techniques developed to characterize recombinant proteins were applied to vaccine research. In a review of the subject, Clapp and colleagues describe how the environment of the protein at the adjuvant surface differs from that in solution, potentially driving structural change (11). Specifically, studies of the rate of acid-catalyzed hydrolysis of glucose-1-phosphate (G1P) adsorbed to aluminum hydroxide concluded that the pH of the microenvironment at the surface of the adjuvant was approximately two pH units higher than that of the bulk solution (12). Differences in polarity and charge density on the adjuvant surface may also result in structural changes in the bound protein (11).

	Spectroscopic methods have been applied to the study of protein structural perturbations resulting from adsorption. Techniques have been well described in the literature and have included fluorescence spectroscopy (13), transmission Fourier-transform infrared (FTIR) spectroscopy (14), FTIR spectroscopy using an attenuated total reflectance (ATR) cell (13, 14, 15), and circular dichroism (CD) (15). Differential scanning calorimetry (DSC) has also been applied (13, 16). In studies of model vaccines, structural changes by FTIR–ATR, fluorescence spectroscopy techniques (13, 15), and DSC (13) were observed by following adsorption.  Studies using transmission FTIR (14) concluded that no structural changes resulted from adsorption in studies of six model proteins. Overall, a review of studies indicates that variation in the buffer concentration, buffer pH, and properties of the antigen itself may influence the structure of the adsorbed protein antigen. In studies of a hepatitis B vaccine, the interaction between the antigen and aluminum hydroxide adjuvant was modified by optimizing the phosphate ion concentration and could possibly ensure that the native concentration of the antigen was maintained (17). While no structural information was presented, the authors concluded that a formulation containing 40 mM phosphate had improved thermal stability by 

 antigen reactivity. Additionally, FTIR–ATR studies determined that the structural changes were dependent on the amount of protein adsorbed, with more native protein observed in samples with maximum adsorption (17). The authors theorize that at lower adsorption concentrations, a monomolecular layer is formed in which the confirmation of the adsorbed protein is affected by the properties of the adjuvant surface and are more denatured. At higher adsorption levels, the protein is more weakly linked and may retain the native structure. While the focus of aluminum-absorbed vaccine characterization studies has protein antigen structure, questions remain about the applicability of studies to individual vaccine formulations and the impact that antigen structural perturbations may have on vaccine effectiveness and stability throughout the shelf life of the product.

	Regulatory guidance for vaccine stability studies

	There is little general guidance on release and stability test methods for aluminum adsorbed vaccines. The FDA document, 

Guidance for Industry, Content and Format of Chemistry, Manufacturing and Controls Information and Establishment Description Information for a Vaccine or Related Product 

(18), states that identity, purity, and potency should be included in drug product release specifications but does not provide specific test methods or guidance. 

) Chapter <1235> Vaccines for Human Use–General Considerations (19) does not provide specific recommendations for release and stability tests beyond general requirements of testing for potency, general safety, sterility, purity, identity, and constituent materials. There was only one aluminum-adsorbed vaccine USP monograph available as an example (Anthrax Vaccine Adsorbed), but this was deleted as of August 2018 (20). The monograph includes tests performed on the sterile filtrate which does not include aluminum. Tests performed on the sterile filtrate include Identity by Western Blot, total protein by using a Bradford assay per 

<1057> (21), and 83kDa Protein using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE). Finished product tests are 

 relative potency, concentration determination of aluminum by atomic absorption spectroscopy, safety, sterility, pH, sodium chloride by ion-selective probe, benzethonium chloride by titration, and a limit test for formaldehyde using UV-Vis spectroscopy.

	While there is not specific guidance on the inclusion of test methods that are potency-indicating, there are parallels that can be drawn from International Council for Harmonization (ICH) Q5E 

Comparability of Biotechnological/Biological Products Subject to Changes

 in their manufacturing process (22). The guidance states that “the manufacturer should consider the limitations of biological assays, such as high variability, that might prevent detection of differences that occur as a result of a manufacturing process change.” However, it also states, “In cases where the biological assay also serves as a complement to physicochemical analysis (e.g., as a surrogate assay for higher order structure), the use of a relevant biological assay with appropriate precision and accuracy might provide a suitable approach to confirm that change in specific higher order structure has not occurred following manufacturing process changes.” While the intent of this analysis is to assess stability rather than manufacturing change, this guidance can be extrapolated to stability test methods. If potency methods lack sufficient accuracy and precision to detect small physical and chemical changes occurring during storage, additional methods may be required to provide early indication of loss of potency and expiry.

	Stanley Hem advocated for the application of preformulation studies, common in the development of biopharmaceuticals, to aluminum adjuvant containing vaccines (23). In addition to characterizing the adjuvant and the free antigen, Hem recommends evaluation of the antigen-adjuvant formulation to understand strength of adsorption as well as the structural and chemical stability of the protein antigens.

	Ideally, test methods to provide stability data for adsorbed vaccine products would not require extensive sample preparation or desorption of the antigen from the adjuvant. There are a variety of different mechanisms suitable for desorption but all share disadvantages. The desorption procedure itself may result in structural changes or additional modifications or impurities in the protein antigen. For analysis in most test methods, desorbed samples will require a buffer exchange step, which will result in solutions with an unknown concentration, which must be determined using a separate assay prior to analysis by stability indicating methods. Lastly, desorption recovery may vary and recovery can decrease in aged samples (15, 24).

	The measurement of unbound antigen by its definition is performed on finished drug product (FDP) without desorption. The intention of the test method is to measure the amount of free protein that was not adsorbed or has desorbed from the aluminum adjuvant. Samples are prepared by centrifugation, and the supernatant is removed for analysis. Typically, chromatography is utilized to quantify the amount of free protein in the sample against a standard curve of the same protein. Chromatographic methodologies vary and may include size-exclusion chromatography (SEC) (25) or reverse-phase high performance liquid chromatography (RP–HPLC). Typical methods can be based on those used for analysis of the antigen bulk drug substance (BDS), which are then optimized for sensitivity and validated using a low-level standard curve.

	Some immunological test methods are feasible in the presence of Alhydrogel or aluminum phosphate and may be used for identification or, in some cases, quantitation without desorption. While Western Blots are not a practical alternative because SDS–PAGE cannot be performed on FDP, slot blots and dot blots eliminate the separation step. In a slot blot or dot blot method, the product is applied directly to a membrane and may be pulled through the membrane using a microfiltration apparatus and vacuum. Additional rinses may be performed as needed to ensure no surface residue remains on the membrane. Following application of samples, standards, and controls to the membrane, the membrane is removed from the apparatus and immunoblotted using standard procedures. Though no published procedures were identified describing the use of dot or slot blots for the analysis of aluminum adsorbed vaccines, the technique was used to quantitate the Hemagglutinin and Neuraminidases in influenza vaccine (26). An advantage of dot and slot blots is that multiple samples can be analyzed simultaneously, and membranes can be cut into strips for immunoblotting using multiple antibodies allowing for the identification of multiple antigens simultaneously.  Zhu demonstrated the feasibility of a direct Alhydrogel formulation immunoassay (DAFIA) for the determination of antigen identity and content (27). In the method, the FDP is added to wells of a black multititer plate. Following centrifugation and washing, the wells are blocked, then probed with primary antibody followed by a secondary antibody tagged with fluorescein. Signal is read with a fluorometer at 485nm/535 nm.

	Concentration measurement of the aluminum adjuvant can be determined directly, using inductively coupled plasma atomic emission spectroscopy (ICP–AES). Traditional methods for protein quantitation such as UV-Vis spectroscopy or RP–HPLC are not suitable for bound antigens. Method 6 of 

 <1057> utilizing o-phthalaldehyde (OPA) (21) has been applied to Alhydrogel vaccines. The OPA reacts with N-terminal amine groups or the amine groups on the side-chains of lysine and results in a fluorescent signal at 340nm/455 nm. Protein concentration of the FDP is compared to a standard curve. The OPA method was applied to Malaria vaccine candidates containing Alhydrogel, and it was determined that for accurate results Alhydrogel must be included in standards at an equivalent concentration as samples (28).  Accuracy was 87–100%, and linearity, though protein dependent, ranged from 25–400 µg/mL. In internal studies, the method had a range of 20–250 µg/mL using a non-linear curve fit and resulted in accuracy of 98–110% and repeatability of ≤2% for samples (see a representative standard curve in 

	HPLC is not suitable for the analysis of intact aluminum-adsorbed FDP because the vaccine molecules would be filtered by the column frit or, if they do pass into the column, would result in column clogging and increased back-pressure. RP–HPLC has been applied to concentration measurement, and to a limited extent, chemical stability of vaccines after desorption (10). While a desorption step is required to perform chromatographic analysis of the intact antigen, peptide mapping provides a powerful tool to verify the identity of FDP as well as evaluate the chemical stability of the antigen following adsorption and during stability. In peptide mapping, the protein is digested with an enzyme such as Lys C or Trypsin to produce a set of peptides. The resulting mixture of peptides can be separated chromatographically and compared visually to a control or the BDS or it can be analyzed by mass spectrometry and compared to the expected masses of peptides based on the primary sequence. The peptide mixture can also be analyzed without separation using matrix-assisted laser desorption mass-spectrometry with time-of-flight detection (MALDI–TOF). Lys C digestion of the desorbed antigens followed by MALD–TOF analysis of a trivalent botulinum vaccine containing Alhydrogel detected oxidation and deamidation reactions in all three protein antigens and reaction rates were accelerated in the presence of the adjuvant (29). Initially a 250 mM succinate buffer, pH 3.5 was utilized to desorb samples, but following incubation, desorption became more difficult, and use of a 4 M Urea pH 7.5 buffer was required. The desorption itself complicates the analysis because, first, it may not be completely reproducible, especially for aged samples, and, second, it may result in additional degradation. In unpublished studies, trypsin was used to successfully digest the antigen bound to Alhydrogel without desorption. Standard digestion procedures of incubation in Tris/HCL buffer, pH 8.0 for four hours at 37 °C were followed by RP–HPLC using an ACN/TFA mobile phase and C18 column. Visually, chromatograms (

) from digested FDP closely matched those of digested BDS, indicating the non-desorbed peptide mapping is a feasible method for the evaluation of antigen stability in FDP. With the increased availability of high mass accuracy/high resolution mass spectrometers, peptide mapping has evolved into multi-attribute methods (MAM), which can replace multiple electrophoretic and chromatographic stability indicating methods for therapeutic proteins (30). The application of MAM to aluminum-adsorbed vaccines has the opportunity to further understanding of protein-antigen stability and improve analysis to support quality by design (QbD) development as well as quality control (QC) release.

	MAM has the potential to replace the electrophoretic and chromatographic stability indicating methods that all require desorption prior to analysis. Desorption methods range from the use of phosphate buffer pH 7.4 (25), phosphate buffer and Zwittergent (9), 250 mM succinate buffer pH 3.5 (29) to harsher conditions such as 20 mM sodium dodecyl sulfate (SDS) or 20 mM cetylpyridium chloride in citrate-phosphate buffer (31), and up to 4M Urea (29). Multiple investigators have noted that desorption is more challenging in aged samples (14, 24). Therefore, development of desorption procedures for stability indicating methods should be performed with aged samples when possible. The selection of desorption method will vary depending on the adjuvant used as well as the intended analysis method and compatibility with the desorption solution. For example, SDS would be a good choice for desorption buffer when preparing samples for methods utilizing SDS in the procedure, such as SDS–PAGE or CE-PAGE. Additionally, SDS does not significantly interfere with RP–HPLC procedures. Urea is often used in sample preparation for imaging capillary electrophoresis (ICE) procedures and is a preferred buffer for preparation of samples for charge separation methods such as ICE and ion-exchange chromatography (IEX). A standard protocol for desorption using SDS involved the following steps:

		Mix buffer, SDS, and vaccine FDP and incubate at 70 °C for 10 minutes.

		Centrifuge, remove supernatant, and determine protein concentration using RP–HPLC. Ensure that SDS concentration in the reference standard matches that in the samples.

		Concentrate using spin-filters and desalt with water wash. Utilize volume to determine approximate concentration prior to analysis.

		A standard protocol for urea desorption follows a similar procedure:

		Mix buffer, urea, and vaccine FDP and shake for 18 hours at refrigerated conditions.

		Centrifuge, remove supernatant, and determine protein concentration using RP–HPLC. Ensure that urea concentration in the reference standard matches that in the samples.

		Dialyze samples to reduce urea concentration.

		Repeat RP–HPLC analysis to determine concentration, concentrate using spin filters and desalt with water wash.

	Following desorption, SDS–PAGE can be performed using standard procedures and commercial gels and reagents. The analysis of a model vaccine pictured in 

 utilized a pre-cast gel (Thermo Invitrogen NuPAGE 4–12% Bis-Tris) with recommended sample and running buffers (NuPAGE LDS Sample Buffer and NuPAGE MOPS SDS Running Buffer). The gel shows results from analysis of SDS-desorbed model vaccine samples that had been stored at 37 °C for 0 to 5 days. Samples are reduced prior to loading the gel and stained using a Coomassie blue stain (NuPAGE Colloidal Blue Staining Kit). Gels were analyzed by densitometry (Bio-Rad GS-800 Densitometer with Quantity One software) for presence of additional bands due to degradation as well as % purity. Purity of the main band ranged from 96% for the Day 0 sample to 90–91% at Days 4 and 5, therefore demonstrating that the desorbed SDS–PAGE method is stability-indicating.

	ICE analysis of a model BSA-Alhydrogel vaccine was performed using an imaged capillary electrophoresis system with detection at 280nm (ICE280 and ICE3 systems, Protein Simple). Samples were prepared using urea desorption and dialysis as described previously and then concentrated using spin filters. Samples, reference material, and a control sample were diluted in a diluent consisting of a mixture of pharmalyte 4–6.5, pharmalyte 3–10, 1 g/mL urea, 0.7% methylcellulose, and pI markers 5.12 and 6.61. Analysis was performed using acidic anolyte (0.1% methylcellulose, 0.08 M H3PO4), basic catholyte (0.1% methylcellulose, 0.10 M NaOH), and separation for 10 minutes at 3000V. The resulting electropherograms were analyzed (ChromPerfect 7 software) for isoform purity. Samples of model vaccine were stored at 37 °C for 0 through 5 days prior to analysis. See 

for an electropherogram overlay showing the isoform profiles for each sample. The main peaks are observed at pI 6.1 and 6.2 in non-degraded samples, but a significant shift toward more acidic isoforms is observed with longer storage at 37 °C. The desorbed ICE method is stability-indicating for detection of changes in isoform profile.

	The nature of aluminum adsorbed vaccines presents challenges to the application of traditional bioanalytical methods commonly used for stability studies of therapeutic proteins. While studies have demonstrated structural and chemical modification of the protein antigen resulting from adsorption, and that adsorption may impact stability, little regulatory guidance is provided beyond the recommendation to include identity, purity, and potency in drug product release testing. Potency methods may not have the necessary accuracy and precision to predict vaccine effectiveness and stability throughout the shelf life of the product. Instead, a panel of tests adapted from traditional therapeutic protein analysis is proposed in 

 Though historically, antigen characterization was performed following desorption, most of the methods proposed can be performed on the FDP directly. As MS-based peptide mapping methods, such as MAM, continue to grow in popularity, the need for desorption followed by electrophoretic and chromatographic methods for the determination of fragmentation or chemical modification may be eliminated.

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	Vol. 43, No. 9
	September 2019
	Pages: 42–52

	When referring to this article, please cite it as W. Saffell-Clemmer and E. Joseph, "Stability Indicating Methods for Aluminum Adsorbed Vaccine Products," 

Articles

The authors provide an introduction to aluminum adsorbed vaccines, review studies of antigen stability, and propose test methods for the analysis of aluminum vaccine release and stability analysis.

Stability Indicating Methods for Aluminum Adsorbed Vaccine Products

The market for pre-filled syringes (PFS) is expected to grow to reach sales of $6.6 million by 2020 (1). Nine out of the top 10 products produced in PFS are biologics (2). The use of PFS as a primary container and delivery system offers significant advantages for biologic products. These advantages include reduced risk of medication errors, simplified and faster preparation and administration, and reduced overfill requirements--which can reduce batch volumes and cost of the drug substance. While advantageous compared to vials, the use of PFS as a primary container adds additional complexity to what are already considered complex products. The PFS product contact materials, and filling and stoppering process, may have an impact on the formation of protein aggregates and particulate matter. As the link between immune response and protein aggregation has been understood over the past decade, FDA has urged product developers to employ risk reduction strategies to better understand the susceptibility of products to aggregation and to use orthogonal techniques to characterize final product (3, 4). PFS product developers must understand the impact the syringe manufacturing process can have on product contact materials and the influence the filling process can have on particle formation and final drug product quality.

	Though vials and PFS are composed of similar materials, such as pharmaceutical-grade glass and rubber, the manufacturing process for syringes and requirements for functional performance increase potential product contract materials. PFS container compatibility issues have been determined to be a root cause of protein aggregation in multiple products (5, 6, 7). The PFS system consists of the barrel, which, on one end has a tip with staked needle, luer lock, or slip tip. The tip or needle is closed with a rubber tip cap or rubber needle cover, often with a rigid plastic cover. The opposite end of the barrel has a flange and is sealed with a rubber plunger stopper. A plunger rod is inserted into the plunger stopper but does not come in contact with the product (

	The production of glass syringes differs significantly from vials. Until recently, most syringe barrels were made from glass, but new options that are growing in popularity include polymers such as cyclic olefin co-polymer (COC) or cyclic olefin polymer (COP). Glass vials are typically made from both Type IA and Type IB borosilicate glass, while glass PFS use only Type IB. Type IA and IB glass differ in chemistry and in thermal properties, which allows Type IB to be formed at lower temperatures, allowing the syringe barrels to be shaped from long tubes of glass. Additionally, the lower temperatures (less than 1200 °C) used for syringe forming versus vial forming (up to 1400 °C) reduce the risk of delamination (8). The glass tubing is produced through several processes that involve heating the glass mixture, pulling the glass, and blowing through a hollow tip to form a tube (9). The tubing is cut into sections, and a flange is formed first using heat and forming tools. The flange region of the barrel has no product contact. A cone is formed at the opposite end by heating the glass while inserting a tungsten probe and compressing the glass around the probe. The tungsten probe is removed, resulting in a fluid path through the syringe tip. If a staked needle is used, it is placed and glue is applied, which is then cured with ultraviolet light.

	Since June 2012, five recalls have been issued as a result of glass delamination in vials, while there have been no reports of delamination in syringes during the same time period (10). A greater concern resulting from the production process of syringe barrels is residual tungsten, which has been identified as a root cause for the formation of protein aggregates in epoetin alfa and an unidentified alpha-helical protein (6). Risk of tungsten-induced aggregation may be greater in formulations with a pH less than seven (6, 11). Additionally, the use of vacuum stoppering may increase the likelihood of tungsten-protein interactions because it may remove the air gap at the tip of the syringe, increasing the contact of the drug product with the “tungsten-rich” cone of the syringe (6).

	The incidence of tungsten-induced aggregation is formulation-specific and may be molecule-specific. In internal studies, a monoclonal antibody (mAb) solution was spiked with sodium tungstate at levels of 1, 10, and 100 ppm and then analyzed by size-exclusion chromatography (SEC), turbidity, and imaging particle analysis. No patterns were observed that indicated measurable interaction of the mAb with sodium tungstate.

	Most PFS are supplied ready-to-fill in a nest or tub format. All glass and most polymer syringe barrels require some level of silicone application to ensure that the plunger stopper moves smoothly during administration. Stoppers and needles may also be siliconized. An advantage of coating barrels with silicone is that it can protect the glass from etching processes resulting from higher pH formulations and further reduce risk of delamination (12). The application of silicone is typically performed by the syringe manufacturer and is usually performed by atomizing viscous silicone fluid using high-pressure air. The silicone may be applied using a fixed needle, which is located at one position during the application process, or a diving needle which applies the silicone while moving within the barrel. The use of the diving needle has been demonstrated to improve the consistency of the silicone coating over the syringe barrel interior and improve the gliding force profile (13). Silicone may be applied as a non-reactive oil, a non-reactive emulsion, or as a reactive or curable silicone fluid. A high-temperature baking process is required to cure silicone emulsions or silicone fluid. Cross-linked silicone is also available in specific low-silicone PFS products.

	Silicone has also been implicated in the formation of protein aggregates and protein-silicone complexes (7). Silicone oil-induced aggregation is more likely to occur in formulations lacking in surfactants, at high protein concentrations, and at formulation pH near the protein’s isoelectric point (pI). The occurrence increases with rising silicone oil concentration. Fewer silicone-related particles can be expected in products filled in PFS with baked-on silicone than with silicone oil. Also, agitation of syringes with headspace resulted in greater particle counts than in containers without headspace (13). United States Pharmacopeia Chapter <1787> classifies silicone particles as intrinsic, but cautions that “careful design, control, and application of the silicone oil are recommended to obtain proper functionality with the minimum amount needed for the shelf life of the product” (14). The chapter warns that protein may be absorbed into silicone oil droplets. The mixture of particle sizes of silicone oil droplets and silicone oil-protein complexes may mimic those of inherent protein particles. Fortunately, silicone oil droplets are easily differentiated by their morphology and optical properties when particulate matter analysis is performed by imaging particle analysis (15).

	Laboratory studies completed at Baxter Biopharma Solutions have indicated that silicone oil-induced aggregation is product-specific. In a typical study, a mAb formulation was spiked with silicone oil levels of 0.5 mg/mL and 1.0 mg/mL and then agitated on a shaker table for four hours at room temperature. Analysis by SEC, turbidity and imaging particle analysis identified no measurable change in aggregation resulting from the addition of silicone oil. In one internal study, the silicone spiked formulation was placed on stability for nine months at real time and accelerated conditions without indication of impact to product quality. Silicone-sensitive products can be filled in low-silicone syringes or a silicone-free polymer syringe. However, even low-silicone syringes can have lot-to-lot variability. In one such case, high particle counts were observed in a batch of syringes filled with product. Micro-flow imaging was used to identify the particles as silicone droplets based on their morphology. The issue was isolated to one syringe manufacturing lot. In an internal laboratory study, syringe samples from a previously used acceptable lot, the suspect lot, and a new lot were filled with placebo solution, allowed to sit for 30 minutes, and then the solution was drained, pooled, and tested for particles by light obscuration. As suspected, the solution from the suspect lot of syringes contained a higher level of particles than the solutions stored in the other two lots of the same syringe assembly.

	PFS filling lines are typically equipped with rotary piston pumps or peristaltic pumps. Historically, piston pumps offered greater accuracy and speed, but advances in peristaltic pump head design, drive motors, and calibration algorithms have narrowed differences in performance for most formulations (16). Accuracy may be decreased when peristaltic pumps are used to dispense viscous formulations greater than 100 cP. Rotary piston, time-pressure, peristaltic, and rolling diaphragm pumps were evaluated for their impact on particle formation in a purified mAb solution (17). The study concluded that while the measured amount of aggregates by SEC was unchanged, use of a piston pump increased levels of proteinaceous particles observed by imaging particle analysis. The author theorized that the increase in particles could be the result of sheer denaturation and concluded that potential for the increase in aggregates may be product-specific (17).

A Baxter product development study provided additional evidence that sensitivity to denaturation during pumping is not only molecule-specific but formulation-specific. Sheer sensitivity was evaluated by stressing a formulated mAb solution at a rate of 1500s

 for up to three hours using a rotary cone viscometer followed by analysis by SEC and micro-flow imaging. A slight increase in aggregate concentration (< 0.2%) was observed through SEC, but it was well within assay variability and a fragment peak increased (<1%) after three hours. Particle counts by imaging particle analysis also increased within expected levels based on prior studies. The same solution was subjected to 25 cycles of repeated circulation through a piston pump without increases in particle count, aggregates, or fragments. After receipt of a new lot of API, however, small visible particulates were observed in process development samples subjected to piston pumping after storage for seven days. An evaluation using imaging particle analysis and microscopic magnification revealed that the particles appeared to be proteinaceous. A comparative study was initiated in which formulated material using the new API was cycled three times through a peristaltic or piston pump, filled in syringes, and placed on stability at accelerated and real-time conditions for three months. Samples exposed to piston pumping developed visible particulates in as little as seven days at accelerated storage temperatures. A significant increase in 1-2 µm subvisible particles was observed in the piston pump cycled samples stored at accelerated conditions throughout the duration of the study. Following the pumping, a follow-up study was performed comparing different buffer systems. All other formulation parameters remained equal, including the pH, the protein concentration, and the concentration of surfactant. The two formulations were exposed to three cycles through a piston pump and placed on stability. An immediate increase in 2-10 µm and 10-25 µm particles was observed in pumped samples of both formulations relative to control. After storage at 5 °C for six weeks, the number of particles counted in the range of 2-10 µm and 10-25 µm increased significantly in formulation two samples exposed to pump cycles. A corresponding increase was not observed in formulation one samples (

) relative to control. After six weeks of 25 °C storage, both the formulation two control and exposed sample had increased particle counts, indicating that the product was not stable in the selected buffer component. No changes were observed by SEC.

	The development of biologic PFS products must be undertaken with a thorough understanding of the impact product contact container materials and filling processes may have on the potential for protein aggregation and product quality. While potential risks can be generalized, the impact of such variables as syringe type and pumping method on the product is both molecule- and formulation-specific. Product-specific studies can allow for the design of specific risk reduction strategies and allow product developers to make scientifically sound decisions while meeting other requirements related to container selection and filling technology such as cost, availability of equipment, dosing accuracy, end user safety, and ease of use.

The Future of Alliances and Partnerships in the Pre-Filled Syringes Market to 2020

,” accessed Jan. 13, 2016.
	2. Drug Development & Delivery, “

SPECIAL FEATURE-Prefilled Syringes & Parenteral Contract Manufacturing: Biologics Present a New Set of Challenges

,” accessed Jan. 13, 2016.
	3. C. Carpenter et al., 

. 98 (4), 1201-1205 (2009). 
	4. A. Rosenberg, D. Verthelyi, and B. Cherney, 

. 101 (10), 3560-3567 (2012).
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	6. W. Liu W, R. Swift, and G. Torraca, PDA, 

. 64, 11-19 (2010).
	7. L. Jones, A. Kaufmann, and R. Middaugh, 

 94 (4) 918-927 (2005).
	8. T. Blankston, “Glass Delamination: Part I. Considerations for Primary Containers,” BD Medical-Pharmaceutical Systems, webcast, Sept. 1, 2011.
	9. British Glass, “

,” accessed Jan. 27, 2017.
	10. FDA, “

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	11. J. Bee et al., 

98 (9), 3290-3301 (2009).
	12. S. Funke et al., 

105 (12), 3520-3531 (December 2016). 
	13. B. Reuter and C. Peterson, 

. 2 (4), 238-244 (2012). 
	14. USP General Chapter 39 <1787>, “Measurement of Subvisible Particulate Matter in Therapeutic Protein Injections” (US Pharmacopeial Convention, Rockville, MD, 2016).
	15. L. Brown and Q. Bernt, “A Comparison of Methods for Quantifying Silicone Droplets in Biologics using Dynamic Imaging Particle Analysis,” presentation at the Workshop on Protein Aggregation and Immunogenicity (Breckenridge, CO, 2014). 
	16. P. Lambert, “

Dispensing Biopharmaceuticals with Piston and Peristaltic Pumps

, Sept. 17, 2008, accessed Feb. 2, 2017.
	17. A. Nayak et al.,

	When referring to this article, please cite it as W. Saffell-Clemmer, "Impact of PFS and Filling Process Selection on Biologic Product Stability," 

The author discusses the impact of prefilled syringe product contact materials and the filling and stoppering process on protein aggregates.

Impact of PFS and Filling Process Selection on Biologic Product Stability

The US Pharmacopeial Convention (USP) released a general announcement on June 24, 2016, stating that a new General Chapter has been proposed, entitled, <1220> The Analytical Procedure Lifecycle, which will be consistent with the concepts of quality by design (QbD) as described in International Council for Harmonization (ICH) Q8 (R2), 9, 10, and 11 (1). In a Stimuli article published in 

 (PF) 39(5), the USP Validation and Verification Expert Panel discussed the application of lifecycle principles described in ICH Q8 to analytical procedures (2, 3).  The article proposes an integration of method validation, transfer, and verification process into the overall lifecycle management process of the product to best align the variability of the analytical procedure with the requirements of the product. Incorporating the method procedure into the lifecycle change management process ensures that the procedure will remain suitable for its intended use. By using QbD concepts in the development and validation of the test procedure, an understanding of method variability-including variability of the test method and reference materials--can be developed, aiding in the understanding of total process variability such that it can be monitored, controlled, and reduced when possible.
	
	As part of the lifecycle process, the panel suggests the establishment of an analytical target profile (ATP), which is similar to the concept of a quality target product profile (QTPP) and describes the criteria for a reportable result. Additionally, the article authors propose the application of quality risk management (QRM) concepts and tools from ICH Q9 to the development and validation of the analytical procedure (4). Suggested tools include process mapping tools to understand potential sources of impact to data quality, and evaluation tools such as the control, noise, experimental (C, N, X) technique. Design of experiment (DoE) studies can be used to evaluate those variables identified as experimental (X) to understand the acceptable ranges for these variables.

	To further understanding of QbD concepts to analytical method development and validation, the tools suggested in the Stimuli article were combined with the guidance in ICH Q2(R1) (5) for this case study, and applied to the validation of a stability-indicating high-performance liquid chromatography (HPLC) method for Protopam drug substance (Baxter) and Protopam chloride for injection meeting ICH Q3A(R2) and ICH Q3B(R2) standards for impurities (6, 7).

	Protopam chloride for injection is used as an antagonist to antichlolinesterases and is indicated as an antidote in the control of over dosage by anticholinesterase drugs and treatment of poisoning resulting from pesticides and chemicals in the organophosphate class that have anticholinesterase activity. It is supplied as a 1000 mg single-dose vial, which is reconstituted prior to injection (8).  The maximum daily dose is 8 g. Based on the calculated maximum daily dose, the drug substance method must have a limit of quantitation (LOQ) ≤ 0.03% and drug product method must have an LOQ ≤ 0.05%. As the method was to be used for drug substance and drug product, the most critical performance criteria for the method was that the LOQ must be ≤ 0.03%, which is equivalent to ≤ 7.5 ng/mL for the test solution as prepared in the method. To meet the performance criteria, the ATP was established as the signal-to-noise ratio (S/N) for each injection of the LOQ solution (7.5 ng/mL) must be ≥ 10, and the percentage of relative standard deviation (RSD) for the peak area in the LOQ solution must be ≤ 20%.

	The assay method from the current USP monograph for pralidoxime chloride was used as a starting point for method optimization (9). Multiple wavelengths were investigated and UV-Vis spectra of pralidoxime chloride and two related substances were obtained. There was no common λ

 between the three related substances and pralidoxime chloride; therefore, the 270 nm detection wavelength specified appeared to be the best compromise for optimal sensitivity. Increased injection volumes were evaluated and multiple columns screened. A 50 µL injection volume and the Spherisorb ODS2 (Waters), 3 µm, 4.6 x 250 mm column, were selected for further development work (

). A preliminary evaluation of LOQ, repeatability, specificity, and linearity was conducted on four HPLC systems and two lots of stationary phase.

	Risk assessment and analytical design space 

	Potential failure modes of the method were examined via discussions with experienced analysts. A fishbone diagram (

	Potential failure causes were assessed using a CNX model (

) to identify those that could be controlled (C), those that were noise (N), and those that required experimentation (X). Instrument malfunction was not listed in the fishbone diagram or table. It must be controlled by system suitability requirements.


	A failure mode effects analysis (FMEA) was conducted using the potential failure mode causes identified. A scale of 1 to 5 was used to rank the severity (S) of the failure, the probability (P) of the failure, and the likelihood that the failure would be detected (D) using the guide in 

. The risk assigned to each possible failure mode was calculated as: Risk = Severity X Probability X Detectability. The category C modes were all related to people and were not unique to the method. The potential failures must be mitigated by training, verification, good distribution practice (GDP), and other aspects of a quality system. The precision section of the method validation demonstrated that these potential failure sources were controlled at that period in time. The potential failure causes identified as N were related to small variations in sample and standard preparation, and were part of the variability to be tested in the intermediate precision section of the validation. The X failure modes were rank ordered in 

	The highest identified X risk was related to standard stability. Stability of the prepared standard was demonstrated for up to 14 days at the assay level in a previous study, but was not evaluated at the impurities level. Stability was assessed as a part of the validation study. The second highest identified risk was related to the risk that the standard or sample could absorb water during weighing. The standard and sample are dried at 105 °C for three hours prior to weighing. The tendency of the dried material to absorb water was evaluated in a simple experiment in which weight gain at ambient conditions was monitored. The average mass increased by 0.19% after four hours, and the method was updated to instruct analysts to weigh standards and samples immediately after removing them from the oven.

	The remaining possible failure causes assigned ranked 3, 4, or 5 were evaluated using a DoE approach (

). Additional chromatographic parameters such as flow rate and injection volume were added to provide a validated space for the method. A eighth fractional factorial design, consisting of 16 assay runs, was constructed in MiniTab to evaluate % acetonitrile (ACN) concentration of the mobile phase, the tetraethylammonium chloride (TEA) concentration in the sample buffer and mobile phase, the sample temperature, the column temperature, the wavelength, the flow rate, and the injection volume. The impact on ATP was assessed by reporting the S/N of the LOQ solution and the % RSD for the peak area in the LOQ solution. The precision of the impurity content and the assay value for the sample at each condition and overall was reported and evaluated. Following the execution of the original DoE study, an additional set of runs was added to explore the effect of higher TEA concentrations and higher wavelengths. The additional studies were intended to evaluate the effect of TEA hydrate form as well as compare variance between old and new columns and the variation between laboratories. All test runs included Protopam Chloride USP standard, Protopam Chloride drug substance, Protopam for Injection from a concluded stability study reconstituted and heat degraded at 80 °C for approximately 24 hours, and a LOQ solution.

	The factorial design was analyzed using MiniTab version 14, using both the LOQ S/N ratio and the % RSD of the peak area for the LOQ injection as the responses. Using the % RSD of the LOQ solution peak area as the response, no significant effects were observed; all factors and all interactions resulted in a response less than the p value. Using the average S/N of the six LOQ injections, two factors were determined to be significant (

). Both TEA and wavelength had p values greater than Alpha. A main effects plot (

) supported that TEA and wavelength are significant. S/N is greatest at 0.4% TEA and a wavelength of 275 nm. An interactions plot and surface plot indicated that there was no interaction between TEA concentration and wavelength.


	To understand if the LOQ solution S/N was optimized, an additional factorial study was executed adding a TEA concentration of 0.5% and wavelength of 280 nm to the study. The additional factorial required four more HPLC runs.

	No significant improvement in the working LOQ solution S/N was obtained by increasing the TEA concentration to 0.5 mg/mL from 0.4 mg/mL. Concentrations of 0.3, 0.35, 0.4, and 0.5 mg/mL resulted in acceptable results for S/N of the LOQ, with higher S/N results obtained for TEA concentrations of 0.4 mg/mL and 0.5 mg/mL. Remaining studies used TEA concentrations of 0.4 mg/mL.

	No significant improvement in the LOQ solution S/N was obtained by increasing the wavelength to 280 nm. In addition, previous studies conducted indicated that there was no single peak maximum in common among all known impurities, but that a wavelength of 270 nm was suitable for all. Wavelengths of 270, 275, and 280 resulted in acceptable results for Protopam chloride at the LOQ. To further understand the optimum wavelength, the combined data sets were reanalyzed evaluating the average total percent impurities as area under the curve (AUC) reported for the heat-degraded product sample. A simple scatter plot (

) of the % impurities as AUC versus wavelength indicated that the amount of impurities detected was reduced at higher wavelengths. Analyzing each data set individually within Minitab analysis software demonstrated that the detection wavelength did have a significant effect on the amount of impurities reported (

	Additional experiments within the robustness study demonstrated that the method was sensitive to TEA type. Tetraethylammonium chloride monohydrate resulted in lower concentration than tetraethylammonium chloride, and the method was updated to specify the tetraethylammonium chloride form.

	A review of DoE meeting the ATP was used to develop system suitability criteria for use in the validation study (

	The method validation was completed following standard procedures in accordance with ICH Q2(R1) and USP <1225> (5, 10). Linearity was demonstrated at assay and impurity levels. Precision was demonstrated for API and drug product. Accuracy was demonstrated through spiking studies, and specificity was shown through a forced degradation study. Robustness was repeated, for wavelength only, to understand if small, 2-nm variations would impact the related substances results. The LOQ was demonstrated as part of the precision study, and range was inferred from the linearity and accuracy results. Sample, standard, and mobile-phase stability were proven for up to seven days.  All acceptance criteria were met.

	The incorporation of QRM concepts and tools from ICH Q9 (4) to the development and validation of a stability indicating method for Protopam chloride for injection resulted in a robust procedure that met its ATP and all acceptance criteria during validation without deviation. DoE studies conducted following risk assessment established two variables that impact the ATP: the TEA concentration utilized for mobile phase and sample buffer and the detector wavelength. The improved understanding of the impact of sources of variability in method performance gained through the risk assessment and DoE study can be a valuable resource for future lifecycle change management process, ensuring that the procedure will remain suitable for its intended use.

	1. USP, General Announcement-General Chapter Prospectus: <1220> The Analytical Procedure Lifecycle, June 24, 2016, www.usp.org/usp-nf/notices/1220-analytical-procedure-lifecycle, accessed July 25, 2016.
	2. USP, “Stimuli to the Revision Process: Lifecycle Management of Analytical Procedures: Method Development, Procedure Performance Qualification, and Procedure Performance Verification,” 

 (ICH, 2009), www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q8_R1/Step4/Q8_R2_Guideline.pdf, accessed July 25, 2016.
	4. ICH, 

 (ICH, 2005), www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q9/Step4/Q9_Guideline.pdf, accessed July 25, 2016
	5. ICH, 

Q2(R1) Validation of Analytical Procedures: Text and Methodology

 (ICH, 2005), www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf, accessed July 25, 2016.
	6. ICH, 

Q3A(R2) Impurities in New Drug Substances

 (ICH, 2006), www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3A_R2/Step4/Q3A_R2__Guideline.pdf, accessed July 25, 2016.
	7. ICH, 

 (ICH, 2006), www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3B_R2/Step4/Q3B_R2__Guideline.pdf, accessed July 25, 2016.
	8. Protopam Chloride (pralidoxime chloride) for Injection FDA Approved Labeling Text dated Sept. 8, 2010, , accessed 25 July 2016.
	9. USP, Monograph for Pralidoxime Chloride, 

.
	10. USP, <1225> Validation of Compendial Procedures, 

The integration of a method validation, transfer, and verification process into the overall lifecycle management process of a product can best align the variability of the analytical procedure with the requirements of the product.

Analytical Method Validation Using QbD and QRM

	When referring to this article, please cite it as W. Saffell-Clemmer and J. Karty, "Application of QbD and QRM to Analytical Method Validation," 

The article proposes an integration of method validation, transfer, and verification process into the overall lifecycle management process.