Evaluation of Particle Techniques for the Characterization of Subvisible Particles From Elastomeric Closure Components

Journal Pre-proof Evaluation of Particle Techniques for the Characterization of Subvisible Particles from Elastomeric Closure Components John Rech, Amber Fradkin, Aaron Krueger, Crystal Kraft, Diane Paskiet PII:

S0022-3549(20)30066-6

DOI:

https://doi.org/10.1016/j.xphs.2020.01.026

Reference:

XPHS 1863

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 11 October 2019 Revised Date:

12 January 2020

Accepted Date: 27 January 2020

Please cite this article as: Rech J, Fradkin A, Krueger A, Kraft C, Paskiet D, Evaluation of Particle Techniques for the Characterization of Subvisible Particles from Elastomeric Closure Components, Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.xphs.2020.01.026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.

Title: Evaluation of Particle Techniques for the Characterization of Subvisible Particles from Elastomeric Closure Components

1

2

2

1

1

Author: John Rech , Amber Fradkin , Aaron Krueger , Crystal Kraft , and Diane Paskiet 1

West Pharmaceutical Services, 530 Herman O. West Drive, Exton, PA 19341

2

KBI Biopharma, 2500 Central Avenue, CO 80301

Abstract: Evaluating a particle profile for parenteral drug products is a well-known challenge due to inevitable variability of results with limited accuracy to actual particle levels present in the product, especially in the subvisible particulate (SbVP) range. It is important to understand the appropriate SbVP counting/characterization technology, methodology capability, and the particle source (intrinsic or extrinsic). Elastomeric closures are prevalent in many types of drug product container closure systems and are a known source of particle contribution. These components need to be considered when establishing a drug product particle profile. In this work, we describe available particle extraction methodology and its applicability in the analysis of elastomeric closure components using multiple detection technologies. Optimum sample preparation and analytical techniques were established to evaluate sub-micron particle and SbVP loads from elastomeric closure components. Additionally, the impact of stopper siliconization and polysorbate 80 interaction on the degree of SbVP in the final drug product was assessed. Keywords: analysis, particle size, drug-delivery system(s), injectable(s), surfactant Abbreviations used: SbVP, subvisible particulate; CQA, critical quality attribute; LO, light obscuration; MFI, microflow imaging; ESZ, electrical sensing zone; SEM, scanning electron microscopy; RMM, resonant mass measurement; RI, refractive index; NMT, not more than; LOD, limit of detection Conflicts of interest: All authors have no known conflict of interest Acknowledgement of funding: West Pharmaceutical Services, Inc. Introduction: Throughout the lifecycle of a biotherapeutic product, it is essential to understand particles and their sources to develop control strategies. Protein formulations, delivery technologies, and processing steps are all sources of a broad range of particulates classified as: inherent (e.g., active pharmaceutical ingredient or formulation); intrinsic (e.g., formulation, process, or container closure system); or extrinsic (e.g., environmental). Particles and particulate matter are important quality and safety considerations in biotherapeutic development. Critical quality attributes 1–5 (CQAs) related to efficacy, potency, clinical safety, and immunogenicity can be affected by particles. The 6,7 presence of visible particles caused 48% of the recalls in sterile drug products from 2010 to 2017. Subvisible 2,3,8–11 particulates (SbVP) are recognized as potential causes of adverse immunogenicity and infusion reactions. Establishing a particle profile for a biopharmaceutical product is a well-known challenge. Instruments have some inherent measurement variability and comparing particle results across different measurement technologies is difficult. It is important to understand the appropriate SbVP characterization technology, methodology capability, and the particle source (inherent, intrinsic and/or extrinsic) to mitigate risk. Sources of particles can be grouped into six main categories: 1) active pharmaceutical ingredient (aggregates and particles); 2) solution and formulation components; 3) packaging components; 4) product packaging interactions; 5) process-generated; and 6) the environment.2 The type of particulate matter found in a product is dependent on the source. It is important to consider all sources in particle evaluations, but the occurrence of particles in sterile drug products originating

2,3,12–15

from packaging components and delivery systems has always been a major concern. This study examines the particle contribution from packaging components, specifically elastomeric closures (stoppers), and how to evaluate the particulates with the most appropriate technology. There are regulatory requirements for the presence of particles in pharmaceutical products, however, there are no defined limits for those originating from the packaging systems. Particulate contamination in drug products can range in size from visible to sub-micron. Figure 1 from USP <1788> describes the size range for visible, SbVP and 16 sub-micron particles. The total contribution of each component individually, and together as a system, should be determined during formulation studies to reduce unexpected risks to product quality. Potential sources of particles attributed to the packaging system include materials of the components, process aids used to assemble the system, secondary packaging, inadequate washing, and contamination from fill-finish processing. Investigating sources of SbVP at the onset of formulation development and throughout a product lifecycle is an important factor to ensure product quality. A comprehensive assessment of particles in pharmaceutical products requires the use of a range of methods. An assessment should be conducted with appropriate methods at each phase of product development and commercialization. In the case of biotherapeutics, there can be a continuum of particle sizes ranging from nanometers to hundreds of micrometers because of inherent protein aggregates and particles, foreign 13 contaminants, and adsorption of protein molecules to foreign particles. 17

18

19

The standards for measuring SbVP particles in parenteral products are USP <788> , EP 2.9.19 , and JP 6.07. These standards outline the use of two procedures for particulate evaluation: the light obscuration particle count test (LO) and microscopy particle count test. Light Obscuration is the preferred method and the microscopy particle count test should be applied when light obscuration is not suitable, i.e. drug products with high viscosity or high opacity. In addition to using these techniques, novel alternative methods can also be used to provide additional information about particulate present in drug product. Some of these alternative methods include micro-flow imaging (MFI), and light microscopy/image analysis (LM/IA). Newer methods continue to emerge, such as resonant mass measurement (RMM), nanoparticle tracking analysis, and advanced LO techniques. Currently, there are no limits for sub-micron particles, but drug product monitoring is required. Overall, a testing strategy for SbVP and sub-micron particles that employs multiple methods can help assure product quality through more accurate particle sizing and counting, as well as better characterization of particle subpopulations. The technologies determined most appropriate for evaluating SbVP particles along with their strengths and weaknesses can be found in Tables 1a and 1b. Consideration of the various factors outlined in these tables is important for each use case. These technologies are typically applied to the evaluation of SbVP in drug product, but only light obscuration is typically used for the evaluation of SbVP in container closure products such as elastomeric closures. Altogether, the diverse types of particulate matter, various sources of contamination, and inherent limitations in characterization techniques underscore the value of using orthogonal testing methods throughout a drug product lifecycle. This process is the responsibility of the drug manufacturer, although packaging suppliers can best support their efforts by providing initial particulate characterization of their components. 21,22

The standard that is Few studies have evaluated the SbVP contribution of primary packaging components. 23 used for testing elastomeric components for particulate is ISO 8871-3. This method uses an extraction to 17 remove loose particulate from stopper surfaces as opposed to USP <788> , which is a direct test of the drug 23 product itself. The methods outlined in ISO 8871-3 served as a starting point for this study to begin the exploration of particulates generated by stoppers using the advanced particulate technologies outlined above. During preliminary studies, we observed that the stopper type selected had a direct contribution to the level of particles that were extracted from the parts. Subsequently, we evaluated various elastomeric stoppers, produced by different treatments and manufacturing technologies, using orthogonal particle analysis methods. The goals for 23 studying the different stoppers were 4-fold: 1) evaluate stopper extractions based on ISO 8871-3 , (contributions

from packaging in the range of 0.1 µm to 100 µm); 2) determine the impact of the stopper treatment on SbVP and sub-micron particle contributions, particularly from silicone-treated stoppers; 3) compare the extraction of SbVP and sub-micron particles with polysorbate 80 versus with water; and 4) evaluate multiple types of particle characterization technologies to determine the most capable and reliable methods. Materials and Methods: Materials Four different samples of the same chlorobutyl stopper formulation were used for the study. See Table 2 for sample descriptions. See Figure 2 for an image describing the parts of the stopper. 24

All mention of water refers to Type 1 water (Millipore) meeting ASTM D1193-91 requirements and filtered through a 0.22 µm terminal filter. Polysorbate 80 was manufactured by Alfa Aesar (VWR cat # L13315-AP, lot number 10191384). The surfactant was prepared at a concentration of 0.03% w/v in water.

Sample Preparation For initial testing, sample extractions were based on ISO 8871-3. All samples were prepared in an ISO 5 cleanroom. An orbital shaker manufactured by Infors (Model: Labotron) was used for all extractions. Light obscuration (LO), microflow imaging (MFI), resonance mass measurement (RMM), and nanoparticle tracking analysis (NTA) 2

Samples were prepared by adding approximately 100 cm (total surface area) of stoppers to a 300 mL Erlenmeyer flask pre-rinsed with water. Water (100 mL) was added to the flask, which was then rotated (using an orbital shaker) at 325 rpm at an orbit of 12 ± 1 mm for 20 seconds. A single sample preparation was made for each stopper type. Samples were analyzed in triplicate. Light microscopy/image analysis (LM/IA) 2

Samples were prepared by adding approximately 400 cm (total surface area) of stoppers to a 400 mL Erlenmeyer flask. Two hundred mL of 0.03% polysorbate 80 solution was added to a flask pre-rinsed with water followed by 0.03% polysorbate 80 solution. The flask was then rotated (using an orbital shaker) at 325 rpm at an orbit of 12 ± 1 mm for 20 seconds. The stopper rinsate was passed through a 25 mm 0.45 µm nitrocellulose filter. An additional 200 mL of polysorbate 80 solution was added to the flask and the process was repeated. Filters were then analyzed by LM/IA. Modified Sample Extraction LO, MFI, RMM and NTA Triplicate samples from each stopper type were extracted with both water and 0.03% polysorbate 80 solution. Extractions were prepared by adding approximately 100 cm2 (total surface area) of stoppers to a 250 mL Erlenmeyer flask pre-rinsed with water. Enough water was added to the flask to just cover the stoppers (approximately 40 mL). The flask was then rotated at 325 rpm at an orbit of 12 ± 1 mm for 20 seconds. This modified preparation was applied to increase the concentration of particles in the sample extractions. The extractions were transferred to a pre-rinsed 125mL high density polypropylene screw-cap bottle (Dynalon, VWR cat # 30620-178), pre-rinsed with water, and analyzed by LO, MFI, RMM, and NTA. The above procedure was also repeated with a 0.03% polysorbate 80 solution.

LM/IA Triplicate samples from each stopper type were extracted with both water and 0.03% polysorbate 80 solution. 2 Extractions were prepared by adding approximately 100 cm (total surface area) of stoppers to a 250 mL Erlenmeyer flask pre-rinsed with water followed by 0.03% polysorbate 80 solution. Enough water to just cover the stoppers (approximately 40 mL) was added to the flask. The flask was then rotated at 325 rpm at an orbit of 12 ± 1 mm for 20 seconds. The stopper rinsate was passed through a 25 mm 0.45 µm nitrocellulose filter. An additional 40 mL of water was added to the flask and the process was repeated. Filters were then analyzed by LM/IA. The above procedure was repeated with 0.03% polysorbate 80 solution.

Experimental Methods Light Obscuration (LO) ®

LO was conducted using a HIAC Royco model 9703 with the HRLD-400 liquid sensor particle counting system. The data were collected using Beckman Coulter, Inc., PharmSpec software version V3.3.0.30040. The system utilized a 10 mL syringe with sampling volume set to 5.00 mL. Prior to analysis, the system was flushed with water. Environmental test blanks were analyzed to identify baseline levels of the extraction method without stoppers. The acceptance criteria for the environmental test blank was ≤ 100 particles ≥ 2 µm, ≤ 5 particles ≥ 10 µm and ≤ 1 particle ≥ 25 µm. Environmental test blanks contained in a 250 mL Erlenmeyer flask were analyzed as part of system suitability by allowing the blank to equilibrate for one minute at room temperature and then aspirating three separate 5 mL aspirates into the instrument. The first aspirate was discarded. The results from the second and third aspirate were averaged. Water environmental test blanks were tested for water-based samples and polysorbate 80 negative environmental test blanks were tested for polysorbate 80-based samples; however, only water blanks were held to acceptance criteria since polysorbate 80 contributes air bubbles to the blank solution. The environmental test blanks were not subtracted from the sample measurements. Prior to each stopper type, environmental test blanks were analyzed. Each sample extraction was performed a total of three times. Sample extractions were transferred to a 250 mL Erlenmeyer flask pre-rinsed with water and allowed to equilibrate at room temperature for one minute. A total of five 5 mL aspirates were collected from each sample extraction. The first aspirate was discarded, and the particle counts from the other four aspirates were averaged. Particle counts in particles/mL were reported and placed in 23 the following size ranges based on ISO 8871-3 : ≥ 2 µm, ≥ 5 µm, ≥ 10 µm, and ≥ 25 µm. Micro Flow Imaging (MFI) Analysis Prior to sample analysis, a system suitability test was performed with an Ezy-Cal 5 µm standard (ThermoFisher 6005, Waltham MA, USA), which was passed according to specifications (2000 particles/mL ± 10%, and 5.010 µm ± 0.035 µm). Water blanks were analyzed before and after each sample to ensure low particle count (≤ 100 particles/mL greater than or equal to 1 µm in size). Samples were gently swirled to ensure homogeneous solutions. For sample analysis, a volume of 0.65 mL was run through the sample cell, with the initial 0.2 mL used to purge the cell and approximately 0.35 to 0.39 mL of the subsequent volume analyzed. The flow rate for analysis was 0.17 mL/min. Particle sizes were reported as equivalent circular diameter (ECD), the diameter of a circle that would have the same area as the imaged particle. Image analysis was performed on particles ≥ 5 µm by manually selecting a representative population of particles for each sample for classification (silicone oil or non-silicone oil). In addition, a digital morphological filter of aspect ratio ≥ 0.85 was used to classify silicone oil versus non-silicone oil for particles ≥ 5 µm. Due to the highly spherical nature of silicone oil droplets, this morphological filter can be 21 applied to distinguish silicone oil with approximately 96% accuracy in solution samples.

Nanoparticle Tracking Analysis (NTA) ®

A Malvern Panalytical NS300 NanoSight NTA instrument using a CMOS camera and a green laser was used for the study. NTA 3.2.16 control software was used. Prior to sample analysis, system suitability was confirmed for accurate sizing using 200 nm polystyrene beads (ThermoFisher 3100A, Waltham MA, USA). Prior to running every sample, the instrument was cleaned using 3% Contrad70, followed by a thorough rinsing using Milli-Q water. The sample was inverted gently six times to ensure adequate suspension of particles, and the sample solution was analyzed neat with no dilution. The following instrument settings were used for sample analysis: acquisition temperature of 25°C, camera shutter of 1300, camera gain of 100, camera histogram high of 11,000, camera histogram low of 250, detection threshold of 6, and syringe pump speed of 10. All other instrument parameters used the auto setting. Each sample was analyzed using five recorded videos of 60 seconds each. Data were plotted using GraphPad Prism 5. Archimedes - Resonant Mass Measurement (RMM) ®

A Malvern Panalytical Archimedes particle metrology system with a MicroH sensor was used with ParticleLab v1.20.17017.1 control software. Prior to sample analysis, system suitability was confirmed for accurate sizing using a 1 µm particle size standard (ThermoFisher 4010A, Waltham MA, USA). Prior to every sample run, the instrument was cleaned using 3% Hellmanex-III, followed by a thorough rinsing using Milli-Q water. Sample was inverted slowly six times to ensure adequate suspension of particles, and the sample solution was analyzed neat with no dilution. A 1.8 mL aliquot was pipetted into a Nalgene cryoware vial (Thermo 5000-0020, Waltham MA, USA) that was placed in the sample chamber. The sample was loaded for 30 seconds prior to the start of data acquisition. Both negatively buoyant and positively buoyant particles were analyzed. Negatively buoyant particles were assumed to be butyl rubber with a density of 1.25 g/mL, and positively buoyant particles were assumed to be silicone oil with a density of 0.965 g/mL. The limit of detection was manually set at 0.012 Hz, and the experiment was terminated automatically by the software when 2000 total particles were analyzed, a measurement time of 45 minutes was reached, or a measured volume of 1.0 µL passed through resonator. Each sample was run a single time with the average and standard deviation of three replicate samples reported. Data were plotted using GraphPad Prism 5. Light Microscopy/Image Analysis (LM/IA) LM/IA was conducted using a Clemex® particle sizing system which consisted of a Leica DM6000 stand and power supply CTR 6000. Reflected light illumination was supplied from an LED light source. A L 2.1 Mp (1600 x 1200) Color CCD camera was used to capture images. A motorized stage with joystick and controller was used to move the filter along a fixed stage pattern. 20x and 2.5x objectives were utilized. The data were collected using Clemex Vision PE software version 8.0.23 (15773). A circular stage pattern was used to analyze the filter at a magnification of 200x for particles in the size range of 5 µm to 100 µm. Although particles smaller than 5 µm were observable, there was not enough contrast to accurately confirm their presence. articles > 100 µm were manually sized using the software at a magnification of 25x. Sizing of particles was based on the longest particle dimension. The software reported results in size ranges of: particles > 5 µm but ≤ 10 µm; > 10 µm but ≤ 25 µm; and > 25 µm. Therefore, cumulative particle counts were calculated in the following size ranges: > 5 µm, > 10 µm, and > 25 µm, in order to compare SbVP results. Particle counts were converted to particles/mL by dividing the number of particles in each size range by the total sample volume used for the analysis (volume of first aliquot plus volume of second aliquot). Samples for each stopper type were analyzed in triplicate and environmental test solutions were analyzed before each triplicate analysis to ensure low particle count (NMT 300 particles ≥ 5.0 µm but < 10.0 µm; 150 particles ≥ 10.0 µm but < 25.0 µm; 70 particles ≥ 25.0 µm but < 50.0 µm; 6 particles ≥ 50.0 µm but < 100.0 µm; and 1 particle ≥ 100.0 µm). Water environmental test blanks were used for water-based samples and 0.03% polysorbate 80 solution environmental test blanks were used for polysorbate 80based samples. The environmental test blank results were not subtracted from the sample measurements.

Results and Discussion: The purpose of this study was to determine the impact that silicone application on rubber elastomers has on SbVP generation in a sample and to determine the most appropriate instrumentation to detect and evaluate the SbVP 23 17 23 that are generated based on ISO 8871-3 testing. There is no consistency between USP <788> and ISO 8871-3 , resulting from the differences between finished drug product testing versus elastomer component testing (Table 23 3). There are no specifications for component SbVP particles per ISO 8871-3 . Particles are counts are reported relative to the surface area of the component rather than particles per volume of drug product. 25

There are several technologies capable of SbVP detection ; however, the ability for instrumentation to perform single particle detection is important because compendia specifications require that drug product results are reported in particles/volume or particle/container. Instrumentation that detects volume-based distributions such as laser diffraction (light scattering), dynamic light scattering, or x-ray sedimentation were not evaluated because they are not capable of single particle detection. 23

As described above in Table 3, different methods require different reporting units. For ISO method 8871-3 , the 2 particle counts are normalized for the surface area of the stopper that is analyzed and the unit of particles/10 cm 17 are reported, whereas, for USP <788> the unit of particles/mL or particles/container is reported. It is important to understand that if comparing methodologies with different units i.e., total particles vs. particles/mL, data should be normalized. For the purposes of this study, the unit of particles/mL was used for normalization across techniques. Sample preparation considerations are important when trying to measure particles in the SbVP and sub23 micron particle size ranges. When using the ISO 8871-3 based sample preparation, it was found that particle 23 concentration was below the optimal limit of detection for the sub-micron techniques. Modifying the ISO 8871-3 based sample preparation by decreasing the solution volume used to prepare extractions resulted in increased 23 particle concentrations. Further, the ISO 8871-3 preparation utilizes a water extraction for the SbVP particle LO analysis and a 0.03% polysorbate 80 solution extraction for the membrane microscopy visible particle analysis. Many biologics contain polysorbate or other surfactants. Since some methodologies, particularly MFI, are capable of differentiating air bubbles that could be generated during the sample preparation, it is more 21 relevant to use surfactant in the sample extraction. Notably, the polysorbate extraction generated substantially more particles than the water extraction for all the technologies except LM/IA. Based on the MFI results, the particles were not air bubbles from the surfactant solution, but rather polysorbate 80 –silicone complexes (Figure 3). 23

The sample preparation environment is also important when using the ISO 8871-3 based extraction. When extracting particles from the surface of components (stoppers), the cleanliness of the testing environment is critical. Preliminary studies of MFI data showed that stoppers that were prepared in a laminar flow hood contained a greater number of extrinsic particles in comparison to stoppers prepared in an ISO 5 cleanroom. The particles present from the ISO 5 cleanroom preparation mainly contained silicone oil particles, which were extracted from the stopper and were not extrinsic (Figure 4).

Initial Test Preparation Results SbVPs Results of the initial testing showed relatively good agreement across instrumentation, except for LM/IA (Figure 5). LM/IA detected more particles than the liquid-based methods for Stopper 1 (Trace Cured Silicone) and Stopper 4 (Untreated). This is most likely because the particulate related to these samples were of a solid nature (rather than silicone oil related) and, therefore, retained on the filter. Additionally, the LM/IA extraction was the only extraction that used 0.03% polysorbate solution instead of purified water. For Stopper 3 (Silicone Oil), MFI and LO detected more particulate than LM/IA, since the predominant particulate in this sample were silicone oil related and not retained on the filter. Particle concentrations for Stopper 2 (Major Cured Silicone) were consistent across instrumentation, with low concentrations of particulate. Sub-micron Particles Initial extrapolation of particle concentrations obtained from the MFI data (Figure 6) suggests that the concentration of particles ≤ 1 µm was too low for accurate quantification using RMM and NTA. To confirm this, the two stopper preparations with the highest particle counts by MFI were analyzed by RMM and NTA. As suspected, they were below the Limit of Quantitation (LOQ) for both sub-micron techniques. For RMM, a background analysis having fewer than one particle per minute detected would be considered a clean blank. The two stopper extraction samples analyzed with RMM counted 36 particles over 88 minutes for one sample and 18 particles over 70 minutes for the other sample, for particles > 250 nm. This resulted in concentrations that were 4 4 4.40 x10 /mL and 1.97 x10 /mL. These values were well below the optimal range of RMM, with too few particles analyzed to obtain reliable statistics on particle size and/or concentration. Similarly, the NTA data resulted in concentrations that were below the optimal concentration range, with too few particles tracked to obtain reliable sizing data. This was compounded by the fact that both techniques analyze such a small sample volume (nL), with lower sample throughput when compared to MFI, LO, and LM/IA techniques. Based upon this data, the ISO 88713-based extraction methods are not appropriate for quantification of particles using RMM and NTA. Modification of the extraction method was required so that a higher concentration of particles was obtained.

Modified Test Preparation Results SbVPs The extraction method was modified by decreasing the extraction volume to increase particle concentration (see sample preparation). After it was confirmed that decreasing the extraction volume increased the particle concentrations, the study was repeated using both purified water and polysorbate 80 as extraction solutions. During the initial testing, LM/IA detected more particulate in Samples 1 and 4. The LM/IA uses 0.03% polysorbate 80 solution for extraction instead of purified water, therefore, 0.03% polysorbate 80 solution was included in the study to assess if the increased particle count for LM/IA was due to the surfactant. The use of 0.03% polysorbate 80 as an extraction solution had a drastic impact on the number of sub-visible particles that were detected. The addition of polysorbate at a concentration of 0.03% polysorbate 80 improves the wettability of the stoppers, which allows for more particles to be removed from the stopper surface. In the repeated study, more particles were detected with the 0.03% polysorbate 80 solution extraction than with the water extraction in all size ranges and with all sub-visible instrumentation (see Figure 7). Sample 3 generated the most particles based on MFI and LO analysis, but not by LM/IA. Particles that were extracted from this sample were mostly silicone oil related. These particle types would not be retained on a membrane and, therefore, not analyzed by LM/IA instrumentation. LM/IA is valuable technology because the entirety of a sample solution is analyzed, so larger particles, which are lower in concentration and more likely to fall out of solution, are more likely to be detected. However, if the detection of semi-solid or liquid particles such as silicone oil is critical, then LM/IA is not recommended. It was also observed that MFI consistently detected the most particles, compared to the other

detection technologies. All the instrumentation that was used demonstrated good repeatability as seen in Figure 7. Sub-micron Particles RMM Adding polysorbate 80 to the extraction solution had a drastic effect on the number of particles extracted, primarily in the amount of positively buoyant particles assumed to be silicone oil. Similar concentrations of negatively buoyant particles were extracted, regardless of the level of stopper siliconization, and are assumed to be butyl rubber. Surprisingly, the samples demonstrated a bimodal distribution for the negatively buoyant particles, with peaks centered at roughly 750 nm and less than 250 nm, that is not seen with the blank samples. This distribution is not dependent on the silicone oil levels because all samples analyzed have this unique distribution, indicating that these negatively buoyant particles likely originated from the stopper material. It can also be seen that there is a more substantial bi-modal distribution for the polysorbate 80 extraction than the water extraction. Stopper 4 (untreated) has the largest bimodal distribution for both polysorbate 80 and water extraction. See Figure 8 for zoomed and offset distributions to highlight differences in samples. Because the graphs are offset, the concentration is not given on the y axis. However, large differences were seen when comparing the amounts of positively buoyant particles. The concentration of positively buoyant particles extracted correlated with the levels of silicone oil on the stoppers. This is evident in both the particle size distributions and the calculated concentrations. RMM analysis demonstrated that Stopper 3 contained the highest amounts of silicone oil, followed by Stopper 4. Stopper 1 and Stopper 2 had slightly lower levels than Stopper 4, and had comparable concentrations to each other, (see Figure 9). Acquisition statistics for the samples demonstrated a relatively high degree of confidence in the data, especially for the polysorbate 80-containing extraction samples. The manually chosen LOD value of 0.012 Hz provided high sensitivity to identify smaller sized particles. Resulting average coincidence values (multiple particles passing through resonator simultaneously) were a bit larger than optimal (< 3%) for the polysorbate 80-containing samples but remained within a good range of between 4-10%. Water-extracted samples had higher levels (10-25%), however this is commonly observed in samples with low particle counts. Likewise, resulting total concentrations 6 (Figure 10) were slightly lower than optimal (< 10 /mL), but still above the 1 particle/minute baseline threshold. Adequate total particles analyzed provided high confidence in the particle size distributions. Generally, more than 100 particles were collected per replicate vial, resulting in cumulative total particle counts of more than 400 total particles in the particle size distributions. Water extractions of Stopper 1 and Stopper 2 had fewer than 400 cumulative particles (336 and 284, respectively), resulting in distributions with more uncertainty. NTA NTA analysis demonstrates that the polysorbate 80 extraction solution had a substantial amount of sub-micron particles. In fact, the concentration of the polysorbate 80 blank was comparable to Stopper 1, Stopper 2, and Stopper 4, albeit with more variability between replicate vials. NTA analysis mirrored the RMM results. Polysorbate 80 extraction of siliconized stoppers resulted in the highest concentration of particles. These results are replicated when water is used as the extraction solution, indicating that siliconized stoppers contain the highest concentration of particles. However, the particle size distributions and the concentration results demonstrate that the addition of polysorbate 80 to the extraction solution had a drastic impact on the concentration of sub-micron particles present, by at least an order of magnitude, (see Figure 11). It must be noted that many of the water extraction samples are near or even below the lower concentration limits of the method.

Conclusion Cleanliness of testing environment is critical to reduce the introduction of extrinsic particles and sample 23 preparation should be optimized for the testing that is being performed. The ISO 8871-3 method was the starting point for this study; however, when evaluating particles in the sub-micron size range, it was found that the method is not optimal because the particle concentration is below the limit of detection for the instrumentation used in this study. It was shown that the water-based extraction removed fewer particles from the stopper surface in comparison to the 0.03% polysorbate extraction. This is especially the case with the SbVPs. Our results confirmed that stopper contribution to particles in the SbVP and sub-micron range do vary depending on the post-processing (silicone treatment) of the stopper, with silicone oil-treated stoppers contributing the most SbVP and sub-micron particulate to test results and the cured silicone/fluoropolymer-treated stoppers contributing the least particulate. The results were consistent across instrumentation except for LM/IA, because silicone particles and other liquid or semi-solid particles pass through the filter and aren’t retained for detection. The silicone that is present on stoppers surfaces can be extracted into the finish product during storage and handling. Therefore, if silicone sensitivity is a critical issue for a given drug product, stopper selection should be carefully considered. In order to understand the possible impact of the presence of air bubbles on the particle count, or the contribution of silicone particulate to the overall particle concentration, it is important to utilize equipment that is capable of differentiating particles. LO will need to be performed because it is required by USP <788> and by ISO 8871-3. The authors found that the ability to characterize particles, however, made MFI the most capable instrumentation for analysis of SbVPs and with the ability to distinguish positively and negatively buoyant particles, RMM was the preferred technique for analysis of sub-micron particulate for its ability to distinguish positively and negatively buoyant particles. An assessment of contributors to particle load is necessary during all phases of drug product development including the sample preparation environment, the silicone treatment of the stoppers, the extraction solvent used, and detection technology. Analyzing particle load on components used in and to administer the drug product enables optimal component selection for the safety and the quality of the finished drug product.

HIAC® is a registered trademark of Hach Company NanoSight® is a registered trademark of Malvern Panalytical, Ltd Archimedes® is a registered trademark of Malvern Panalytical Ltd. Clemex® is a registered trademark of Les Technologies Clemex Inc./Clemex Technologies, Inc.

Acknowledgements - We thank Allison Radwick for writing assistance, proofreading, and language editing. Funding – The authors received no financial support for the research, authorship, and/or publication of this article. References

1.

Walpot H, Frank RP, Burchard WG, Agternkamp C, Muller FG, Mittermayer C KG. Particulate Contamination of Intravenous Solutions and Drug Additives During Long- Term Intensive Care. Anaesthesist. 1989;38:617– 621.

2.

Langille SE. Particulate Matter in Injectable Drug Products. PDA Journal of Pharmaceutical Science and Technology. 2013;67(3):186-200. doi:10.5731/pdajpst.2013.00922

3.

Bukofzer S, Ayres J, Chavez A, et al. Industry Perspective on the Medical Risk of Visible Particles in Injectable Drug Products. PDA Journal of Pharmaceutical Science and Technology. 2015;69(1):123139. doi:10.5731/pdajpst.2015.01037

4.

Benlabed M, Perez M, Gaudy R, et al. Clinical implications of intravenous drug incompatibilities in critically ill patients. Anaesthesia Critical Care and Pain Medicine. 2019;38(2):173-180. doi:10.1016/j.accpm.2018.04.003

5.

Kotarek J, Stuart C, De Paoli SH, et al. Subvisible Particle Content, Formulation, and Dose of an Erythropoietin Peptide Mimetic Product Are Associated with Severe Adverse Postmarketing Events. Journal of Pharmaceutical Sciences. 2016;105(3):1023-1027. doi:10.1016/S00223549(15)00180-X

6.

Johns J, Golfetto P, Bush T, et al. Achieving ’ ’ Zero ’ ’ Defects for Visible Particles in Injectables. 2018. doi:10.5731/pdajpst.2018.009027

7.

Shabushnig J, Lankers M, Ayres J, Cherris R, Miller R VR and WR. Industry Eyes Future of Visual Inspection. PDA Letter. 2019;54:20-26.

8.

Doessegger L, Mahler H, Szczesny P, Rockstroh H, Kallmeyer G. The Potential Clinical Relevance of Visible Particles in Parenteral Drugs. Journal of Pharmaceutical Sciences. 2012;101(8):2635-2644. doi:10.1002/jps.23217

9.

Kretsinger J, Frantz N, Hart SA, et al. Expectations for Phase-Appropriate Drug Substance and Drug Product Speci fi cations for Early-Stage Protein Therapeutics. 2019;108:1442-1452. doi:10.1016/j.xphs.2018.11.042

10.

Li J, Krause ME, Chen X, et al. Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective. The AAPS Journal. 2019;21(3). doi:10.1208/s12248-019-0312-3

11.

Christie M, Torres RM, Kedl RM RT and CJ. Recombinant Murine Growth Hormone Particles are More Immunogenic with Intravenous than Subcutaneous Administration. J Pharm Sci. 2014;103(1):128-139.

12.

Turco S and Davis NM. Glass particles in intravenous injections. The New England Journal of Medicine. 1972;287:1204-1205.

13.

Kijanka G, Bee JS, Korman SA, et al. Submicron Size Particles of a Murine Monoclonal Antibody Are More Immunogenic Than Soluble Oligomers or Micron Size Particles Upon Subcutaneous Administration in Mice. Journal of Pharmaceutical Sciences. 2018;107(11):2847-2859. doi:10.1016/j.xphs.2018.06.029

14.

Jack T, Brent BE, Boehne M, et al. Analysis of particulate contaminations of infusion solutions in a pediatric intensive care unit. Intensive Care Medicine. 2010;36(4):707-711. doi:10.1007/s00134010-1775-y

15.

Madsen RE, Cherris RT, Shabushnig JG, Hunt DG. Visible Particulates in Injections—A History and a Proposal to Revise USP General Chapter Injections <1>. Pharmacopeial Forum. 2009;35(5):13831387.

16.

USP <1788> Methods for Detection of Particulate Matter in Injections and Ophthalmic Solutions.

17.

USP <788> Particulate Matter in Injections. US Pharmacopeia. 2017;USP 40—NF(35th Edition):Rockville, MD.

18.

2.9.19 Particulate Contamination: Subvisible particles, Method 1. Light obscuration particle count test. In: European Pharmacopeia. Vol 9th Editio. ; 2016.

19.

6.07 Insoluble Particulate Matter Test for Injections. The Japanese Pharmacopeia. 2016;The 17th E.

20.

Dean C. Ripple, Joshua R. Waymet MJC. Development of Standards for the Optical Detection of Protein. Amer Pharm Rev. 2011;14:90-96.

21.

Sharma DK, King D, Oma P, Merchant C. Micro-Flow Imaging: Flow Microscopy Applied to Subvisible Particulate Analysis in Protein Formulations. The AAPS Journal. 2010;12(3):455-464. doi:10.1208/s12248-010-9205-1

22.

Pollo M, Mehta A, Torres K, Thorne D, Zimmermann D, Kolhe P. Contribution of Intravenous Administration Components to Subvisible and Submicron Particles Present in Administered Drug Product. Journal of Pharmaceutical Sciences. 2019;108(7):2406-2414. doi:10.1016/j.xphs.2019.02.020

23.

Internal Standard ISO 8871-3: Elastomeric Parts for Parenterals and for Devices for Pharmaceutical Use — Part 3: Determination of Released-Particle Count. In: International Organization for Standardization. Vol First Edit. ; 2003.

24.

ASTM Standard 1193-91 Standard Specification for Reagent Water. In: ASTM International. ; 2018.

25.

Zölls S, Tantipolphan R, Wiggenhorn M, Winter G, Jiskoot W FW and HA. Particles in therapeutic protein formulations, Part 1: overview of analytical methods. J Pharm Sci. 2012;10(1):914-935. doi:10.1002/jps.23001

Table 1a. Summary of Particle Techniques used for Stopper Evaluation

Detection Method

Optical microscopy

Light obscuration (LO)/ Single particle optical sensing (SPOS)

Dynamic flow imaging

Information Collected

Size Range

Utility/Considerations for Elastomer Evaluation

2 µm - 150 µm

Silicone particles will be excluded

• •

Particle size Surface characteristics

•

Particle detection by extinction of light to sensor 2 µm - 150 µm Detection of Individual particles

Industry standard for drug product and compendia test

Particle Concentration Particle morphology Counts and sizes particles using images captured as a solution is passed through a flow cell

Morphology information allows characterization of particles

•

• • •

1 µm - 100 µm

Nanoparticle Particle size distribution, and Particle generation often Tracking Analysis concentration via light scattering and 0.030 µm - 1 µm below LoD for extraction (NTA) Brownian motion properties sample.

Resonant mass measurement (RMM)

• • • •

Particle mass distribution Particle size distribution 0.05 µm - 5 µm, Particle buoyancy sensor Measures frequency changes of a dependent suspended microchannel as particles pass through

•

Particle generation often below LoD, • Can distinguish silicone oil from elastomer

Table 1b. Strengths and Weaknesses of Particle Techniques used for Stopper Evaluation

Detection Method

Advantages

Optical microscopy

• Entire sample is filtered • Complete sample measurement

Disadvantages • Liquid /semisolid particles lost, • long analysis time

Light obscuration (LO)/ Single particle optical sensing (SPOS)

• Can analyze larger volumes faster • • Robust results due to circular • diameter • Easiest to use- no operator error, no interpretation of data • • High resolution

Dynamic flow imaging

• High throughput, reasonable sample volume (~1 mL/analysis) • Higher sensitivity for transparent particles • Classification of particles based on morphological parameters calculated from images

Nanoparticle Tracking Analysis (NTA)

• • High resolution tracking of • individual nanoparticles • • Observe small changes in particle size distributions •

• Can undercount/size particles with RI close to that of solution matrix (≤ 0.5) • Difficulty distinguishing particles based on morphological parameters below 5 µm

• •

Resonant mass measurement (RMM)

No characterization Results are in circular diameter inaccurate size information for non-spherical particles All inclusions detected, including air bubbles

• Can distinguish between positive and negative buoyant particles such as silicone oil and protein • Compatible with high • concentration/high viscosity •

Small volume analyzed Susceptible to user subjectivity Viscosity and refractive index can influence results Experienced user necessary Small volume analyzed Assume single density for positive and negative particles – heterogeneous particles not sized accurately Experienced user necessary Filtration necessary for samples with large particles

Table 2. Sample descriptions Stopper number Stopper 1

Silicone level Trace Cured Silicone

Stopper 2

Major Cured Silicone

Stopper 3 Stopper 4

Silicone Oil Untreated

Description Top of flange and plug laminated with fluoropolymer, silicone polymer coating on bottom of flange Bottom of flange and plug laminated with fluoropolymer, silicone polymer coating on top of flange Uncoated stopper coated with silicone oil Stopper that had not been coated with silicone (silicone is still used in stopper manufacturing, but stopper has not been intentionally coated with silicone)

Table 3. Comparison of USP <788>18 and ISO 8871-327

Sampling Specifications Size Ranges

Method 1/ LO

Blank Limits

Requirements

Size Ranges Method 2/ Membrane Blank Limits Microscopy Requirements

USP <788> Evaluation of drug product by testing directly or with appropriate dilution/sample treatment Specified by USP > 10 µm > 25 µm NMT 25 particles > 10 µm in 25mL of blank solution Method 1 (LO) is preferred. When Method 1 cannot be applied or specifications cannot be met, Method 2 (microscopy) can be used > 10 µm, > 25 µm (evaluated at 100x) NMT 20 particles > 10 µm NMT 5 particles > 25 µm Method 2 (microscopy) is used when Method 1 (LO) cannot be applied or specifications cannot be met.

ISO-8871-3 Evaluation of extraction solution created by rotating stopper and purified water on an orbital shaker Agreed upon with customer > 2 µm but < 5 µm > 5 µm but < 10 µm > 10 µm but < 25 µm > 25 µm NMT 100 particles > 2 µm per 5 mL of blank solutoin The LO method is used for the detection of SbVP <25 µm in size > 25 µm but < 50 µm > 50 µm but < 100 µm > 100 µm (evaluated at 50x) NMT 5 particles > 25 µm but < 50 µm NMT 1 particle > 50 µm but < 100 µm 0 particles > 100 µm The Microscope Method is used for particles > 25 µm

Figure 1: Size range of particles as specified in USP <1788>

Figure 2. Image describing the parts of the stopper Image Copyright West Pharmaceutical Services 2020

Figure 3. Polysorbate 80 -silicone complex MFI images

ISO 5 cleanroom prep

Laminar flow hood prep

Figure 4. MFI comparison of particles extracted from stoppers prepared in an ISO 5 cleanroom vs. a laminar flow hood

A) Stopper 1 ≥ 2 µm

B) Stopper 2 ≥ 5 µm

≥ 10 µm

≥ 25 µm

≥ 2 µm 100 000

MFI LO LM/IA 10 000

1 000

100

10

Cumulative Concentration (particles/mL)

Cumulative Concentration (particles/mL)

100 000

1

Cumulative Concentration (particles/mL)

≥ 10 µm

100

10

1

MFI LO LM/IA 10 000

1 000

100

10

≥ 25 µm

≥ 2 µm

≥ 5 µm

≥ 10 µm

≥ 25 µm

100 000

MFI LO LM/IA

1 000

≥ 25 µm

D) Stopper 4 ≥ 5 µm

Cumulative Concentration (particles/mL)

≥ 2 µm

10 000

≥ 10 µm

1

C) Stopper 3 100 000

≥ 5 µm

MFI LO LM/IA 10 000

1 000

100

10

1

Figure 5. Summary of SbVP particle concentrations (≥ 2 micron) for initial test preparation via ISO 88713, as determined by MFI, LO and LM/IA. Each bar is an average of three sample extractions (standard deviations shown). The results are displayed in cumulative counts.

Figure 6. Extrapolated MFI Data < 1 micron – shows projected concentrations in sub-micron size-range are below the NTA and RMM range or limit of detection.

B) Stopper 2

A) Stopper 1 ≥ 2 µm

≥ 5 µm

≥ 10 µm

≥ 2 µm

≥ 25 µm

10 000

1 000

100

10

Cumulative Concentration (particles/mL)

Cumulative Concentration (particles/mL)

MFI LO LM/IA

1

≥ 10 µm

≥ 25 µm MFI LO LM/IA

10 000

1 000

100

10

1 PS80

Water

PS80

Water

PS80

Water

Water

PS80

PS80

Water

PS80

Water

PS80

Water

PS80

D) Stopper 4

C) Stopper 3 ≥ 2 µm

≥ 5 µm

≥ 10 µm

100 000

≥ 2 µm

≥ 25 µm MFI LO LM/IA

10 000

1 000

100

10

1

≥ 5 µm

≥ 10 µm

100 000

Cumulative Concentration (particles/mL)

Water

Cumulative Concentration (particles/mL)

≥ 5 µm

100 000

100 000

≥ 25 µm MFI LO LM/IA

10 000

1 000

100

10

1

Water

PS80

Water

PS80

Water

PS80

Water

PS80

Water

PS80

Water

PS80

Water

PS80

Water

PS80

Figure 7. Summary of SbVP concentrations (≥ 2 micron) for modified test preparation using both purified water and 0.03% polysorbate 80 solution (tween) extractions, as determined by MFI, LO, and LM/IA. Each bar is an average of results for three extractions (standard deviations shown).

Stopper 1 - PS80 Stopper 2 - PS80 Stopper 3 - PS80 Particle Concentration Graphs Off-set

Stopper 4 - PS80 PS80 Extraction Blank

Stopper 1 - Water Stopper 2 - Water Stopper 3 - Water Stopper 4 - Water Water Extraction Blank

0.25

0.50

0.75

1.00

1.25

1.50

Diameter, uM

Figure 8. Zoomed (0.2 to 1.50 µm) particle size distributions for negatively buoyant particles using water and 0.03% polysorbate 80 solution (tween) extractions for Stoppers 1-4, as determined by RMM. Data is offset to highlight distribution differences.

Figure 9. Zoomed (0.5-2.0 µm) particle size distributions for positively buoyant particles using water and 0.03% polysorbate 80 solution (tween) extractions for Stoppers 1-4, as determined by RMM. Data is offset to highlight distribution differences

Positive Buoyant Negative Buoyant

1.75 0.5 0.4 0.3 0.2

Stopper #4

Stopper #3

Stopper #2

Stopper #1

Water Extractions

Extraction Blank

PS80 Extractions

Extraction Blank

Stopper #4

Stopper #3

0.0

Stopper #2

0.1 Stopper #1

Mass Concentration, (µ µ g/mL)

2.00

Figure 10. Average mass concentrations of positive buoyant and negative buoyant particles in Stoppers 1-4, as determined by RMM.

Concentration (particles/mL)

Total Concentrations - PS80 Extraction 3×10 8

2.5×10 8 2×10 8

Stopper 1

1.5×10 8

Stopper 2

1×10 8

Stopper 3

5×10 7

Stopper 4 Extraction Blank

0 1 2 3

1 2 3

1 2 3

1 2 3

1 2 3 4

Extraction Replicate

Concentration (particles/mL)

Total Concentrations - Water Extraction 3×10 8

2.5×10 8 2×10 8

Stopper 1

1.5×10 8

Stopper 2

1×10 8

Stopper 3

5×10 7

Stopper 4 Extraction Blank

0 1 2 3

1 2 3

1 2 3

1 2 3

1 2 3 4

Extraction Replicate Figure 11. Total particle concentrations of 0.03% polysorbate 80 solution (tween) and water extractions for Stoppers 1-4, as determined by NTA.

Figure 1: Size range of particles as specified in USP <1788> Figure 2. Image describing the parts of the stopper Image Copyright West Pharmaceutical Services 2020 Figure 3. Polysorbate 80 -silicone complex MFI images ISO 5 cleanroom prep

Laminar flow hood prep

Figure 4. MFI comparison of particles extracted from stoppers prepared in an ISO 5 cleanroom vs. a laminar flow hood Figure 5. Summary of SbVP particle concentrations (≥ 2 micron) for initial test preparation via ISO 8871-3, as determined by MFI, LO and LM/IA. Each bar is an average of three sample extractions (standard deviations shown). The results are displayed in cumulative counts.

Figure 6. Extrapolated MFI Data < 1 micron – shows projected concentrations in sub-micron size-range are below the NTA and RMM range or limit of detection. Figure 7. Summary of SbVP concentrations (≥ 2 micron) for modified test preparation using both purified water and 0.03% polysorbate 80 solution (tween) extractions, as determined by MFI, LO, and LM/IA. Each bar is an average of results for three extractions (standard deviations shown). Figure 8. Zoomed (0.2 to 1.50 µm) particle size distributions for negatively buoyant particles using water and 0.03% polysorbate 80 solution (tween) extractions for Stoppers 1-4, as determined by RMM. Data is offset to highlight distribution differences.

Figure 9. Zoomed (0.5-2.0 µm) particle size distributions for positively buoyant particles using water and 0.03% polysorbate 80 solution (tween) extractions for Stoppers 1-4, as determined by RMM. Data is offset to highlight distribution differences Figure 10. Average mass concentrations of positive buoyant and negative buoyant particles in Stoppers 1-4, as determined by RMM.

Figure 11. Total particle concentrations of 0.03% polysorbate 80 solution (tween) and water extractions for Stoppers 1-4, as determined by NTA.