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Subvisible Particulate Contamination in Cell Therapy Products—Can We Distinguish?
Journal of Pharmaceutical Sciences xxx (2019) 1-4
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org
Rapid Communication
Subvisible Particulate Contamination in Cell Therapy ProductsdCan We Distinguish? Ilona Vollrath 1, 2, Roman Mathaes 1, Ahmad S. Sediq 1, Dhananjay Jere 1, € rg Huwyler 2, Hanns-Christian Mahler 1, * € rg 1, Jo Susanne Jo 1 2
Drug Product Services, Lonza AG, Hochbergerstrasse 60A, 4057 Basel, Switzerland Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2019 Revised 30 August 2019 Accepted 6 September 2019
Cell therapy products represent an exciting new class of medicinal products, which must be parenterally administered. Thus, compliance with parenteral preparation guidelines is required. One requirement for parenteral products is the characterization of particle contaminations. As cell-based products are turbid suspensions, containing particles, the cells, characterization and control of foreign particle impurities remain a challenge. Within this study, we evaluated a flow imaging microscopy method for the detection and characterization of subvisible particle contaminations in cell-based products. We found that flow imaging microscopy is a potential method where subvisible particle contaminations can be differentiated from the cells in cell therapy products. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: biotechnology cell line(s) injectable(s) particle size analytical chemistry
Introduction Cell therapy products represent an exciting new class of medicinal products. They offer new opportunities to treat and cure diseases, which have only limited treatment options with traditional therapeutic modalities.1 The specific nature of cellbased products, for example, short shelf life or hold times or their sensitivity to many stress factors, pose new challenges for the development and manufacturing of these products compared to other biologics.2,3 Nevertheless, cell therapy products need to fulfill critical quality requirements to assure patient safety.4-10 Cell therapy products must be parenterally administered, that is, typically by injection or infusion into the human body. Hence, compliance with requirements for parenteral products is required. For example, particle contaminations need to be identified, characterized, and controlled during drug product development and routine manufacturing, given the potential safety concern of particulates when administered parenterally. Particulate matter is usually categorized in visible particles and subvisible particles (SvP). For the latter, the harmonized monographs Ph.Eur 2.9.19 and
* Correspondence to: Hanns-Christian Mahler (Telephone: þ41-61-316-8322). E-mail address: [email protected] (H.-C. Mahler).
USP <788>, and additionally, USP <787> or <789> provide guidance on specifications and test methods.11 Harmonized pharmacopoeial SvP methods are light obscuration method and a light microscopic method. Cell-based products containdby definitiondsubvisible as well as visible particles, the cells. Characterization and control of foreign particle impurities remain a significant challenge by light obscuration, the primary pharmacopoeial method for SvP given that light obscuration is unable to discriminate cells from extrinsic particulates. In addition, the limits provided in the Ph.Eur. 2.9.19 are not applicable for products such as suspensions and cell therapy products, where the cells are counted as particulates in the test. Health authorities acknowledge the analytical challenges associated with cell therapies. Therefore, different analytical endpoints or requirements for cell-based therapies were discussed in guidelines or new regulations. For example, the “Guidelines on GMP specific to ATMPs”12 suggests for “visible particle testing”: “As cells in suspension are not clear solutions, it is acceptable to replace the particulate matter test by an appearance test (e.g. color), provided that alternative measures are put in place, such as controls of particles from materials (e.g. filtration of raw material solutions) and equipment used during manufacturing, or the verification of the ability of the manufacturing process to
https://doi.org/10.1016/j.xphs.2019.09.002 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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produce low particle products with simulated samples (without cells)”. This approach satisfies the immediate needs to ensure market supply. However, the guideline also demonstrates the urgent need to foster analytical development and improvements for cell-based products to ensure a similar level of quality compared to other biologic parenteral products. In particular, testing for particulate matter (contamination) in cell therapy products is a commonly overlooked gap in the drug product control strategy. Flow imaging microscopy techniques, micro-flow imaging (MFI) and FlowCAM, have been evaluated as alternative methods to light obscuration for SvP testing of biologics, for example, antibodies or vaccines.13-16 Even though MFI and FlowCAM are based on the same measurement principle, they differ in resolution and particle parameters.17 However, in some cases, MFI may provide more accurate particle determinations.15,16,18 Flow imaging methods also provide the benefit of morphological assessments; hence, the differentiation of different particle species may be achievable. Wu et al19 and Farrell et al20 used MFI to study cell aggregation and confluency on microcarriers, respectively. Sediq et al18 showed that flow imaging microscopy techniques provide an alternative method to hemocytometry or automated cell counting for total cell concentration and viability determination.21 Within this study, we investigated FlowCAM, as it provides higher resolution images and more particle parameters compared to MFI, as an alternative to light obscuration for the characterization of SvP contaminations in cell-based products. The method was challenged with typical SvP contaminants and cells to assess the capability of the FlowCAM to differentiate particle contaminants and impurities from actual cells. Materials and Methods Study Design Cell suspensions with 50,000 cells/mL suspended in phosphate buffered saline (PBS) were measured by light obscuration and flow imaging microscopy. Dispersions/suspensions of silicone oil, rubber stopper abrasion, or glass particles in PBS were measured by both methods to simulate foreign particle impurities. In addition, a cell suspension was stressed (vortexed) to induce cell debris, which are a potential SvP impurity in cell therapy products. Finally, foreign particle impurity dispersion/suspension was spiked into cell suspensions to assess the method capability in discriminating cells from foreign particle impurities. Materials Human neuroblastoma cells SK-N-AS (ATCC® CRL2137™) were used as a model cell line for this study. Dulbecco's Modified Eagle Medium high glucose with pyruvate (Gibco™), PBS (Gibco™), fetal bovine serum (Gibco™), and Trypsin-EDTA 0.25% (Gibco™) were purchased from Fisher Scientific AG (Reinach, Suisse). PenicillinStreptomycin and nonessential amino acids were ordered from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The waterdilutable Dow Corning 365, 35% Dimethicone NF emulsion was purchased from DuPont (Wilmington). Cell Culture SK-N-AS cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 0.1 mM nonessential amino
acids. The cells were cultured at 37 C and 5% CO2 atmosphere in a humidified incubator up to approximately 80% confluency. The cells were harvested by Trypsin-EDTA. Cell concentrations were determined using a NucleoCounter NC-200 (ChemoMetec A/S, Allerød, Denmark). Preparation of Solutions After harvest, the cells were resuspended in PBS at a concentration of 50,000 cells/mL to yield the cell suspension. The cell concentration of 50,000 cells/mL was determined using the NucleoCounter NC-200 (ChemoMetec A/S, Allerød, Denmark). Cell debris was generated by vortexing the cell suspension for approximately 5 min. Glass particles of a defined size (~150 mm) were pestled to a fine glass powder. A suspension of glass particles in PBS was prepared. The silicone oil droplets were produced by mixing 2 drops of the Dow Corning emulsion to PBS. To generate rubber stopper particles, stoppers were cut into small pieces and with the addition of PBS solution, pestled. For the measurement, the PBS containing the rubber stopper abrasion was collected and further diluted in PBS. Solutions containing cells and the previously described extrinsic particulates were achieved by comixing the cell suspension (4 mL) with 1 mL of each foreign impurity dispersion/suspension followed by mixing gently. Light Obscuration Particle determination by light obscuration method was performed using a PAMAS SBSS instrument (Partikelmess- und Analysesysteme GmbH, Rutesheim, Germany) equipped with an HCBLD-50/50 detector, a 1 mL syringe and a pressurizable sample chamber. Before sample analysis, an system suitability test was performed for count verification using the 5 mm particle count STD (COUNT-CAL Count Precision Size Standard; Thermo Fisher Scientific, Waltham, MA). Sizing accuracy is ensured by recalibrating the instrument every 6 months. Four measurements of 0.4 mL sample volume were performed, from which the first 0.4 mL was discarded (preflush volume). The following 3 0.4 mL measurements were averaged to obtain the final reported result. Flow Imaging Microscopy Characterization of SvP was performed using Flow Imaging Microscopy using a FlowCAM VS1 (Fluid Imaging Technologies, Yarmouth, ME) with an 80 mm field of view flow cell, applying a proprietary method we developed. Before sample analysis, an system suitability test for particle size and count using a 5 mm particle size standard and 5 mm particle count standard (COUNTCAL Count Precision Size Standard; Thermo Fisher Scientific, Waltham) was conducted. The samples were run at a flow rate of 0.1 mL/min controlled by a syringe pump. Images were taken with a Sony XCD-SX90 camera at 22 fps (10 lens). Results and Discussion The cell suspension was prepared at a cell concentration of 50,000 cells/mL. When analyzed quantitatively by using light obscuration, only about 30,000 cells/mL was detected (Fig. 1). FlowCAM detected even less cells (15,000-25,000 cells/mL). The discrepancy between the expected 50,000 cells/mL and the lower actual measured cell count could have several reasons. The automated cell counting is based on image cytometry, whereas the flow imaging microscopy method depends a lot on the focus, thus out-
I. Vollrath et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-4
3
Figure 1. Particle counts per mL of particles larger than 5 mm (exemplary) determined by light obscuration (orange) and flow imaging microscopy (blue).
of-focus cells are not counted. Sediq et al18 found FlowCAM to have a lower precision for cell counting compared to automated cell counting. In addition, cells can clump together or several cells are detected as one cell by light obscuration and FlowCAM. The cell suspension containing (spiked in) particle impurities showed higher particle counts compared to the pure cell suspension, which suggests that both the cells and the spiked in particulates were counted for both methods (Fig. 1). As light obscuration only provides information about particle counts and the size distribution, it does not allow characterization of unknown particles in a cell suspension. In contrast to the compendial light obscuration method, the aim of the flow imaging method is used to provide morphological characterization of SvP’s by image analyzing algorithms. The flow imaging microscopy results confirmed cell clumping (upper part of Fig. 2). In addition, morphological differences between the foreign particle impurities and the cells were observed. The cells were circular shaped with a rough interior, although of different morphology and appearance compared to silicone oil particles. Cell debris, by contrast, formed coil-like structures with a low intensity. The silicone oil particles appeared as circular ring structures, whereas the glass particles were angled polygonal shaped. Finally, the rubber stopper abrasion was seen as a coil-like, dark particle of an irregular form. The morphological differences enabled differentiation of cells from foreign particle impurities. Figure 3 shows exemplarily images of the cell suspension spiked with silicone oil, and by the differences defined previously, we can see that both silicone oil particles and cells were recognized to be present. These distinct morphology differences were reliable identified by the operator and may enable automated algorithms to classify SvPs impurities in cell therapy products. From our experience, flow imaging microscopy algorithms show the best performance when assessed on a product-specific basis. Cells in cell therapy products may exhibit significant differences in their morphological properties including size and granularity (associated to the measured total light intensity of a particle). Similarly, the flow imaging microscopy algorithms should be developed with potential impurities from the representative GMP manufacturing process. The findings of the present study may not be transferred to other cell therapy products and SvP impurities without verification. In addition, cell therapy products may comprise new classes of SvP impurities compared to traditional
therapeutic protein or small molecule drug products. For example, cell therapy products may feature unwanted lipoprotein particles, cellular contamination, cell debris or other SvP associated to the process/equipment used upstream of the final drug product because of significant differences in the manufacturing process and the absence of a sterile filtration process step, compared to protein therapeutics. As the cells are counted as particles in both methods, the particle concentration by itself would not help to assess for foreign particle contaminations in cell therapy product. Furthermore, the
Figure 2. Representative images of cells, cell debris and the foreign impurities, glass particles, silicone oil, and rubber stopper abrasion taken by flow imaging microscopy.
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Figure 3. Example images of a cell suspension spiked with silicon oil as foreign impurity.
FlowCAM instrument provides 30 describing parameters (e.g., circularity, intensity, with, edge gradient, etc.) for each counted particle. Further work is in progress to implement automated image analyzing algorithms for flow imaging. For the differentiation of cells versus unwanted particle contaminants, further work is certainly required and suggested. Conclusion Within this study, we were assessing SvP in cell therapy products, comparing cell suspensions and using solutions containing artificially product foreign particulate contaminants. We found, that testing for SvP contamination by light obscurationdas expecteddwas solely not providing sufficient insights into product quality of cell suspensions with regards to SvP. Using the flow imaging microscopy method, we found that SvP contaminationsdand cell debrisdcan be differentiated from actual cells in cell therapy products by the analyst. While the flow imaging microscopy method is currently not suggested for quality control purposes for traditional drug products,17,22 the method may offer value in a process assessment related to minimizing potential particulate contaminations and support product development and the particle control strategy for cell therapy products. References 1. Fischbach MA, Bluestone JA, Lim WA. Cell-based therapeutics: the next pillar of medicine. Sci Transl Med. 2013;5(179):179ps7. 2. Campbell A, Brieva T, Raviv L, et al. Concise review: process development considerations for cell therapy. Stem Cells Transl Med. 2015;4(10):1155-1163. 3. Heathman TR, Nienow AW, McCall MJ, et al. The translation of cell-based therapies: clinical landscape and manufacturing challenges. Regen Med. 2015;10(1):49-64.
4. Kellathur SN, Lou H-X. Cell and tissue therapy regulation: worldwide status and harmonization. Biologicals. 2012;40(3):222-224. € mhild A, Kaiser D, et al. Good manufacturing practices 5. Abou-El-Enein M, Ro (GMP) manufacturing of advanced therapy medicinal products: a novel tailored model for optimizing performance and estimating costs. Cytotherapy. 2013;15(3):362-383. 6. Abou-El-Enein M, Bauer G, Medcalf N, et al. Putting a price tag on novel autologous cellular therapies. Cytotherapy. 2016;18(8):1056-1061. 7. Martín PG, Martinez AR, Lara VG, Naveros BC. Regulatory considerations in production of a cell therapy medicinal product in Europe to clinical research. Clin Exp Med. 2014;14(1):25-33. € der B, et al. Towards a commercial process for 8. Kaiser AD, Assenmacher M, Schro the manufacture of genetically modified T cells for therapy. Cancer Gene Ther. 2015;22(2):72. 9. Wuchter P, Bieback K, Schrezenmeier H, et al. Standardization of Good Manufacturing Practiceecompliant production of bone marrowederived human mesenchymal stromal cells for immunotherapeutic applications. Cytotherapy. 2015;17(2):128-139. 10. Migliaccio G, Pintus C. Role of the EU framework in regulation of stem cellbased products. In: Mesenchymal Stem Cells-Basics and Clinical Application II. Berlin, Heidelberg: Springer; 2012:287-299. 11. Singh SK, Afonina N, Awwad M, et al. An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics. J Pharm Sci. 2010;99(8):3302-3321. 12. Commission, E.. Guidelines on Good Manufacturing Practices Specific to Advanced Therapy Medicinal Products. Brussels: EurdraLex; 2017:4. 13. Werk T, Volkin DB, Mahler H-C. Effect of solution properties on the counting and sizing of subvisible particle standards as measured by light obscuration and digital imaging methods. Eur J Pharm Sci. 2014;53:95-108. 14. Frahm GE, Pochopsky AW, Clarke TM, Johnston MJ. Evaluation of microflow digital imaging particle analysis for sub-visible particles formulated with an opaque vaccine adjuvant. PLoS One. 2016;11(2):e0150229. € lls S, Weinbuch D, Wiggenhorn M, et al. Flow imaging microscopy for pro15. Zo tein particle analysisda comparative evaluation of four different analytical instruments. AAPS J. 2013;15(4):1200-1211. €lls S, Wiggenhorn M, et al. Microeflow imaging and resonant 16. Weinbuch D, Zo mass measurement (archimedes)ecomplementary methods to quantitatively differentiate protein particles and silicone oil droplets. J Pharm Sci. 2013;102(7):2152-2165. 17. Rios Quiroz A, Finkler C, Huwyler J, Mahler HC, Schmidt R, Koulov AV. Factors governing the accuracy of subvisible particle counting methods. J Pharm Sci. 2016;105(7):2042-2052. 18. Sediq AS, Klem R, Nejadnik MR, Meij P, Jiskoot W. Label-free, flow-imaging methods for determination of cell concentration and viability. Pharm Res. 2018;35(8):150. 19. Wu L, Martin T, Li Y, et al. Cell aggregation in thawed haematopoietic stem cell products visualised using micro-flow imaging. Transfus Med. 2012;22(3):218220. 20. Farrell CJ, Cicalese SM, Davis HB, et al. Cell confluency analysis on microcarriers by micro-flow imaging. Cytotechnology. 2016;68(6):2469-2478. 21. Polchow B, Kebbel K, Schmiedeknecht G, et al. Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks. J Transl Med. 2012;10(1):98. 22. Matter A, Koulov A, Singh S, et al. Variance between different light obscuration and flow imaging microscopy instruments and the impact of instrument calibration. J Pharm Sci. 2019;108:2397-2405.
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org
Rapid Communication
Subvisible Particulate Contamination in Cell Therapy ProductsdCan We Distinguish? Ilona Vollrath 1, 2, Roman Mathaes 1, Ahmad S. Sediq 1, Dhananjay Jere 1, € rg Huwyler 2, Hanns-Christian Mahler 1, * € rg 1, Jo Susanne Jo 1 2
Drug Product Services, Lonza AG, Hochbergerstrasse 60A, 4057 Basel, Switzerland Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2019 Revised 30 August 2019 Accepted 6 September 2019
Cell therapy products represent an exciting new class of medicinal products, which must be parenterally administered. Thus, compliance with parenteral preparation guidelines is required. One requirement for parenteral products is the characterization of particle contaminations. As cell-based products are turbid suspensions, containing particles, the cells, characterization and control of foreign particle impurities remain a challenge. Within this study, we evaluated a flow imaging microscopy method for the detection and characterization of subvisible particle contaminations in cell-based products. We found that flow imaging microscopy is a potential method where subvisible particle contaminations can be differentiated from the cells in cell therapy products. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: biotechnology cell line(s) injectable(s) particle size analytical chemistry
Introduction Cell therapy products represent an exciting new class of medicinal products. They offer new opportunities to treat and cure diseases, which have only limited treatment options with traditional therapeutic modalities.1 The specific nature of cellbased products, for example, short shelf life or hold times or their sensitivity to many stress factors, pose new challenges for the development and manufacturing of these products compared to other biologics.2,3 Nevertheless, cell therapy products need to fulfill critical quality requirements to assure patient safety.4-10 Cell therapy products must be parenterally administered, that is, typically by injection or infusion into the human body. Hence, compliance with requirements for parenteral products is required. For example, particle contaminations need to be identified, characterized, and controlled during drug product development and routine manufacturing, given the potential safety concern of particulates when administered parenterally. Particulate matter is usually categorized in visible particles and subvisible particles (SvP). For the latter, the harmonized monographs Ph.Eur 2.9.19 and
* Correspondence to: Hanns-Christian Mahler (Telephone: þ41-61-316-8322). E-mail address: [email protected] (H.-C. Mahler).
USP <788>, and additionally, USP <787> or <789> provide guidance on specifications and test methods.11 Harmonized pharmacopoeial SvP methods are light obscuration method and a light microscopic method. Cell-based products containdby definitiondsubvisible as well as visible particles, the cells. Characterization and control of foreign particle impurities remain a significant challenge by light obscuration, the primary pharmacopoeial method for SvP given that light obscuration is unable to discriminate cells from extrinsic particulates. In addition, the limits provided in the Ph.Eur. 2.9.19 are not applicable for products such as suspensions and cell therapy products, where the cells are counted as particulates in the test. Health authorities acknowledge the analytical challenges associated with cell therapies. Therefore, different analytical endpoints or requirements for cell-based therapies were discussed in guidelines or new regulations. For example, the “Guidelines on GMP specific to ATMPs”12 suggests for “visible particle testing”: “As cells in suspension are not clear solutions, it is acceptable to replace the particulate matter test by an appearance test (e.g. color), provided that alternative measures are put in place, such as controls of particles from materials (e.g. filtration of raw material solutions) and equipment used during manufacturing, or the verification of the ability of the manufacturing process to
https://doi.org/10.1016/j.xphs.2019.09.002 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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I. Vollrath et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-4
produce low particle products with simulated samples (without cells)”. This approach satisfies the immediate needs to ensure market supply. However, the guideline also demonstrates the urgent need to foster analytical development and improvements for cell-based products to ensure a similar level of quality compared to other biologic parenteral products. In particular, testing for particulate matter (contamination) in cell therapy products is a commonly overlooked gap in the drug product control strategy. Flow imaging microscopy techniques, micro-flow imaging (MFI) and FlowCAM, have been evaluated as alternative methods to light obscuration for SvP testing of biologics, for example, antibodies or vaccines.13-16 Even though MFI and FlowCAM are based on the same measurement principle, they differ in resolution and particle parameters.17 However, in some cases, MFI may provide more accurate particle determinations.15,16,18 Flow imaging methods also provide the benefit of morphological assessments; hence, the differentiation of different particle species may be achievable. Wu et al19 and Farrell et al20 used MFI to study cell aggregation and confluency on microcarriers, respectively. Sediq et al18 showed that flow imaging microscopy techniques provide an alternative method to hemocytometry or automated cell counting for total cell concentration and viability determination.21 Within this study, we investigated FlowCAM, as it provides higher resolution images and more particle parameters compared to MFI, as an alternative to light obscuration for the characterization of SvP contaminations in cell-based products. The method was challenged with typical SvP contaminants and cells to assess the capability of the FlowCAM to differentiate particle contaminants and impurities from actual cells. Materials and Methods Study Design Cell suspensions with 50,000 cells/mL suspended in phosphate buffered saline (PBS) were measured by light obscuration and flow imaging microscopy. Dispersions/suspensions of silicone oil, rubber stopper abrasion, or glass particles in PBS were measured by both methods to simulate foreign particle impurities. In addition, a cell suspension was stressed (vortexed) to induce cell debris, which are a potential SvP impurity in cell therapy products. Finally, foreign particle impurity dispersion/suspension was spiked into cell suspensions to assess the method capability in discriminating cells from foreign particle impurities. Materials Human neuroblastoma cells SK-N-AS (ATCC® CRL2137™) were used as a model cell line for this study. Dulbecco's Modified Eagle Medium high glucose with pyruvate (Gibco™), PBS (Gibco™), fetal bovine serum (Gibco™), and Trypsin-EDTA 0.25% (Gibco™) were purchased from Fisher Scientific AG (Reinach, Suisse). PenicillinStreptomycin and nonessential amino acids were ordered from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The waterdilutable Dow Corning 365, 35% Dimethicone NF emulsion was purchased from DuPont (Wilmington). Cell Culture SK-N-AS cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 0.1 mM nonessential amino
acids. The cells were cultured at 37 C and 5% CO2 atmosphere in a humidified incubator up to approximately 80% confluency. The cells were harvested by Trypsin-EDTA. Cell concentrations were determined using a NucleoCounter NC-200 (ChemoMetec A/S, Allerød, Denmark). Preparation of Solutions After harvest, the cells were resuspended in PBS at a concentration of 50,000 cells/mL to yield the cell suspension. The cell concentration of 50,000 cells/mL was determined using the NucleoCounter NC-200 (ChemoMetec A/S, Allerød, Denmark). Cell debris was generated by vortexing the cell suspension for approximately 5 min. Glass particles of a defined size (~150 mm) were pestled to a fine glass powder. A suspension of glass particles in PBS was prepared. The silicone oil droplets were produced by mixing 2 drops of the Dow Corning emulsion to PBS. To generate rubber stopper particles, stoppers were cut into small pieces and with the addition of PBS solution, pestled. For the measurement, the PBS containing the rubber stopper abrasion was collected and further diluted in PBS. Solutions containing cells and the previously described extrinsic particulates were achieved by comixing the cell suspension (4 mL) with 1 mL of each foreign impurity dispersion/suspension followed by mixing gently. Light Obscuration Particle determination by light obscuration method was performed using a PAMAS SBSS instrument (Partikelmess- und Analysesysteme GmbH, Rutesheim, Germany) equipped with an HCBLD-50/50 detector, a 1 mL syringe and a pressurizable sample chamber. Before sample analysis, an system suitability test was performed for count verification using the 5 mm particle count STD (COUNT-CAL Count Precision Size Standard; Thermo Fisher Scientific, Waltham, MA). Sizing accuracy is ensured by recalibrating the instrument every 6 months. Four measurements of 0.4 mL sample volume were performed, from which the first 0.4 mL was discarded (preflush volume). The following 3 0.4 mL measurements were averaged to obtain the final reported result. Flow Imaging Microscopy Characterization of SvP was performed using Flow Imaging Microscopy using a FlowCAM VS1 (Fluid Imaging Technologies, Yarmouth, ME) with an 80 mm field of view flow cell, applying a proprietary method we developed. Before sample analysis, an system suitability test for particle size and count using a 5 mm particle size standard and 5 mm particle count standard (COUNTCAL Count Precision Size Standard; Thermo Fisher Scientific, Waltham) was conducted. The samples were run at a flow rate of 0.1 mL/min controlled by a syringe pump. Images were taken with a Sony XCD-SX90 camera at 22 fps (10 lens). Results and Discussion The cell suspension was prepared at a cell concentration of 50,000 cells/mL. When analyzed quantitatively by using light obscuration, only about 30,000 cells/mL was detected (Fig. 1). FlowCAM detected even less cells (15,000-25,000 cells/mL). The discrepancy between the expected 50,000 cells/mL and the lower actual measured cell count could have several reasons. The automated cell counting is based on image cytometry, whereas the flow imaging microscopy method depends a lot on the focus, thus out-
I. Vollrath et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-4
3
Figure 1. Particle counts per mL of particles larger than 5 mm (exemplary) determined by light obscuration (orange) and flow imaging microscopy (blue).
of-focus cells are not counted. Sediq et al18 found FlowCAM to have a lower precision for cell counting compared to automated cell counting. In addition, cells can clump together or several cells are detected as one cell by light obscuration and FlowCAM. The cell suspension containing (spiked in) particle impurities showed higher particle counts compared to the pure cell suspension, which suggests that both the cells and the spiked in particulates were counted for both methods (Fig. 1). As light obscuration only provides information about particle counts and the size distribution, it does not allow characterization of unknown particles in a cell suspension. In contrast to the compendial light obscuration method, the aim of the flow imaging method is used to provide morphological characterization of SvP’s by image analyzing algorithms. The flow imaging microscopy results confirmed cell clumping (upper part of Fig. 2). In addition, morphological differences between the foreign particle impurities and the cells were observed. The cells were circular shaped with a rough interior, although of different morphology and appearance compared to silicone oil particles. Cell debris, by contrast, formed coil-like structures with a low intensity. The silicone oil particles appeared as circular ring structures, whereas the glass particles were angled polygonal shaped. Finally, the rubber stopper abrasion was seen as a coil-like, dark particle of an irregular form. The morphological differences enabled differentiation of cells from foreign particle impurities. Figure 3 shows exemplarily images of the cell suspension spiked with silicone oil, and by the differences defined previously, we can see that both silicone oil particles and cells were recognized to be present. These distinct morphology differences were reliable identified by the operator and may enable automated algorithms to classify SvPs impurities in cell therapy products. From our experience, flow imaging microscopy algorithms show the best performance when assessed on a product-specific basis. Cells in cell therapy products may exhibit significant differences in their morphological properties including size and granularity (associated to the measured total light intensity of a particle). Similarly, the flow imaging microscopy algorithms should be developed with potential impurities from the representative GMP manufacturing process. The findings of the present study may not be transferred to other cell therapy products and SvP impurities without verification. In addition, cell therapy products may comprise new classes of SvP impurities compared to traditional
therapeutic protein or small molecule drug products. For example, cell therapy products may feature unwanted lipoprotein particles, cellular contamination, cell debris or other SvP associated to the process/equipment used upstream of the final drug product because of significant differences in the manufacturing process and the absence of a sterile filtration process step, compared to protein therapeutics. As the cells are counted as particles in both methods, the particle concentration by itself would not help to assess for foreign particle contaminations in cell therapy product. Furthermore, the
Figure 2. Representative images of cells, cell debris and the foreign impurities, glass particles, silicone oil, and rubber stopper abrasion taken by flow imaging microscopy.
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Figure 3. Example images of a cell suspension spiked with silicon oil as foreign impurity.
FlowCAM instrument provides 30 describing parameters (e.g., circularity, intensity, with, edge gradient, etc.) for each counted particle. Further work is in progress to implement automated image analyzing algorithms for flow imaging. For the differentiation of cells versus unwanted particle contaminants, further work is certainly required and suggested. Conclusion Within this study, we were assessing SvP in cell therapy products, comparing cell suspensions and using solutions containing artificially product foreign particulate contaminants. We found, that testing for SvP contamination by light obscurationdas expecteddwas solely not providing sufficient insights into product quality of cell suspensions with regards to SvP. Using the flow imaging microscopy method, we found that SvP contaminationsdand cell debrisdcan be differentiated from actual cells in cell therapy products by the analyst. While the flow imaging microscopy method is currently not suggested for quality control purposes for traditional drug products,17,22 the method may offer value in a process assessment related to minimizing potential particulate contaminations and support product development and the particle control strategy for cell therapy products. References 1. Fischbach MA, Bluestone JA, Lim WA. Cell-based therapeutics: the next pillar of medicine. Sci Transl Med. 2013;5(179):179ps7. 2. Campbell A, Brieva T, Raviv L, et al. Concise review: process development considerations for cell therapy. Stem Cells Transl Med. 2015;4(10):1155-1163. 3. Heathman TR, Nienow AW, McCall MJ, et al. The translation of cell-based therapies: clinical landscape and manufacturing challenges. Regen Med. 2015;10(1):49-64.
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