BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention is directed to a method of characterizing sub-visible particles in a cell-based suspension, the method comprising subjecting the cell-based suspension to flow imaging microscopy, collecting images of the sub-visible particles in the cell-based suspension, and then characterizing the sub-visible particles in the images.

Background of the Invention

[0002] Cell therapy products represent an exciting new class of medicinal products, offering new opportunities to treat and cure diseases which have only limited treatment options with traditional therapeutic modalities (Fischbach, M.A., et al, Cell-based therapeutics: the next pillar of medicine. Sci Transl Med, 2013. 5(179): 179ps7). The specific nature of cell-based products, e.g. 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 biologies (Campbell, A., et al, Concise Review: Process Development Considerations for Cell Therapy. Stem Cells Transl Med, 2015. 4(10): 1155-63). Nevertheless, cell therapy products need to fulfill critical quality requirements to assure patient's safety.

[0003] Cell-therapy products must be parenterally administered, i.e. by injection, infusion or implant into the human body. Hence, compliance with parenteral preparation guidelines is required. For example, particle contaminations need to be characterized and controlled during drug product development and routine manufacturing, given the potential safety concern. Particulate matter is usually categorized in visible particles and sub-visible particles (SvP). For latter, the harmonized monographs Ph.Eur 2.9.19 and USP <788>, and additionally USP <787> or <789> provide guidance on specifications and test methods. Harmonized pharmacopoeial SvP methods are: light obscuration method and a light microscopic method. [0004] Cell-based products contain 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. 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” suggests for“visible particle testing”:

[0005] “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 produce low particle products with simulated samples (without cells)”.

See, Commission, E., EurdraLex Volume 4: Guidelines on Good Manufacturing Practices specific to Advanced Therapy Medicinal Products, E. Commission,

Editor. 2017.

[0006] 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, SvP testing for cell therapy products is a commonly overlooked gap in the drug product control strategy.

[0007] Flow imaging microscopy techniques, Micro-Flow Imaging (MFI) and FlowCAM, have been evaluated as alternative methods to light obscuration for SvP testing of biologies, e.g. antibodies or vaccines (T. Werk, Eur. J. Pharma Sciences, 53:95-108 2014); G.E. Frahm et al, PlosONE 77(2):e0150229 (2016), S. Zolls, AAPS Journal 75(4): 1200-1211 (2013), D. Weinbuch, J. Pharma Sciences 102(7):2\52-2165 (2013). Even though MFI and FlowCAM are based on the same measurement principle, they differ in resolution and particle parameters. However, MFI provides more accurate numbers of particle concentration (Zolls 2013; Weinbuch 2013, Sediq et al, Pharm Res, 2018. 35(8): 150). [0008] Wu et al (2012) and Farrell et al (2016) used MFI to study cell aggregation and confluency on microcarriers, respectively. Sediq 2018 showed that flow imaging microscopy techniques provide an alternative method to hemocytometry or automated cell counting for total cell concentration and viability determination.

BRIEF SUMMARY OF THE INVENTION

[0009] In some embodiments, the invention is directed to a method of characterizing sub-visible particles in a cell -based suspension, the method comprising subjecting the cell-based suspension to flow imaging microscopy, collecting images of the sub-visible particles in the cell-based suspension, and then characterizing the sub-visible particles in the images.

[0010] In some embodiments, the sub-visible particles comprise biological cells, cellular debris, glass particles, silicone oil, rubber, plastic, or combinations thereof. In some embodiments, the method further comprises providing a ratio of biological cells to cellular debris, glass particles, silicone oil, rubber, plastic, or combinations thereof.

[0011] In some embodiments, the cell-based suspension is subjected to flow imaging technology at a flow rate of 0.01 mL/min to 1 mL/min. In some embodiments, the cell-based suspension is subjected to flow imaging technology at a flow rate of 0.05 mL/min to 0.5 mL/min.

[0012] In some embodiments, the images are collected at a rate of 1 frame per second to 100 frames per second. In some embodiments, the images are collected at a rate of 5 frames per second to 40 frames per second. In some embodiments, the images are collected at a rate of 20 frames per second to 30 frames per second.

[0013] In some embodiments, the cell-based suspension has a biological cell concentration of about 10,000 cells/mL to 100,000 cells/mL. In some embodiments, the cell-based suspension has a biological cell concentration of about 40,000 cells/mL to 60,000 cells/mL.

[0014] In some embodiments, the biological cell is a neuroblastoma cell, stem cell, hematopoietic cell, muscle cell, lymphocyte, dendritic cell, or pancreatic cell. [0015] In some embodiments, the cell-based suspension has a volume of about 1 mL to about 100 mL. In some embodiments, the cell-based suspension has a volume of about 2 mL to about 10 mL.

[0016] In some embodiments, the sub-visible particles are characterized according to circularity, intensity, width, and edge gradient.

[0017] In some embodiments, the images collected of sub-visible particles are stored on a computer readable medium.

[0018] In some embodiments, the collecting images and the characterizing the sub-visible particles in the images are performed by the same device.

[0019] Further embodiments, features, and advantages of the embodiments, as well as the structure and operation of the various embodiments, are described in detail below with reference to accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0020] FIGS. 1A-1B show particle count per ml of particles larger than 5 pm determined by (a) light obscuration and (b) flow imaging microscopy.

[0021] FIG. 2 shows representative images of cells, cell debris, glass particles, silicone oil and rubber stopper abrasion obtained by flow imaging microscopy.

[0022] FIG. 3 shows example images taken from a cell suspension spiked with silicone oil as a foreign impurity.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention is directed to a method of characterizing sub-visible particles in a cell-based suspension using flow imaging microscopy. More specifically, the invention is directed to a method of characterizing sub-visible particles in a cell-based suspension, the method comprising subjecting the cell-based suspension to flow imaging microscopy, collecting images of the sub-visible particles in the cell-based suspension, and then characterizing the sub-visible particles in the images. [0024] As described herein, the cell-based suspension comprises a biological cell. In some embodiments, the biological cell is a therapeutic cell. In some embodiments, the biological cell is a neuroblastoma cell, stem cell, hematopoietic cell, muscle cell, lymphocyte, dendritic cell, or pancreatic cell. In some embodiments, the biological cell is an adult stem cell, an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, an undifferentiated cell. In some embodiments, the biological cell is a blood cell, e.g., an erythrocyte, a leukocyte (e.g., a lymphocyte, monocyte granulocyte), or a thrombocyte. In some embodiments, blood cell is a neutrophil, eosinophil or basophil. In some embodiments, the cell-based suspension comprises a combination of one or more of the blood cells listed herein.

[0025] Cell-based suspensions can comprise contaminants of various sizes. In some embodiments, the contaminants are visible. In some embodiments, the contaminants are sub-visible. While not being limited, the present disclosure is especially suitable for characterizing contaminants in the sub-visible scale. In some embodiments, any contaminants detectable thru flow imaging microscopy can be characterized according to the present disclosure. In some embodiments, the sub-visible has a size of from 0.1 pm to 500 pm, from 0.5 pm to 100 pm, from 1 pm to 50 pm, or from 2 pm to 50 pm. In some embodiments, the sub- visible particles comprise biological cells, cellular debris, glass particles, silicone oil, rubber, plastic, or combinations thereof. The foregoing list is not exhaustive and is used for example only as one of skill in the art can recognize that other contaminants of different compositions and origins can also be present and are contemplated by the present disclosure.

[0026] In some embodiments, each of the various types of sub-visible particles are quantitated.

E.g., in some embodiments, the quantity of biological cells, cellular debris, glass particles, silicone oil, rubber, plastic, or combinations thereof can be determined. In some embodiments, this quantity of the biological cell, or any of the listed contaminants, can be used to determine the suitability of the cell-based suspension for its intended purpose. In some embodiments, the method further comprises providing a ratio of biological cells to cellular debris, glass particles, silicone oil, rubber, plastic, or combinations thereof. E.g., in some embodiments, a ratio of a biological cell, e.g., a therapeutic cell, and a contaminant, e.g., cellular debris, glass particles, silicone oil, rubber, plastic, or combinations thereof, are determined. In some embodiments, this ratio can be utilized to determine the suitability of the cell-based suspension for its intended purpose. In some embodiments, the above described quantity or ratios can be compared to a standard reference to determine the suitability of the cell-based suspension for its intended purpose. In some embodiments, the above described quantity or ratios can be during the processing of a cell-based suspension to determine and/or characterize contaminants that may arise in one or more processing steps in the manufacture of the cell-based suspension.

[0027] In some embodiments, the present disclosure provides a method to determine the presence or absence of one or more specific contaminants. In other words, in some embodiments the sub-visible particles are not quantitated but rather the method provides a way to determine the existence or absence of one or more specific contaminants. For example, in some embodiments, the disclosure provides a method of determining the existence of a contaminant, e.g., cellular debris, glass particles, silicone oil, rubber, and/or plastic in a cell-bases suspension.

[0028] The present invention provides a method of characterizing sub-visible particles. In some embodiments, the term“characterizing” as used herein can include identifying the sub- visible particles. In some embodiments, the term“characterizing” as used herein can include distinguishing sub-visible particles into different categories according to one or more criteria. In some embodiments, the term“characterizing” as used herein can include identifying a sub-visible particle as belonging to a specific category of contaminant, e.g., identifying a sub-visible particle as cellular debris, glass particles, silicone oil, rubber, or plastic.

[0029] Various flow imaging devices are known in the art, e.g., FlowCam (Fluid Imaging Technologies, Inc, Scarborough, ME). The skilled artisan is familiar with how to operate the imaging devices described herein. For example, various flow rates can be used in the imagine of the cell-based suspension. In some embodiments, the cell-based suspension is subjected to flow imaging technology at a flow rate of 0.01 mL/min to 1 mL/min. In some embodiments, the cell-based suspension is subjected to flow imaging technology at a flow rate of 0.05 mL/min to 0.5 mL/min. [0030] The flow imaging devices can utilize various different imaging means and mechanisms to obtain images of the cells and contaminants in the cell-based suspension. In some embodiments, the rate the images are obtained can be optimized. In some embodiments, the images are collected at a rate of 1 frame per second to 100 frames per second. In some embodiments, the images are collected at a rate of 5 frames per second to 40 frames per second. In some embodiments, the images are collected at a rate of 20 frames per second to 30 frames per second. In some embodiments, the images are obtained continuously, and the information processed accordingly.

[0031] Various concentrations of biological cells can be present in the cell-based suspension. In some embodiments, the cell-based suspension has a biological cell concentration of about 10,000 cells/mL to 100,000 cells/mL. In some embodiments, the cell-based suspension has a biological cell concentration of about 40,000 cells/mL to 60,000 cells/mL.

[0032] Various volumes of cell-based suspensions can be utilized according to the present invention. In some embodiments, the cell-based suspension has a volume of about 1 mL to about 100 mL. In some embodiments, the cell-based suspension has a volume of about 2 mL to about 10 mL. In some embodiments, only a representative portion of the cell- based suspension is subjected to flow imaging microscopy.

[0033] Various characteristics of the sub-visible particles can be used in characterizing the particles. For example, in some embodiments, the sub-visible particles are characterized according to circularity, intensity, width, and edge gradient. Commercially available software is available to aid in the characterization of the particles. Alternatively, in some embodiments, an individual can review the images of the particles and characterize the particles according to any desired methodology. In some embodiment, the present disclosure provides for an alternative to light obscuration for the characterization of sub- visible particles, e.g., contaminants, in cell-based products. The methods described herein are suitable to sufficiently differentiate particle impurities from actual cells.

[0034] Various means can be employed to obtain and collect the images of the sub-visible particles. For example, in some embodiments, the images collected of sub-visible particles are stored on a computer readable medium. In some embodiments, the images are analyzed and characterized concurrently with the image collection process. [0035] In some embodiments, the collecting images and the characterizing the sub-visible particles in the images are performed by the same device.

[0036] Further embodiments, features, and advantages of the embodiments, as well as the structure and operation of the various embodiments, are described in detail below with reference to accompanying drawings.

[0037] It should be appreciated that the particular implementations shown and described herein are examples and are not intended to otherwise limit the scope of the application in any way.

[0038] The published patents, patent applications, websites, company names, and scientific literature referred to herein are hereby incorporated by reference in their entireties to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

[0039] As used in this specification, the singular forms“a,”“an” and“the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term“about” is used herein to mean approximately, in the region of, roughly, or around. When the term“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0040] Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present application pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of ordinary skill in the art. EXAMPLES

Example 1 Study design

[0041] Cell suspensions with 50' 000 cells/ml suspended in phosphate buffer 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. Additionally, one cell suspension sample was stressed (vortexed) to induce cell debris and evaluate their impact on particle counts and characterization of impurities. Finally, foreign particle impurity dispersion/suspension were spiked in cell suspensions to assess the method capability in discriminating cells from foreign particle impurities.

Materials

[0042] Human neuroblastoma cells SK-N-AS (ATCC® CRL2137™) were used as a model cell line for this study. Dulbecco' s Modified Eagle Medium (DMEM) high glucose with pyruvate (Gibco™), phosphate buffered saline (Gibco™), fetal bovine serum (Gibco™), and Trypsin-EDTA 0.25% (Gibco™) were purchased from Fisher Scientific AG (Reinach, Suisse). Penicillin-Streptomycin and non-essential amino acids were ordered from Sigma- Aldrich (Merck KGaA, Darmstadt, Germany).

Cell culture

[0043] SK-N-AS cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 pg/ml streptomycin and 0.1 mM non-essential amino acids. The cells were cultured at 37°C and 5% CCh 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, Allerod, Denmark). Preparation of suspensions

[0044] After harvest, the cells were re-suspended in PBS at a concentration of 50' 000 cells/ml to yield the cell suspension. Cell debris were generated by vortexing one part of the cell suspension for approximately 5 min.

[0045] Glass particles of a defined size (~ 150 pm) 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 silicone oil (Dow coming) to PBS.

[0046] 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.

[0047] Suspensions containing cells and the above-described extrinsic particulates were achieved by co-mixing the cell suspension (4 ml) d with 1 ml of each foreign impurity dispersion/suspension followed by gently mixing.

Light Obscuration

[0048] Particle determination by light obscuration method was performed using a PAMAS SBSS instrument (Partikelmess- und Analysesysteme GmbH, Rutesheim, Germany) equipped with an HCB-LD-50/50 detector, a 1 ml syringe and a pressurizable sample chamber. Four measurements of 0.4 ml sample volume at a flow rate of about 0.1 mL/min and 1 bar were performed. The first 0.4 mL were discarded (pre-flush volume). The following 3 x 0.4 mL measurements were averaged to obtain the final reported result.

Flow Imaging Microscopy

[0049] Flow Imaging Microscopy was used as an alternative method to characterize SvP.

Measurements were performed on a FlowCAM VS1 (Fluid Imaging Technologies, Yarmouth, ME, USA) with an 80 pm field of view (FOV) flow cell. 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 (lOx lens).

Results and Discussion [0050] The cell suspension was prepared at a cell concentration of 50' 000 cells/ml. When analyzed by using light obscuration, only about 30Ό00 cells per ml were detected (FIG. 1A). FlowCAM detected even less cells (15Ό00 - 25Ό00 cells per ml) (FIG. IB). 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-of-focus cells are not counted. Sediq et al (2018) 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 particle impurities (spiked in) 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).

[0051] 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 flow imaging microscopy method enables a morphological characterization of SvP by image analyzing algorithms. The flow imaging microscopy results confirm cells clumps (FIG. 2/Cells, pure cell suspension). Additionally, morphological differences between the foreign particle impurities and the cells were detectable. The cells were circular shaped with a rough interior, though of different morphology and appearance compared to silicone oil particles. Cell debris, in 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. Lastly, the rubber stopper abrasion was seen as a coil-like, dark particle of an irregular form.

[0052] The morphological differences enabled differentiation of cells from foreign particle impurities. FIG. 3 exemplarily shows images of the cell suspension spiked with silicone oil and by the differences defined above we can see that e.g. particles 2, 4, 6, 7 are silicone oil particles, whereas particles 3, 5, 8, 59, 66 are cells.

[0053] As the cells are counted as particles in both methods, the particle concentration by itself would not help to characterize foreign particle impurities in cell therapy product. Furthermore, the 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 the differentiation of cells versus unwanted particle impurities.

[0054] Within this study we were measuring SvP in cell therapy products, comparing cell suspensions and using solutions containing foreign particulate contaminants. We found, that testing for SvP contamination by light obscuration is not expected to solely provide sufficient insights into product quality of cell suspensions with regards to SvP, and obviously, suggest that traditional limits for SvP cannot be applied. Using the flow imaging microscopy method, we found that SvP contaminations can be differentiated in cell therapy products. Whilst the flow imaging microscopy method is currently not suggested for quality control purposes for traditional drug products, the method may offer value in a particle control strategy for cell therapy products.

[0055] It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of any of the embodiments.

[0056] It is to be understood that while certain embodiments have been illustrated and described herein, the claims or items are not to be limited to the specific forms or arrangement of parts described and shown. In the specification, there have been disclosed illustrative embodiments and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0057] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.