Method and system for validating flow cytometry measurements

Abstract

To provide an improved verification method for verifying the performance of a fluorescence-based analysis system.SOLUTION: (a) negatively staining cells in a first portion of a standard sample comprising target cells expressing a target cell marker; (b) positively staining target cells in a second portion of the standard sample; (c) passing the first and second portions of the standard sample through an instrument to obtain a fluorescence measurement indicative of target cell concentration; (d) based on the fluorescence measurement obtained in (c), each diluted sample having a nominal concentration of target cells, each nominal cell concentration being greater than the concentration of target cells indicated by the fluorescence measurement of the negatively stained first portion in (a); Preparing a dilution series comprising a plurality of diluted samples, wherein the nominal concentration in each diluted sample is different from the nominal concentration in each of the remaining diluted samples; (e) passing the dilution series of (d) through the instrument and obtaining a fluorescence measurement; and (f) comparing the nominal cell concentration of (d) and the fluorescence measurement of (e) for each diluted sample to quantify the performance of the staining method in the instrument.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 013098, filed April 21, 2020, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates to fluorescence-based analytical methods and systems, and in particular to validation methods for assessing the reliability and reproducibility of measurements made by fluorescence-based analytical instruments. [Background technology]

[0003] Fluorescence-based cell analysis systems, such as flow cytometry and fluorescence-activated cell sorting (FACS), are powerful tools for measuring various cellular elements, such as cell surface receptors and intracellular components, in test samples. In FACS, antibody-conjugated fluorescent dyes (fluorescent dyes) are used to stain samples to signal the presence and quantity of elements, such as cell receptors, or other cell markers, peptides, and nucleic acids. The development of multiple different fluorescent dyes, each with a unique, distinguishable emission spectrum, makes it possible to use two or more fluorescent dyes simultaneously. As a result of their combined power and versatility, flow cytometry and FACS are commonly used in the drug development process, for example, for immunophenotyping, receptor expression or occupancy, and other functional assays.

[0004] While powerful, flow cytometry and FACS methods are generally more difficult to validate than other analytical methods for a variety of reasons. These include a lack of protocol standardization, a lack of reference materials, and the use of complex and sensitive instrumentation. The monoclonal and polyclonal antibodies typically used for staining are not standardized and can vary substantially in quality and performance. Therefore, it is incumbent on researchers to carefully evaluate their reagents to ensure they are working as intended. However, researchers have little guidance for validating a given method and instrument. For example, the U.S. FDA defines validation simply as the evaluation of a method's suitability for its intended use and recommends that a validation testing process must ensure that the assay meets predetermined specifications, is performed reliably, and is suitable for its intended use. The FDA guidelines acknowledge that a one-size-fits-all validation regulation is inappropriate and do not provide specific guidance for validating assays. The lack of standard or accepted regulatory guidelines and reference materials for validation is, unfortunately, an issue that is not adequately addressed in the literature. The challenge of method validation becomes even greater when the target cells or target markers of interest are expected to be present in the sample at very low concentrations. There is a need for improved validation methods for verifying the performance of fluorescence-based analytical methods and systems. Summary of the Invention [Means for solving the problem]

[0005] Various aspects of the present disclosure include a method for validating measurements of a stained target cell population in a test sample by a fluorescence-based analytical instrument, comprising: (a) negatively staining or negatively staining cells in a first portion of a standard sample containing target cells expressing a target cell marker; (b) positively staining or positively staining target cells in a second portion of the standard sample; (c) passing the first portion of the standard sample through the instrument to obtain a fluorescence measurement indicative of the target cell concentration in the first portion of the standard sample, and passing the second portion of the standard sample through the instrument. (d) preparing a dilution series including a plurality of diluted samples, each having a nominal concentration of target cells based on the fluorescence measurements obtained in (c), each nominal cell concentration being greater than the concentration of target cells indicated by the fluorescence measurements of the negatively stained first portion in (a), and the nominal concentration in each diluted sample being different from the nominal concentration in each of the remaining diluted samples; (e) passing the series of diluted samples of (d) through the instrument to obtain a series of fluorescence measurements including a fluorescence measurement for each diluted sample; and (f) comparing the nominal cell concentration of (d) with the fluorescence measurement of (e) for each diluted sample to quantify the performance of the staining method in the instrument. The instrument may be, for example, a flow cytometer.

[0006] In another aspect, the disclosure provides a method for validating flow cytometric measurements of a stained target cell population in a test sample, comprising: (a) negatively staining or negatively staining cells in a first portion of a standard sample containing target cells expressing a target cell marker; (b) positively staining or positively staining target cells in a second portion of the standard sample; (c) passing both the first and second portions of the standard sample through a flow cytometer to obtain fluorescence measurements indicative of target cell concentrations in each of the first portion of the standard sample and the second portion of the standard sample; and (d) measuring the fluorescence intensity of the target cells in (c). Based on the obtained fluorescence measurements, the method includes preparing a series of diluted samples, each having a nominal concentration of target cells that varies systematically in the dilution series, wherein the nominal cell concentration of at least one diluted sample is greater than the concentration of target cells indicated by the fluorescence measurements of the negatively stained first portion in (a); (e) passing the series of diluted samples of (d) through a flow cytometer to obtain a series of fluorescence measurements including a fluorescence measurement for each diluted sample; and (f) comparing the nominal cell concentration of (d) with the fluorescence measurements of (e) for each diluted sample to quantify the performance of the staining method in the flow cytometer.

[0007] Any of the disclosed methods may include one or more of the following configurations. The comparison in (f) may include, for example, performing statistical calculations on the difference between the nominal cell concentration and the fluorescence measurement value of each diluted sample to determine at least one of the linearity, range, precision, accuracy, limit of detection (LOD), and lower limit of quantitation (LLOQ) for the instrument and staining method. Determining the LLOQ may include, for example, identifying the concentration of target cells associated with a predetermined criterion of precision and a predetermined criterion of accuracy. The negative staining of the target cells in the first portion of the standard sample in (a) may include introducing into the first portion of the standard sample: (i) a fluorescent dead cell exclusion dye; (ii) a nonspecific antibody conjugated to a fluorescent dye, for example, as an isotype control; and (iii) a specific antibody capable of specifically binding to a target marker (antigen) in target cells that is not conjugated to a fluorescent dye. The nonspecific antibody conjugated to the fluorescent dye for negative staining may include an antibody lacking the ability to specifically bind to a cellular antigen, such as IgD, IgG, IgA, IgM, or IgE. The negatively stained first portion may be prepared without a cell depletion step. Positive staining of the target cells in the second portion of the standard sample (b) may involve introducing into the second portion of the standard sample (i) a fluorescent dead cell exclusion dye (the same dye used for negative staining) and (ii) the same specific antibody used for negative staining, but not conjugated to the fluorescent dye, but rather conjugated to the same fluorescent dye used for negative staining (the fluorescent dye is conjugated to the nonspecific antibody). In any of the methods, the fluorescent dead cell exclusion dye used for staining may be selected from nucleic acid-binding dyes, propidium iodide, DAPI, DRAQ7, 7-AAD, TO-PRO-3, and amine-reactive dyes. The fluorescent dyes used may be known fluorescent dyes, such as, but not limited to, allophycocyanin (APC), APC C750, APC AF700, brilliant violet (BV) 421, BV510, hilite 7 (H7), BV605, BV650, PE CF594, fluorescein isothiocyanate (F ITC), R-phycoerythrin (PE or R-PE), PE-Cy7 (PE linked to the cyanine dye Cy7), APC-Cy7 (APC linked to the cyanine dye Cy7), APC-H7 (APC linked to the Cy analog Hilite 7 (H7)). The target cell marker may be selected from, for example, CD3, CD4, and CD8 as T cell markers, CD19 as a B cell marker, CD235a as an erythrocyte marker, CD56 as a natural killer (NK) cell marker, CD14 as a monocyte marker, and CD66b as a granulocyte marker. In one embodiment, the target cell population is a T cell population and the target cell marker is CD3.

[0008] The method can further include multiplexing by negative staining and positive staining for two or more different target markers on target cells, or optionally for staining for two or more different target markers on two or more target cells. Negative staining can further include introducing two or more specific antibodies into a first portion of the standard sample, each specific antibody capable of specifically binding to one of the two or more target markers and not conjugated to a fluorescent dye. Positive staining can further include introducing two or more specific antibodies into a second portion of the standard sample, each specific antibody being conjugated to a fluorescent dye.

[0009] The calculation of (f) may be performed by a processor coupled to the instrument or flow cytometer. The test sample may contain target cells present at a concentration of 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, or 0.02% or less. The dilution series may include diluted samples having the highest concentrations of target cells, with the highest concentrations being 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.02%. In any of the methods, the series of diluted samples may include at least two, three, four, five, six, seven, eight, nine, or ten diluted samples.

[0010] In another aspect, the present disclosure contemplates a non-transitory computer-readable medium comprising instructions for a computer processing device to perform the comparison of step (f) of any of the disclosed methods or to perform any of the statistical calculations to determine any one or more of the linearity, range, precision, accuracy, limit of detection (LOD), and lower limit of quantitation (LLOQ) for the instrument and staining method.

[0011] In yet another aspect, the present disclosure provides a system for validating fluorescence measurements on a test sample made by a fluorescence-based instrument, the system comprising: a fluorescence-based instrument; and a computer coupled to the fluorescence-based instrument, the computer comprising a computer-readable medium containing instructions for the computer processing device as described above. In the system, the fluorescence-based instrument may be a flow cytometer.

[0012] In yet another aspect, the disclosure describes a kit including reagents for staining cells in a standard sample for validating a fluorescence-based analytical method for analyzing target cells in a test sample, the kit including: (i) a negative staining reagent including (a) a fluorescent dead cell exclusion dye, (b) a non-specific antibody conjugated to the fluorescent dye, and (c) an unconjugated specific antibody capable of specifically binding to a target marker (antigen) on the target cells; (ii) a positive staining reagent including (a) a fluorescent dead cell exclusion dye and (b) a specific antibody conjugated to the fluorescent dye; and instructions for preparing a dilution series including a plurality of diluted samples each having a nominal concentration of target cells that varies systematically in the dilution series, (a) negatively staining a first portion of the standard sample, (b) positively staining a second portion of the standard sample, and (c) the nominal cell concentration of at least one diluted sample is greater than the concentration of target cells indicated by the fluorescence measurement of the negatively stained first portion.

[0013] Other aspects and configurations of the present disclosure are detailed below.

[0014] Reference to color drawings The application file contains at least one photograph executed in color. Copies of this patent application publication with color photograph(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0015] [Figure 1] 1 provides a schematic diagram of a method according to the present disclosure. [Figure 2] 1 is a FACS plot showing typical results obtained with a negatively stained sample. Events shown were gated for viable cells based on forward and side scatter and PI (to exclude dead cells). [Figure 3] 1 is a FACS plot showing typical results obtained with a positively stained sample. Events shown were gated for viable cells based on forward and side scatter and PI (to exclude dead cells). [Figure 4] 1 shows the procedural flow for preparation of linearity samples. [Figure 5] 1 shows the procedural flow for preparing a dilution series. [Figure 6] 1 shows the method qualification run by two operators on three different occasions as specified in Example 2. [Figure 7] Residual plots of a) untransformed data and b)-f) untransformed nominal and estimated CD3% (outcomes) for different log-transformed data. [Figure 8] The percentage of estimated CD3% compared to nominal CD3% is shown. The dotted line indicates a recovery rate of 100±30%. [Figure 9] 1 shows a regression analysis of the estimated linearity samples. DETAILED DESCRIPTION OF THE INVENTION

[0016] Analytical method validation is important for assessing the reliability and reproducibility of fluorescence-based analytical measurements. For such measurements to be useful, they must be reproducible. For example, fluorescence-activated cell sorting (FACS) is a specialized type of fluorescence-based analysis and a powerful method for sorting mixtures of biological cells into distinct populations based on the fluorescent signal of each cell. In contrast to flow cytometry, which is performed simply to count and sort cells, FACS provides both qualitative and quantitative analysis using flow cytometry data. Because the quality and performance of antibodies used in analytical techniques vary, it is important to properly validate such fluorescence-based analytical measurements made using FACS.

[0017] One validation approach relies on preparing a dilution series of multiple diluted or spiked samples, each with a known or nominal concentration of target cells or target markers. For example, serial dilutions are performed by spiking a known number of target cells to systematically generate multiple spiked samples with known (nominal) cell concentrations ranging from higher concentrations, e.g., 50%, to lower concentrations, e.g., 1%. The spiked samples are run through a cytometer, and fluorescence measurements are obtained for each spiked sample. The spiked samples are compared to the cytometer measurements. The accuracy of the instrument and staining method is determined by statistical calculations, such as calculating the correlation between nominal and measured values, determining the slope of the relationship, determining R-sq, etc. By staining (one or more) replicate non-spiked samples, acquiring them, and running them through the cytometer to obtain a measure of positive events, the limit of detection (LOD) and / or limit of quantification (LOQ) of the FACS can be calculated, as known. Thus, the assay being validated can be an assay used to detect and quantify the concentration of cells in a sample.

[0018] To accurately prepare a series of spiked samples, at least two samples are required: one or more samples each containing a known (nominal) concentration of target cells or target markers, and one sample containing almost no target cells or target markers (approximately 0 concentration). Typically, more than two spiked samples are used. For example, validation of an instrument and staining method using five levels involves preparing a series of spiked samples containing five different nominal concentrations of target cells or target markers (e.g., 2%, 4%, 6%, 8%, and 10%) and one sample containing 0%. To prepare a series of spiked samples, a standard containing a known, relatively higher concentration of the target cells or target marker of interest is serially and systematically diluted to prepare a series of spiked samples with lower concentrations. However, in many cases, preparing spiked samples is not easy because commercially available starting standards are not available. For example, when the analyte of interest is expressed in cells, e.g., CD3 + In the case of cells (T cells), no such standard standard exists, i.e., a known concentration, e.g., 10% CD3, can be diluted to prepare spiked samples with lower concentrations of 8%, 6%, 4%, and 2%. + It is not possible to simply purchase standard reference materials containing 0% or less of the target cells. In theory, it would be possible to use commercially available cell sorters to prepare a series of spiked samples with successively lower concentrations. However, when the estimated concentration of target cells or target markers is low, for example, below about 0.1% or even as low as about 0.01%, such methods are expensive and time-consuming.

[0019] The present disclosure solves the technical problem of providing negative standards and spiked samples for situations where a standard reference material does not exist, and provides for the preparation of spiked samples of less than 0.1%, which is useful for validation when target cells or target markers are present at concentrations of less than 0.1%. For example, depending on the instrument and staining used, the LLOQ can be less than 0.01%. Thus, the present disclosure relates to methods and systems for validating measurements of stained target cell populations in test samples using fluorescence-based analytical instruments, such as flow cytometers.

[0020] I. Fluorescence-Based Analytical Instruments Fluorescence-based analytical instruments include any analytical instrument capable of measuring a fluorescent signal as an indicator of the presence of a target analyte or target cell type in a sample. Depending on the instrumentation and method used, the instrument may identify the presence of a test sample based on the intensity of the fluorescent signal. It may be possible to quantify the amount of a target analyte or cell type in a sample. One example of a fluorescence-based analytical instrument is a flow cytometer. FACS (fluorescence-activated cell sorting) is a different, but related, method based on flow cytometry. Flow cytometry is a method that uses a flow cytometer to examine and determine the expression of molecules both inside and outside cells, defining and characterizing distinct and single cell types. Flow cytometry can also be used to determine other parameters, such as cell size, volume, and purity of isolated cell samples. FACS is a flow cytometry technique that utilizes specific antibodies labeled with fluorescent dyes to obtain expression data and sort cell samples by a number of variables.

[0021] FACS can be performed using any one of many types of flow cytometers, including conventional flow cytometers, acoustic focusing flow cytometers, cell sorters, or imaging flow cytometers. Most common conventional cytometers use a sheath fluid to focus the sample stream, as well as common lasers such as 488 nm (blue), 405 nm (violet), 532 nm (green), 552 nm (green), 561 nm (yellow-green), 640 nm (red), and 355 nm (ultraviolet). In acoustic focusing flow cytometers, ultrasound is used to focus cells for analysis while preventing sample clogging and allowing for larger sample inputs. A cell sorter is a type of conventional flow cytometer that allows the user to retrieve the sample after processing. Cells positive for a desired parameter can be separated from cells negative for the parameter. An imaging cytometer is a conventional cytometer combined with fluorescence microscopy, all for rapid analysis of samples for morphology and multiparameter fluorescence at both the single cell and cell population levels. A computer with a processing unit that controls the instrument's functions is usually linked to the flow cytometer. The methods and systems described herein can be applied to the use of any of the flow cytometers described herein.

[0022] II. Method The methods described herein address the technical challenge of providing negative standards and low-level spiked samples in situations where target cells or target markers are expected to be present in the sample at concentrations below 0.1%, or even lower, and where no standard reference standard exists, thus allowing reliable validation of fluorescence-based measurements, for example by flow cytometer (FACS).

[0023] Figure 1 is a schematic diagram of the method. Depending on whether negative or positive staining is being performed, positively and negatively stained standard samples are prepared using an antibody capable of specifically binding to a target cell marker (specific antibody), an "isotype" (nonspecific antibody), and a fluorescent dye conjugated to the specific or nonspecific antibody. More specifically, a standard sample is obtained and divided into two portions. The first portion of the standard sample is negatively stained with (i) a fluorescent dead cell exclusion dye, (ii) a nonspecific antibody conjugated to a fluorescent dye, e.g., as an isotype control, and (iii) a specific antibody capable of specifically binding to a target marker (antigen) on the target cells. For negative staining, the specific antibody is not conjugated to a fluorescent dye. A nonspecific antibody conjugated to a fluorescent dye for negative staining is an antibody capable of binding to cells that do not have a specific epitope for that antibody, i.e., it binds to cells without specific binding such as the binding of an antibody to a specific epitope on the cell, and therefore, the nonspecific antibody also has a high dissociation constant (Kd) for the marker, as described in more detail below. Any one of the nonspecific immunoglobulins IgD, IgG, IgA, IgM, or IgE can be used for the nonspecific antibody.

[0024] Positively stain a second portion of the standard sample with a fluorescent dead cell exclusion dye (the same dye used for the negative staining step) and the same specific antibody used for negative staining, but not conjugated, but rather conjugated to the same fluorescent dye used for negative staining (the fluorescent dye is conjugated to a non-specific antibody).

[0025] The target cell marker can be any known cell marker, for example, any known marker for T cells, B cells, NK cells, erythrocytes, granulocytes, or monocytes. T cell markers include, but are not limited to, CD3, CD4, CD8, CD69, CD71, and CD25. B cell markers include, but are not limited to, IL-6, CD19, CD25, CD30, CD27, CD38, CD78, CD138, and CD319. In one embodiment, for example, the target marker can be selected from CD3 as a T cell marker, CD19 as a B cell marker, CD235a as an erythrocyte marker, CD56 as a natural killer (NK) cell marker, CD14 as a monocyte marker, and CD66b as a granulocyte marker. It will be understood that validation of two or more markers of a cell population can be easily performed by applying the method to multiple samples derived from the same standard using appropriate specific antibodies in each case. Non-limiting examples include: To validate two different staining protocols for two different markers for T cell populations, the method can be performed once using an antibody that specifically binds CD3 and a second time using an antibody that specifically binds CD4.

[0026] It will be understood that the selection of the target marker results in the selection of a specific one for staining. Specific binding of an antibody to a target marker refers to the antibody's ability to recognize one or more epitopes of the marker, such that the selected antibody exhibits high binding affinity to the marker and, preferably, low binding affinity to other cross-reactive species. Generally, the binding affinity of a molecule is described using the dissociation (or equilibrium) constant (Kd). The lower the dissociation constant, the greater the binding affinity or degree of binding of bound antibody A to analyte (marker) B. Thus, a specific antibody for a marker exhibits a low dissociation constant (Kd) for the marker. Methods for determining Kd for a given antibody are very well described in the literature, as further described below. Antibodies that produce insufficient signals at low concentrations have high variability (noise), making them difficult to distinguish from higher measured cross-reactivity. As used herein, a "specific antibody" refers to an antibody having a Kd for a target marker of 10 nM or less, 7 nM or less, 5 nM or less, 1 nM or less, 0.5 nM or less, 0.15 nM or less, or 0.1 nM or less, or less than 0.1 nM. As a non-limiting example, it is possible to select a specific antibody having specific binding to a target marker with a Kd of 5 nM or less, for example, 2 nM or less, preferably 1 nM or less, more preferably 0.7 nM or less. The Kd value describing the binding affinity of an antibody considered to be specific for a target marker can be provided by a commercial supplier of a commercially available antibody, or can be determined by well-known methods, such as, but not limited to, fluorescence titration, competitive ELISA, calorimetry, such as isothermal titration calorimetry (ITC), flow cytometry titration analysis (FACS titration), and surface plasmon resonance (BIAcore). Such methods are well described in the literature. (See, e.g., Goodrich and Kugel, 2007, Binding and Kinetics for Molecular Biologists, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA; De See Jong, LAA et al., J. Chromatogr. B829(1-2):1-25 (2005); Heinrich, L. et al., J. Immunol. Methods 352(1-2):13-22 (2010); Williams, MA and Daviter, T. (eds.) 2013, Protein-Ligand Interactions, Methods and Applications, Springer, New York, NY, USA).

[0027] Thus, a specific antibody of the present disclosure is any antibody with a Kd of 1 nM or less, 0.5 nM or less, 0.15 nM or less, or 0.1 nM or less for any of the target cell markers, including, but not limited to, any of the cell markers described herein: CD3, CD4, CD8, CD69, CD71 and CD25, IL-6, CD19, CD25, CD30, CD27, CD38, CD78, CD138 and CD319, CD235a, CD56, CD14, and CD66b. Specific antibodies can be polyclonal, monoclonal, chimeric, humanized, or fully human. They can be single-chain or multi-chain antibodies. Suitable specific antibodies can be, and commonly are, purchased from commercial suppliers, but monoclonal antibodies can also be produced from hybridomas or in host cells that contain and then express vectors containing nucleic acid sequences encoding the antibodies. Thus, the specific antibodies described herein can be produced by transforming a host cell with at least one nucleic acid molecule encoding the specific antibody; expressing the nucleic acid molecule in the host cell; and isolating the specific antibody, all using materials and techniques well known in the art. Commercial Supply of Specific Antibodies Against Various Cell Markers These include, but are not limited to, BD Biosciences, Takara Bio USA (e.g., providing monoclonal anti-CD3 (OKT3)); BioX Cell (Lebanon, NH; e.g., providing anti-human CD19 monoclonal antibodies); Thermo-Fisher Scientific Inc.; BioLegend Inc.; and Bio-Rad Antibodies, among others.

[0028] The fluorescent dead cell exclusion dye used in both the negative and positive staining steps can be any dye that allows for the discrimination of live cells from dead or dying cells and is also known as a viability dye. For example, viability dyes include dyes that are not membrane-permeable, i.e., do not pass through the cell membrane. For example, a nucleic acid-binding dye that does not pass through the cell membrane will selectively stain accessible nucleic acids (e.g., DNA) in dead and dying cells, but will not stain inaccessible nucleic acids inside live cells with intact membranes. Alternatively, the dead cell exclusion or viability dye can be a protein-binding dye (amine-reactive dye) rather than a nucleic acid-binding dye. Protein-binding dyes bind to live and dead cells, but dead and dying cells with disrupted membranes are more strongly stained by dyes that reach intracellular proteins, and therefore, dead and dying cells exhibit higher fluorescence. Therefore, nucleic acid-binding or amine-reactive dyes can be used to exclude dead cells by gating on a population that is less stained, including live cells. Dead cell exclusion dyes include, but are not limited to, many readily commercially available nucleic acid binding and amine reactive dyes, such as propidium iodide, DAPI, DRAQ7, 7-AAD, TO-PRO-3, live / dead fixable dyes, eFluor fixable dyes, Horizon dyes, Biolegend Zombie dyes, and Ghost dyes. Amine reactive dyes include M1420MP, M1410, D6105, P130, P6114, A10168, M10165, D10161, D374, D126, D1421, B30250, H185, H1428, H1193, P30253, A30000, A30100, C10164, P10163, S6110, D2184, D2183, and D3834.

[0029] The fluorescent dye may be any one having a characteristic, visible emission spectrum, such as, but not limited to, any one of the many known fluorescent dyes currently commercially available, such as allophycocyanin (APC), APC C750, APC AF700, brilliant violet (BV) 421, BV510, hilite7 (H7) BV605, BV650, PE CF594, fluorescein isothiocyanate (FITC), R-phycoerythrin (PE or R-PE), PE-Cy7 (PE linked to the cyanine dye Cy7), APC-Cy7 (APC linked to the cyanine dye Cy7), APC-H7 (APC linked to the Cy analogue Hilite7 (H7)).

[0030] The target marker binding sites in the negatively stained sample (isotype stained sample) are blocked using an unconjugated version of the specific antibody, i.e., the specific antibody to which the fluorescent dye is not conjugated or covalently bound. The positively stained sample is then used to prepare at least two, and usually three or more, spiked samples by spiking the target nominal cell concentration using the negatively stained cells. The series of spiked samples is then analyzed on a flow cytometer to obtain measured concentrations for each spiked sample from the instrument. The data (nominal and measured concentrations for each spiked sample) are plotted and statistically analyzed for correlation, and validation parameters, such as linearity, precision, accuracy, LOD, and / or LLOQ, are calculated. To determine at least one of linearity, range, precision, accuracy, LOD, and LLOQ for the instrument and staining method, a computer processor linked to the instrument, for example, calculates the correlation between each nominal cell concentration and the fluorescence measurements for each diluted sample. It will be appreciated that analyzing the comparison of nominal cell concentration data and measured cell concentration data to quantify the performance of the staining method in any of the methods can be performed by a processing device configured with instructions to perform one or more standard statistical tests of difference. Determining the LLOQ can include, for example, identifying a concentration of target cells associated with a predetermined standard of precision and a predetermined standard of accuracy.

[0031] Example 1 below illustrates a method used to determine the concentration of viable T cells in a cell product sample using propidium iodide as the dead cell exclusion dye, an anti-CD3 antibody, IgG as the nonspecific antibody, and allophycocyanin as the fluorescent dye. However, it should be understood that any one of the dead cell exclusion dyes, cell marker (CD3) and specific antibody (anti-CD3) pairs, nonspecific antibodies (IgG), and / or fluorescent dyes (APC) in the examples can be substituted as described herein. After negative and positive staining, the negatively stained cells are used to dilute the positively stained sample to the planned, highest desired spike level (the highest nominal concentration of target cells). Serial dilutions are then prepared to generate diluted samples with systematically varying nominal cell concentrations. The dilution series can include any multiple. While theoretically, two diluted samples could be used, in practice, at least three diluted samples are prepared, each with a systematically varying nominal concentration, preferably for ease of analysis. A dilution series can include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more diluted (spiked) samples. As a non-limiting example, the nominal concentration of each diluted (spiked) sample can vary from 0.1% down to 0.003%, although it will be understood that the selection of the nominal value will vary depending on a range of factors, such as the expected concentration of target cells or target markers in the sample, the number of spiked samples being used, the instrumentation, the staining protocol being used, etc.

[0032] It will be further appreciated that the present methods and systems address the problem of providing negative samples for test samples that may contain target cells present at concentrations of 2% or less, 1.5% or less, 1% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.25% or less, 0.2% or less, 0.15% or less, 0.1% or less, 0.05% or less, 0.02% or less, or 0.01% or less. Equivalently, a dilution series may include diluted samples having the highest concentrations of target cells, where the highest concentrations are 2%, 1.5%, 1%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, 0.05%, 0.02%, or 0.01%. For example, the method provided herein can be used to determine target cells or target markers that are considered to be residues or impurities in sample.For example, in the composition of natural killer cells, CD3+ cells or T cells can be considered to be residues or impurities.This method may enable determination of residues or impurities at very low levels, for example, less than 2%, less than 1.5%, less than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%, less than 0.05%, less than 0.02%, or less than 0.01%.

[0033] III.System The present disclosure also provides systems and devices that include any one or more fluorescence-based instruments and associated system components for implementing the disclosed methods and various aspects and configurations thereof. A system can include, for example, one or more fluorescence-based instruments, one or more computer processing devices coupled to the instrument(s), and optionally one or more of any cell sample, dyes, fluorescent dyes, cell markers, antibodies, or any combination thereof. For example, the present disclosure provides systems and devices that can analyze data generated by an instrument as described herein.

[0013] The present invention also contemplates a non-transitory computer-readable medium comprising processor-executable instructions for a processor to perform either data analysis or statistical calculations on the instrument. A system for validating fluorescence measurements made by a fluorescence-based instrument can include a fluorescence-based instrument, a computer coupled thereto, and a computer-readable medium comprising processor-executable instructions. As further detailed herein above, the instrument can be a flow cytometer that can be used to perform FACS.

[0034] IV. Kit The present disclosure also contemplates kits and devices containing any one or more cell samples, dyes, fluorescent dyes, cell markers, specific and / or non-specific antibodies, buffers, diluents, or any combination thereof for performing any of the validation methods described herein. The kits can include any one or more of the reagents in the amounts required for negatively and / or positively staining cells in a standard sample, for example, to validate a fluorescence-based analytical method for analyzing target cells in a test sample. The kits can include components required for negative staining, including any one or more of a fluorescent dead cell exclusion dye capable of specifically binding to a target marker (antigen) in the target cells, a non-specific antibody conjugated to a fluorescent dye, and an unconjugated specific antibody as described herein. The kits can include components required for positive staining, including a fluorescent dead cell exclusion dye and any one or more of a specific antibody conjugated to a fluorescent dye as described herein.

[0035] It will be understood that when a target cell marker is selected, the specific antibody for both negative and positive staining will be the same. Similarly, the fluorescent dye in the kit for both negative and positive staining will be the same. The kit may include written instructions, in hard copy or electronic format, for negative and positive staining of standard samples and for preparing a dilution series as described herein above. The instructions may additionally include one or more instructions for using the kit to perform any of the validation methods described herein; preparing a standard sample; and / or reading, analyzing, and interpreting the results of the validation method. The kit may usefully include one or more containers for preparing and / or containing any of the reagents, as well as components for labeling them.

[0036] definition Since various changes could be made in the above-described devices, kits, and methods without departing from the scope of the present invention, it is intended that all matter contained in the above description and in the examples given below be interpreted as illustrative and not limiting.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. References include: The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Margham, The Harper Collins Dictionary of Biology (1991), provide those of skill in the art with general definitions of many of the terms used in this invention. As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0038] When introducing elements of the present disclosure or its preferred embodiment(s), the article The words "a," "an," "the," and "said" are intended to mean that there are one or more of an element. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0039] The term "comprising" means "including, but not necessarily limited to"; it specifically indicates an open-ended inclusion or membership in the combination, group, series, etc. so described. As used herein, the terms "comprising" and "including" are inclusive and / or open-ended and do not exclude additional, unrecited elements or method steps. The term "consisting essentially of" is more restrictive than "comprising," but less restrictive than "consisting of." Specifically, the term "consisting essentially of" limits membership to certain materials or steps, and those that do not materially affect the essential characteristics of the claimed invention. [Example]

[0040] Any publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0041] The following examples are included to illustrate the present disclosure. Those skilled in the art should recognize that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure. However, those skilled in the art should, in light of the present disclosure, recognize that many changes can be made in the present disclosure and still obtain the same or similar results without departing from the spirit and scope of the present disclosure, and therefore, all matter shown should be interpreted as illustrative and not limiting. [Example]

[0042] Generation of spiked samples for validation of flow cytometry analysis In this example, spiked samples were generated for validation of flow cytometry analysis of "rare event targets" for determination of the LLOQ (lowest level of quantification) for residues or impurities present in the samples.

[0043] I. Method Figure 1 is a schematic diagram of the method. Positively stained and negatively stained cell samples were prepared using a "fluorochrome-conjugated target marker" (specific antibody) and an "isotype" (non-specific antibody), respectively. The target marker binding site in the negatively stained sample (isotype-stained sample) was blocked by using an unconjugated version of the specific antibody, i.e., an antibody to which the fluorochrome was not covalently bound. The positively stained sample was then spiked into the target nominal cell percentage using the negatively stained cells. The spiked sample was analyzed using a flow cytometer to obtain the measured percentage. The data (nominal and measured percentages of the spiked sample) were then used for statistical analysis to calculate validation parameters such as linearity, precision, accuracy, and LLOQ.

[0044] II. Sample Preparation This example describes the validation of the viable T cell percentage of the cell product using the following staining panel: To be:

[0045] [Table 1]

[0046] a) Preparation of negatively stained samples The cell test sample was divided into two parts. One part was stained according to the manufacturer's protocol using a designed staining panel: a combination of fluorescent dye-conjugated antibodies and dead cell exclusion dyes required to gate on the viable cell population of interest. Isotype controls were used in the staining mixture for the target cell population. In this example, the staining mixture used was as follows:

[0047] [Table 2]

[0048] After staining, unconjugated antibodies to the target marker were added to the final cell solution. In this example, unconjugated antibodies were purified with anti-CD3 antibodies. As shown in Figure 2, the FACS plots demonstrated the results obtained with negatively stained samples. Events shown here were gated on viable cells based on forward and side scatter and PI (to exclude dead cells).

[0049] b) Preparation of positively stained samples From the cell test sample in step 1, another portion of the cells according to the manufacturer's protocol was stained using the designed staining panel:

[0050] [Table 3]

[0051] FACS plots demonstrated the results obtained with positively stained samples, as shown in Figure 3. Events shown here were gated on live cells based on forward and side scatter and PI (to exclude dead cells).

[0052] c) Preparation of low level T cell spike samples Using the negatively stained cells from step 1), the positively stained samples were diluted to the highest planned spike level (nominal concentration of target cells), and then serial dilutions were prepared to systematically generate diluted samples of various nominal T cell percentages.

[0053] III. Sample Analysis Spiked samples were prepared as described above and used to obtain fluorescence measurements from a flow cytometer, each corresponding to a nominal concentration of CD3+ cells in the spiked sample. Routine statistical analysis of each nominal concentration and the corresponding fluorescence measurement from the flow cytometer was performed to assess the accuracy and reliability of the measurements, as described elsewhere herein.

[0054] This example describes a novel preparation of negatively stained cells using fluorescent dye-conjugated isotype and unconjugated antibodies. Typically, target cell population-depleted cells prepared using magnetic beads or cell sorters are used to prepare negative populations for spiked sample preparation. However, these depletion techniques rarely achieve up to 99.9% depletion of the target population. As a result, the negative population contributes at least 0.1% background, making it impossible to prepare spiked samples with less than 0.1%. Validating fluorescence-based analytical methods, such as FACS, with an LLOQ of less than 0.1% is challenging. This example demonstrates the effective preparation of negative populations with target cell concentrations less than 0.1%, enabling the preparation of spiked samples with a LLOQ of less than 0.1% for validation. Depending on the instrumentation and staining panel used, this method was able to provide an LLOQ of less than 0.01%. [Example]

[0055] Qualification Protocol for T Cell Quantification Methods of K-NK Drug Products This method qualification protocol provides a procedure (Protocol Reference: ATM-3343) for the qualification of T cell quantification methods for the quantification of CD3+-expressing cells in stimulated expanded natural killer cell compositions (K-NK drug products (DPs)), such as those disclosed in PCT Publication WO2018160673A1, the disclosure of which is incorporated herein by reference in its entirety. K-NK DPs were CD3+-depleted donor lymphocyte preparations in which NK cells were expanded to large numbers and high densities in vitro using 21-41 bbl plasma membrane (PM21) particles, such as those described in WO2018160673A1. The primary impurity in K-NK DPs was residual CD3+-expressing cells, which could induce graft-versus-host disease if administered to patients above clinically relevant limits. To detect and provide quantification of low concentrations of residual CD3+ expressing cells in K-NK DPs, a FACS-based method was developed to detect and / or quantify only viable CD3+ cell content, as described herein. The definitions provided in Tables 1 and 2 are used in the following examples.

[0056] [Table 4]

[0057] [Table 5]

[0058] A healthy donor PBMC (RM-PBMC225398) was used as a "positive control" sample to verify CD3+ staining and set the CD3+ gate for flow cytometry analysis. The PBMC were prepared by density gradient centrifugation. This positive control had no associated acceptance criteria, but was utilized for gating and verification of CD3-APC antibody addition and reactivity.

[0059] The test article utilized for this qualification was a cryopreserved NK cell sample, "20035 d14." The 20035 d14 sample was generated by further processing of NK cells expanded using the PM21 particle platform and was obtained after Day 7, and the 20035 d14 sample exhibited a K-NK product. After obtaining, these cells were stored in the laboratory for 7 days. On Day 14, 5 x 10 cells were cultured in one vial. 7 The cells were cryopreserved at 1000 x 1000 cells. This material did not contain Plasmalyte or HSA; however, the material was cryoformulated in CS10, and samples were washed in cell staining buffer (CSB) and resuspended in CSB prior to assay staining. Thus, regardless of initial formulation, the matrix of all test samples was standardized prior to assay. Performance testing during method development determined that the relative mean viable CD3+ content was 1.4% (n=8) for 20,035 d14 samples.

[0060] The instrument used was an Attune™ Classic Acoustic Focusing Flow Cytometer and the software used to collect and process the data was Attune Cytometric Software v2.1 and JMP v14.

[0061] The analytical method and technique were designed to detect and quantify low concentrations of CD3+ expressing cells. Briefly, cell-rich samples were washed multiple times in FACS staining buffer and then stained with CD3-APC antibody to detect CD3+ expressing cells and propidium iodide (PI) for dead cells. The samples were then resuspended to a predetermined concentration and acquired using an Attune Flow Cytometer. Flow cytometry data were processed using Attune Cytometric Software v2.1. To obtain a resolution with precise and accurate quantification at 0.01% cell content, 1 × 10 6Viable MNCs were obtained. Flow cytometry gating strategy was performed for MNC- * Singlet- * Survival MNC- * CD3+ was defined as viable CD3+. Qualification was designed to demonstrate the suitability of the method to detect and quantify the percentage of CD3+ expressing cells. Reported parameters included: % viable CD3+ cells.

[0062] Test sample preparation: For linearity, precision / accuracy, and accuracy analysis, the cryopreserved test sample 20035 d14 was divided into two preparations. One aliquot (1 x 10 8 aliquot (2 × 10 cells) was stained with IgG-APC and unlabeled CD3 antibody to generate a mock CD3- sample, and the other aliquot (2 × 10 7 Cells (0.001 cells) were stained with CD3-APC. To assess the linearity and relative precision of the method, an isotype-stained (IgG-APC) sample was used as a diluent and matrix sample for spiking with the fully stained sample. An unlabeled CD3 antibody was used to block the CD3-binding site of CD3-APC to prevent any carryover from the spiked sample. Prior to acquisition, the cell density was adjusted to achieve an event rate of approximately 1,000 events per second according to ATM-3343-0. A dilution scheme was generated using the concentrations of the initial positively stained CD3+ spiked sample and the negatively stained IgG-APC-stained sample. The CD3-APC-stained sample was divided into two samples: 1) one for determining the CD3+ percentage (%) and 2) one that served as the spiked sample. Similarly, one aliquot (1 / 10 of the preparation) of the IgG-APC-stained sample was used to determine the CD3+ percentage (%), and the remainder of the sample was used as a diluent in the linearity scheme.

[0063] Sample preparation: Samples were prepared according to ATM-3343 and scaled up as described in Table 3, including staining of positive control samples, IgG-APC samples, and CD3-APC samples. 8Because 2 × 10 cells were stained with IgG-APC, the reagents used were scaled up 10-fold. Additionally, a molar equivalent of unlabeled CD3 antibody was added to block CD3 binding sites. 7 Because CD3-APC staining was performed on 2 x 10 cells, the reagent used for ATM-3343 was doubled. No additional backup tubes were used. Each serially diluted sample contained 2 x 10 6 The samples had the same cell concentration of 1.00% / mL (minimum volume of 2 mL). The procedure for preparing both the linearity sample and the dilution series is shown in Figures 4 and 5, respectively. A four-fold dilution series was performed to five levels, with the dilution series reaching 1.00%. The dilution range of 0.004% was included. Spiked samples were prepared according to Table 4. In each case, one positive control sample, one IgG-APC sample and one CD3-APC sample were prepared and obtained.

[0064] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0065] [Table 7]

[0066] Data sets for the qualification parameters linearity, accuracy / trueness, precision, and specificity were generated from three cases run by two operators as shown in Figure 6. One test sample per case was processed according to the dilution scheme described above, and qualification was performed according to the scheme shown in Table 5 below. Samples were processed according to ATM-3343 (serial dilutions were equivalent to test samples). First, dilution level 5 was obtained, followed by dilution level 4, and so on up to the first dilution level. One data point per level per case was generated. For this qualification activity, one operator was assigned to one level for a total of six data points per dilution level. Three measurements were taken per day.

[0067] [Table 8]

[0068] Relative precision / relative accuracy: Relative precision or relative accuracy was assessed using the mean value of the test material to the corresponding nominal relative CD3+ percentage (%) per dilution level. Intermediate precision was determined for each case and analyst per dilution level.

[0069] The range of the method was determined from the dilution linear activity. The lower limit of quantitation was determined from the linear activity as the lowest dilution at which the level of precision and relative accuracy was adequate.

[0070] The antibodies and vital staining methods utilized for ATM-3343 are shown in Table 6. The average of six measurements (one per operator per case) was used for comparison for each sample type.

[0071] [Table 9]

[0072] The range of the method was determined from the dilution linear activity. The lower limit of quantitation was determined from the linear activity as the lowest dilution at which the level of precision and relative accuracy was adequate.

[0073] Linearity: Linear regression analysis was performed to determine R values ​​by fitting the measured relative proportions of all assay operators to the nominal relative proportions as fixed effects in a linear mixed model using JMP. Recovery rates for test samples at each level from the linearity assessment were compared to the nominal relative proportions when set as random effects and in a linear mixed model with operator interaction using JMP.

[0074] Precision (inter-assay variance): Variance estimates for intermediate precision were calculated by fitting relative proportions to a linear mixed mode in JMP with operator set as a random effect and nominal relative proportions as fixed effects.

[0075] Analysis from precision and accuracy analysis using linear mixed models generated using JMP The method range was determined. The limit of quantitation was determined from precision and accuracy analyses using linear mixed models generated using JMP. The LOQ was determined as the lowest sample concentration that met adequate precision and accuracy criteria.

[0076] No acceptance criteria were established for method qualification, but target criteria are listed in Table 7.

[0077] [Table 10] [Example]

[0078] CD3+ T cell quantification assay for K-NK drug products Objective: This Example reports the results of the ATM-3343 method qualification, which tested accuracy, precision, linearity, limit of quantitation, and range. Qualification parameters and procedures were presented in Example 2. These procedures were designed to qualify sensitive analytical outcomes from ATM-3343 and define the limit of quantitation for the % CD3+ residual T-cell impurity in K-NK DP. This Example also summarizes method qualification data generated by two operators on three occasions, as specified in Example 2. Regression analysis results comparing observed and predicted target measures were used to define outcomes for the qualification parameters in Example 2.

[0079] Materials and Reagents: The materials and reagents described in ATM-3343 were utilized for the execution of the qualification protocol as detailed in Example 2. Table 8 lists the materials and reagents used.

[0080] [Table 11]

[0081] The instruments used were the Classic Attune™ Acoustic Focusing Cytometer and Centrifuge (Thermo Scientific). The software used was Attune Cytometric Software v2.1 and JMP v.14.3.0.

[0082] Cryopreserved PBMC-225398 samples were used as positive controls to verify CD3+ gating and set the CD3+ gate for flow cytometry analysis. Preparation of PBMC-225398 was described in Example 2.

[0083] Test Article Materials: Cryopreserved batch 20035 d14 NK cells were utilized as the test article for this method qualification. Preparation of 20035 d14 cells for this method qualification is described in Example 2. 20035 d14 cells were expanded with PM21 particles after CD3+ depletion and utilized in a standardized sample matrix in this assay, resulting in K-NK DP. Batch 20035 d14 had a mean CD3+ content (%) of 1.389%, determined as part of method performance testing using a draft version of ATM-3343 (test article analyzed on two occasions with two sample vials and two replicates for n=8). 1.389% CD3+ was the expected (nominal) value for the test article.

[0084] Arrows from top to bottom table section) and were subsequently included in the accompanying statistical analysis.

[0085] Methodology: Method qualification to test linearity, precision / accuracy, and accuracy was described in Example 2. Briefly, frozen collected NK20035 d14 samples were stained with isotype-APC and CD3-APC antibodies according to ATM-3343. Isotype-stained cells were also mock-stained to prepare sample matrix diluent for CD3+ T cell spiking. These mock-stained cells were left unlabeled due to blocking of any CD3-APC binding of any carryover antibody solution during CD3+ T cell spiking. The samples were treated with purified CD3 antibodies. Therefore, serial dilutions for CD3+ T cell linearity estimation were prepared in the K-NK DP sample matrix according to the requirements set forth in Example 2 (Figure 6). Data analysis was performed using JMP v14.3.0 statistical analysis software. Nominal (predicted) and estimated (resulted) CD3+ percentage (%) values ​​were collated from reports by two analysts on three occasions each. The test article used for spiking required analysis with ATM-3343 to determine the CD3+ content of the spiked mock-stained preparations, generating measurements in each case. Therefore, six measurements were performed according to the test method, and these data points were included in the method qualification data analysis as an additional level (Level 0) used to evaluate linearity, precision, accuracy, and range. A residual plot of untransformed and Log (Log10, Log, Logist, Logit percentage, and Logit) transformed nominal and estimated CD3% was examined to select a regression model (Figure 7). In residual plot analysis, the untransformed data showed a biased spread of data, with smaller spread at lower levels and wider spread at higher levels. Therefore, the data were transformed for further analysis. Log10 transformation was the most convenient transformation resulting in improved spread of data in residual plots. Therefore, Log10-transformed data of "nominal" and "estimated" percentages of CD3 to three decimal places were selected for further analysis. Results reported in tables are presented to three decimal places.

[0086] [Table 12]

[0087] Results: Precision was analyzed using a linear mixed model with "operator" and "case" as random factors. Recovery was calculated according to the following formula:

number

[0088] The relative precision acceptance target criteria shown in Example 2 was 100±30%. The recovery rates (%) for the nominal CD3 percentage (%) dilution levels, i.e., 0.016% to 1.389%, were within the 100±30% acceptance criteria. The nominal CD3 percentage (%) at a CD3+ T cell level of 0.004% did not meet the precision acceptance criteria (Table 10 and Figure 8).

[0089] [Table 13]

[0090] Precision: Precision was analyzed using a linear mixed model in JMP v14.3.0 by using "operator" and "case" as random factors. Due to time limitations, sequential replicate measurements of the same sample were not obtained, as specified in Example 2. Because level 5 (nominal 0.004% CD3+ T cells) did not meet the precision / accuracy acceptance criteria, the nominal and estimated data at this level were excluded from the precision analysis. The acceptance criteria, as specified in Example 2, were to report a CV percentage (%) for precision and a CV of less than 20% for intermediate precision. The CV percentage results for "operator," "case," "residual," and intermediate precision were 0.288%, 2.386%, 4.819%, and 5.386%, respectively (Table 11). Therefore, levels 0 to 4 (1.389% to 0.016% CD3+ T cells) met the acceptance criteria for precision analysis.

[0091] [Table 14]

[0092] Linearity: To determine the linearity of CD3+ quantification, CD3- APC stained cells were spiked and serially diluted in K-NK DP sample matrix as shown in Figure 5. Five levels of CD3+ T cell percentage (% CD3+ T cells) were predicted (Levels 1–5, or 1.000%–0.004% CD3+ T cells). The sample matrix consisted of mock cells treated with isotype-APC and purified unlabeled human CD3 antibody (negative staining). Unlabeled (unconjugated) CD3 antibody was included to block any CD3 staining from carryover of CD3-APC antibody from the spiked sample into the sample matrix diluent. As an additional level (Level 0) for assessing linearity, undiluted test article data generated for the spike were included; therefore, linearity was assessed from 1.389%–0.004% CD3+ T cells. Because the estimated results from the nominal Level 5 (0.004% CD3+ T cells) did not meet the precision acceptance criteria, this level was excluded from the linearity analysis in the regression model. The regression plot and residuals from the prediction plot are shown in Figure 9. The linearity target criterion given in Example 2 was R > 0.8. The R observed in the regression analysis 2 was 0.999 (Table 12). Therefore, the linearity of CD3+ T cells from levels 0 to 4, 1.389% to 0.016%, met the target acceptance criteria.

[0093] [Table 15]

[0094] Range: A linear mixed model generated in JMP v14.3.0 was used to determine the range for quantification of CD3+ T cell percentage (%) from precision and accuracy analyses. Precision was analyzed for five dilution levels (1–5) in a 4-fold dilution scheme within the K-NK DP sample matrix, with an expected range of 1.000%–0.004% CD3+ T cells. The inclusion of undiluted test article added additional levels, increasing the upper range to 1.389% CD3+ T cells. The predicted quantification of CD3+ T cell percentage (%) for levels (0–4) was 1.389%–0.016% CD3+ T cells, but met the precision acceptance criteria (100% ± 30%). Level 5 (0.004% CD3+ T cells) did not meet the precision acceptance criteria. Therefore, the range of this method was defined as the quantification of CD3+ T cells from 1.389%–0.016% CD3+ T cells. Percentages of CD3+ T cells below this quantification range should be reported as BLOQ (below the lower limit of quantification), whereas percentages of CD3+ T cells above this quantification range should be reported as above the ULOQ (upper limit of quantification). To report results within this quantification range, a minimum of 1 × 10 6 Assay validity criteria had to be met to obtain viable single cells.

[0095] K-NK DP consisted of NK cells expanded to high density in vitro from a CD3+ T cell-depleted PBMC population using PM21 particles. Impurities in K-NK DP were likely residual CD3+ T cells after depletion, which may persist at levels lower than the defined limits of pre-established analytical assays. Therefore, to detect low levels (<0.3% CD3+ T cells) of residual CD3+ T cells as potential impurities in K-NK DP, we developed the flow cytometry-based ATM-3343 assay. ATM-3343 combines blocking and washing steps to specifically stain and capture CD3+ T cells. This method utilizes the human TruStain FcX reagent, CD3, for the specific detection and quantification of the percentage of CD3+ T cells in K-NK DP. -APC and isotype-APC reagents were utilized. A reference PBMC population was utilized as a positive control. Given the requirement for low-level CD3+ quantification, ATM-3343 was used to measure 1 x 10 CD3+ from K-NK DP samples to meet statistical expectations. 6 It was stipulated that a single survival event be acquired.

[0096] Data on qualification parameters such as accuracy, precision, linearity, and range were generated by two operators on each of three occasions. In each case, five levels of a given CD3+ concentration (%) were obtained, as well as standards and isotype-APC stained cells or CD3-APC stained cells (Table 8). In each case, K-NK These dilution levels were prepared by spiking CD3+ T cells in a mock (isotype and unlabeled CD3 treated) sample matrix of DP cells. Based on ATM-3343 and the sequential gating strategy shown in Example 2 (total events > MNCs > single cells > viable cells > CD3+), 1 × 10 T cells were collected from each sample. 6 Flow cytometry acquisition parameters were set to acquire viable single cells.

[0097] [Table 16]

[0098] Summary: This method qualification was performed according to the procedures described in Example 2, "Method Qualification Protocol for ATM-3343 T Cell Quantification." The K-NK Drug Product (DP) consisted of NK cells expanded in vitro to high density from a CD3+ T cell-depleted donor PBMC population. 1x10 T cell counts were determined by flow cytometry. 6 ATM-3343 was used to detect and quantify low frequencies of residual CD3+ T cells within a population of viable single cells. The data evaluated in this Method Qualification Report were acquired by two analysts on three occasions each. Evaluation of the accuracy, precision, and linearity parameters presented in Example 2 demonstrated the suitability of ATM-3343 for its intended use: to detect and quantify residual CD3+ T cells in K-NK DP. ATM-3343 was precise and accurate for measuring different levels of CD3+ T cell impurities in K-NK DP within the range of 0.016% to 1.389% (Table 14). An R of 0.999 was observed for quantification of the CD3+ percentage (%) in this range. 2 The values ​​met the K-NK NP sample matrix linearity acceptance criteria, thereby establishing the method's limit of quantitation (LOQ) of 0.016% detection of CD3+ T cells. Detection of CD3+ T cells by ATM-3343 was specific, reliably distinguishing positive events from the background noise of isotype staining. ATM-3343 required 1 x 10 detection from the K-NK DP to meet efficacy criteria. 6 A single event of 1 × 10 viable events was required to be obtained. Overall, the protocol used was appropriate for the determination of CD3+ T cell impurities in K-NK DP. Percentages of CD3+ T cells below or above the quantification range of 0.016% to 1.389% should have been reported as below the BLOQ (lower limit of quantification) or above the ULOQ (upper limit of quantification), respectively. 6 This quantitative range reported from the acquisition of survival single events could be set as the validity and system suitability criterion.

[0099] [Table 17]

[0100] Conclusions: ATM-3343 was qualified to quantify low (≥0.016%) CD3+ T cell percentages (%) in K-NK DP. Precision, precision, and linearity for the range of quantification of CD3+ T cell percentages (%) between 0.016% and 1.389% were determined to be acceptable according to qualification parameters. ATM-3343 assay validity and system suitability criteria were required to reliably measure CD3+ T cell impurities in K-NK DP.

Claims

1. 1. A method for validating fluorescence-based analytical instrument measurements of a stained target cell population in a test sample, comprising: a. negatively staining or negatively staining cells in a first portion of a standard sample containing target cells expressing a target cell marker; b. positively staining or positively staining the target cells in the second portion of the standard sample; c. passing a first portion of the standard sample through the instrument to obtain a fluorescence measurement indicative of the target cell concentration in the first portion of the standard sample, and passing a second portion of the standard sample through the instrument to obtain a fluorescence measurement indicative of the target cell concentration in the second portion of the standard sample; d. preparing a dilution series including a plurality of diluted samples, each having a nominal concentration of target cells based on the fluorescence measurements obtained in (c), each nominal cell concentration being greater than the concentration of target cells indicated by the fluorescence measurements of the negatively stained first portion in (a), and the nominal concentration in each diluted sample being different from the nominal concentration in each of the remaining diluted samples; e. passing the series of diluted samples of (d) through the instrument to obtain a series of fluorescence measurements, including a fluorescence measurement for each diluted sample; f. For each diluted sample, compare the nominal cell concentration in (d) with the fluorescence measurements in (e) to quantify the performance of the staining method in the instrument. The method comprising:

2. 2. The method of claim 1, wherein the comparison in (f) comprises performing statistical calculations on the difference between the nominal cell concentration and the fluorescence measurement value of each diluted sample for each diluted sample to determine at least one of linearity, range, precision, accuracy, limit of detection (LOD), and lower limit of quantitation (LLOQ) for the instrument and staining method.

3. 2. The method of claim 1, wherein negative staining of the target cells in the first portion of the standard sample in (a) comprises introducing into the first portion of the standard sample (i) a fluorescent dead cell exclusion dye, (ii) a nonspecific antibody conjugated to a fluorescent dye, and (iii) a specific antibody capable of specifically binding to a target marker in the target cells.

4. 4. The method of claim 3, wherein positive staining of target cells in the cells in the second portion of the standard sample of (b) comprises introducing into the second portion of the standard sample (i) a fluorescent dead cell exclusion dye, and (ii) a specific antibody of claim 3 conjugated to the fluorescent dye of claim 3.

5. The method of claim 3 or 4, wherein the fluorescent dead cell exclusion dye is selected from nucleic acid binding dyes, propidium iodide, DAPI, DRAQ7, 7-AAD, TO-PRO-3 and amine reactive dyes.

6. The method of claim 3 , wherein the fluorescent dye-conjugated non-specific antibody comprises an antibody that lacks the ability to specifically bind to a cellular antigen.

7. 8. The method of claim 7, wherein the fluorescent dye-conjugated non-specific antibody comprises any one of IgD, IgG, IgA, IgM, or IgE conjugated to a fluorescent dye.

8. The fluorescent dyes were allophycocyanin (APC), APC C750, APC AF700, brilliant violet (BV) 421, BV510, hilite 7 (H7) BV605, BV650, PE CF594, fluorescein isothiocyanate (FITC), R-phycoerythrin (PE or R-PE), PE-Cy7 (cyanine dye Cy7), and FITC.

9. The method of any one of claims 3 to 8, wherein the APC is selected from the group consisting of APC-Cy7 (PE linked to Cy7), APC-Cy7 (APC linked to the cyanine dye Cy7), and APC-H7 (APC linked to the Cy analogue Hilite7 (H7)).

9. 9. The method of claim 1, wherein the target cell marker is selected from CD3 as a T cell marker, CD19 as a B cell marker, CD235a as an erythrocyte marker, CD56 as a natural killer (NK) cell marker, CD14 as a monocyte marker, and CD66b as a granulocyte marker.

10. The method of claim 1 , wherein the calculation of (f) is performed by a processing device to which the device is coupled.

11. 3. The method of claim 2, wherein determining the LLOQ comprises identifying a concentration of target cells associated with a predetermined criterion of precision and a predetermined criterion of accuracy.

12. 10. The method of claim 1, wherein the test sample comprises target cells present at a concentration of 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, or 0.02% or less.

13. 13. The method of claim 12, wherein the dilution series comprises diluted samples having the highest concentrations of target cells, the highest concentrations being 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.02%.

14. 5. The method of claim 4, wherein the negatively stained first portion of the test sample is prepared in the absence of a cell depletion step.

15. The method of claim 1 , wherein the fluorescence-based instrument is a flow cytometer.

16. 1. A method for validating flow cytometric measurements of stained target cell populations in a test sample, comprising: a. negatively staining or negatively staining cells in a first portion of a standard sample containing target cells expressing a target cell marker; b. positively staining or positively staining the target cells in the second portion of the standard sample; c. passing both the first portion and the second portion of the standard sample through a flow cytometer to obtain fluorescence measurements indicative of target cell concentrations in each of the first portion of the standard sample and the second portion of the standard sample; d. preparing a series of diluted samples based on the fluorescence measurements obtained in (c), each having a nominal concentration of target cells that varies systematically in the dilution series, the nominal cell concentration of at least one diluted sample being greater than the concentration of target cells indicated by the fluorescence measurements of the negatively stained first portion in (a); e. passing the series of diluted samples of (d) through a flow cytometer to obtain a series of fluorescence measurements, including a fluorescence measurement for each diluted sample; f. For each diluted sample, compare the nominal cell concentration in (d) with the fluorescence measurements in (e) to quantify the performance of the staining method in the flow cytometer. The method comprising:

17. The comparison in (f) involves performing statistical calculations on the difference between the nominal cell concentration and the fluorescence measurement value of each diluted sample to determine at least one of the linearity, range, precision, accuracy, limit of detection (LOD), and lower limit of quantitation (LLOQ) for the instrument and staining method.

17. The method of claim 16, comprising determining

18. 17. The method of claim 16, wherein the negative staining of the target cells in the first portion of the standard sample in (a) comprises introducing into the first portion of the standard sample (i) a fluorescent dead cell exclusion dye, (ii) a non-specific antibody conjugated to a fluorescent dye, and (iii) a specific antibody not conjugated to a fluorescent dye that is capable of specifically binding to a target marker in the target cells.

19. 19. The method of claim 18, wherein positive staining of target cells in cells in the second portion of the standard sample of (b) comprises introducing into the second portion of the standard sample (i) a fluorescent dead cell exclusion dye, and (ii) a specific antibody of claim 18 conjugated to a fluorescent dye of claim 18.

20. 20. The method of claim 18 or 19, wherein the fluorescent dead cell exclusion dye is selected from nucleic acid binding dyes, propidium iodide, DAPI, DRAQ7, 7-AAD, TO-PRO-3 and amine reactive dyes.

21. 20. The method of claim 18, wherein the non-specific fluorochrome-conjugated antibody comprises an antibody that lacks the ability to specifically bind to a cellular antigen.

22. 22. The method of claim 21, wherein the non-specific fluorochrome-conjugated antibody comprises any one of IgD, IgG, IgA, IgM, or IgE conjugated to a fluorochrome.

23. 23. The method of any one of claims 18 to 22, wherein the fluorescent dye is selected from allophycocyanin (APC), APC C750, APC AF700, brilliant violet (BV) 421, BV510, hilite 7 (H7) BV605, BV650, PE CF594, fluorescein isothiocyanate (FITC), R-phycoerythrin (PE or R-PE), PE-Cy7 (PE linked to the cyanine dye Cy7), APC-Cy7 (APC linked to the cyanine dye Cy7), APC-H7 (APC linked to the Cy analogue Hilite 7 (H7)).

24. 24. The method of any one of claims 16 to 23, wherein the target cell marker is selected from CD3 as a T cell marker, CD19 as a B cell marker, CD235a as an erythrocyte marker, CD56 as a natural killer (NK) cell marker, CD14 as a monocyte marker, and CD66b as a granulocyte marker.

25. The method of any one of claims 16 to 23, wherein the target cell population is a T cell population and the target cell marker is CD3.

26. 17. The method of claim 16, wherein the calculation of (f) is performed by a processing device to which the device is coupled.

27. 18. The method of claim 17, wherein determining the LLOQ comprises identifying a concentration of target cells associated with a predetermined criterion of precision and a predetermined criterion of accuracy.

28. 20. The method of claim 18, wherein the test sample comprises target cells present at a concentration of 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, or 0.02% or less.

29. The dilution series consisted of diluted samples with the highest concentration of target cells, with the highest concentrations being 2%, 1%, 29. The method of claim 28, comprising a diluted sample that is 0.5%, 0.2%, 0.1%, 0.05%, or 0.02%.

30. 30. The method of any one of claims 1 to 29, wherein the series of diluted samples comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 diluted samples.

31. 28. A non-transitory computer-readable medium comprising instructions for a computer processing device to perform the comparison of (f) of claim 1 or claim 16, or to perform the statistical calculation of claim 2, 12, 17, or 27.

32. 32. A system for validating fluorescence measurements on a test sample taken by a fluorescence-based instrument, the system comprising: a fluorescence-based instrument; and a computer coupled to the fluorescence-based instrument, the computer comprising the computer-readable medium of claim 31.

33. 33. The system of claim 32, wherein the fluorescence-based instrument is a flow cytometer.

34. 1. A kit comprising reagents for staining cells in a standard sample for validating a fluorescence-based analytical method for analyzing target cells in a test sample, the kit comprising: (i) a negative staining reagent comprising (a) a fluorescent dead cell exclusion dye, (b) a non-specific antibody conjugated to the fluorescent dye, and (c) a specific antibody not conjugated to the fluorescent dye that is capable of specifically binding to a target marker on the target cells; (ii) a positive staining reagent comprising (a) a fluorescent dead cell exclusion dye, and (b) a specific antibody conjugated to the fluorescent dye; and (iii) instructions for preparing a dilution series including a plurality of diluted samples, each having a nominal concentration of target cells varying in the dilution series, including: (a) negatively staining a first portion of the standard sample; (b) positively staining a second portion of the standard sample; and (c) a nominal cell concentration of at least one diluted sample greater than the concentration of target cells indicated by the fluorescence measurements of the negatively stained first portion. The kit comprising:

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