WO2019210306A1

WO2019210306A1 - Blood preparation

Info

Publication number: WO2019210306A1
Application number: PCT/US2019/029666
Authority: WO
Inventors: Lynn MARKOWITZ
Original assignee: Momenta Pharmaceuticals , Inc.
Priority date: 2018-04-27
Filing date: 2019-04-29
Publication date: 2019-10-31

Abstract

The disclosure relates to a method of preparing blood for a flow cytometry assay by freezing the blood in a preservative

Description

BLOOD PREPARATION

TECHNICAL FIELD

The disclosure relates to methods of preparing blood for biological assays.

BACKGROUND

Storing and transporting whole blood specimens is necessary for clinical and diagnostic analysis. There are many challenges associated with the storage and transport of blood, including reduction in the stability of samples as a result of fluctuating temperatures during storage and transit. For example, whole blood samples collected at remote clinical sites may have to be transported long distances to laboratories for analysis. Refrigeration of samples over long distances can be difficult to ensure and expensive. Furthermore, cryopreservation of whole blood samples can result in the lysis of blood cells and destruction of the samples.

SUMMARY

In one aspect, the disclosure related to a method of preparing blood for a biological assay, the method comprising the steps of: providing ethylene diamine tetra acetic acid (EDTA) and diazolidinyl urea; providing a blood sample; adding the blood sample to the EDTA and diazolidinyl urea to create a mixture; and freezing the mixture.

In some embodiments, the frozen mixture is thawed and tested in a biological assay. In some embodiments, the blood is incubated with EDTA and diazolidinyl urea prior to freezing. In some embodiments, the blood is incubated with EDTA and diazolidinyl urea for 15 to 60 minutes prior to freezing. In some embodiments, the ratio of volume of the EDTA and diazolidinyl urea to a combined volume of the blood and the EDTA and diazolidinyl urea is about 1 : 100 to about 2: 100. In some embodiments, the blood is stabilized when added to the EDTA and diazolidinyl urea to create a mixture, and wherein the stabilization comprises incubating the mixture for 15 to 60 minutes.

In some embodiment, the mixture is frozen at or around -70°C. In some embodiments, the mixture is frozen for at least 12 hours prior to testing in a biological assay. In some embodiments, the mixture is frozen for 24 hours prior to testing in the biological assay. In some embodiments, the mixture is frozen for at least 8 days prior to testing in the biological assay.

In some embodiments, the biological assay is a flow cytometry assay. In some embodiments, the flow cytometry assay is used to determine receptor occupancy by an antibody. In some embodiments, the antibody is M281.

In some embodiments, the blood is added to a Cyto-Chex™ BCT storage tube. In some embodiments, the contents of the Cyto-Chex™ BCT storage tube are transferred to a second tube prior to freezing.

In some embodiments, the blood is drawn from a subject who was administered a monoclonal antibody. In some embodiments, the blood is drawn from a subject who was administered M281.

In some embodiments, the frozen mixture is transported for testing using a biological assay.

In another aspect, the disclosure related to a method of preparing blood for a biological assay, the method comprising the steps of: providing ethylene diamine tetra acetic acid (EDTA) and diazolidinyl urea; providing blood; adding the blood to the EDTA and diazolidinyl urea to create a mixture; freezing the mixture; thawing the mixture; and testing the thawed mixture in the biological assay. In some embodiments, the biological assay is a flow cytometry assay to test receptor occupancy by an antibody.

In some embodiments, the blood is incubated with EDTA and diazolidinyl urea prior to freezing. In some embodiments, the blood is incubated with EDTA and diazolidinyl urea for 15 to 60 minutes prior to freezing. In some embodiments, the ratio of volume of the EDTA and diazolidinyl urea to a combined volume of the blood and the EDTA and diazolidinyl urea is about 1 : 100 to about 2: 100. In some embodiments, the blood is stabilized when added to the EDTA and diazolidinyl urea to create a mixture, and wherein the stabilization comprises incubating the mixture for 15 to 60 minutes.

In some embodiments, the mixture is frozen at or around -70°C. In some embodiments, the mixture is frozen for at least 12 hours prior to testing in a biological assay. In some embodiments, the mixture is frozen for 24 hours prior to testing in the biological assay. In some embodiments, the mixture is frozen for at least 8 days prior to testing in the biological assay. In some embodiments, the blood is added to a Cyto-Chex™ BCT storage tube. In some embodiments, the contents of the Cyto-Chex™ BCT storage tube are transferred to a second tube prior to freezing.

In some embodiments, the antibody is M281. In some embodiments, the blood is drawn from a subject who was administered the antibody. In some embodiments, the blood is drawn from a subject who was administered M281.

In another aspect, the disclosure relates to a method of freezing a blood sample comprising: providing a blood sample; providing a preservative; mixing the blood sample with the preservative to form a mixture, and freezing the mixture.

In some embodiments, the preservative is diazolidinyl urea. In some

embodiments, the mixture further comprises adding an anticoagulant to the mixture prior to freezing. In some embodiments, the anticoagulant is EDTA.

In another aspect, the disclosure relates to a method of performing a biological assay comprising: mixing ethylene diamine tetra acetic acid (EDTA), diazolidinyl urea, and blood; freezing the mixture; and performing the biological assay.

In some embodiments, the mixture is transported after the mixture is frozen, but before the biological assay is performed. In some embodiments, the mixture is stored in freezing conditions after the mixture is frozen, but before the biological assay is performed. In some embodiments, the freezing conditions are at or around -70°C.

In some embodiments, the biological assay is a flow cytometry assay to test receptor occupancy by an antibody. In some embodiments, the blood comprises a monoclonal antibody.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG 1 depicts a bar chart showing the results of a flow cytometry assay testing receptor occupancy by an antibody using fresh and cryopreserved blood samples that were fixed using the methods described herein. DETAILED DESCRIPTION

The disclosure relates to the discovery that a blood sample that is added a preservative such as diazolidinyl urea, for example in a Ctyo-Chex™ BCT tube, can be frozen, thawed, and subsequently used in a biological assay, e.g., a receptor occupancy flow cytometry assay. Freezing the blood sample in these tubes does not damage the blood sample, e.g., by lysing blood cells or reducing the stability of the blood sample, so that it cannot be used in an assay. Freezing blood sample in these tubes also does not impair the effectiveness of biological assay testing. Rather, freezing blood with diazolidinyl urea, can improvesignals produced in biological assays, such as flow cytometry assays, e.g., by increasing the signals generated in the assay. Further, freezing blood with diazolidinyl urea , can improve the stability of the blood. In some

embodiments, blood is added to a preservative such as diazolidinyl urea, for example in a Ctyo-Chex™ BCT tube, incubated for a period of time, e.g., 5, 10 , 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 or more minutes, and then transferred to another vessel, e.g., a tube, vial or container that is designed to be stored and/or transported for long periods of time at freezing temperatures. In some embodiments, blood is drawn from a subject and added to a container (e.g., a tube, vial, receptacle, etc.) containing at least one anticoagulant and at least one preservative. In some embodiments, the blood is drawn from a subject and added to a container (e.g., a tube, vial, receptacle, etc.) containing at least one anticoagulant, at least one preservative, and polyacrylic acid or a suitable acid having a pH ranging from about one to about seven. In some embodiments, the preservative increases the stability of the blood. In some embodiments, the anticoagulant can be ethylene diamine tetra acetic acid (EDTA), e.g., K3EDTA. In some embodiments, the preservative can be diazolidinyl urea. In some embodiments, the blood is added to a container (e.g, a vial, tube, receptacle, etc.) containing EDTA, e.g.,

K3EDTA, and diazolidinyl urea. In some embodiments, the container is a freezing container. In some embodiments, the blood is added to a Cyto-Chex™ BCT storage tube (Streck Laboratories, Inc.). In some embodiments, the blood is added to a tube as described in ETS 2004/0137417, which is herein incorporated by reference in its entirety. In some embodiments, the preservative is selected from the group consisting of diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5dimethylhydantoin, dimethylol urea, 2-bromo-2.-nitropropane-l,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymethyl-l-laza-3, 7-dioxabicyclo [3.3.0] octane, 5-hydroxymethyl-l- 1 aza-3,7dioxabicyclo [3.3.0] octane, 5 -hydroxypoly[methyleneoxy] methyl- 1 - 1 aza- 3,7dioxabic yclo [3.3.0] octane, quaternary adamantine and combinations thereof.

In some embodiments, a mixtures described herein containing a blood sample are frozen for some period of time prior to testing in a biological assay, e.g., a receptor occupancy flow cytometry assay. In some embodiments, the mixtures described herein are stored at 0°C or at a lower temperature prior to testing, e.g., at -l0°C, -20°C, -30°C, - 40°C, -50°C, -60°C, -70°C, or -80°C. In some embodiments, the mixtures described herein are stored for at least 2 hours at 0°C or at a lower temperature, e.g., -70°C, prior to testing, e.g., for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10, days, 11 days, 12 days, 13 days, 14 days, 20 days, 30 days, or more. In some embodiments, the frozen mixture containing blood and a preservative, e.g., diazolidinyl urea, is stored and/or transported for testing using a biological assay.

In some embodiments, blood is drawn from a subject who has been administered a therapeutic agent, e.g., an antibody, and then the blood is added to a preservative, as described herein. In some embodiments, the therapeutic agent is a monoclonal antibody. In some embodiments, the antibody is an anti-FcRn antibody. In some embodiments, the therapeutic agent is M281, as described in EiS 2017/0052200, US 2018/0016334, and in WO 2017/059276, which are incorporated by reference herein in their entirety. In some embodiments, the blood is drawn from a pregnant woman

In some embodiments, the antibody, e.g., M281, binds to an intracellular receptor, e.g., FcRn receptor. Thus, the antibody can bind to an intracellular target such as a receptor. The antibody can bind to an intracellular target within a sample fixed with diazolidinyl urea (e.g., in a Ctyo-Chex™ tube, frozen for a period of time, thawed, and subsequently tested using a flow cytometry assay for receptor occupancy. Thus, intracellular binding to a target receptor can be detected in fixed blood cells that have been preserved with diazolidinyl urea and frozen using flow cytometry, rather than just detection of cell surface antibody-receptor binding. In some embodiments, the methods as described herein are useful in improving receptor occupancy assays, such that the assays can be used to adjust the dosages and/or administration schedule of a given therapeutic antibody. For example, the methods can make it easier to transport a blood sample for testing because blood can be frozen with the preservative, thereby reducing the possibility that the sample will be degraded prior to testing compared to samples kept at room temperature. The methods also increase signal levels in flow cytometry assays (as shown in Example 1), and can make it easier to detect and measure assay signals, e.g., weak assay signals. In some embodiments, the methods described herein can be used to adjust the dosages and/or administration schedule of a therapeutic antibody in a subject that has lost or gained weight during the course of treatment with the antibody (e.g., in a pregnant woman that is administered the antibody). In some embodiments, the methods described herein are used to measure exposure in a subject to anti-FcRn antibodies, e.g., blood is prepared as described herein, frozen, and used in a flow cytometry assay to measure exposure to an anti-FcRn antibody.

As used herein, a“biological assay” can be any assay used to measure or determine a biological activity. In some embodiments, a biological can be a diagnostic assay. In some embodiments, a biological assay can be a flow cytometry assay. In some embodiments, a biological assay can be a receptor occupancy assay.

In some embodiments, blood can be drawn from a subject who was administered a therapeutic agent, e.g., M281, at a clinical site, prepared and frozen as described herein, and then shipped or stored a long period of time at freezing temperatures, e.g., shipped to a distant laboratory site for analysis. Therefore, it is anticipated that the methods described herein will be useful in the conduct of clinical studies and in diagnostic studies.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: Whole Blood Collection and Preparation for Receptor

Occupancy Flow Cytometry Assay

Blood drawn from a healthy subject was collected in K3EDTA tubes. The samples were then spiked with labeled-M28l at 50% (at a concentration of 1 ng/ml) or 100% (at a concentration of 2 ng/ml) and incubated for 30 minutes at 37°C. The M281 was labeled with Dylight-650 (Thermo Scientific). One sample was not spiked with labeled-M28l to serve as a fluorescence minus one (FMO) control for flow cytometry. Other labeled antibodies for cell surface markers were added to the samples, including antibodies for CD20, CD3, CD45, CD14. The blood samples were then transferred to Cyto-Chex™ BCT tubes (Streck, Catalog Number 11-716-358) and allowed to incubate at room temperature for 20 minutes to 1 hour for fixation. Aliquots of the blood was then transferred to plastic tubes to test the effects of different storage conditions on the outcome of a flow cytometry assay to test the occupancy of the FcRn receptor by M281. Aliquots of blood (non-spiked or spiked with 50% or 100% with labeled M281) were tested right away (at 0 hours) without having been frozen. Other aliquots of blood were frozen at -70°C for 24 hours or 8 days prior to testing (at 24 hours and 8 days, respectively). Frozen samples were thawed in a warm water bath prior to testing them in a flow cytometry receptor occupancy assay.

FIG. 1 shows the results of a standard flow cytometry receptor occupancy assay using fresh blood or blood that had been frozen for 24 hours or 8 days before testing. Freezing the blood samples resulted in greater signals in the flow cytometry assay (MEF) relative to the signals detected using fresh blood samples that had never been frozen. In addition, freezing blood for a longer period of time (8 days versus 24 hours) prior to testing the blood using this assay resulted in greater fluorescence signal. Spiking the blood with 50% and 100% M281 (compound X in Fig. 1) reduced the signal, as expected. The FMO control samples (non-spiked with M281 or spiked with 50% or 100% M281) produced low signal intensity, as anticipated.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing blood for a biological assay, the method comprising the steps of:

providing ethylene diamine tetra acetic acid (EDTA) and diazolidinyl urea; providing a blood sample;

adding the blood sample to the EDTA and diazolidinyl urea to create a mixture; and

freezing the mixture.

2. The method of claim 1, wherein the frozen mixture is thawed and tested in a

biological assay.

3. The method of claim 1, wherein the blood is incubated with EDTA and diazolidinyl urea prior to freezing.

4. The method of claim 3, wherein the blood is incubated with EDTA and diazolidinyl urea for 15 to 60 minutes prior to freezing.

5. The method of claim 1, wherein the ratio of volume of the EDTA and diazolidinyl urea to a combined volume of the blood and the EDTA and diazolidinyl urea is about 1 : 100 to about 2: 100.

6. The method of claim 1, wherein the blood is stabilized when added to the EDTA and diazolidinyl urea to create a mixture, and wherein the stabilization comprises incubating the mixture for 15 to 60 minutes.

7. The method of claim 1, wherein the mixture is frozen at or around -70°C.

8. The method of claim 1, wherein the mixture is frozen for at least 12 hours prior to testing in a biological assay.

9. The method of claim 1, wherein the mixture is frozen for 24 hours prior to testing in the biological assay.

10. The method of claim 1, wherein the mixture is frozen for at least 8 days prior to testing in the biological assay.

11. The method of claim 1, wherein the biological assay is a flow cytometry assay.

12. The method of claim 11, wherein the flow cytometry assay is used to determine receptor occupancy by an antibody.

13. The method of claim 12, wherein the antibody is M281.

14. The method of claim 1, wherein the blood is added to a Cyto-Chex™ BCT storage tube.

15. The method of claim 14, wherein the contents of the Cyto-Chex™ BCT storage tube are transferred to a second tube prior to freezing.

16. The method of claim 1, wherein the blood is drawn from a subject who was

administered a monoclonal antibody.

17. The method of claim 1, wherein the blood is drawn from a subject who was

administered M281.

18. The method of claim 1, wherein the frozen mixture is transported for testing using a biological assay.

19. A method of preparing blood for a biological assay, the method comprising the steps of: providing ethylene diamine tetra acetic acid (EDTA) and diazolidinyl urea; providing blood;

adding the blood to the EDTA and diazolidinyl urea to create a mixture;

freezing the mixture;

thawing the mixture; and

testing the thawed mixture in the biological assay.

20. The method of claim 19, wherein the biological assay is a flow cytometry assay to test receptor occupancy by an antibody.

21. The method of claim 19, wherein the blood is incubated with EDTA and diazolidinyl urea prior to freezing.

22. The method of claim 19, wherein the blood is incubated with EDTA and diazolidinyl urea for 15 to 60 minutes prior to freezing.

23. The method of claim 19, wherein the ratio of volume of the EDTA and diazolidinyl urea to a combined volume of the blood and the EDTA and diazolidinyl urea is about 1 : 100 to about 2: 100.

24. The method of claim 19, wherein the blood is stabilized when added to the EDTA and diazolidinyl urea to create a mixture, and wherein the stabilization comprises incubating the mixture for 15 to 60 minutes.

25. The method of claim 19, wherein the mixture is frozen at or around -70°C.

26. The method of claim 19, wherein the mixture is frozen for at least 12 hours prior to testing in a biological assay.

27. The method of claim 19, wherein the mixture is frozen for 24 hours prior to testing in the biological assay.

28. The method of claim 19, wherein the mixture is frozen for at least 8 days prior to testing in the biological assay.

29. The method of claim 19, wherein the blood is added to a Cyto-Chex™ BCT storage tube.

30. The method of claim 29, wherein the contents of the Cyto-Chex™ BCT storage tube are transferred to a second tube prior to freezing.

31. The method of claim 20, wherein the antibody is M281.

32. The method of claim 20, wherein the blood is drawn from a subject who was

administered the antibody.

33. The method of claim 20, wherein the blood is drawn from a subject who was

administered M281.

34. A method of freezing a blood sample comprising:

providing a blood sample;

providing a preservative;

mixing the blood sample with the preservative to form a mixture; and

freezing the mixture.

35. The method of claim 34, wherein the preservative is diazolidinyl urea.

36. The method of claim 34, further comprising adding an anticoagulant to the mixture prior to freezing.

37. The method of claim 36, wherein the anticoagulant is EDTA.

38. A method of performing a biological assay comprising:

mixing ethylene diamine tetra acetic acid (EDTA), diazolidinyl urea, and blood; freezing the mixture; and

performing the biological assay.

39. The method of claim 38, wherein the mixture is transported after the mixture is

frozen, but before the biological assay is performed.

40. The method of claim 38, wherein the mixture is stored in freezing conditions after the mixture is frozen, but before the biological assay is performed.

41. The method of claim 40, wherein the freezing conditions are at or around -70°C.

42. The method of claim 38, wherein the biological assay is a flow cytometry assay to test receptor occupancy by an antibody.

43. The method of claim 38, wherein the blood comprises a monoclonal antibody.