Method for preparing sterilized composition for stabilizing extracellular nucleic acid

Abstract

The present invention provides a method for preparing a sterilized composition for stabilizing extracellular nucleic acids. Also provided are sterilizable compositions, wherein the compositions in sterilized form are suitable for stabilizing extracellular nucleic acid populations of biological samples. Other useful methods, devices, kits and uses are also provided. Other sterilizable and sterilized compositions described herein are also suitable for stabilizing intracellular nucleic acids (e.g., intracellular DNA such as genomic DNA and / or intracellular RNA) and cell characteristics, such as cell surface proteins and / or cell morphology.

Description

[0001] This application is a divisional application of international application date November 21, 2016, international application number PCT / EP2016 / 078330, which entered the Chinese national phase on May 18, 2018, application number 201680067712.2, and invention name “Method for preparing a sterilized composition for stabilizing extracellular nucleic acids”. Technical Field

[0002] The present invention provides a method for preparing a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample. Also provided are sterilizable compositions, wherein the compositions in sterilized form are suitable for stabilizing an extracellular nucleic acid population of a biological sample. Other useful methods, devices, kits and uses are also provided. Specifically, sterilizable and sterilized compositions suitable for stabilizing cell-containing samples such as blood samples are provided. In embodiments, these compositions are suitable for stabilizing intracellular nucleic acids (e.g., intracellular DNA and / or intracellular RNA), cell surface features and / or cell morphology. Background Art

[0003] Extracellular nucleic acids have been identified in blood, plasma, serum and other body fluids. Extracellular nucleic acids found in corresponding samples have a certain degree of resistance to degradation because they are protected from the influence of nucleases (for example, because they are secreted in the form of protein lipid complexes, combined with proteins or contained in vesicles). In many medical conditions, malignant tumors and infection processes, there are changes in extracellular nucleic acids such as DNA and / or RNA levels, such as increasing, which is interesting, especially for screening, diagnosis, prognosis, monitoring disease progression, identifying potential therapeutic targets and monitoring treatment responses. In addition, the increase of fetal DNA / RNA in maternal blood is being used to determine, for example, gender identity, assess chromosomal abnormalities and monitor pregnancy-related complications. Therefore, extracellular nucleic acids are particularly useful for non-invasive diagnosis and prognosis and can be used as, for example, diagnostic markers in many application fields such as non-invasive prenatal genetic testing, oncology, transplantation medicine or many other diseases, and therefore have diagnostic relevance (such as fetus or tumor-derived nucleic acids). However, extracellular nucleic acids are also found in healthy humans. In addition to mammalian extracellular nucleic acids derived from, for example, tumor cells or fetuses, cell-containing samples can also include other target nucleic acids that are not included in cells. Important non-limiting examples are pathogen nucleic acids such as viral nucleic acids. Preserving the integrity of viral nucleic acids in cell-containing samples, such as blood samples in particular, during transport and handling is also crucial for subsequent analysis and viral load monitoring. Common applications and analytical methods for extracellular nucleic acids are described in, for example, the following documents: WO 97 / 035589, WO 97 / 34015, Swarup et al., FEBS Letters 581 (2007) 795-799, Fleischhacker Ann. NY Acad. Sci. 1075: 40-49 (2006), Fleischhacker and Schmidt, Biochmica et Biophysica Acta 1775 (2007) 191-232, Hromadnikova et al. (2006) DNA and Cell biology, Vol. 25, No. 11, pp. 635-640; Fan et al. (2010) Clinical Chemistry 56: 8.

[0004] Although the analysis of extracellular nucleic acids is highly interesting, it is difficult to find their analysis due to several reasons. Only low concentrations of extracellular nucleic acids are usually included in the sample. In addition, extracellular nucleic acids are often circulated as fragments of 500nt, 300nt (when indicating size and thus indicating chain length, the term "nt" also includes "bp" in the case of DNA) or even smaller (circulating nucleosomes). For the cell-free DNA (ccfDNA) of the circulation in plasma, the average length is often only about 140-170bp. In addition, the actual target extracellular nucleic acids that are considered to be identified for diagnostic purposes are also usually only a small part of the total extracellular nucleic acids. About ccfDNA, depending on, for example, pregnancy status or tumor grading, there are usually only thousands of amplifiable copies per ml of blood. Specifically, tumor-specific DNA fragments are very rare, and the concentrations included are often 1000-fold lower than the "normal" extracellular nucleic acid background. This low concentration has challenged the stabilization of samples and the subsequent separation of extracellular nucleic acids from stabilized samples. Furthermore, in addition to the degradation that occurs in serum and possibly plasma, a major problem associated with the analysis of circulating cell-free nucleic acids (cfNA), such as cfNA from tumors or fetal sources, is that after sample collection, the extracellular DNA (and RNA) may be diluted with genetic material from damaged or decaying cells. After sample collection, during ex vivo incubation, usually within a relatively short time after sample collection, cellular nucleic acids are released from the cells contained in the sample due to cell disruption. Once cell lysis begins, the lysed cells release a large amount of additional nucleic acids that become mixed with the extracellular nucleic acids, and it becomes increasingly difficult to recover the extracellular nucleic acids for testing.

[0005] If the sample contains a large number of cells, as is the case, for example, with whole blood samples, the release of intracellular nucleic acids after sample collection is particularly a problem. With regard to blood samples, the lysis of white blood cells in particular is a problem, since they release a large amount of genomic DNA in addition to RNA. Red blood cells do not contain genomic DNA. Therefore, the stabilization of circulating nucleic acids in whole blood must include mechanisms to stabilize blood cells in order to prevent contamination of the extracellular nucleic acid population with cellular genomic DNA and also RNA during stabilization. In particular, the dilution of extracellular nucleic acids, in particular rare target extracellular nucleic acids, is a problem and must be prevented. These issues are discussed in the prior art (see, for example, Chiu et al. (2001), Clinical Chemistry 47: 9 1607-1613; Fan et al. (2010) and US 2010 / 0184069). In addition, the amount and recoverability of available extracellular nucleic acids can be significantly reduced after a period of time due to degradation.

[0006] To address these issues and the need for efficient stabilization of extracellular nucleic acids, a number of methods, compositions and devices for stabilizing extracellular nucleic acid populations have been developed.

[0007] Stabilization using formaldehyde or formaldehyde releasers has been described in US 7,332,277, US 7,442,506 and US 2011 / 0111410. Methods for stabilizing blood samples have also been described in, for example, US 2010 / 0184069 and US 2010 / 0209930. However, the use of formaldehyde or formaldehyde releasers has disadvantages. Formaldehyde is toxic. Moreover, these stabilizers can chemically modify biomolecules such as proteins and nucleic acids. Therefore, they can jeopardize the effectiveness of extracellular nucleic acid separation by inducing crosslinking between nucleic acid molecules or between proteins and nucleic acids. This can especially reduce the number of nucleic acid molecules that can be amplified or obtained for diagnosis. This is a huge disadvantage because the number of ccfDNA in plasma is limited to only several thousand copies per ml of blood.

[0008] In addition to extracellular nucleic acids, other nucleic acids are also important biomarkers in the field of diagnosis. For example, the transcripts (particularly mRNA and miRNA) spectra of the genome are widely used as biomarkers in molecular in vitro diagnosis, and provide an understanding of normal biological and pathological processes, in order to predict the consequences of the disease and indicate the individualized treatment process. Therefore, the spectrum analysis of nucleic acids, especially RNA, is important in disease diagnosis, prognosis and in clinical trials for biomarker discovery. However, if there are no preventive measures in the stabilization of the sample to be analyzed, the sample will undergo changes during transportation and storage, which may seriously change the expression spectrum of the target molecule (see, for example, Rainen et al., 2002; Baechler et al., 2004). Therefore, gene expression, especially blood cell gene expression, is sensitive to the in vitro treatment of the sample. If the expression spectrum is changed due to the treatment of the sample, the subsequent analysis cannot reflect the original condition of the sample, and therefore does not reflect the original condition of the patient, but measures the artificial spectrum generated during sample processing, transportation and storage. Therefore, an optimized stabilization method is needed to stabilize the expression spectrum, thereby allowing reliable analysis. In particular, it is necessary to stabilize the blood sample so as to allow the analysis of the blood cell gene expression spectrum.

[0009] Many known techniques for stabilizing expression profiles are based on cell lysis (e.g., PAXgene blood RNA tubes, US 6,617,170, US 7,270,953, Kruhoffer et al., 2007). The disadvantage of various methods is that the stabilization causes complete cell lysis. The destruction of cells causes intracellular nucleic acids to become mixed with extracellular nucleic acids, which prevents the two nucleic acid populations from being analyzed separately. Moreover, the destruction of cells makes it impossible for any cell sorting or cell enrichment to perform cell analysis, respectively. Therefore, for some applications, sample collection and stabilization systems are required that retain the morphology of cells while stabilizing nucleic acids. In order to address the demand for simultaneous cell stabilization and nucleic acid stabilization, a stabilization system based on the use of formaldehyde releasers has been developed (e.g., cell-free RNA BCT (blood collection tubes), US 2011 / 0111410). However, it is very difficult to separate nucleic acids from the corresponding stabilized samples because the formaldehyde releasers used interfere with the subsequent nucleic acid separation process. Therefore, similar problems as described above for extracellular nucleic acids will occur.

[0010] In order to overcome the above-mentioned shortcomings, stabilization techniques that do not need to use cross-linking agents such as formaldehyde or formaldehyde releasers have been developed. These stabilization techniques are disclosed in, for example, WO 2013 / 045457 A1, WO 2014 / 146780 A1, WO 2014 / 146782 A1, PCT / EP2015 / 055699 (WO 2015 / 140218), WO 2014 / 049022 A1, WO 2013 / 045458A1, WO 2013 / 045432 A1 and WO 2014 / 146781 A1. Described technology particularly uses caspase (caspase) inhibitors, which are used alone or in combination with other stabilization compounds such as primary, secondary or tertiary amides to stabilize extracellular nucleic acid populations. These stabilization techniques reduce the extracellular nucleic acid populations to be polluted by intracellular nucleic acids, particularly by genomic DNA, after sample collection and stabilization. WO 2014 / 049022, WO 2014 / 146780, WO 2014 / 146782 and WO2014 / 146781 also teach corresponding stabilization compositions suitable for stabilizing intracellular nucleic acids (particularly gene expression profiles). In addition, these applications disclose stabilization compositions that allow subsequent analysis of stabilized cells, such as cell surface features and / or cell morphology. In addition, WO 2008 / 145710 describes a method for stabilizing cell-containing samples such as blood or tissue samples, which stabilizes, for example, cells, transcriptomes, genomes and proteomes. Although these applications provide effective stabilization techniques that ensure, for example, the stabilization of extracellular nucleic acids, they do not specifically address the sterilization and sterility aspects of the disclosed stabilization compositions and devices.

[0011] However, the use of these stabilizing compositions in sterilized form offers several advantages. For example, the shelf life of the composition can be increased. Moreover, when the sample is contacted with the sterilized composition for stabilization, the storage time of the stabilized sample can be further increased in certain applications. Sterilized compositions suitable for stabilizing extracellular nucleic acid populations are particularly useful if a biological sample is collected, for example, from a human or an animal and the biological sample is contacted directly with the stabilizing composition. This is the case, for example, when the stabilizing composition is contained in a sample collection device, such as a blood collection device or tube, which may also be subject to regulatory requirements. The same applies to the case of using a stabilizing composition that also stabilizes intracellular nucleic acids.

[0012] Therefore, in a large number of applications, from a safety and / or regulatory perspective, providing a sterile stabilized composition provides significant advantages. However, although it is desirable to sterilize the composition used to stabilize extracellular and / or intracellular nucleic acids and the device comprising such a composition, the sterilization process causes several problems. On the one hand, sterilization should be carried out under sufficiently strict conditions to ensure the sterility of the resulting composition or device. On the other hand, the application of such sterilization conditions may interfere with the relevant properties of the stabilized composition. For example, it has been found that conventional sterilization methods can significantly reduce the stabilization performance of the composition in the extracellular nucleic acid population of a stable biological sample. In addition, the measures for implementing sterilization should ideally not interfere with more downstream applications, such as the separation of stabilized extracellular nucleic acids and the processing or analysis of separated nucleic acids. The same applies to the case of using a stabilized composition that also stabilizes intracellular nucleic acids.

[0013] Among the available sterilization methods, irradiation sterilization offers advantages. Irradiation operates without excessive heat and does not expose the product to be sterilized to toxic chemicals. The forms of irradiation commonly used for sterilization purposes generally include ionizing irradiation forms, such as gamma irradiation, X-rays and electron beam irradiation. For example, according to ISO specifications (particularly ISO11137-1:2006, ISO11137-2:2012, ISO11137-3:2006), sterilization by gamma irradiation can be performed as part of the production process of disposable medical devices, such as the production process of sample collection devices such as blood collection tubes. Sterilization of disposable medical devices by electron beam irradiation is also regulated by ISO standards 1137 and 13409.

[0014] For sterilization by irradiation, for example gamma irradiation, a sufficiently high irradiation dose should be chosen to ensure the desired sterility level. Also, in the production of compositions and devices for sample collection, such as, in particular, blood collection devices or tubes, the minimum requirements for sterility and irradiation dose set out in the relevant ISO standards should be met. In general, sterilization for medical applications must ensure that the probability that a single unit is not sterile after sterilization is not higher than 10%. -6 . 10 -6 The probability of viable microorganisms on an article produces a "sterility assurance level" (SAL) of 1 in 1,000,000 - the probability of a single viable microorganism being present on the article after sterilization. Gamma irradiation is performed and guaranteed to have a SAL of 10 -6 The determination of the minimum dose is standardized by several ISO standards (ISO11137-1:2006, ISO11137-2:2012, ISO11137-3:2006). -6 The minimum dose of SAL required depends on the initial bioburden of the product.

[0015] It is also often desirable to sterilize with an irradiation dose that exceeds the minimum sterilization dose. For example, from a practical point of view of the workflow of the irradiation equipment, it is often more convenient to irradiate at a given higher dose. In addition, selecting an irradiation dose that exceeds the minimum sterilization dose can provide important advantages because, depending on the dose applied, simplified microbiological tests can be performed to confirm the sterility of the product. Depending on the irradiation dose applied, the product can be labeled as sterile with reduced or even no microbiological tests. For example, a relevant lower boundary for sterilizing sample collection devices such as blood collection tubes is irradiation with a dose of about 7.8kGy or 8kGy. Doses ≥ 8kGy are generally sufficient to eliminate low bioburden levels. In the case of elevated bioburden levels, higher doses may be required to achieve sterility. If the tube is irradiated with a dose of 15kGy, simplified microbiological tests can often be applied to determine and confirm sterility. In general, 25kGy can achieve a sterility assurance level (SAL) of 10 -6 Even with elevated bioburden levels, it is possible to achieve sterility with a reduced probability of sterility (e.g., SAL of 10 -5 or 10 -4 ) to achieve bioburden reduction. Therefore, blood collection tubes irradiated with a (comparatively high) dose of 25 kGy can be labeled sterile without the need for microbiological testing, which is a significant advantage.

[0016] However, irradiation, such as gamma irradiation, X-ray or electron beam irradiation, results in the formation of free radicals. Once the applied irradiation dose is higher than the maximum irradiation dose, the free radicals formed during irradiation can lead to a reduction in the potency or loss of the active ingredients of the sterilized product. The maximum irradiation dose can be defined as the sterilization dose that does not affect the integrity of the product, i.e., the maximum dose that does not cause degradation effects. For example, gamma irradiation is not widely used for aqueous drug products and drugs with protein components ( Isomedix Services, TechTip 01:09 / 07, Rev 1).

[0017] In the case where the initial bioburden of the product determines that the minimum sterilizing dose is higher than the maximum tolerable dose, several measures have been proposed. These measures include improving the production process, such as using clean and sterilized components and equipment, filtration, etc. However, implementing these measures is costly and may not always be feasible to use sterile components. Although these measures may help to achieve a lower minimum sterilizing dose, the sterilization of the final product is still needed. Other strategies recorded for mitigating degradation include cooling or temperature-controlled irradiation, adjusting the dose rate, minimizing water content or irradiating in dry or solid form and using an additive that serves as a radiation protectant. However, these strategies often cannot be applied in a universal manner, and the implementation of appropriate strategies also depends on the product or composition to be sterilized.

[0018] It is therefore a well-known problem that the conditions required to achieve sterilization, in particular irradiation sterilization, may lead to degradation of the active components of the stabilizing composition and thus may reduce or even eliminate the ability of the stabilizing composition to stabilize the extracellular nucleic acid population after sterilization. The same applies when using a stabilizing composition that also stabilizes intracellular nucleic acids and / or cell characteristics; said stabilizing properties must be maintained after sterilization.

[0019] The inventors have discovered that compositions containing caspase inhibitors for stabilizing the extracellular nucleic acid population in a sample exhibit significantly reduced performance after irradiation sterilization. Of course, such a negative impact of the sterilization process on the stabilizing properties of the stabilized composition is undesirable. Although measures can be taken to reduce the minimum sterilization dose required for a given product, these measures are generally insufficient to ensure the sterility of the final product itself, and reducing the minimum sterilization dose does not address the situation where it is desired to apply an irradiation dose that exceeds the maximum sterilization dose due to, for example, the reasons mentioned above. Many of the strategies discussed above for mitigating degradation were developed for pharmaceutical products and are not easily transferable to nucleic acid-related applications because different considerations apply.

[0020] An appropriate strategy for sterilizing compositions suitable for stabilizing nucleic acid populations in biological samples needs to ensure adequate stabilization functionality after sterilization and ideally not interfere with further downstream processes such as nucleic acid isolation and / or analysis. Appropriate stabilization performance after sterilization is particularly relevant to extracellular nucleic acid populations that are often contained in biological samples at low concentrations.

[0021] Therefore, the object of the present invention is to overcome at least one disadvantage of the above-mentioned prior art. In particular, the object of the present invention is to provide a method for preparing a sterilized composition, the sterilized composition comprises a caspase inhibitor and is also suitable for stabilizing the extracellular nucleic acid population of a biological sample, such as a cell-containing biological sample, after sterilization. Another object of the present invention is to provide a corresponding sterilizable composition, wherein the composition in sterilized form is suitable for stabilizing the extracellular nucleic acid population of such a biological sample. Another object of the present invention is to prevent the degradation of one or more active ingredients contained in the stabilizing composition, such as a caspase inhibitor, during irradiation sterilization to maintain the stabilization characteristics in the sterilized composition. Therefore, it is desirable and therefore an object of the present invention to maintain one or more stabilizing characteristics of the sterilizable composition in the sterilized composition. Another object is to provide a corresponding sterilization technology for a stabilizing composition containing a caspase inhibitor, the stabilizing composition stabilizes, for example, i) cells contained in a biological sample, ii) intracellular nucleic acids contained (such as DNA and / or RNA, particularly for stabilizing the gene expression profile of the contained cells) and / or iii) cell-associated proteins such as cell surface proteins. Summary of the invention

[0022] The present invention is based on the surprising discovery that if at least one compound selected from thiols (e.g., preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives is included in a composition containing a caspase inhibitor suitable for stabilizing the extracellular nucleic acid population of a biological sample (also referred to herein as a "stabilizing composition"), the stabilizing composition can be effectively protected from degradation during radiation sterilization. Surprisingly, one or more of these compounds can be included in the stabilizing composition for degradation protection without adversely affecting the extracellular nucleic acid stabilizing properties. Moreover, the inclusion of at least one of these compounds in the stabilizing composition advantageously does not interfere with downstream applications, such as the separation and / or analysis of the stabilized extracellular nucleic acid population. These compounds thus protect at least one active component in the composition, such as in particular a caspase inhibitor, from degradation during radiation sterilization, respectively, and effectively retain the stabilizing characteristics of the stabilizing composition after sterilization.

[0023] The method of the present invention significantly promotes the composition suitable for stabilizing the extracellular nucleic acid population of biological samples by irradiation such as gamma irradiation, electron beam irradiation or X-ray sterilization. It allows the preparation of a sterilized stabilization composition based on a stabilization composition comprising one or more caspase inhibitors and optionally one or more other stabilizers, which are disclosed in WO 2013 / 045457 A1, WO 2014 / 146780 A1, WO 2014 / 146782 A1, PCT / EP2015 / 055699, WO 2014 / 049022 A1, WO 2013 / 045458 A1 and WO 2014 / 146781 A1, which are incorporated by reference herein. As discussed, these applications also disclose stabilization compositions suitable for stabilizing intracellular nucleic acids such as intracellular DNA (e.g., genomic DNA) and / or intracellular RNA (particularly gene expression profiles) of contained cells. The method of the invention allows in particular the preparation of a sterilized stabilization composition based on the highly effective and advantageous stabilization composition comprising one or more caspase inhibitors disclosed in PCT / EP2015 / 055699, which is incorporated herein by reference. The stabilization composition can be conveniently prepared and / or sterilized directly in the sample collection device.

[0024] By using compounds selected from thiols, water-soluble vitamins, and vitamin E or derivatives thereof, it is also possible to prepare liquid compositions that can be sterilized by irradiation. Sterilized compositions in liquid form, especially in aqueous form, are highly advantageous. In the stabilization of, for example, blood samples, the use of sterilized liquid or aqueous stabilizing compositions allows, for example, to reduce hemolysis, thereby improving the stabilization effect. Using the method of the present invention, it is possible to prepare and sterilize liquid stabilizing compositions, including those with a mild pH, especially a weakly acidic pH. Sterilization can be carried out without having to return to a strongly alkaline or strongly acidic pH. Weakly acidic pH helps to avoid or reduce the appearance of precipitates in the stabilizing composition during sterilization.

[0025] The present invention also allows for the preparation of sterilized stabilized compositions having an extended shelf life compared to non-sterilized stabilized compositions and compared to sterilized compositions sterilized in the absence of the aforementioned compounds.

[0026] According to a first aspect, the present invention thus provides a method for producing a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample, the method comprising:

[0027] a) providing a composition comprising:

[0028] i. at least one caspase inhibitor, and

[0029] ii. at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives; and

[0030] b) irradiating the composition for sterilization.

[0031] According to a second aspect, the present invention provides a method for stabilizing an extracellular nucleic acid population contained in a cell-containing biological sample, the method comprising:

[0032] a) i) obtaining a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample according to the method of the first aspect of the invention, or ii) obtaining a composition according to the sixth aspect of the invention in sterilized form; and

[0033] b) contacting the cell-containing biological sample with the sterilized composition for stabilization.

[0034] According to a third aspect, the present invention provides a method for isolating extracellular nucleic acids from a stabilized cell-containing biological sample, the method comprising:

[0035] a) stabilizing the cell-containing biological sample according to the method of the second aspect of the invention; and

[0036] b) Isolation of extracellular nucleic acids.

[0037] According to a fourth aspect, the present invention provides a method for processing and / or analyzing extracellular nucleic acids, the method comprising:

[0038] a) isolating extracellular nucleic acids from a stabilized cell-containing biological sample according to the method of the third aspect of the invention; and

[0039] b) processing and / or analyzing the isolated extracellular nucleic acids.

[0040] According to a fifth aspect, the present invention provides a method for producing a sterilizable composition, wherein the composition in sterilized form is suitable for stabilizing the extracellular nucleic acid population of a biological sample, the method comprising:

[0041] a) preparing a composition comprising:

[0042] i. at least one caspase inhibitor, and

[0043] ii. at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives; and optionally

[0044] b) sterilizing the composition.

[0045] This method can be used to prepare the composition according to the sixth aspect.

[0046] According to a sixth aspect, the present invention provides a sterilizable composition, wherein the composition in sterilized form is suitable for stabilizing the extracellular nucleic acid population of a biological sample, wherein the composition is a composition as provided in step a) of the method according to the first aspect of the present invention. It comprises i. at least one caspase inhibitor, and ii. at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives. The sterilizable composition according to the sixth aspect may be a composition as provided in step a) of the method according to the first aspect of the present invention, which may be sterilized in an embodiment to provide a sterilized composition.

[0047] According to a seventh aspect, the present invention provides a sample collection device, such as a container, preferably a sample collection tube, comprising the sterilizable composition according to the sixth aspect of the present invention.

[0048] According to an eighth aspect, the present invention provides a kit comprising the composition according to the sixth aspect of the present invention or the sample collection device according to the seventh aspect of the present invention.

[0049] According to a ninth aspect, the present invention relates to the use of at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives in a composition suitable for protecting an extracellular nucleic acid population that is suitable for stabilizing a biological sample or its components during radiation sterilization.

[0050] Other aspects of the invention are provided in the detailed description that follows.

[0051] The present technology allows the preparation of sterilizable and sterilized compositions suitable for stabilizing extracellular nucleic acid populations in biological samples in a convenient, reliable, safe and cost-effective manner. Such stabilized compositions are particularly advantageous for applications in which biological samples such as blood samples are collected from humans or animals. In these applications, the sterility of the stabilized compositions and devices is highly preferred from a safety point of view. In addition, according to the present invention, non-toxic compounds can be used as radiation protectants, which also contribute to safety. In an embodiment, the sterilizable and sterilized compositions provided are also suitable for stabilizing intracellular nucleic acids (e.g., intracellular DNA, such as genomic DNA and / or intracellular RNA), particularly the gene expression profiles of the cells contained therein. In other embodiments, the sterilizable and sterilized compositions provided are suitable for stabilizing cell-containing samples, so that the cells contained in the stabilized samples can be analyzed, for example, based on cell surface features and / or cell morphology.

[0052] In particular, the present invention allows the preparation of irradiation-sterilized liquid stabilized compositions comprising caspase inhibitors. Sterilization of liquids by irradiation often interferes with the activity of the product to be sterilized. The inventors have found that incorporating at least one compound selected from thiols (preferably N-acetylcysteine ​​and / or glutathione), water-soluble vitamins, and vitamin E or derivatives thereof into a stabilized composition comprising a caspase inhibitor protects the stabilized composition from irradiation-induced degradation while not interfering with the stabilizing properties of the stabilized composition. This provides a sterilized composition that maintains the stabilizing characteristics. These beneficial effects are not seen when using other compounds used as free radical scavengers. N-acetylcysteine, ascorbic acid and glutathione in reduced form have been found to be particularly effective.

[0053] Providing a liquid sterile composition is particularly useful. For example, it allows rapid and uniform mixing of the stabilizing composition and a liquid biological sample, such as whole blood or a blood-derived sample, such as plasma or serum. Moreover, the use of a liquid composition, preferably an aqueous composition, reduces hemolysis.

[0054] Furthermore, sterilizing the stabilized composition according to the method of the present invention can further improve the storage properties of the stabilized composition and allow for an extended shelf life compared to a non-sterilized composition and a composition sterilized in the absence of at least one of a thiol (preferably N-acetylcysteine, glutathione), a water-soluble vitamin, and vitamin E or a derivative thereof, which is another advantage.

[0055] According to the following description and claims, other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art. However, it should be understood that the following description, claims and specific examples, while indicating the preferred embodiments of the present application, are given only as illustrations. By reading the following, various changes and modifications within the spirit and scope of the disclosed invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 : Effect of gamma irradiation on the stabilizing properties of a composition containing a caspase inhibitor. The effect was analyzed based on ccfDNA levels in plasma. The figure shows the mean change and standard deviation (x-fold change) in the copy number of 66 bp and 500 bp fragments of the 18S rDNA gene in non-stabilized EDTA blood samples (left) and stabilized blood samples drawn into tubes containing a stabilizing composition containing a caspase inhibitor (non-sterile or sterilized with different doses of gamma irradiation) after storage.

[0057] Figure 2: HPLC standard curve prepared with different concentrations of pure caspase inhibitors dissolved in DMSO.

[0058] Figure 3 : N-acetylcysteine, ascorbic acid, gallic acid and tannic acid were added to the stabilization composition containing caspase inhibitors. Average change in the copy number of 66 bp and 500 bp fragments of the 18S rDNA gene in stabilized blood after storage (x-fold change).

[0059] Figure 4 : Addition of different concentrations of ascorbic acid, N-acetylcysteine ​​and glutathione (reduced form) to the stabilization composition containing a caspase inhibitor. Average change (x-fold change) in the copy number of 66 bp and 500 bp fragments of the 18S rDNA gene in stabilized blood after storage.

[0060] Figure 5 : Addition of different concentrations of ascorbic acid, N-acetylcysteine ​​and glutathione to the stabilization composition containing a caspase inhibitor. Average total copy number of a 500 bp fragment of the 18S rDNA gene in stabilized blood after storage.

[0061] Figure 6 : Addition of different combinations of ascorbic acid, N-acetylcysteine ​​and glutathione to the stabilizing composition containing a caspase inhibitor. Average change in the copy number of the 66 bp and 500 bp fragments of the 18S rDNA gene in stabilized blood after storage (x-fold change).

[0062] Figure 7 : Effect of gamma irradiation on ccfDNA levels in stabilized blood with added scavengers. Mean change in the copy number of the 66 bp and 500 bp fragments of the 18S rDNA gene in blood samples stabilized with irradiated stabilization reagents after storage (x-fold change).

[0063] Fig. 8A , 8B: Effect of addition of N-acetylcysteine ​​to the stabilizing agent and increasing doses of gamma irradiation on ccfDNA levels in plasma. Mean change and standard deviation (x-fold change) of the copy number of 66 bp and 500 bp fragments of the 18S rDNA gene in unstabilized EDTA blood (left) and in stabilized blood samples drawn into tubes containing 0.5, 1.0, 2.0 and 4.0 mg / ml N-acetylcysteine, non-sterile or sterilized with different doses of gamma irradiation; ccfDNA levels were determined after storage of the stabilized samples.

[0064] Fig. 9: Average total copy number of a 500 bp fragment of the 18S rDNA gene in blood samples from 8 donors and stored at room temperature for 5 days. The blood samples were stabilized using stabilizing agents containing (left bar) or not containing (third bar from left) vitamin B6 as a scavenger. These stabilizing agents have been sterilized. As controls, stabilizing agents with vitamin B6 but not sterilized (second bar from left) and unstabilized EDTA blood (fourth bar from left) were included.

[0065] Fig.10 : Average total copy number of a 500 bp fragment of the 18S rDNA gene in blood samples from 6 donors and stored at room temperature for 5 days. Blood samples were stabilized with stabilization reagents containing different caspase inhibitors. Left bar: Stabilization reagent containing the caspase inhibitor Boc-D-(OMe)-FMK (non-sterile); second bar from the left: Stabilization reagent containing the caspase inhibitor Boc-D-(OMe)-FMK and N-acetylcysteine ​​as a scavenger (sterilized); third bar from the left: Stabilization reagent containing the caspase inhibitor Z-VAD(OMe)-FMK (non-sterile); fourth bar from the left: Stabilization reagent containing the caspase inhibitor Z-VAD(OMe)-FMK and N-acetylcysteine ​​as a scavenger (sterilized); fifth bar from the left: PAXgene blood ccfDNA tube; sixth bar from the left: EDTA blood that has not been stabilized. DETAILED DESCRIPTION

[0066] As described above, it is desirable to provide a sterilized composition suitable for stabilizing an extracellular nucleic acid population, particularly a ccfDNA population, of a biological sample for a variety of reasons. This is also applicable to stabilizing intracellular nucleic acids, such as intracellular DNA (e.g., genomic DNA) and / or intracellular RNA, and particularly suitable for stabilizing gene expression profiles. Without wishing to be bound by theory, the inventors have found that sterilization by irradiation, particularly gamma irradiation, can lead to degradation of the main component of the stabilizing agent, the caspase inhibitor. Depending on the irradiation energy and time, up to 95% of the caspase inhibitor may be degraded during the sterilization process. The degradation of the caspase inhibitor during sterilization results in a loss of performance of the stabilizing composition. In an embodiment, when such a sterilized stabilizing composition is used to stabilize a blood sample, this effect can be seen by increasing the release of genomic DNA from blood cells. Therefore, a method is needed to prevent one or more active ingredients in a stabilizing composition comprising one or more caspase inhibitors, such as, in particular, the caspase inhibitor, from becoming substantially degraded during a sterilization process involving irradiation, particularly during sterilization by gamma irradiation.

[0067] The inventors have found that only certain compounds are able to protect the stabilized composition during sterilization involving irradiation, such as sterilization by gamma irradiation. These compounds include thiols, such as, in particular, N-acetylcysteine ​​and glutathione (particularly in reduced form), water-soluble vitamins such as ascorbic acid, and vitamin E or its derivatives, such as trolox. Among the numerous candidate compounds tested, only these compounds were found to protect the caspase inhibitors (and optional other degradation-sensitive components) contained in the stabilized composition from substantial degradation during irradiation sterilization, but without adversely affecting the stabilization properties of the stabilized composition. This advantageous combination of effects is shown in the examples of stabilizing ccfDNA populations in blood samples. For ease of reference, thiols, such as, in particular, N-acetylcysteine ​​and glutathione (particularly in reduced form), water-soluble vitamins such as ascorbic acid, and vitamin E or its derivatives, such as trolox, are sometimes collectively referred to herein as "compounds of the present invention."

[0068] Other compounds, such as tannic acid and gallic acid, which are known to act as free radical scavengers in, for example, pharmaceutical compositions, have been found to interfere with the stabilization properties of compositions containing caspase inhibitors suitable for stabilizing extracellular nucleic acid populations after sterilization. Therefore, the sterilized compositions obtained containing these compounds are not suitable for stabilizing extracellular nucleic acid populations in samples. Moreover, other compounds described in the art that act as free radical scavengers, such as mannitol, isopropanol or Tween-80, have not been found to mediate appropriate radiation protection of compositions containing caspase inhibitors suitable for stabilizing extracellular nucleic acid populations contained in biological samples.

[0069] As demonstrated in the examples, the protective effect mediated by the compounds of the invention is obtained over a wide range of sterilization conditions and a wide range of irradiation doses. Advantageously, the compounds are effective at weakly acidic pH and advantageously do not require return to strongly acidic or alkaline pH conditions during sterilization.

[0070] Therefore, the inventors have found that by including at least one compound of the present invention in a composition containing a caspase inhibitor suitable for stabilizing the extracellular nucleic acid population of a biological sample before sterilization, the composition can be effectively protected from degradation during sterilization. Surprisingly, it is possible to add these protective compounds without substantially adversely affecting the performance of the composition in stabilizing the extracellular nucleic acid population. The resulting sterilized composition also allows downstream applications, such as separation and / or analysis of the contained stabilized extracellular nucleic acid population. This is also applicable to stabilized compositions containing caspase inhibitors that are additionally suitable for stabilizing intracellular nucleic acids such as intracellular DNA and / or intracellular RNA and are particularly suitable for stabilizing gene expression profiles. The present invention has thus made an important contribution to this area.

[0071] The most preferred compound used in the present invention is N-acetylcysteine. It provides excellent protection during sterilization, for example by irradiation, in particular gamma irradiation. At the same time, as demonstrated in the examples, it does not interfere with the stabilization properties, in particular the performance of the stabilization composition in terms of ccfDNA stabilization.

[0072] A. Methods for making sterilized compositions

[0073] The above findings can be advantageously applied in methods of making a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample and comprising one or more caspase inhibitors.

[0074] Thus, according to a first aspect, the present invention provides a method for producing a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample, the method comprising:

[0075] a) providing a composition comprising:

[0076] i. at least one caspase inhibitor, and

[0077] ii. at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives; and

[0078] b) irradiating the composition for sterilization.

[0079] As used herein, the term "extracellular nucleic acid" refers in particular to nucleic acids that are not contained in cells. The corresponding extracellular nucleic acids are also often referred to as cell-free nucleic acids. These terms are used as synonyms in this article. Therefore, extracellular nucleic acids are usually present outside of cells or outside of many cells in a sample. The term "extracellular nucleic acid" refers to, for example, extracellular RNA and extracellular DNA. Examples of typical extracellular nucleic acids found in a cell-free fraction (corresponding portion) of a biological sample such as a body fluid include, but are not limited to, mammalian extracellular nucleic acids such as extracellular tumor-related or tumor-derived DNA and / or RNA, other extracellular disease-related DNA and / or RNA, epigenetically modified DNA, fetal DNA and / or RNA, small interfering RNA such as miRNA and siRNA, and non-mammalian extracellular nucleic acids such as viral nucleic acids, pathogen nucleic acids released into the extracellular nucleic acid population by, for example, prokaryotes (such as bacteria), viruses, eukaryotic parasites or fungi. The extracellular nucleic acid population usually contains a certain amount of intracellular nucleic acids released from damaged or dying cells. For example, the extracellular nucleic acid population present in blood usually contains intracellular globulin mRNA released from damaged or dying cells. This is a natural process that occurs in vivo. This intracellular nucleic acid present in the extracellular nucleic acid population can even be used as a control in subsequent nucleic acid detection methods. The stabilization method described in the aforementioned application particularly reduces the risk of significantly increasing the amount of intracellular nucleic acids such as genomic DNA contained in the extracellular nucleic acid population due to in vitro treatment of the sample after collecting the cell-containing sample. Therefore, the change of the extracellular nucleic acid population caused by in vitro treatment is reduced and can even be prevented. In this article, it refers to the extracellular nucleic acid obtained as the extracellular nucleic acid of circulation or the cell-free nucleic acid of circulation obtained from circulating body fluids such as blood or lymph. When used in this article, the term extracellular nucleic acid can particularly refer to mammalian extracellular nucleic acid. Examples include but are not limited to disease-related or disease-derived extracellular nucleic acids, such as tumor-related or tumor-derived extracellular nucleic acids, due to inflammation, necrosis or injury, especially trauma and released extracellular nucleic acids, related to other diseases and / or due to other diseases and released extracellular nucleic acids, or derived from the extracellular nucleic acids of the fetus. Stabilizing ccfDNA and thus stabilizing the ccfDNA population is particularly preferred. The term "extracellular nucleic acids" described herein also refers to extracellular nucleic acids obtained from other cell-containing biological samples, in particular biological samples other than body fluids. Typically, a sample contains more than one species or type of extracellular nucleic acids.

[0080] As used herein, the term "extracellular nucleic acid population" refers in particular to a collection of different extracellular nucleic acids contained in a cell-containing sample. Cell-containing samples generally include characteristic and thus unique extracellular nucleic acid populations. Therefore, the type, species, ratio and / or amount of one or more extracellular nucleic acids contained in the extracellular nucleic acid population of a particular sample can be an important sample feature. According to one embodiment, the extracellular nucleic acid population refers to extracellular DNA, particularly cell-free circulating DNA. As described above, it is important to stabilize and thus substantially retain the extracellular nucleic acid population in the state when sample collection, because the composition and / or amount of one or more extracellular nucleic acids contained in the extracellular nucleic acid population of the sample can provide valuable medical, prognostic or diagnostic information. Therefore, it is advantageous if the extracellular nucleic acid population spectrum is effectively stabilized within a predetermined stable period. Stabilization techniques described herein reduce contamination after sample collection and stabilization, and therefore reduce dilution of the extracellular nucleic acid population by intracellular nucleic acids, particularly by genomic DNA. Thus achieving substantially retaining the extracellular nucleic acid population. As shown in the examples, in the case of blood samples or samples derived from blood, when using a stabilizing composition comprising a caspase inhibitor as described herein, the changes in the amount, quality and / or composition of the contained extracellular nucleic acids in the extracellular nucleic acid population (shown here as the extracellular DNA population), in particular the changes attributable to the increased release of genomic DNA, are greatly reduced during the stabilization period, compared to a sample that has not been stabilized or a corresponding sample that has been stabilized, for example, by EDTA. According to one embodiment, in the case of a blood sample (for example a blood sample stabilized with 1.5 mg EDTA / ml) or a sample derived from blood, the increase in genomic DNA from T0 (stabilization point) to the end of the stabilization period (preferably 48 hours, 72 hours or 96 hours after T0) is reduced by at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to a sample that has not been stabilized or a corresponding sample that has been stabilized, for example, by EDTA.

[0081] In an embodiment, the sterilized composition provided is also suitable for stabilizing intracellular nucleic acids, such as intracellular DNA (e.g., genomic DNA) and / or intracellular RNA. In particular, it can be suitable for stabilizing the gene expression profile of the contained cells, thereby allowing reliable gene expression profile analysis. By stabilizing and thus retaining the gene transcription profile of the contained cells, the change of the gene expression profile during the stable period is reduced or even prevented. Therefore, the sterilized stabilization composition provided allows the analysis of the extracellular and intracellular nucleic acid populations in the stabilized sample, if necessary.

[0082] In an embodiment, the sterilized stabilization composition can also be used to analyze cells contained in the stabilized sample by stabilizing the cells contained in the biological sample so that they remain substantially intact. Stabilization of cell properties such as cell surface characteristics and / or cell morphology is advantageous. It allows analysis and separation of specific cells contained in the stabilized sample, for example, blood cells or circulating tumor cells contained in a blood sample.

[0083] The biological sample can be a cell-containing biological sample or a suspected cell-containing biological sample. The biological sample can be selected from body fluids and cell-containing samples derived from body fluids. Examples include, but are not limited to, whole blood, samples derived from blood such as plasma or serum, buffy coat, urine, sputum, tears, lymph, amniotic fluid, sweat, juice, cerebrospinal fluid, ascites, milk, feces, bronchial lavage fluid, saliva, amniotic fluid, nasal secretions, vaginal secretions, semen, wound secretions, cell culture and swab samples. According to one embodiment, the cell-containing biological sample is a body fluid, a body secretion or a body excretion, preferably a body fluid, most preferably urine, lymph, blood, buffy coat, plasma or serum. In particular, the cell-containing biological sample can be a circulating body fluid, such as blood or lymph. Preferably, the cell-containing biological sample is a blood sample, sometimes also referred to as whole blood. The blood sample is preferably not diluted or graded before stabilization. According to one embodiment, the blood sample is peripheral blood. It is also preferred that the biological sample may be plasma or serum. According to one embodiment, the cell-containing biological sample is obtained from a human. The cell-containing biological sample comprises or is suspected of comprising extracellular nucleic acids in the extracellular part of the sample.

[0084] It should be understood that in the method according to the first aspect of the present invention, the composition provided in step a) and irradiated in step b) has not yet come into contact with the biological sample to be stabilized. The original stabilized composition (i.e., the reagent without the biological sample) is irradiated for sterilization. Therefore, the composition provided in step a) and irradiated in step b) has not yet come into contact with the biological sample, and thus does not contain the biological sample in steps a) and b). Thus, the composition provided in step a) and irradiated in step b) does not contain cells and / or cell fragments. Thus, the composition provided in step a) and irradiated in step b) does not contain body fluids and / or cell-containing samples derived from body fluids.

[0085] The method will now be described in more detail. In doing so, steps a) and b) of the method will first be described. When discussing step a), emphasis will be placed on further describing the advantageous compounds described according to feature ii of step a). Then, an exemplary composition provided, for example prepared, in step a) will be further described.

[0086] Step a) providing a composition

[0087] Step a) comprises providing a composition comprising at least one caspase inhibitor and at least one compound selected from thiols (preferably N-acetylcysteine ​​and / or glutathione), water-soluble vitamins, and vitamin E or its derivatives. Such a composition can be produced and prepared using the method according to the fifth aspect.

[0088] The provided compositions containing caspase inhibitors are suitable for stabilizing the extracellular nucleic acid population of a biological sample, in particular ccfDNA. Other additives that can be additionally included in the composition to enhance the stabilizing effect are known to the skilled person and are also described herein.

[0089] The form of the composition provided in step a) is not particularly limited. For example, the composition may be provided in solid form. If desired, the composition may be provided in lyophilized form. Where provided in solid form, e.g. to prepare the composition, it may be modified prior to carrying out method step b) to provide a liquid composition. For example, the solid composition may be dissolved in a liquid prior to irradiation to provide a liquid composition for irradiation step b), which is preferably an aqueous composition.

[0090] Preferably, the composition provided in step a) is in liquid form. Preferably, the composition is prepared in aqueous form. For example, the components of the composition may be contacted, preferably mixed, with a suitable solvent to produce a liquid composition. The liquid composition is preferably an aqueous composition. Water may be used as a solvent.

[0091] As used herein, providing or preparing in "liquid form" may particularly mean providing or preparing a liquid composition. The liquid composition may be a homogeneous mixture of only a single phase, but it is also within the scope of the present invention that the liquid composition comprises solid components such as precipitates. As used herein, providing or preparing in "aqueous form" may particularly mean providing or preparing an aqueous liquid composition. An "aqueous" composition comprises water. According to one embodiment, an "aqueous" composition comprises water as the main component (% v / v).

[0092] Without wishing to be bound by theory, the preparation of liquid, particularly aqueous compositions, provides several advantages when used to stabilize extracellular nucleic acid populations in biological samples. Liquid compositions allow, for example, rapid and convenient mixing with liquid samples containing or suspected of containing extracellular nucleic acids to be stabilized. Examples of liquid samples are blood samples, plasma samples, and serum samples. Furthermore, when stabilizing blood samples, it is particularly preferred to prepare a stabilizing composition containing water, because this embodiment significantly reduces hemolysis. Thus, preparing a stabilizing composition containing water provides advantages when the stabilizing composition is subsequently used to stabilize a blood sample.

[0093] The components of the composition prepared in step a) may be contained, respectively dissolved in a solvent such as water or a buffer, the buffer comprising, for example, a biological buffer such as MOPS, TRIS, PBS, etc. In addition, the components of the stabilized composition may be dissolved in a polar aprotic solvent such as dimethyl sulfoxide (DMSO), or the stabilized composition may comprise a polar aprotic solvent such as dimethyl sulfoxide (DMSO). It has also been found that DMSO supports the protection of the stabilized composition during sterilization conferred by the compounds of the invention, particularly when used in combination with vitamin E or a derivative thereof such as trolox.

[0094] Using the method of the present invention, it is possible to protect the caspase inhibitor contained in the stabilizing composition during a sterilization process involving irradiation. This is also advantageous under mild pH conditions. There is no need to return to strongly acidic or alkaline pH conditions. The at least one compound selected from thiols (preferably N-acetylcysteine ​​and / or glutathione), water-soluble vitamins, and vitamin E or its derivatives is active under mild pH conditions. This includes the use of ascorbic acid for protection. The use of mild pH conditions such as weakly acidic conditions is advantageous because it prevents the single components of the stabilizing reagent from precipitating at lower temperatures particularly well. Thereby, it is also possible to mix with the liquid sample even more quickly, which helps to reduce hemolysis and thereby improve the stabilization effect. Preferably, the composition has a weakly acidic pH.

[0095] According to one embodiment, the stabilizing composition provided in step a) has a pH selected from pH 4.0 to 7.0, pH 4.1 to 6.9, pH 4.2 to 6.8, pH 4.3 to 6.6, pH 4.4 to 6.3, pH 4.5 to 6.0, or pH 4.5 to 5.5.

[0096] According to one embodiment, the composition is provided in a sample collection device, or the composition is filled into the sample collection device before the irradiation step b). The sample collection device may be a device according to the seventh aspect of the present invention. The sample collection device is suitable for sterilization involving irradiation.

[0097] Feature i, at least one caspase inhibitor

[0098] The stabilization composition provided in step a) comprises at least one caspase inhibitor.

[0099] The caspase inhibitor may be a caspase inhibitor as described in WO 2013 / 045457 A1 or WO 2013 / 045458 A1. The caspase inhibitors disclosed therein are incorporated herein by reference. As described in these prior art documents, caspase inhibitors are highly effective in stabilizing the extracellular nucleic acid population (e.g., extracellular DNA) of biological samples such as blood samples, both alone and in combination with other stabilizers.

[0100] Preferably, the caspase inhibitor is cell-penetrable.Members of the caspase gene family play an important role in apoptosis.The substrate preference or specificity of each caspase has been used to develop peptides that successfully compete for caspase binding.By coupling caspase-specific peptides with, for example, aldehydes, nitrile or ketone compounds, it is possible to produce reversible or irreversible caspase activation inhibitors.For example, peptides derived from fluoromethyl ketone (FMK), such as Z-VAD-FMK, serve as effective irreversible inhibitors without adding cytotoxic effects.Inhibitors synthesized with benzyloxycarbonyl groups (BOC) on N-terminal and O-methyl side chains show that cell permeability is improved.Other suitable caspase inhibitors are synthesized to have phenoxy groups at the C-terminal.An example is Q-VD-OPh, which is a cell-penetrable, irreversible broad-spectrum caspase inhibitor, which is even more effective than caspase inhibitor Z-VAD-FMK in preventing apoptosis and thus supporting stabilization.

[0101] According to one embodiment, the caspase inhibitor is a pan-caspase inhibitor, and is thus a broad-spectrum caspase inhibitor. According to one embodiment, the caspase inhibitor comprises a peptide or protein or is composed of a peptide or protein. According to one embodiment, the caspase inhibitor comprises a modified caspase-specific peptide. Preferably, the caspase-specific peptide is modified with an aldehyde, nitrile or ketone compound. According to one embodiment, the caspase-specific peptide is modified with an O-phenoxy (OPh) or fluoromethylketone (FMK) group, preferably at the carboxyl end. Suitable caspase inhibitors comprising proteins or peptides or consisting of proteins or peptides and caspase inhibitors comprising modified caspase-specific peptides are disclosed in Table 1 of WO 2013 / 045457, and are incorporated herein by reference. The table provides examples of suitable caspase inhibitors. Without wishing to be bound by theory, it is believed that the compounds of the invention are particularly active for the protection of caspase inhibitors comprising peptides, in particular caspase inhibitors that are peptides modified with functional groups. The compounds of the invention are particularly suitable for the protection of peptide caspase inhibitors modified with an O-phenoxy (OPh) group, preferably at the carboxyl terminus, and / or with a glutamine (Q) group, preferably at the N-terminus. An example of such a caspase inhibitor is Q-VD-OPh.

[0102] According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh, Z-VAD (OMe) -FMK and Boc-D- (OMe) -FMK. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh and Z-VAD (OMe) -FMK. In a preferred embodiment, Q-VD-OPh, which is a broad-spectrum inhibitor of caspases, is used for stabilization. Q-VD-OPh is cell-permeable and inhibits cell death caused by apoptosis. Q-VD-OPh is non-toxic to cells even at very high concentrations and contains a carboxyl-terminal phenoxy group conjugated to the amino acids valine and aspartic acid. It is equally effective in preventing apoptosis mediated by the following three major apoptotic pathways: caspase-9 and caspase-3, caspase-8 and caspase-10, and caspase-12 (Caserta et al., 2003).

[0103] As shown in the examples, the compounds of the invention are effective over a wide range of concentrations and protect the caspase inhibitor contained in the stabilizing composition during irradiation. Within the concentration range tested for the protective compounds of the invention, protection was achieved over a wide range of caspase inhibitor concentrations contained in the stabilizing composition, and the degree of protection conferred was largely independent of the caspase inhibitor concentration tested.

[0104] The stabilizing composition comprises one or more caspase inhibitors, in particular caspase inhibitors comprising modified caspase-specific peptides such as Q-VD-OPh, in an amount sufficient to produce a stabilizing effect on the extracellular nucleic acid population contained in the biological sample. According to one embodiment, the stabilizing composition comprises a caspase inhibitor at a concentration such that the final concentration of the caspase inhibitor produced after the stabilizing composition is contacted with the expected volume of the sample to be stabilized is 0.1 μM to 25 μM, 0.5 μM to 20 μM, 1 μM to 17 μM, 2 μM to 16 μM, and more preferably 3 μM to 15 μM. As can be seen from the examples, final concentrations of about 5 μM, about 10 μM, and about 15 μM were found to be very suitable. As also explained above, although what is referred to here is the concentration that produces the final concentration of the caspase inhibitor after the stabilizing composition is contacted with the expected volume of the sample to be stabilized, it should be understood that in the method according to the first aspect of the present invention, the composition provided in step a) and irradiated in step b) has not yet come into contact with the biological sample to be stabilized.

[0105] According to one embodiment, the stabilizing composition and thus the stabilizing agent comprises a caspase inhibitor selected from the following concentrations: 0.35 μg / ml to 70 μg / ml, 0.7 μg / ml to 63 μg / ml, 1.74 μg / ml to 59 μg / ml, 10.5 μg / ml to 56 μg / ml, or 15 μg / ml to 50 μg / ml, 20 μg / ml to 45 μg / ml, 25 μg / ml to 40 μg / ml, and 30 μg / ml to 38 μg / ml. The concentration may be selected from 0.7 μg / ml to 45 μg / ml and 1.74 μg / ml to 40 μg / ml.

[0106] According to one embodiment, the stabilizing composition and thus the stabilizing agent comprises a caspase inhibitor at a concentration selected from: 0.68 μM to 136 μM, 1.36 μM to 122.5 μM, 3.38 μM to 114.72 μM, 20.4 μM to 109 μM, or 29.2 μM to 97.2 μM, 38.9 μM to 87.5 μM, 48.6 μM to 77.8 μM, and 58.3 μM to 74 μM. The concentration may be selected from 20.4 μM to 97.2 μM and 29.2 μM to 87.5 μM.

[0107] The above concentrations of the caspase inhibitor in a mixture comprising a stabilizing composition (reagent) and a biological sample to be stabilized and the stabilizing composition (reagent) itself are suitable for use with a single caspase inhibitor and a combination of caspase inhibitors. When using a pan-caspase inhibitor, particularly a modified caspase-specific peptide such as Q-VD-OPh and / or Z-VAD (OMe) -FMK, the above concentrations are particularly suitable. Another example of a modified caspase-specific peptide is Boc-D-(OMe) -FMK. The above concentrations are suitable, for example, for stabilizing blood samples. Suitable concentration ranges for each caspase inhibitor and / or other cell-containing biological samples can be determined by a technician, for example, by testing the corresponding caspase inhibitors of different concentrations in the test assay described in the examples.

[0108] Feature ii, at least one compound selected from thiols (preferably N-acetylcysteine ​​and / or glutathione), water-soluble vitamins, and vitamin E or its derivatives

[0109] The composition provided in step a) additionally comprises at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives. The composition can therefore be sterilized by irradiation (e.g., by gamma irradiation), wherein the sterilized composition substantially maintains its stabilizing properties and thus maintains performance. Without wishing to be bound by theory, these compounds contribute to the protection of the stabilized composition to be irradiated, in particular the caspase inhibitors contained therein. At the same time, these compounds do not interfere with the stabilizing effect, thereby ensuring the functionality of the composition in stabilizing extracellular nucleic acid populations, such as, in particular, ccfDNA. Surprisingly, the compounds of the present invention achieve these balanced effects. As shown in the examples, many other free radical scavengers do not confer protection against irradiation and / or interfere with the stabilizing properties of the composition and are therefore unsuitable.

[0110] The at least one compound contained in the stabilizing composition may be selected from N-acetylcysteine, glutathione, preferably glutathione in reduced form, ascorbic acid, B vitamins, and / or water-soluble vitamin E derivatives. The at least one compound contained in the stabilizing composition may be selected from N-acetylcysteine, glutathione, preferably glutathione in reduced form, ascorbic acid, and / or water-soluble vitamin E derivatives. Preferably, the at least one compound is selected from N-acetylcysteine, glutathione, preferably glutathione in reduced form, ascorbic acid, and / or trolox. More preferably, the at least one compound is selected from N-acetylcysteine, glutathione, preferably glutathione in reduced form, vitamin B6, and / or ascorbic acid. More preferably, the at least one compound is selected from N-acetylcysteine, glutathione, preferably glutathione in reduced form, and / or ascorbic acid. N-acetylcysteine ​​and ascorbic acid have been found to be particularly effective, as has glutathione, especially glutathione in reduced form. Vitamin B6 has also been found to be useful. Most preferred according to the invention is N-acetylcysteine.

[0111] As shown in the examples, the compounds of the invention can be included in the compositions and are effective over a wide range of concentrations.

[0112] According to one embodiment, the stabilizing composition comprises at least one compound selected from thiols, which are preferably N-acetylcysteine ​​and / or glutathione (preferably in reduced form), water-soluble vitamins, and vitamin E or its derivatives, in a concentration of less than 20 mg / ml, preferably 15 mg / ml or less, 10 mg / ml or less, 12 mg / ml or less, 7 mg / ml or less, 3 mg / ml or less, 1.5 mg / ml or less, or 0.75 mg / ml or less. Preferably, the composition comprises at least 0.05 mg / ml, at least 0.1 mg / ml, at least 0.2 mg / ml or at least 0.3 mg / ml of the at least one compound. According to one embodiment, the composition comprises at least one compound selected from N-acetylcysteine, glutathione (preferably in reduced form), ascorbic acid, B vitamins such as vitamin B6, and trolox at these concentrations. According to one embodiment, the composition comprises at least one compound chosen from N-acetylcysteine, glutathione (preferably in reduced form), ascorbic acid and trolox in these concentrations.

[0113] The at least one compound of the present invention may be included in a concentration such that at least 15%, at least 20%, at least 35% or at least 50% of the caspase inhibitor contained in the stabilized composition before irradiation sterilization is present in the stabilized composition after sterilization. Preferably, the at least one compound of the present invention may be included in a concentration such that at least 60%, at least 70%, at least 80%, or at least 90%, more preferably at least 80% of the caspase inhibitor contained in the stabilized composition before irradiation sterilization is present in the stabilized composition after sterilization. This can be determined, for example, using HPLC analysis as done in the examples. As shown in the examples, the degree of degradation of the caspase inhibitor after irradiation and the percentage of the caspase inhibitor still present in an undegraded form depend on the irradiation dose. Exemplary compound concentrations for achieving a desired percentage of remaining caspase inhibitor after irradiation with a given irradiation dose are also shown in the examples.

[0114] The at least one compound of the present invention may be included in a concentration as described below, in which it can exert a protective effect on the caspase inhibitor during sterilization as defined above, while substantially not interfering with the stabilizing properties of the stabilizing composition. According to one embodiment, the stabilizing effect of the stabilizing composition comprising the protective compound of the present invention is at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the composition that is otherwise identical but does not contain the protective compound. The stabilizing effect may even be increased by adding the protective compound. For example, the stabilizing effect of the stabilizing composition comprising the protective compound of the present invention may be 120%, 115%, 110% or 105% of the composition that is otherwise identical but does not contain the protective compound. Preferably, the stabilizing composition comprising the protective compound of the present invention and the composition that is otherwise identical but does not contain the protective compound are compared with each other in an unsterilized or unirradiated form. Tests for determining the stabilizing properties of stabilizing compositions with and without target compounds are provided in the examples and are described in the experimental procedures section therein. The extracellular nucleic acids in the sample can be quantified using a real-time PCR assay and the described increase in copy number over time can be used to determine the stabilizing effect of a given stabilizing composition. Thus, in one embodiment, the stabilizing effect of a given stabilizing composition can be determined by measuring the ratio of total ccfDNA on day 0 immediately after blood drawing and after storage at room temperature for, for example, 6 days, as described in the Examples.

[0115] According to one embodiment, the at least one compound of the present invention is included in a concentration as described below, in which it can exert a protective effect on the caspase inhibitor during sterilization as defined above, while substantially not reducing the amount of detectable target extracellular nucleic acids in the stabilized sample. In one embodiment, when stabilizing a sample with a composition comprising the protective compound, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the target extracellular nucleic acids detected in a sample stabilized with a composition otherwise identical but not comprising the protective compound is detected. The amount of extracellular nucleic acids can be determined, for example, using real-time PCR and determining the absolute copy number, as shown in the Examples.

[0116] The preparation of compositions comprising more than one compound of the invention is also provided and included. In this case, the composition may contain each contained compound at the concentration indicated above. Alternatively, the concentration indicated above refers to the total concentration of all compounds of the invention contained in the composition. Suitable concentrations of individual compounds and combinations of compounds can be determined by a skilled person in accordance with the technical information provided herein. As shown in the examples, combining the compounds of the invention can further improve the protection of the stabilized composition during sterilization compared to the use of individual compounds. This beneficial effect tends to be more pronounced at high irradiation doses and is already achieved when relatively low concentrations of each individual compound are used. The embodiments of the invention are particularly contemplated herein. Figure 6 and the exemplary combinations and concentrations described in Table 7 for use.

[0117] In the case where the composition is initially provided in solid form, the composition can be made into liquid form before irradiation. The liquid composition can contain the at least one compound in the aforementioned concentration. According to this embodiment, the solid composition contains the one or more compounds of the present invention in an amount sufficient to produce a liquid composition containing the one or more compounds in the concentration as described above and below after solvation.

[0118] The various components of the present invention that may be included in the composition for protection will now be explained in more detail.

[0119] According to one embodiment, the provided composition, for example the composition prepared in step a), comprises at least one thiol. As used herein, "thiol" may in particular be, for example, Mercaptans as defined in Lexikon Chemie, 10th edition 1999, Georg Thieme Verlag Stuttgart, New York.

[0120] Particularly preferred thiols are N-acetylcysteine ​​and glutathione. A highly suitable and very preferred component for the purposes of the present invention is N-acetylcysteine. "N-acetylcysteine" as used herein is preferably "N-acetyl-L-cysteine". Therefore, "N-acetylcysteine" as used herein preferably indicates the compound "N-acetyl-L-cysteine". Therefore, when "N-acetylcysteine" is mentioned in the present text, the corresponding disclosure also applies to "N-acetyl-L-cysteine", unless the context dictates otherwise. Whenever "N-acetylcysteine" is mentioned, N-acetyl-L-cysteine ​​is preferred. N-acetyl-D-cysteine ​​can also be used. Like glutathione, N-acetylcysteine ​​is a thiol. When used herein, "N-acetylcysteine" can in particular be as Pschyrembel Klinisches N-acetylcysteine ​​as defined in (258. Auflage, Walter de Gruyter, Berlin, New York, 1998).

[0121] It has been found that N-acetylcysteine ​​effectively protects stabilizing compositions comprising one or more caspase inhibitors, in particular caspase inhibitors containing caspase-specific peptides such as Q-VD-OPh, during irradiation sterilization, such as gamma irradiation sterilization. At the same time, N-acetylcysteine ​​advantageously does not substantially interfere with the stability of extracellular nucleic acid populations, such as ccfDNA. Therefore, in a preferred embodiment, the stabilizing composition comprises N-acetylcysteine. N-acetylcysteine ​​also allows a mild pH, such as a weakly acidic pH, for the stabilizing composition. Suitable pH values ​​are described above. Advantageously, the stabilizing composition comprising N-acetylcysteine ​​can be irradiated and sterilized in liquid form, including aqueous form. Advantageously, it is not necessary to irradiate the composition in solid or frozen form to maintain the stabilizing properties of the composition containing the caspase inhibitor.

[0122] The stabilizing composition may comprise N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, or 0.1 mg / ml to 2 mg / ml. Preferably, the composition comprises at least 0.2 mg / ml, or at least 0.3 mg / ml N-acetylcysteine. Particularly preferred are compositions comprising 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml.

[0123] The stabilized composition may comprise N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml, and is sterilized by irradiation in a liquid form, preferably an aqueous form.

[0124] It has been found that N-acetylcysteine ​​provides particularly good protection of the one or more caspase inhibitors contained in the stabilized composition, while ensuring good performance of the stabilized composition in downstream applications. The aforementioned N-acetylcysteine ​​concentrations are effective and produce particularly good results. As shown in the examples, N-acetylcysteine ​​at all tested concentrations (0.5 mg / ml, 1 mg / ml, 2 mg / ml, 4 mg / ml and 10 mg / ml) provides excellent protection of the stabilized composition during irradiation. At the same time, even higher concentrations of N-acetylcysteine ​​did not interfere with the performance of the stabilized composition in subsequent applications.

[0125] In the caspase inhibitor-containing composition, N-acetylcysteine ​​concentrations of 0.2 mg / ml to 1 mg / ml or 0.3 mg / ml to 0.8 mg / ml are particularly preferred. Such compositions were found to provide irradiated / sterilized stabilized compositions with particularly good storage properties after sterilization. In particular, excellent stability was observed at the end of the shelf life. Using these concentrations, the shelf life of the sterilized stabilized composition can be advantageously extended.

[0126] According to one embodiment, the stabilizing composition comprises N-acetylcysteine ​​and optionally one or more compounds selected from glutathione, water-soluble vitamins, and vitamin E or its derivatives. According to one embodiment, the stabilizing composition comprises N-acetylcysteine ​​and may also comprise one or more of glutathione, preferably glutathione in reduced form, ascorbic acid, B vitamins, preferably vitamin B6, and water-soluble vitamin E derivatives, preferably trolox. In particular, the composition may comprise N-acetylcysteine ​​and may also comprise one or more of glutathione, preferably glutathione in reduced form, ascorbic acid, and water-soluble vitamin E derivatives, preferably trolox. Thus, it is also considered that the stabilizing composition of step a) comprises N-acetylcysteine ​​and also comprises glutathione, preferably glutathione in reduced form, and / or ascorbic acid. According to one embodiment, the composition comprises N-acetylcysteine ​​and glutathione, preferably glutathione in reduced form. While all of the above combinations have been found to be very useful for protecting stabilized compositions comprising one or more caspase inhibitors to be sterilized by irradiation, N-acetylcysteine ​​alone has been found to produce excellent results. Combinations of N-acetylcysteine ​​and B vitamins such as, in particular, vitamin B6 are also contemplated.

[0127] According to one embodiment, the stabilizing composition provided in step a) comprises glutathione. As shown in the examples, the thiol glutathione, in particular the reduced form of glutathione, is also suitable for protecting the composition during irradiation sterilization and does not substantially adversely affect the stabilizing properties of the composition containing the caspase inhibitor. Glutathione exists in both reduced and oxidized forms. In the reduced form, the thiol group of the cysteine ​​contained in the compound is able to provide a reducing equivalent. As used herein, "glutathione" may in particular be a Pschyrembel Klinisches Glutathione as defined in (258. Auflage, Walter de Gruyter, Berlin, New York, 1998).

[0128] The composition may comprise glutathione, preferably in reduced form, in a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml. The composition may comprise glutathione, preferably in reduced form, in a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml, and may be irradiated in a liquid form, preferably an aqueous form.

[0129] The skilled person will appreciate that after irradiation and / or sterilization, the amount of reduced glutathione in the stabilized composition may decrease, since some or all of the glutathione may be oxidized during the process. Therefore, the concentration of reduced glutathione indicated above refers specifically to the concentration in the composition before irradiation sterilization.

[0130] As shown in the examples, glutathione, particularly in reduced form, is very effective in protecting stabilized compositions comprising one or more caspase inhibitors. The reduced form of glutathione delivers particularly good protection to the stabilized composition. Relatively low concentrations and mild pH conditions, particularly weakly acidic conditions, can be used in this embodiment. Advantageously, it is possible to irradiate and sterilize the composition in liquid form. There is no need to irradiate the composition in solid or frozen form in order to maintain the stabilizing properties of the composition. As shown in the examples, a protective effect on the caspase inhibitors can be observed at glutathione concentrations as low as 0.05 mg / ml. At slightly higher concentrations, for example at least 0.1 mg / ml or at least 0.2 mg / ml, a more pronounced effect is observed. Concentrations of 0.5 mg / ml and 1 mg / ml produce particularly good results.

[0131] As shown in the examples, water-soluble vitamins such as ascorbic acid are also active in protecting stabilized compositions comprising one or more caspase inhibitors. In addition, the examples show that water-soluble vitamin B6 is active in protecting stabilized compositions comprising one or more caspase inhibitors. Therefore, a stabilized composition comprising at least one water-soluble vitamin can be provided in step a) for protection. Derivatives of water-soluble vitamins are also considered and can be used herein. As used herein, "derivatives of water-soluble vitamins" can particularly be derivatives that have the same activity as the corresponding vitamins as free radical scavengers. In one embodiment, the water-soluble vitamins are selected from vitamin C and B vitamins. In one embodiment, the water-soluble vitamins are selected from ascorbic acid and B vitamins. In an embodiment, the B vitamins are vitamin B6. When used herein, vitamin B6 refers in particular to pyridoxine. Thus, any disclosure given herein about "vitamin B6" is generally particularly applicable to embodiment pyridoxine and refers to embodiment pyridoxine. Thus, all disclosures about vitamin B6 described in this application are generally applicable to and specifically refer to the preferred embodiment pyridoxine, even if not explicitly stated. In an embodiment, vitamin B6 can be selected from pyridoxine, pyridoxal, pyridoxamine and / or pyridoxal phosphate.

[0132] The water-soluble vitamin may be selected from ascorbic acid, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pyridoxine (vitamin B6), folate or folic acid, vitamin B12 (cobalamin), biotin and pantothenic acid.

[0133] Ascorbic acid is preferably used as the water-soluble vitamin. Ascorbic acid can be represented by the following formula:

[0134]

[0135] As used herein, "ascorbic acid" may include stereoisomers, such as L-ascorbic acid, D-ascorbic acid, L-isoascorbic acid, D-isoascorbic acid. As used herein, "ascorbic acid" may in particular be as described in PschyrembelKlinisches Ascorbic acid as defined in (258.Auflage, Walter de Gruyter, Berlin, New York, 1998). It should be understood that derivatives of ascorbic acid are also contemplated and can be used herein. As used herein, "derivatives of ascorbic acid" may in particular be derivatives that, like ascorbic acid, have activity as free radical scavengers.

[0136] For ease of reading, in the following, reference is made to "water-soluble vitamins" without also explicitly mentioning derivatives thereof. Also, reference is made to "ascorbic acid" without explicitly mentioning derivatives of ascorbic acid. However, unless the context dictates otherwise, disclosures relating to water-soluble vitamins (e.g., regarding concentration ranges) always also apply to derivatives of water-soluble vitamins, and unless the context dictates otherwise, disclosures relating to ascorbic acid (e.g., regarding concentration ranges) always also apply to derivatives of ascorbic acid.

[0137] The stabilized composition may comprise at least one water-soluble vitamin in a concentration of less than 20 mg / ml, for example 0.1 mg / ml to 15 mg / ml, 0.2 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml, preferably selected from ascorbic acid, vitamin B1, vitamin B2, vitamin B3, vitamin B6, folate or folic acid, vitamin B12, biotin and pantothenic acid, more preferably ascorbic acid. In an embodiment, the stabilized composition comprises a B vitamin in one of the aforementioned concentrations, for example vitamin B6. In particular, it may comprise, for example, a B vitamin in a concentration of 0.1 mg / ml to 15 mg / ml or 0.1 mg / ml to 14 mg / ml, preferably vitamin B6. It was found that vitamin B6 protects the stabilized composition comprising at least one caspase inhibitor during sterilization already at relatively low concentrations, for example at a concentration of about 2 mg / ml. Therefore, B vitamins and in particular vitamin B6 can also be used at relatively low concentrations, for example 0.1 mg / ml to 11 mg / ml, 0.2 mg / ml to 7.5 mg / ml or even 0.2 mg / ml to 2 mg / ml. It is also more economical to use B vitamins such as vitamin B6 at correspondingly low concentrations.

[0138] The composition may comprise at least one water-soluble vitamin, preferably selected from ascorbic acid, vitamin B1, vitamin B2, vitamin B3, vitamin B6, folate or folic acid, vitamin B12, biotin and pantothenic acid, more preferably ascorbic acid, in a concentration of less than 20 mg / ml, for example 0.1 mg / ml to 15 mg / ml, 0.2 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml, and may be irradiated in liquid form, preferably aqueous form, for sterilization. In an embodiment, the stabilized composition comprises a B vitamin, for example vitamin B6, in one of the aforementioned concentrations. In particular, B vitamins, preferably vitamin B6, may be included, for example, in a concentration of 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 14 mg / ml, 0.1 mg / ml to 11 mg / ml, 0.2 mg / ml to 7.5 mg / ml or 0.2 mg / ml to 2 mg / ml.

[0139] The stabilizing composition may comprise ascorbic acid at a concentration of less than 20 mg / ml, for example, 0.1 mg / ml to 15 mg / ml, 0.2 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml.

[0140] The composition may comprise ascorbic acid at a concentration of less than 20 mg / ml, e.g., 0.1 mg / ml to 15 mg / ml, 0.2 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml, and may be sterilized by irradiation in liquid form, preferably aqueous form.

[0141] As shown in the examples, it was found that various concentrations of ascorbic acid, such as 1 mg / ml, 2 mg / ml, 5 mg / ml and 10 mg / ml, protected the stabilized composition comprising at least one caspase inhibitor within a wide range of irradiation doses applied during sterilization. Within the concentrations tested, the protective effect tended to increase as the ascorbic acid concentration increased. It was most obvious at a concentration of about 5 mg / ml to about 10 mg / ml. Although it has been found that ascorbic acid can protect the stabilized composition during sterilization when used in a wide range of concentrations, it is preferred to use ascorbic acid at a concentration of less than 20 mg / ml. Particularly suitable ascorbic acid concentrations are those concentrations (e.g., 2, 4 or 5 mg / ml) within the range of 0.5 mg / ml to 15 mg / ml, such as 2 mg / ml to 11 mg / ml, as indicated above. Surprisingly, ascorbic acid is also protective at these relatively low concentrations, which advantageously also allows the use of ascorbic acid at a mild pH, such as a weakly acidic pH. Advantageously, it is possible to irradiate and sterilize the composition in liquid form. Surprisingly, it is not necessary to irradiate the composition in solid or frozen form in order to maintain the stabilizing properties of the composition. In addition, the use of ascorbic acid at a concentration of less than 20 mg / ml, preferably 15 mg / ml or less, advantageously prevents the inhibitory effect of ascorbic acid on the stabilization of ccfDNA in plasma.

[0142] The composition may comprise ascorbic acid in combination with one or more other compounds of the invention. As explained in the discussion of N-acetylcysteine, the composition comprising a caspase inhibitor may, for example, comprise ascorbic acid and N-acetylcysteine ​​for protection. Similarly, the composition may comprise ascorbic acid and glutathione, preferably glutathione in reduced form. Figure 6 Exemplary combinations are shown in the examples of FIG. 7 and are specifically contemplated herein.

[0143] As shown in the examples, vitamin E and its derivatives also provide protection during irradiation sterilization for stabilized compositions containing one or more caspase inhibitors. Vitamin E and its derivatives, particularly water-soluble derivatives such as trolox, also show protective activity, although the degree of protection mediated by, for example, trolox is lower than that observed for N-acetylcysteine, ascorbic acid or glutathione. Water-soluble derivatives of other fat-soluble vitamins, such as water-soluble derivatives of vitamins D, K and A, are also contemplated herein; the derivatives may, in particular, have activity as free radical scavengers, like the corresponding fat-soluble vitamins.

[0144] "Vitamin E" as used herein may in particular be as described in Pschyrembel Klinisches (258.Auflage, Walter de Gruyter, Berlin, New York, 1998) as defined in Vitamin E. As used herein, a "derivative of Vitamin E" may in particular be a derivative that has activity as a free radical scavenger like Vitamin E. As used herein, a "derivative of Vitamin E" may in particular be a water-soluble Vitamin E derivative that has activity as a free radical scavenger. An example of a particularly preferred water-soluble Vitamin E derivative is trolox. Trolox may be referred to as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

[0145] Thus, a composition comprising vitamin E or a derivative thereof can be prepared. The composition may comprise a water-soluble vitamin E derivative. Preferably, the water-soluble vitamin E derivative is trolox. Throughout the application, disclosures relating to "vitamin E or a derivative thereof" are particularly applicable to water-soluble vitamin E derivatives including trolox, unless the context dictates otherwise. Whenever "vitamin E or a derivative thereof" is mentioned, water-soluble vitamin E derivatives are preferred, such as, in particular, trolox.

[0146] Vitamin E or a derivative thereof, for example preferably a water-soluble vitamin E derivative, more preferably trolox, can be used in the stabilizing composition in a concentration of 0.5 mg / ml to 5 mg / ml, 0.6 mg / ml to 4 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml, or 1 mg / ml to 1.4 mg / ml. Thus, the composition can, for example, comprise trolox in a concentration of 0.5 mg / ml to 5 mg / ml, 0.6 mg / ml to 4 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml, or 1 mg / ml to 1.4 mg / ml.

[0147] The composition comprising at least one caspase inhibitor may comprise vitamin E or a derivative thereof, preferably a water-soluble vitamin E derivative, more preferably trolox, at a concentration of 0.5 mg / ml to 5 mg / ml, 0.6 mg / ml to 4 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml or 1 mg / ml to 1.4 mg / ml, and is irradiated sterilized in liquid form, preferably aqueous form.

[0148] The composition may contain a water-soluble vitamin E derivative, preferably trolox, at a concentration of 0.5 mg / ml to 5 mg / ml, 0.6 mg / ml to 4 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml, or 1 mg / ml to 1.4 mg / ml, and is sterilized by irradiation in a liquid form, preferably an aqueous form. As shown in the examples, it was found that a vitamin E derivative at a concentration of about 1.25 mg / ml provided protection of the stabilized composition during irradiation. There, the vitamin E derivative trolox has been tested.

[0149] When more than one compound is combined, the concentrations and concentration ranges of each compound disclosed herein may also be used. For example, when more than one compound is combined, the ranges described in Table A below may be used. Exemplary combinations and concentration ranges of N-acetylcysteine, glutathione, and ascorbic acid are shown in Table A. Figure 6 The above examples are shown in Table 7 of the neutralization examples and are specifically contemplated herein. As can be seen from the examples, when used in combination, relatively low concentrations of the individual compounds are still protective during irradiation sterilization. Thus, when one or more of the following compounds are combined, concentrations of, for example, about 0.3 mg / ml to 4 mg / ml can be used for each of N-acetylcysteine ​​and ascorbic acid, and a concentration range of, for example, about 0.1 mg / ml to 1.3 mg / ml can be used for glutathione.

[0150] Exemplary preferred compounds of the present invention as well as advantageous concentration ranges included in the stabilizing composition provided in step a) are disclosed in the following table.

[0151]

[0152] Exemplary preferred compounds of the present invention as well as advantageous concentration ranges included in the stabilizing composition provided in step a) are also disclosed in the following table.

[0153]

[0154] Other characteristics of the composition prepared or provided in step a)

[0155] In addition to the at least one caspase inhibitor and the at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives as described above, the stabilization composition provided in step a) may also comprise additional components. These additional components may be included in particular to support the stabilizing effect on the extracellular nucleic acid population.

[0156] Other components that may be included and advantageous compositions that may be provided, for example prepared, in step a) are described below. Of course, the preferred embodiments discussed above, especially those related to features i. and ii., apply, and the headings and subsections provided herein are only used for ease of reading.

[0157] Advantageous compositions comprising one or more caspase inhibitors suitable for stabilizing extracellular nucleic acid populations are disclosed in WO 2013 / 045457 A1, WO 2014 / 146780 A1, WO 2014 / 146782 A1, PCT / EP2015 / 055699, WO 2014 / 049022 A1, WO 2013 / 045458 A1 and WO 2014 / 146781 A1. These references to which it refers describe several stabilizing compositions for stabilizing extracellular nucleic acid populations. The extracellular nucleic acid stabilizing effect of the described stabilizing compositions is based on the use of caspase inhibitors or one or more other compounds that can be used in combination with caspase inhibitors for stabilization. WO2014 / 049022 and WO2014 / 146781 also teach stabilization compositions containing caspase inhibitors suitable for stabilizing intracellular nucleic acids (e.g., intracellular DNA and / or intracellular RNA, particularly suitable for stabilizing gene expression profiles). In addition, these applications disclose stabilization compositions that allow subsequent analysis of stabilized cells, such as cell morphology and / or cell surface characteristics. The stabilization compositions described in these references that contain one or more caspase inhibitors disclosed therein are incorporated herein by reference, and the stabilization compositions described in PCT / EP2015 / 055699 are particularly preferred. These compositions containing caspase inhibitors can be sterilized with the method of the present invention. Therefore, it is preferred to provide a composition comprising one or more caspase inhibitors disclosed in one of the aforementioned references in step a), wherein the composition also comprises at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione, preferably wherein glutathione is in reduced form), water-soluble vitamins, and vitamin E or its derivatives, and N-acetylcysteine ​​is highly preferred. The same applies to the case where a stabilization composition as disclosed in WO 2008 / 145710 (which allows stabilization of, for example, the genome, transcriptome and proteome) is combined with a caspase inhibitor to provide a composition containing a caspase inhibitor, wherein the composition further comprises at least one compound selected from the group consisting of thiols (preferably N-acetylcysteine ​​or glutathione, preferably wherein the glutathione is in reduced form), water-soluble vitamins, and vitamin E or a derivative thereof, N-acetylcysteine ​​being highly preferred.

[0158] The stabilizing composition provided, e.g. prepared, in step a) may therefore also comprise one or more of the following:

[0159] a) anticoagulants and / or chelating agents;

[0160] b) poly(ethylene oxide) polymers; and / or

[0161] c) at least one primary, secondary or tertiary amide.

[0162] Suitable concentrations of anticoagulants, chelating agents, poly(ethylene oxide) polymers and primary, secondary and tertiary amides are disclosed in PCT / EP2015 / 055699.

[0163] Subsequently, exemplary suitable concentrations of each agent, if present in the stabilizing composition, are indicated.

[0164] The stabilizing composition may be a liquid. The indicated concentrations are particularly preferred for stabilizing blood samples. For example, 0.5 ml to 2.5 ml, 0.5 ml to 2 ml, preferably 1 ml to 2 ml or 1 ml to 1.5 ml of a liquid stabilizing composition may be used. Such a stabilizing composition comprising a stabilizer in the concentration indicated below may be used to stabilize, for example, 10 ml of blood.

[0165] For example, the composition may also include an anticoagulant, preferably a chelating agent, more preferably EDTA, such as K2EDTA. In the case where the sample to be stabilized is blood, this embodiment is particularly useful. According to one embodiment, the composition includes an anticoagulant or a chelating agent, preferably EDTA, and a concentration selected from 9.5mM to 1100mM, 20mM to 750mM, 50mM to 600mM, 75mM to 550mM, 100mM to 500mM, 125mM to 450mM, 130mM to 300mM and 140mM to 250mM. According to one embodiment, the concentration is 100mM to 250mM.

[0166] In one embodiment, the composition provided in step a) comprises at least one poly(ethylene oxide) polymer. As described in detail in PCT / EP2015 / 055699, to which it is referenced, poly(ethylene oxide) polymers exhibit advantageous stabilizing properties when intended to stabilize extracellular nucleic acids. They are particularly effective when used in combination with caspase inhibitors and can improve the stabilizing effect. Therefore, it is advantageous for the stabilized composition to be sterilized to additionally comprise a poly(ethylene oxide) polymer. Such a composition may have one or more of the following features:

[0167] a) the poly(oxyethylene) polymer is polyethylene glycol, preferably unsubstituted polyethylene glycol;

[0168] b) the composition comprises a poly(ethylene oxide) polymer which is a high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500;

[0169] c) the composition comprises at least one poly(oxyethylene) polymer having a molecular weight of less than 1500, preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less, more preferably the molecular weight is within the range selected from 100 to 800, 150 to 700, 200 to 600 and 200 to 500;

[0170] d) the composition comprises a poly(oxyethylene) polymer which is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500, and at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer, wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less; and / or

[0171] e) the composition comprises a poly(oxyethylene) polymer as a high molecular weight poly(oxyethylene) polymer and a poly(oxyethylene) polymer as a low molecular weight poly(oxyethylene) polymer, wherein the molecular weight of the high molecular weight poly(oxyethylene) polymer is within the range selected from 1500 to 50000, 1500 to 40000, 2000 to 30000, 2500 to 25000, 3000 to 20000, 3500 to 15000 and 4000 to 12500 and / or wherein the molecular weight of the low molecular weight poly(oxyethylene) polymer is 1000 or less and wherein preferably the molecular weight is within the range selected from 100 to 1000, 150 to 800, 150 to 700, 200 to 600, 200 to 500 and 200 to 400.

[0172] According to one embodiment, the stabilizing composition comprises a high molecular weight poly(oxyethylene) polymer, preferably polyethylene glycol, at a concentration selected from 0.4% to 35% (w / v), 0.8% to 25% (w / v), 1.5% to 20% (w / v), 2.5% to 17.5% (w / v), 3% to 15% (w / v), 4% to 10% (w / v) and 3% to 5% (w / v). Suitable concentrations can be determined by a skilled person and may depend in particular on whether the high molecular weight poly(oxyethylene) glycol is used alone or in combination with other poly(oxyethylene) polymers, such as low-order poly(oxyethylene) polymers, and the amount of the stabilizing composition used to stabilize a certain amount of a cell-containing sample, such as by volume. When a high molecular weight poly(oxyethylene) polymer is used alone, examples of suitable concentration ranges include, but are not limited to, concentrations selected from the group consisting of 2.2% to 33.0% (w / v), 4.4% to 22.0 (w / v)%, 6.6% to 16.5% (w / v), and 8.8% to 13.2% (w / v). When a high molecular weight poly(oxyethylene) polymer is used in combination with a low molecular weight poly(oxyethylene) polymer, examples of suitable concentration ranges include, but are not limited to, concentrations selected from the group consisting of 0.4% to 30.7%, 0.8% to 15.3%, 1% to 10%, 1.5% to 7.7%, 2.5% to 6%, 3.1% to 5.4%, and 3% to 4%.

[0173] According to one embodiment, the stabilizing composition comprises a low molecular weight poly(ethylene oxide) polymer, preferably polyethylene glycol, in a concentration selected from 0.8% to 92.0%, 3.8% to 76.7%, 11.5% to 53.7%, 19.2% to 38.3%, 20% to 30% and 20% to 27.5%. According to one embodiment, the concentration is 11.5% to 30%. Depending on whether the low molecular weight poly(ethylene oxide) polymer is a liquid, the aforementioned concentration refers to (w / v) or (v / v). As demonstrated in the examples of PCT / EP2015 / 055699, low molecular weight poly(ethylene oxide) polymers can effectively support the stabilization of cell-containing samples, particularly when used in combination with one or more other stabilizers described therein.

[0174] According to one embodiment, the stabilizing composition prepared or provided in step a) comprises at least one primary, secondary or tertiary amide. Use one or more of these amides to support the stabilization of the extracellular nucleic acid population, as demonstrated in the applications WO 2013 / 045457 A1, WO 2014 / 146780 A1, WO 2014 / 146782 A1, PCT / EP2015 / 055699, WO 2014 / 049022 A1, WO 2013 / 045458 A1 or WO 2014 / 146781 A1 to which it refers. WO2014 / 049022 and WO 2014 / 146781 also teach that such amides are suitable for stabilizing intracellular nucleic acids (particularly gene expression profiles). In addition to the caspase inhibitors used for stabilization, preferably in addition to at least one poly (ethylene oxide) polymer, one or more of these amides can also be used.

[0175] According to one embodiment, the composition thus comprises one or more compounds of formula 1

[0176]

[0177] Wherein R1 is a hydrogen group or an alkyl group, preferably a C1-C5 alkyl group, a C1-C4 alkyl group or a C1-C3 alkyl group, more preferably a C1-C2 alkyl group, R2 and R3 are the same or different and are selected from a hydrogen group and a hydrocarbon group arranged in a straight chain or branched manner with a carbon chain length of 1 to 20 atoms, preferably an alkyl group, and R4 is an oxygen, sulfur or selenium group, preferably R4 is oxygen.

[0178] Combinations of one or more compounds of Formula 1 may also be used. In embodiments where R1 is an alkyl group, it is preferred that the chain length of R1 is 1 or 2. R2 and / or R3 of the compound of Formula 1 are identical or different and are selected from hydrogen groups and hydrocarbon groups, which are preferably alkyl groups. According to one embodiment, both R2 and R3 are hydrogen. According to one embodiment, one of R2 and R3 is hydrogen and the other is a hydrocarbon group. According to one embodiment, R2 and R3 are identical or different hydrocarbon groups. The hydrocarbon group R2 and / or R3 may be independently selected from alkyl (including short-chain alkyl and long-chain alkyl), alkenyl, alkoxy, long-chain alkoxy, cycloalkyl, aryl, haloalkyl, alkylsilyl, alkylsiloxy, alkylene, alkylenediyl, arylene, carboxylate and carbonyl (for these groups, see, for example, WO2013 / 045457, pages 20-21, incorporated herein by reference). The chain length n of R2 and / or R3 may in particular have values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. According to one embodiment, the carbon chain length of R2 and R3 is 1-10, preferably 1 to 5, more preferably 1 to 2. According to one embodiment, R2 and / or R3 is an alkyl group, preferably a C1-C5 alkyl group. Preferably, the compound of formula 1 is a carboxylic acid amide, whereby R4 is oxygen. It may be a primary, secondary or tertiary carboxylic acid amide.

[0179] According to one embodiment, the compound of formula 1 is an N,N-dialkylcarboxylic acid amide. Preferred R1, R2, R3 and R4 groups are as described above. The use of the corresponding compound of formula 1 has the following advantages: the intracellular nucleic acid in the cell-containing sample can be additionally stabilized, such as in particular RNA, such as mRNA and / or miRNA transcripts. Additional stabilization of intracellular nucleic acids, in particular gene transcript levels, is advantageous because it allows, for example, subsequent analysis of target transcripts or transcript profiles in the contained cells. According to one embodiment, the compound of formula 1 is selected from N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide and N,N-diethylformamide. The corresponding thio analogs containing sulfur instead of oxygen as R4 are also suitable. Preferably, at least one compound of formula 1 is used, which is not a toxic agent according to the GHS classification. According to one embodiment, the compound of formula 1 is an N,N-dialkylpropionamide, such as N,N-dimethylpropionamide.

[0180] And, for example, the composition may include one or more compounds of formula 1'

[0181]

[0182] wherein R1 is a hydrogen group or an alkyl group, preferably a C1-C5 alkyl group, more preferably a methyl group, R2 and R3 are the same or different hydrocarbon groups having a carbon chain length of 1-20 atoms and arranged in a branched or branched manner, and R4 is an oxygen, sulfur or selenium group.

[0183] Formula 1' is encompassed by formula 1 discussed above and, in contrast thereto, defines that R2 and R3 are identical or different hydrocarbon groups (not hydrogen). Apart from this, the groups R1 to R4 correspond to those discussed above for formula 1 and this reference also applies to the disclosure herein.

[0184] Preferably, the composition comprises butanamide and / or N,N-dialkylpropionamide, more preferably N,N-dimethylpropionamide.

[0185] According to one embodiment, the stabilizing composition comprises one or more primary, secondary or tertiary amides in a concentration selected from 0.4% to 38.3%, 0.8% to 23.0%, 2.3% to 11.5%, 3.8% to 9.2%, 5% to 15% and 7.5% to 12.5%. Depending on whether the primary, secondary or tertiary amide is liquid, the aforementioned concentration refers to (w / v) or (v / v). As discussed, primary, secondary or tertiary carboxylic acid amides are preferred.

[0186] The composition may also include DMSO. For example, the composition may include 0.05% to 2.5% (v / v) DMSO, 0.1% to 1.8%, or 0.2% to 1.4% DMSO. DMSO is a suitable solvent and has been found to support protection of the stabilized composition during irradiation, more particularly gamma irradiation. This effect is particularly observed when DMSO is used in combination with trolox, but is not limited thereto.

[0187] As explained above, the composition may initially be provided in solid form. It may then be brought into liquid form prior to irradiation. The solid composition may comprise the caspase inhibitor, one or more compounds of the invention, and optional other components discussed herein, in amounts sufficient to produce, after solvation, a composition comprising the caspase inhibitor, the one or more compounds, and optional other components in concentrations as described above and below.

[0188] According to non-limiting exemplary embodiments, a solid composition comprising 0.001% to 0.01% (w / w) caspase inhibitor, for example 0.005% to 0.009% (w / w) caspase inhibitor can be prepared. According to the composition described in this exemplary embodiment, about 0.05 to about 0.5% (w / w), for example 0.1% to 0.4% (w / w) of the compound of the present invention, for example thiol, particularly N-acetylcysteine, can be included in the exemplary solid composition. If there is a high molecular weight poly (ethylene oxide) polymer, its exemplary concentration is, for example, 3.25% to 10% (w / w), such as 5% to 8.5% (w / w). If there is a low molecular weight poly (ethylene oxide) polymer, its exemplary concentration is, for example, 20% to 70% (w / w), such as 40% to 65% (w / w). If a primary, secondary or tertiary amide is present, an exemplary concentration is, for example, 15% to 30% (w / w), such as 18% to 25% (w / w). If an anticoagulant and / or chelating agent is present, an exemplary concentration is, for example, 6% to 24% (w / w), such as 8% to 15% (w / w).

[0189] Preferred compositions to be sterilized comprise:

[0190] a) one or more caspase inhibitors,

[0191] b) one or more compounds selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives as discussed above, wherein preferably the composition comprises N-acetylcysteine,

[0192] c) at least one poly(ethylene oxide) polymer as a stabilizer, which is preferably a high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500,

[0193] and optionally one or more, preferably two or more, other additives selected from the following:

[0194] d) at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the first poly(oxyethylene) polymer, which is preferably a high molecular weight poly(oxyethylene) polymer, wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less;

[0195] e) at least one primary, secondary or tertiary amide;

[0196] f) Anticoagulants and / or chelating agents.

[0197] Preferably, the stabilizing composition comprises one or more compounds of the invention in concentrations as disclosed above in the discussion of feature ii, more preferably as disclosed above in Table A. The stabilizing composition may comprise one or more compounds as disclosed above in Table B. The use of N-acetylcysteine ​​is particularly preferred.

[0198] By stabilizing the cells and extracellular nucleic acid populations contained in cell-containing biological samples such as blood, plasma and / or serum, the corresponding stabilizing composition is particularly effective in stabilizing the sample. The extracellular nucleic acid population contained in the cell-containing biological sample substantially retains the state displayed when the biological sample is in contact with the stabilizing composition during the stabilization period. As shown in, for example, the embodiments of the present application and the embodiments of PCT / EP2015 / 055699, the release of genomic DNA and other intracellular nucleic acids is significantly reduced. Compared with the extracellular nucleic acids separated from the samples that have never been stabilized, the extracellular nucleic acids separated from the corresponding stabilized samples contain significantly less intracellular nucleic acids, particularly fragmented genomic DNA contamination. The corresponding stabilizing composition allows the stabilized sample, such as blood, to be stored and / or processed, such as transported, at room temperature for several days without substantially damaging the quality of the sample, and accordingly without damaging the extracellular nucleic acid population contained therein.

[0199] Another preferred composition to be sterilized comprises:

[0200] a) one or more caspase inhibitors, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide, even more preferably a caspase inhibitor selected from Q-VD-OPh, Z-VAD(OMe)-FMK and Boc-D-(OMe)-FMK, most preferably Q-VD-OPh;

[0201] b) one or more compounds selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins (preferably vitamin B6 or ascorbic acid), and vitamin E or its derivatives as discussed above, wherein preferably the composition comprises N-acetylcysteine,

[0202] c) at least one poly(oxyethylene) polymer as a stabilizer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol;

[0203] and optionally one or more, preferably two, other additives selected from the following:

[0204] d) at least one primary, secondary or tertiary amide;

[0205] e) Anticoagulants and / or chelating agents.

[0206] Preferably, the stabilizing composition comprises one or more compounds of the invention in concentrations as disclosed above in discussing feature ii, more preferably as disclosed above in Table A or Table B. The use of N-acetylcysteine ​​is particularly preferred.

[0207] In an embodiment, the at least one poly(oxyethylene) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(oxyethylene) polymers, preferably low molecular weight poly(oxyethylene) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(oxyethylene) polymers having a molecular weight of at least 1500.

[0208] Another example of a preferred stabilizing composition having excellent stabilizing and sterilizing properties comprises:

[0209] a) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide, most preferably Q-VD-OPh;

[0210] b) N-acetylcysteine, glutathione (preferably in reduced form) and / or ascorbic acid, preferably N-acetylcysteine, more preferably N-acetylcysteine ​​in a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml;

[0211] c) at least one high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500, preferably in the range of 2000 to 40000, more preferably 2500 to 30000, 2500 to 25000 or 3000 to 20000;

[0212] d) one or more compounds of formula 1, preferably wherein the composition comprises butanamide and / or N,N-dialkylpropionamide, more preferably N,N-dimethylpropionamide;

[0213] e) at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer used, and wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) having a molecular weight of 1000 or less, preferably a molecular weight in the range of 200 to 800 or 200 to 600;

[0214] f) Optional anticoagulants and / or chelating agents, preferably EDTA.

[0215] Another example of a preferred stabilizing composition having excellent stabilizing and sterilizing properties comprises:

[0216] a) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide, even more preferably most preferably Q-VD-OPh;

[0217] b) N-acetylcysteine, glutathione (preferably in reduced form) and / or ascorbic acid, preferably N-acetylcysteine, more preferably N-acetylcysteine ​​in a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml;

[0218] c) at least one poly(oxyethylene) polymer as a stabilizer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol;

[0219] d) one or more compounds of formula 1, preferably wherein the composition comprises butanamide and / or N,N-dialkylpropionamide, more preferably N,N-dimethylpropionamide;

[0220] e) Optional anticoagulants and / or chelating agents, preferably EDTA.

[0221] In an embodiment, the at least one poly(oxyethylene) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(oxyethylene) polymers, preferably low molecular weight poly(oxyethylene) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(oxyethylene) polymers having a molecular weight of at least 1500.

[0222] The stabilized composition provided in step a) may have one or more of the following characteristics:

[0223] a) it is capable of stabilizing cells and reducing the release of genomic DNA from cells contained in said cell-containing biological sample into the cell-free portion of said sample;

[0224] b) it is capable of reducing the degradation of nucleic acids, particularly genomic DNA, present in the sample being stabilized;

[0225] c) it is capable of reducing or preventing the contamination of the extracellular DNA population contained in the biological sample with genomic DNA originating from cells contained in the stabilized sample;

[0226] d) it is capable of reducing or preventing the contamination of the extracellular nucleic acid population contained in the biological sample by intracellular nucleic acids originating from cells contained in the stabilized sample;

[0227] e) the stabilization composition comprises an additive at a concentration that does not cause the additive to induce or promote cell lysis;

[0228] f) the stabilization composition does not contain a cross-linking agent that induces protein-DNA and / or protein-protein cross-links, such as formaldehyde, formalin, paraformaldehyde or formaldehyde-releasing agents;

[0229] g) the stabilization composition does not contain toxic agents;

[0230] h) it is capable of stabilizing the extracellular nucleic acid population comprised in the cell-containing biological sample without refrigeration, preferably at room temperature, for a period of time selected from at least three days, at least four days, at least five days or at least six days; and / or

[0231] i) The composition does not comprise a cell-containing sample to be stabilized, preferably blood.

[0232] In particular, it is preferred that the composition does not contain a cross-linking agent that induces protein-DNA and / or protein-protein cross-linking. Cross-linking agents that induce protein-DNA and / or protein-protein cross-linking are, for example, formaldehyde, formalin, paraformaldehyde or formaldehyde releasers. This provides important advantages over known prior art stabilization reagents and methods involving the use of cross-linking agents such as formaldehyde, formaldehyde releasers, etc. Cross-linking agents cause intermolecular or intramolecular covalent bonds between nucleic acid molecules or between nucleic acids and proteins. This effect can lead to a reduced recovery of such stabilized and partially cross-linked nucleic acids after purification or extraction from complex biological samples. Since the concentration of circulating nucleic acids in, for example, whole blood samples is already relatively low, any measures that further reduce the yield of such nucleic acids should be avoided. This may be particularly important when detecting and analyzing very rare nucleic acid molecules derived from malignant tumors or from a developing fetus in the first trimester of pregnancy. Therefore, it is preferred that a formaldehyde releaser is not included in the sterilized stabilization composition, and accordingly, the formaldehyde releaser is not used additionally for stabilization. Thus, according to one embodiment, the stabilizing composition does not contain a crosslinking agent such as formaldehyde or a formaldehyde releaser, and accordingly the crosslinking agent is not used additionally for stabilization. In addition, as described, the stabilizing composition preferably does not contain any additives that induce nucleated cell or total cell lysis, such as, for example, chaotropic salts.

[0233] Compositions comprising the caspase inhibitor Q-VD-OPh, dimethylpropionamide, PEG, EDTA such as K2EDTA and one or more of the compounds of Table A above are very suitable and have excellent sterilization and stabilization properties. The same applies to compositions comprising the caspase inhibitor Q-VD-OPh, dimethylpropionamide, PEG, EDTA such as K2EDTA and one or more of the compounds of Table B above. Most preferably, the composition comprises N-acetylcysteine. Compositions comprising ascorbic acid and compositions comprising glutathione (preferably reduced form) are also preferred. Trolox has also been found to provide protection during sterilization. However, other compounds are more preferred.

[0234] As discussed, in embodiments, the stabilization composition containing a caspase inhibitor is also suitable for stabilizing intracellular nucleic acids, particularly intracellular DNA (e.g., genomic DNA) and / or intracellular RNA. In particular, it can be suitable for stabilizing intracellular RNA, and is preferably suitable for stabilizing the gene transcription profile of the contained cells. Therefore, the intracellular nucleic acids separated from the corresponding stabilized samples are very suitable, for example, for gene expression profiling and other analytical methods, which require accurate representation of the in vivo transcript levels in the stabilized samples. In addition, advantageously, if necessary, such stabilization allows the stabilized extracellular nucleic acids to be separated from the same stabilized sample separately from the stabilized intracellular nucleic acids. The corresponding composition can include, for example, at least one primary or secondary carboxylic acid amide and / or at least one tertiary amide, preferably N, N-dialkyl propionamide, more preferably N, N-dimethyl propionamide (see, for example, WO 2014 / 049022 and WO 2014 / 146781). After sample stabilization, intracellular RNA is protected from degradation, and in addition, changes in the gene transcription profile of the contained cells are suppressed. Therefore, the stabilization particularly reduces in vitro degradation and minimizes gene induction. It is advantageous to stabilize the gene transcript levels of the contained cells, because the generation of new transcripts and the degradation of existing transcripts in the stabilized sample are inhibited compared to the unstabilized sample, thereby substantially "freezing" the gene transcription profile of the contained cells after stabilization. Therefore, the stabilization is also applicable to stabilize the transcriptome by maintaining the transcript levels in the state they display when the sample is collected and stabilized. The term transcriptome refers in particular to a collection of all RNA molecules (including mRNA, rRNA, tRNA and other non-coding RNAs, such as miRNA) produced in one or a group of cells. In an embodiment, a cell-containing biological sample such as a blood sample can be stabilized for at least three days without substantially changing the transcript level. By reducing RNA degradation and minimizing changes in gene expression such as particularly gene induction or downregulation, the gene transcription profile is particularly stabilized. Thus, the in vivo gene expression profile present during collection and corresponding stabilization is retained. In addition, the quality and integrity of the RNA can be maintained, thereby providing an accurate representation of the in vivo transcript levels during sample collection and corresponding sample stabilization. Retaining the in vivo gene transcription profile after stabilization allows, for example, gene expression profiling or other analytical methods that require accurate representation of the transcript levels using a corresponding stabilized sample. However, even if desired, it is often not necessary to stabilize or stabilize all transcript levels equally well. The stabilization and performance characteristics of a specific or new target transcript should be verified, as is typically done with prior art techniques for stabilizing gene transcription profiles. Stabilization of a gene transcription profile or specific transcript levels can be determined, for example, based on marker genes established for analyzing the stabilization of the gene transcription profile.According to one embodiment, the gene transcription profile of the contained cells or the stabilization of the transcript level cause one or more, preferably two or more marker genes selected from c-fos, IL-1 β, IL-8 and p53 to be stabilized for at least 12h, at least 24h or at least 48h after stabilization. These marker genes are identified as providing very unstable transcripts during storage, and therefore are raised or lowered in the case of no suitable stabilization after sample collection. Therefore, the transcript levels of these genes are suitable as markers to analyze whether the stabilization of gene transcription levels has been achieved. The stabilizing effect can be analyzed using the real-time RT-PCR assay described in the embodiment. According to one embodiment, the transcript levels of one or more of these marker genes change no more than 1.5CT values ​​between T0 (stabilization point) and the end of the stable period, preferably no more than 1.25CT values, more preferably no more than 1CT values. Preferably, the corresponding stabilizing effect of at least 12h, at least 24h, at least 48h, at least 72h or at least 96g is achieved. Preferably, corresponding stabilizing properties are achieved with at least the marker genes c-fos, IL8 and IL-1β and preferably with all the aforementioned marker genes. As demonstrated in W2014 / 049022 and W2014 / 146781, various amides such as primary and / or secondary carboxylic acid amides achieve corresponding stabilizing properties.

[0235] In an embodiment, the sterilized stabilization composition is capable of retaining the characteristics of the cells contained in the sample to be stabilized, for example, retaining the cell surface characteristics and / or cell morphology of the contained cells. Due to stabilization, the cells remain intact. This allows, for example, to separate cells from the stabilized sample after a stabilization period, and the cells separated from the stabilized sample are suitable for analysis. For example, intracellular nucleic acids such as DNA and / or RNA can be separated from the contained cells and can be analyzed. In addition, retaining the cells in the stabilized sample opens up the possibility of sorting or capturing cells or even enriching specific cells such as tumor cells, which can then be specifically analyzed. For example, circulating tumor cells can be separated and their gene expression profiles can be analyzed. In addition, cell morphology and / or cell markers, particularly cell surface markers, can be analyzed to characterize the obtained cells. In addition, intracellular nucleic acids can be separated from the enriched specific cells. For example, RNA can be isolated from the cells. The transcript level stabilization properties of the stabilization composition described herein advantageously allow the use of isolated RNA for gene expression profiling and other important analyses. Therefore, such stabilization is advantageous in molecular diagnosis of, for example, cancer or other diseases, because it allows enrichment of cells and then extraction of nucleic acids from the enriched cells, thereby increasing the probability of detecting rare events of circulating tumor cells, for example in a cell-containing sample, for example in a blood sample. This also increases the probability of identifying specific biomarkers, in particular rare biomarkers, in the sample. According to one embodiment, the morphology of the cells is retained during a stabilization period of preferably at least 12 h, at least 24 h and more preferably at least 48 h. This allows analysis and optionally characterization of the contained cells based on their morphology. According to one embodiment, the morphology of the nucleated cells is retained.

[0236] According to one embodiment, when the sterilized stabilization composition is used, the morphology of the lymphocytes contained in the blood sample is preserved during the stabilization period. According to one embodiment, the cell surface epitopes of the cells are retained. According to one embodiment, cell surface proteins such as CD proteins are retained. The retention of cell surface epitopes and cell surface proteins is an advantage because it allows the contained cells to be characterized and / or separated based on these cell surface characteristics. In particular, it allows the analysis of tumor markers present on the cell surface or the separation of specific cells based on the markers. Suitable stabilization compositions for this purpose are described, for example, in WO 2014 / 049022 and WO 2014 / 146781 to which they refer.

[0237] In an embodiment, the stabilizing composition used is suitable for stabilizing a proteome.

[0238] Step b) irradiating the composition for sterilization

[0239] The method further comprises irradiating the stabilizing composition in step b) for sterilization. As explained above, the composition is irradiated before contacting the composition with the biological sample. The object is to provide a sterilized stabilizing composition comprising a caspase inhibitor, which can be used to stabilize the extracellular nucleic acid population in the biological sample, for example by contacting the sample with the sterilized composition.

[0240] Several forms of irradiation are suitable and can be used. Forms of irradiation suitable for sterilizing compositions and devices are known to the skilled person. For example, ionizing radiation can be used, and forms of ionizing radiation suitable for sterilizing compositions and devices are also known to the skilled person. Examples of ionizing radiation are gamma irradiation, electron beam irradiation, X-rays and ionizing ultraviolet radiation. For the purpose of carrying out the present invention, gamma irradiation, electron beam irradiation and X-rays are preferred; gamma irradiation and X-rays are more preferred. Most preferably, irradiation is carried out by gamma irradiation.

[0241] Gamma radiation doses are measured in kilograys (kGy), which quantify the amount of radiation energy absorbed. One gray is one joule of radiation energy absorbed by one kilogram of material (1 kGy = 1 joule / gram).

[0242] The composition can be irradiated with an irradiation dose of 5kGy to 35kGy, 6kGy to 30kGy, 7kGy to 26kGy or about 8kGy to about 25kGy, for example, about 8kGy to about 15kGy or about 15kGy to about 25kGy. The irradiation dose of about 8kGy to about 15kGy provides the advantage of relatively gentle irradiation conditions, which further supports the maintenance of the stabilization properties in the irradiated composition. It is equally feasible and particularly cost-effective to irradiate with an irradiation dose of about 15kGy to about 25kGy, which is an important advantage for large-scale production. The composition can be irradiated with an irradiation dose of about 8kGy to about 25kGy, about 8kGy to about 15kGy or about 15kGy to about 25kGy by gamma irradiation. The composition can be irradiated by gamma irradiation at an irradiation dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, or about 8 kGy to about 25 kGy, such as about 8 kGy to about 15 kGy or about 15 kGy to about 25 kGy.

[0243] The irradiation dose can be applied over a wide range of irradiation times. Although it has been found that irradiation time is not a critical parameter for irradiating the stabilized solution, from a practical and economic point of view, it may be preferred to select a relatively short irradiation time. The skilled person is familiar with the selection of suitable irradiation times. Exemplary ranges include 1 second to 1 hour, 1 second to 30 minutes, 2 seconds to 10 minutes, or 5 seconds to 1 minute.

[0244] Typically, the composition is irradiated with a sufficiently high radiation dose to ensure that the composition is sterilized.

[0245] As used herein, "sterility" or "sterilized" may be defined in particular by the probability of viable microorganisms on a product after sterilization. This probability may be referred to as the sterility assurance level (SAL). For example, 10 -6 SAL is often used for sterilization of medical devices. As used herein, 10 -6 A SAL of 10 may particularly refer to a probability of 1 in 1,000,000 of finding a non-sterile unit, such as a non-sterile composition or device, after sterilization. As used herein, "sterility" or "sterilized" may particularly refer to a SAL of 10 -4 or lower, more preferably 10 -5 or lower, most preferably 10 -6 According to one embodiment, "sterility" or "sterilized" means "free from viable microorganisms".

[0246] Suitable validation methods for demonstrating that a sterilization process routinely imparts a selected SAL are known to the skilled person and for irradiation are set out, for example, in ANSI / AAMI / ISO 11137:2006.

[0247] Thus, irradiation sterilization can result in a composition or device with a sterility assurance level (SAL) of 10 -4 or lower, more preferably 10 -5 or lower, most preferably 10 -6 Or lower. The irradiation may meet the requirements of ISO standards ISO11137-1:2006, ISO11137-2:2012 and / or ISO11137-3:2006.

[0248] According to a preferred embodiment, sterility is achieved by irradiating with an irradiation dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy or about 8 kGy to about 25 kGy, for example about 8 kGy to about 15 kGy or about 15 kGy to about 25 kG, particularly preferably about 8 kGy to about 15 kGy, wherein the irradiation is preferably carried out by gamma irradiation.

[0249] There is no limitation on the form in which the composition is irradiated. Thus, the composition can be irradiated, for example, in solid or liquid form. However, for all embodiments described herein, it is preferred that the composition be irradiated sterilized in liquid form, preferably aqueous form.

[0250] It is found that the at least one compound selected from N-acetylcysteine, glutathione, water-soluble vitamins, and vitamin E or its derivatives is particularly active in protecting liquids, especially aqueous stabilization compositions containing one or more caspase inhibitors, especially containing one or more peptide caspase inhibitors. This is surprising because liquid compositions are often found to be difficult to sterilize without impairing the function of the composition in the prior art (e.g., in the pharmaceutical field). It is desirable to irradiate a liquid form, especially an aqueous form, of an extracellular nucleic acid population suitable for stabilizing a biological sample and containing one or more caspase inhibitors. This allows the provision of a sterilized stabilization composition that can be easily handled and mixed with a biological sample, especially a liquid biological sample such as blood, plasma or serum.

[0251] If the composition is irradiated in liquid form, preferably aqueous form, it is possible to first provide, for example prepare, the composition in solid form in step a), which is then dissolved in a suitable solvent to provide a liquid, preferably aqueous composition, which is then irradiated for sterilization. This can be done, for example, by dissolving the composition before or during step b).

[0252] According to one embodiment, the stabilized composition is irradiated in a non-solid state. According to one embodiment, the stabilized composition is irradiated in a non-frozen state.

[0253] The stabilized composition comprising at least one caspase inhibitor may be sterilized in a sample collection device, such as a container. The container may be a tube, more preferably a blood collection tube. The container is typically made of a material suitable for irradiation. For example, the container, such as a blood collection tube, may comprise or consist of polyethylene terephthalate. The collection device may be a device according to the seventh aspect of the invention.

[0254] Irradiating the composition for sterilization can provide a sterilized composition. Therefore, the method of the first aspect of the present invention is preferably a method for producing a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample, the method comprising:

[0255] a) providing a stabilizing composition as described above; and

[0256] b) sterilizing the composition by irradiation.

[0257] Other details and embodiments of the stabilized composition are as described above and with reference to the corresponding disclosures. In an embodiment, the sterilized composition is suitable for additional stabilization of intracellular nucleic acids (particularly intracellular RNA and / or intracellular DNA, such as genomic DNA). In an embodiment, the sterilized composition is suitable for additional stabilization of cell characteristics, such as cell surface characteristics and / or cell morphology. This is in accordance with the disclosures above that also apply here. The composition is preferably sterilized in liquid form, more preferably in aqueous form. This is in accordance with the disclosures above.

[0258] According to the specific implementation of the first aspect of the present invention

[0259] Suitable and preferred embodiments of the method according to the first aspect of the present invention are described below.

[0260] According to one embodiment of the first aspect of the present invention, there is provided a method for producing a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample, the method comprising:

[0261] a) providing a liquid, preferably aqueous, composition comprising:

[0262] i. at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide. The caspase-specific peptide can be modified with an aldehyde, nitrile or ketone compound. According to one embodiment, the caspase-specific peptide is modified with an O-phenoxy (OPh) or fluoromethylketone (FMK) group, preferably at the carboxyl end. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh, Boc-D-(OMe)-FMK and Z-VAD(OMe)-FMK. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh and Z-VAD(OMe)-FMK, most preferably Q-VD-OPh.

[0263] ii. one or more of the following compounds:

[0264] - N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0265] - reduced form of glutathione at a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml, and / or

[0266] - ascorbic acid in a concentration of less than 20 mg / ml, for example 1 mg / ml to 15 mg / ml, 0.2 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml,

[0267] - B vitamins, preferably vitamin B6, in a concentration of 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 14 mg / ml, 0.1 mg / ml to 11 mg / ml, 0.2 mg / ml to 7.5 mg / ml, or 0.2 mg / ml to 2 mg / ml,

[0268] The composition optionally further comprises one or more components selected from the following:

[0269] iii. at least one poly(oxyethylene) polymer as a stabilizer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol,

[0270] iv. at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide,

[0271] v. an anticoagulant and / or a chelating agent, preferably EDTA; and

[0272] b) irradiating the composition to sterilize, preferably by gamma irradiation.

[0273] In an embodiment, the at least one poly(ethylene oxide) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(ethylene oxide) polymers, preferably low molecular weight poly(ethylene oxide) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(ethylene oxide) polymers having a molecular weight of at least 1500.

[0274] The composition may be sterilized by irradiating the composition.

[0275] According to another embodiment of the first aspect of the present invention, there is provided a method for producing a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample, the method comprising:

[0276] a) providing a liquid, preferably aqueous, composition comprising:

[0277] i. at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide. The caspase-specific peptide can be modified with an aldehyde, nitrile or ketone compound. According to one embodiment, the caspase-specific peptide is modified with an O-phenoxy (OPh) or fluoromethylketone (FMK) group, preferably at the carboxyl end. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh, Boc-D-(OMe)-FMK and Z-VAD(OMe)-FMK. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh and Z-VAD(OMe)-FMK, most preferably Q-VD-OPh.

[0278] ii. one or more of the following compounds:

[0279] - N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0280] - reduced form of glutathione at a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml, and / or

[0281] - ascorbic acid in a concentration of less than 20 mg / ml, for example 1 mg / ml to 15 mg / ml, 0.2 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml,

[0282] The composition optionally further comprises one or more components selected from the following:

[0283] iii. at least one high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500, preferably in the range of 2000 to 40000, more preferably 2500 to 30000, 2500 to 25000 or 3000 to 20000;

[0284] iv. at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide,

[0285] v. at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer used, and wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) having a molecular weight of 1000 or less, preferably a molecular weight in the range of 200 to 800 or 200 to 600, and

[0286] vi. an anticoagulant and / or a chelating agent, preferably EDTA; and

[0287] b) irradiating the composition to sterilize, preferably by gamma irradiation.

[0288] The composition may be sterilized by irradiating the composition.

[0289] According to another embodiment of the first aspect of the present invention, there is provided a method for producing a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample, the method comprising:

[0290] a) providing a liquid composition, which is preferably aqueous, comprising:

[0291] i. at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide as described above, most preferably Q-VD-OPh,

[0292] ii. at least one compound as defined in Table A or Table B above, preferably wherein the composition comprises N-acetylcysteine,

[0293] The composition optionally further comprises one or more components selected from the following:

[0294] iii. at least one high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500, preferably in the range of 2000 to 40000, more preferably 2500 to 30000, 2500 to 25000 or 3000 to 20000;

[0295] iv. at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide,

[0296] v. at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer used, and wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) having a molecular weight of 1000 or less, preferably a molecular weight in the range of 200 to 800 or 200 to 600, and

[0297] vi. an anticoagulant and / or a chelating agent, preferably EDTA; and

[0298] b) irradiating the composition to sterilize, preferably by gamma irradiation.

[0299] The stabilized composition may be sterilized by irradiating the composition.

[0300] According to a preferred embodiment, the present invention provides a method for producing a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample, the method comprising:

[0301] a) providing an aqueous composition comprising:

[0302] i. at least one pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide as described above, most preferably Q-VD-OPh,

[0303] ii. N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0304] And the composition optionally further comprises:

[0305] iii. at least one poly(oxyethylene) polymer as a stabilizer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol,

[0306] iv. at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide, and

[0307] v. optional anticoagulant and / or chelating agent, preferably EDTA; and

[0308] b) irradiating the composition for sterilization, preferably by gamma irradiation, more preferably using a dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, about 8 kGy to about 25 kGy, or about 8 kGy to about 15 kGy.

[0309] In an embodiment, the at least one poly(ethylene oxide) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(ethylene oxide) polymers, preferably low molecular weight poly(ethylene oxide) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(ethylene oxide) polymers having a molecular weight of at least 1500.

[0310] The stabilized composition may be sterilized by irradiating the composition.

[0311] According to another preferred embodiment, the present invention provides a method for producing a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample, the method comprising:

[0312] a) providing an aqueous composition comprising:

[0313] i. at least one pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide as described above, most preferably Q-VD-OPh,

[0314] ii. N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0315] And the composition optionally further comprises:

[0316] iii. at least one high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500, preferably in the range of 2000 to 40000, more preferably 2500 to 30000, 2500 to 25000 or 3000 to 20000;

[0317] iv. at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide,

[0318] v. at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer used, and wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) having a molecular weight of 1000 or less, preferably a molecular weight in the range of 200 to 800 or 200 to 600, and

[0319] vi. optional anticoagulants and / or chelating agents, preferably EDTA; and

[0320] b) irradiating the composition for sterilization, preferably by gamma irradiation, more preferably using a dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, about 8 kGy to about 25 kGy, or about 8 kGy to about 15 kGy.

[0321] The stabilized composition may be sterilized by irradiating the composition.

[0322] According to one embodiment, in the above preferred embodiments, the aqueous composition provided in step a) comprises glutathione in a reduced form in a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml, and / or comprises ascorbic acid in a concentration of less than 20 mg / ml, for example 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml.

[0323] In addition to or as an alternative to N-acetylcysteine, the composition may comprise glutathione and ascorbic acid in reduced form. However, as explained before, it is advantageous for the composition to comprise N-acetylcysteine.

[0324] According to another preferred embodiment, the present invention provides a method for producing a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample, the method comprising:

[0325] a) providing an aqueous composition comprising:

[0326] i. Caspase inhibitor Q-VD-OPh,

[0327] ii. N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0328] The composition optionally further comprises:

[0329] iii. at least one poly(oxyethylene) polymer as a stabilizer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol,

[0330] iv. at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide, and

[0331] v. optional anticoagulant and / or chelating agent, preferably EDTA; and

[0332] b) sterilizing the composition by gamma irradiation, preferably using a dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, about 8 kGy to about 25 kGy, or about 8 kGy to about 15 kGy.

[0333] In an embodiment, the at least one poly(ethylene oxide) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(ethylene oxide) polymers, preferably low molecular weight poly(ethylene oxide) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(ethylene oxide) polymers having a molecular weight of at least 1500.

[0334] According to another preferred embodiment, the present invention provides a method for producing a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample, the method comprising:

[0335] a) providing an aqueous composition comprising:

[0336] i. Caspase inhibitor Q-VD-OPh,

[0337] ii. N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0338] The composition optionally further comprises:

[0339] iii. at least one high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500, preferably in the range of 2000 to 40000, more preferably 2500 to 30000, 2500 to 25000 or 3000 to 20000;

[0340] iv. at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide,

[0341] v. at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer used, and wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) having a molecular weight of 1000 or less, preferably a molecular weight in the range of 200 to 800 or 200 to 600, and

[0342] vi. an anticoagulant and / or a chelating agent, preferably EDTA; and

[0343] b) the composition is sterilized by gamma irradiation, more preferably using a dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, about 8 kGy to about 25 kGy, or about 8 kGy to about 15 kGy.

[0344] According to one embodiment, the composition provided in step a) and irradiated in step b) does not contain tannic acid and does not contain gallic acid. According to one embodiment, the composition also does not contain mannitol, isopropanol and / or Tween-80 or other compounds that may interfere with the stabilizing effect. According to one embodiment, the composition has a weakly acidic pH.

[0345] According to one embodiment, the stabilizing composition comprises the caspase inhibitor Q-VD-OPh at a concentration selected from 0.35 μg / ml to 70 μg / ml, 0.7 μg / ml to 63 μg / ml, 1.74 μg / ml to 59 μg / ml, 10.5 μg / ml to 56 μg / ml, or 15 μg / ml to 50 μg / ml, 20 μg / ml to 45 μg / ml, 25 μg / ml to 40 μg / ml and 30 μg / ml to 38 μg / ml.

[0346] B. Methods for Stabilizing Extracellular Nucleic Acid Populations

[0347] According to a second aspect, the present invention provides a method for stabilizing an extracellular nucleic acid population contained in a cell-containing biological sample, the method comprising:

[0348] a) producing a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample according to the method of the first aspect of the invention; and

[0349] b) contacting the cell-containing biological sample with the sterilized composition.

[0350] Also contemplated is a method for stabilizing an extracellular nucleic acid population contained in a cell-containing biological sample, the method comprising:

[0351] a) obtaining the composition according to the sixth aspect of the present invention, wherein the composition is sterilized, or obtaining the sample collection device according to the seventh aspect of the present invention, wherein the composition contained in the sample collection device is sterilized; and

[0352] b) contacting the cell-containing biological sample with a sterilized composition.

[0353] Suitable and preferred embodiments of the stabilizing composition and of the compound for sterilization protection are as described above, reference being made to the above disclosure which also applies here.According to a preferred embodiment, the extracellular nucleic acid population to be stabilized is ccfDNA.

[0354] After contacting the cell-containing biological sample with the composition, the resulting mixture may, for example, comprise:

[0355] a) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably Q-VD-OPh, preferably in a concentration ranging from 0.1 μM to 20 μM, 0.2 μM to 18 μM, 0.5 μM to 17 μM, 3 μM to 16 μM, more preferably in a range of 3 μM to 12 μM;

[0356] and one or more of the following

[0357] b) N-acetylcysteine ​​in a concentration range selected from 0.05 mM to 13.4 mM, 0.09 mM to 8.9 mM, 0.09 mM to 6.7 mM, 0.09 mM to 4.5 mM, 0.09 mM to 1.8 mM, 0.18 mM to 0.9 mM, or 0.27 mM to 0.71 mM;

[0358] c) glutathione, preferably in reduced form, in a concentration range selected from 0.01 mM to 4.2 mM, 0.02 mM to 3.15 mM, 0.03 mM to 2.1 mM, 0.06 mM to 1.68 mM, 0.13 mM to 1.26 mM, 0.17 mM to 0.95 mM or 0.17 mM to 0.61 mM;

[0359] d) a water-soluble vitamin, preferably ascorbic acid, in a concentration range selected from less than 16.5 mM, such as 0.08 mM to 12.4 mM, 0.83 mM to 10.72 mM, 1.24 mM to 9.9 mM, or 1.65 mM to 9.1 mM;

[0360] e) Vitamin E or a derivative thereof, preferably a water-soluble vitamin E derivative, more preferably trolox, in a concentration range selected from 0.29 mM to 2.9 mM, 0.44 mM to 1.74 mM, 0.52 mM to 1.16 mM, or 0.58 mM to 0.81 mM.

[0361] It is also contemplated that the resulting mixture may comprise B vitamins, preferably vitamin B6, in a concentration range selected from 0.08 mM to 12.9 mM, 0.08 mM to 12 mM, 0.1 mM to 9.4 mM, 0.17 mM to 6.4 mM, or 0.17 mM to 1.7 mM.

[0362] The use of N-acetylcysteine ​​is preferred.

[0363] After the cell-containing biological sample has been contacted with the sterilized stabilizing composition, the resulting mixture may comprise one or more of the following:

[0364] a) one or more compounds of Formula 1 in a concentration range of 0.25% to 5%, 0.3% to 4%, 0.4% to 3%, 0.5% to 2.5% or 0.75% to 2%;

[0365] b) at least one poly(oxyethylene) polymer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol;

[0366] c) a chelating agent, more preferably EDTA.

[0367] In an embodiment, the at least one poly(ethylene oxide) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(ethylene oxide) polymers, preferably low molecular weight poly(ethylene oxide) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(ethylene oxide) polymers having a molecular weight of at least 1500.

[0368] Thus, after the cell-containing biological sample has been contacted with the sterilized stabilizing composition, the resulting mixture may comprise one or more of the following:

[0369] a) one or more compounds of Formula 1 in a concentration range of 0.25% to 5%, 0.3% to 4%, 0.4% to 3%, 0.5% to 2.5% or 0.75% to 2%;

[0370] b) a high molecular weight poly(ethylene oxide) polymer in a concentration range selected from 0.1% to 3% (w / v), 0.2% to 2.5% (w / v), 0.25% to 2% (w / v), 0.3% to 1.75% (w / v) and 0.35% to 1.5% (w / v) or selected from 0.25% to 1.5% (w / v), 0.3% to 1.25% (w / v), 0.35% to 1% (w / v) and 0.4% to 0.75% (w / v);

[0371] c) a low molecular weight poly(ethylene oxide) polymer in a concentration range selected from 0.5% to 10%, 1.5% to 9%, 1.75% to 8%, 2% to 7% and 2.5% to 6%;

[0372] d) a chelating agent, more preferably EDTA.

[0373] According to one embodiment, the cell-containing sample is a blood sample. In this embodiment, stabilization preferably comprises the use of an anticoagulant, which may be a chelating agent, such as EDTA.

[0374] As described herein, in an embodiment, the sterilized composition is suitable for additionally stabilizing intracellular nucleic acids such as intracellular DNA (e.g., genomic DNA) and / or RNA. According to one embodiment, due to the stabilization, the degradation of nucleic acids present in the cell-containing sample is reduced. In particular, intracellular RNA is stabilized. Preferably, the transcriptome and / or transcript levels in the cells contained in the sample are stabilized. Details are as described above. According to one embodiment, the transcript levels of one or more marker genes selected from c-fos, IL-1β, IL-8 and p53 are stabilized for at least 12h, at least 24h, and preferably at least 48h after stabilization. According to one embodiment, after the stabilizing composition is contacted with a blood sample, the transcript levels of one or more marker genes selected from c-fos, IL-1β, IL-8 and p53 are stabilized for at least 12 hours, at least 24 hours, and preferably at least 48 hours after stabilization, and wherein, in one embodiment, the volume ratio of the stabilizing composition to the cell-containing sample is selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5.

[0375] In an embodiment, the sterilized composition is suitable for additionally stabilizing cell characteristics, such as cell surface characteristics and / or cell morphology. Importantly, the stabilized cells remain intact and can thus be separated from the stabilized sample. This is referred to the disclosure above. In one embodiment, the stabilization method has one or more of the following features:

[0376] a) said stabilization allows the isolation of cells from the stabilized sample;

[0377] b) the cell-containing sample is a blood sample, and wherein the cells contained in the blood sample are stabilized;

[0378] c) the cell-containing sample is a blood sample, and the leukocytes therein are stabilized;

[0379] d) preserve cell morphology;

[0380] e) retain the morphology of nucleated cells;

[0381] f) the sample is a blood sample and the lymphocytes and / or monocytes contained therein are stabilized;

[0382] g) retaining cell surface epitopes; and / or

[0383] h) Retention of cell surface proteins.

[0384] According to one embodiment, the sample is a blood sample, and the morphology and / or cell surface epitopes on leukocytes, preferably lymphocytes, are preserved therein. As described above, the characteristics of other cells contained in the blood sample, such as circulating tumor cells, if present, may also be preserved.

[0385] After the stabilization period, the method may include one or more of the following:

[0386] a) subjecting the stabilized sample to a nucleic acid analysis and / or detection method;

[0387] b) isolating extracellular nucleic acids from the stabilized sample;

[0388] c) isolating extracellular nucleic acids from the stabilized sample and analyzing and / or detecting the isolated nucleic acids;

[0389] d) removing cells contained in the stabilized sample;

[0390] e) removing cells contained in the stabilized sample before performing nucleic acid isolation, analysis and / or detection steps;

[0391] f) removing cells from the stabilized sample and isolating extracellular nucleic acids from a cell-free or cell-poor fraction of the stabilized sample;

[0392] g) storing (i) the stabilized sample, (ii) the stabilized sample from which cells have been removed and / or (iii) cells removed from a sample;

[0393] h) removing and discarding cells from said stabilized sample; and / or

[0394] i) removing cells from said stabilized sample and isolating nucleic acids from the cells removed from said stabilized sample;

[0395] j) removing cells from the stabilized sample, and isolating biomolecules such as proteins and / or metabolites, nucleic acids from the cells removed from the stabilized sample;

[0396] k) removing cells from the stabilized sample, and isolating nucleic acids and biomolecules, such as proteins and / or metabolites, from the cells removed from the stabilized sample;

[0397] 1) removing cells from the stabilized sample and isolating extracellular nucleic acids from a cell-free or cell-poor fraction of the stabilized sample using a size-selective nucleic acid isolation method.

[0398] Thus, the cell-containing biological sample stabilized using the method of the present invention can be analyzed in a nucleic acid analysis and / or detection method and / or can be further processed. Stabilization of the biological sample can be followed by a nucleic acid analysis technique, or nucleic acids can be first isolated from the stabilized sample. Details about nucleic acid isolation and analysis are also described below in conjunction with the third aspect of the present invention, and with reference to the disclosure.

[0399] In addition, as described, cells can be removed from the stabilized sample and analyzed, and / or nucleic acids can be isolated from the obtained cells. This allows, for example, identification of tumor cells. According to one embodiment, intracellular RNA is isolated from cells contained in the stabilized sample and analyzed. According to one embodiment, the method comprises qualitative or quantitative analysis of one or more gene transcripts. In addition, in embodiments, the morphology or cell surface characteristics of the cells can be analyzed.

[0400] According to one embodiment, the cell-containing sample is a blood sample, and the sterilized stabilizing composition achieves stabilization of leukocytes. This allows separation of leukocytes from the stabilized sample. Leukocytes are stabilized if at least one type of blood cells contained is stabilized during the stabilization period, and the stabilization period is preferably at least 12h, more preferably at least 24h, and more preferably at least 48h. According to one embodiment, lymphocytes and / or monocytes contained in the blood sample are stabilized. In one embodiment, the sterilized composition does not induce or promote the lysis of nucleated cells contained in the cell-containing sample. Therefore, stabilization is not based on cell lysis. Preferably, when the cell-containing sample is blood and the target nucleic acid is extracellular nucleic acid, in particular extracellular RNA, the sterilized stabilizing composition prevents hemolysis. Most of the causes of in vitro hemolysis are related to sample collection. However, in vitro hemolysis also usually occurs during the ex vivo storage of blood samples if appropriate stabilization methods are not used. Depending on the target extracellular nucleic acid, hemolysis may be a considerable problem. If the target extracellular nucleic acid is DNA, hemolysis is less of a problem because red blood cells do not contain nuclei and therefore do not contain genomic DNA. Therefore, red blood cells do not release intracellular DNA during hemolysis. When the target extracellular nucleic acid is DNA, in particular the lysis or decay of white blood cells is a problem because in this case, genomic DNA is released in addition to intracellular RNA. Therefore, when the target extracellular nucleic acid is extracellular DNA, the lysis of white blood cells must be prevented in particular. White blood cells may differ from each other in their stability characteristics. Thus, certain types of white blood cells are more stable than other types. However, in general, white blood cells are significantly more stable than red blood cells. Therefore, the lysis of red blood cells does not necessarily indicate that the white blood cells are lysed. The different susceptibility of white blood cells and red blood cells to lysis is also used in the art, for example, to specifically lyse red blood cells while retaining white blood cells to allow, for example, to collect white blood cells. However, if the target extracellular nucleic acid is RNA, hemolysis and therefore red blood cell lysis do constitute a problem. Mature red blood cells also do not contain RNA, however, their precursors (reticulocytes) contain RNA. Reticulocytes account for about 0.5% to 1% of red blood cells and contain a large amount of globulin RNA. Therefore, in particular when the target extracellular nucleic acid is RNA, in order to reduce the dilution of the extracellular nucleic acid population, in particular the extracellular RNA population, by globin mRNA, the lysis of erythrocytes and therefore reticulocytes during storage should be prevented / reduced. In one embodiment, hemolysis can be effectively prevented / reduced by the sterilized stabilizing composition (for a stabilizing composition containing a suitable caspase inhibitor that can be sterilized using the teachings of the present invention, see, for example, WO 2014 / 146781).Thus, the extracellular nucleic acid population is substantially retained, and in addition, the stabilized blood sample, in particular the plasma or serum obtained from the stabilized blood sample, is generally also suitable for other standard laboratory analyses due to the prevention of hemolysis and cell lysis. In addition, preventing leukocyte lysis allows the separation and analysis of the corresponding cells. In particular, it allows the separation of intracellular nucleic acids, such as intracellular RNA, from the leukocytes or other retained cells contained in the stabilized sample. Moreover, when stabilizing cells and intracellular nucleic acids including anucleated cells (e.g. reticulocytes), the transcriptome of such anucleated cells can also be analyzed, for example.

[0401] C. Methods for Isolating Extracellular Nucleic Acids

[0402] According to a third aspect, the present invention provides a method for isolating extracellular nucleic acids from a stabilized cell-containing biological sample, the method comprising:

[0403] a) stabilizing the cell-containing biological sample according to the method of the second aspect of the invention; and

[0404] b) Isolation of extracellular nucleic acids.

[0405] In step a), the extracellular nucleic acid population contained in the cell-containing sample is stabilized according to the method described in the second aspect of the invention, wherein a sterilized stabilization composition as described above is used. This reference is made to the disclosure above which also applies here.

[0406] If the cell-containing biological sample contains a large number of cells, such as, for example, in the case of using whole blood, the cells are preferably separated from the rest of the sample in order to obtain a cell-free, corresponding cell-reduced or cell-poor portion of the stabilized sample, from which the extracellular nucleic acid is then separated in step b). Thus, according to one embodiment, cells are removed from the cell-containing sample between step a) and step b). This intermediate step can be eliminated if a sample such as plasma or serum containing only a small amount of residual cells is processed and / or the target extracellular nucleic acid is DNA. Due to the stabilization of the present invention, the genomic DNA released from the contained cells during the stabilization period is reduced or even prevented, and in addition, due to the caspase inhibitor, the fragmentation of the genomic DNA is reduced. Due to the significantly larger size, the unfragmented genomic DNA can be distinguished from the smaller extracellular DNA. This, for example, allows the selective separation of extracellular DNA by using a size-selective separation scheme even in the presence of unfragmented genomic DNA. However, in order to improve the results, it is preferred to remove cells (or potential remaining cells) from the stabilized sample before isolating the extracellular nucleic acids in step b) to reduce the contamination of the extracellular nucleic acid population by intracellular nucleic acids that would otherwise be released from the cells during nucleic acid separation. If the target extracellular nucleic acid is RNA, it is also advantageous to remove the contained cells because it may be difficult to distinguish intracellular RNA from extracellular RNA, and in addition, dilution of the extracellular RNA can be prevented thereby. If the target extracellular nucleic acid is DNA, a cell removal step before step b) is also generally advantageous and therefore preferred because it allows the use of standard nucleic acid separation procedures in step b).

[0407] Depending on the type of cell-containing biological sample, cells, including residual cells, can be separated and removed, for example, by centrifugation, for example, at a medium speed, or, if a centrifugation step is to be avoided, by using means other than centrifugation, such as filtration, sedimentation or binding to a surface on, for example, (optionally magnetic) particles. Corresponding cell separation methods are well known in the prior art and therefore do not need to be described in detail. Corresponding cell removal steps can also be easily included in automated sample preparation protocols. If desired, the corresponding removed cells can also be further processed. The cells can, for example, be stored, analyzed and / or biomolecules such as nucleic acids or proteins can be separated from the removed cells. In addition, intracellular nucleic acids (e.g., intracellular DNA and / or RNA) can be separated from the contained cells, for example, after the cells are separated from the remaining sample. For example, intracellular RNA can be separated and used for gene expression analysis. For these embodiments, it is preferred that the sterilized composition also stabilizes intracellular nucleic acids, such as RNA and / or cell characteristics, particularly as discussed above. This is with reference to the corresponding disclosure.

[0408] Furthermore, it is within the scope of the present invention to include other intermediate steps to work-up the stabilized sample.

[0409] In step b), extracellular nucleic acids are separated, preferably from the cell-free, corresponding cell-poor part of the stabilized sample, such as from the supernatant or, when the stabilized cell-containing sample is a blood sample, from plasma and / or serum. In order to separate extracellular nucleic acids, any known nucleic acid separation method suitable for separating nucleic acids from the stabilized sample, the corresponding cell-poor sample obtained, can be used. Examples of corresponding purification methods include, but are not limited to, extraction, solid phase extraction, silica-based purification methods, purification based on magnetic particles, phenol-chloroform extraction, chromatography, anion exchange chromatography (using anion exchange surface), electrophoresis, filtration, precipitation and combinations thereof. Specific separation of specific target extracellular nucleic acids, such as by using a suitable probe coupled to a solid carrier and capable of achieving sequence-specific binding to specifically separate specific target extracellular nucleic acids, is also within the scope of the present invention. Any other nucleic acid separation techniques known to the technician can also be used. Exemplary methods for separating extracellular nucleic acids are described in WO 2013 / 045432 A1, which is incorporated herein by reference. The extracellular nucleic acid separation method of WO 2013 / 045432 A1 can be applied to the method according to the third aspect of the present invention.

[0410] D. Methods for processing and / or analyzing extracellular nucleic acids

[0411] According to a fourth aspect, the present invention provides a method for processing and / or analyzing extracellular nucleic acids, the method comprising:

[0412] a) isolating extracellular nucleic acids from a stabilized cell-containing biological sample according to the method of the third aspect of the invention; and

[0413] b) processing and / or analyzing the isolated extracellular nucleic acids.

[0414] The method according to the third aspect for separating extracellular nucleic acids in step a) is described above, and reference is also applicable to the corresponding disclosure herein. In step b), the separated extracellular nucleic acids are then analyzed and / or further processed. This can be accomplished using a suitable assay and / or analytical method. For example, they can be identified, modified, contacted with at least one enzyme, amplified, reverse transcribed, cloned, sequenced, contacted with a probe, detected (their presence or absence) and / or can be quantified. The corresponding method is well known in the prior art and is widely used in medical treatment, diagnosis and / or prognosis fields, so as to analyze extracellular nucleic acids (also referring to the detailed description in the background of the present invention). Therefore, after separating extracellular nucleic acids in step a), they can be analyzed to, for example, identify the presence, absence or severity of a disease state, which includes, but is not limited to, numerous tumor diseases, particularly premalignant tumors and malignant tumors such as tumors or cancers of different forms. For example, in many application areas, including but not limited to screening corresponding to non-invasive prenatal genetic testing, disease screening, screening for point mutations, trisomy, chromosomal aberrations, pathogen screening, oncology, cancer screening, early cancer screening, cancer treatment monitoring, genetic testing (genotyping), infectious disease detection, injury diagnosis, trauma diagnosis, transplant medicine or many other diseases, the separated extracellular nucleic acids can be analyzed to detect diagnostic and / or prognostic markers (e.g., extracellular nucleic acids of fetal or tumor origin), and therefore have diagnostic and / or prognostic relevance. According to one embodiment, the separated extracellular nucleic acids are analyzed to identify and / or characterize disease or fetal characteristics. Therefore, as discussed above, the separation method described herein can also include step c) nucleic acid analysis and / or processing.

[0415] Thus, according to one embodiment, the isolated extracellular nucleic acids are analyzed in step b) to identify, detect, screen, monitor or rule out a disease and / or at least one fetal characteristic.

[0416] E. Methods for Making Sterilizable Compositions

[0417] According to a fifth aspect, the present invention provides a method for producing a sterilizable composition, wherein the composition in sterilized form is suitable for stabilizing the extracellular nucleic acid population of a biological sample, the method comprising:

[0418] a) preparing a composition comprising:

[0419] i. at least one caspase inhibitor, and

[0420] ii. at least one compound selected from the group consisting of thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives.

[0421] The method may further comprise:

[0422] b) sterilizing the composition.

[0423] The method can be used to make sterilizable compositions, such as those provided in step a) of the first aspect of the present invention, or sterilizable compositions according to the sixth aspect of the present invention. The order in which the components of the composition are contacted with each other is not limited. For example, but not as a limitation, the composition in step a) can be prepared in the following manner: first prepare a composition comprising one or more caspase inhibitors, and then add the at least one compound selected from thiol (preferably N-acetylcysteine ​​and / or glutathione), water-soluble vitamins, and vitamin E or its derivatives. If necessary, it is also possible to add the at least one compound selected from thiol (preferably N-acetylcysteine ​​and / or glutathione), water-soluble vitamins, and vitamin E or its derivatives before adding the one or more caspase inhibitors. When preparing a liquid composition (e.g., an aqueous composition), a liquid (e.g., water) can be provided first for convenience, but this is not required. Similarly, the composition in step a) can be prepared, for example, by contacting the various components of the composition to be prepared with each other simultaneously or in any order. Therefore, the components of the composition can contact each other. They are preferably mixed to produce a liquid composition, preferably an aqueous composition. The liquid composition may be a homogeneous mixture of only a single phase, but it is also within the scope of the present invention that the liquid composition contains solid components such as precipitates. Step a) of the method according to the fifth aspect of the present invention may correspond to step a) of the method according to the first aspect of the present invention. This refers to the above disclosure. There, suitable and preferred embodiments of the stabilized composition and achievable stabilization characteristics are also described.

[0424] According to optional step b), the composition is then sterilized. Sterilization can be achieved by irradiation or other suitable means. In particular, sterilization can be achieved as described above in conjunction with the method of the first aspect of the invention. This is referred to the disclosure above.

[0425] F. Sterilizable Compositions

[0426] According to a sixth aspect, the present invention provides a sterilizable composition, wherein the composition in sterilized form is suitable for stabilizing the extracellular nucleic acid population of a biological sample. The composition is a composition provided in step a) of the method of the first aspect of the present invention. Suitable sterilizable compositions of the present invention have been described above when discussing the method according to the first aspect, with reference to the corresponding disclosure. With particular reference to the disclosure of the details of the composition above, in particular the components included discussed above, its suitable and preferred embodiments, and suitable and preferred concentrations and other features. In an embodiment, the sterilizable composition is suitable for additional stable intracellular nucleic acids (particularly intracellular RNA and / or intracellular DNA (e.g., genomic DNA)). In an embodiment, the sterilizable composition is suitable for additional stable cell features, such as cell surface features and / or cell morphology. The details are described above, with reference to the disclosure above.

[0427] Preferably, the composition is sterilized. As discussed, sterilization by irradiation, such as gamma irradiation, is preferred. According to one embodiment, the composition is a sterilized composition obtainable and / or obtained by the method according to the first aspect of the invention. Therefore, a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample prepared by the method according to the first aspect of the invention is contemplated and is preferred.

[0428] Preferably, the composition is a liquid, more preferably the composition is aqueous.

[0429] The sterilizable composition of the sixth aspect of the present invention can be used to stabilize the extracellular nucleic acid population in a cell-containing biological sample, preferably a blood sample. In an embodiment, the sterilizable composition can be used to additionally stabilize intracellular nucleic acids (particularly intracellular RNA and / or intracellular DNA), preferably gene expression profiles. In an embodiment, the sterilizable composition can be used to additionally stabilize cell characteristics, for example, cell surface characteristics and / or cell morphology. This is with reference to the disclosure above.

[0430] Advantageously, the composition is used in sterilized form.

[0431] G. Collection device

[0432] According to a seventh aspect of the present invention, a collection device for collecting a cell-containing biological sample, preferably a blood sample, is also provided. The collection device is subsequently also referred to as a container.

[0433] The device comprises the composition according to the sixth aspect of the present invention. Therefore, the composition contained in the device can be the composition provided in step a) of the method according to the first aspect of the present invention. For details, refer to the disclosure above.

[0434] Thus, in one embodiment, the collection device comprises a composition, which is preferably liquid, more preferably aqueous, and which comprises:

[0435] a) one or more caspase inhibitors,

[0436] b) one or more compounds selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives as discussed above, wherein preferably the composition comprises N-acetylcysteine,

[0437] c) at least one poly(ethylene oxide) polymer as a stabilizer, which is preferably a high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500,

[0438] and optionally one or more, preferably two or more, other additives selected from the following:

[0439] d) at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the first poly(oxyethylene) polymer, the first poly(oxyethylene) polymer being preferably a high molecular weight poly(oxyethylene) polymer, wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less;

[0440] e) at least one primary, secondary or tertiary amide;

[0441] f) Anticoagulants and / or chelating agents.

[0442] Preferably, the stabilizing composition comprises one or more compounds of the invention in concentrations as disclosed above in the first aspect of the invention when discussing feature ii, more preferably as disclosed above in Table A. Suitable compounds and concentrations are also shown in Table B. Particularly preferred is the use of N-acetylcysteine.

[0443] Thus, in one embodiment, the collection device comprises a composition, which is preferably liquid, more preferably aqueous, and which comprises:

[0444] a) one or more caspase inhibitors, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide, even more preferably a caspase inhibitor selected from the group consisting of: Q-VD-OPh, Z-VAD(OMe)-FMK and Boc-D-(OMe)-FMK, most preferably Q-VD-OPh;

[0445] b) one or more compounds selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins (preferably vitamin B6 or ascorbic acid), and vitamin E or its derivatives as discussed above, wherein preferably the composition comprises N-acetylcysteine,

[0446] c) at least one poly(oxyethylene) polymer as a stabilizer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol;

[0447] and optionally one or more, preferably two, other additives selected from the following:

[0448] d) at least one primary, secondary or tertiary amide;

[0449] e) Anticoagulants and / or chelating agents.

[0450] In an embodiment, the at least one poly(ethylene oxide) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(ethylene oxide) polymers, preferably low molecular weight poly(ethylene oxide) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(ethylene oxide) polymers having a molecular weight of at least 1500.

[0451] Preferably, the stabilizing composition comprises one or more compounds of the invention in concentrations as disclosed above in discussing feature ii, more preferably as disclosed above in Table A or Table B. The use of N-acetylcysteine ​​is particularly preferred.

[0452] In another embodiment, the collection device comprises a stabilizing composition, which is preferably liquid, more preferably aqueous, and which comprises:

[0453] a) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide. The caspase-specific peptide can be modified with an aldehyde, nitrile or ketone compound. According to one embodiment, the caspase-specific peptide is modified with an O-phenoxy (OPh) or fluoromethylketone (FMK) group, preferably at the carboxyl terminus. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh, Boc-D-(OMe)-FMK and Z-VAD(OMe)-FMK. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh and Z-VAD(OMe)-FMK, most preferably Q-VD-OPh.

[0454] b) one or more of the following compounds:

[0455] - N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0456] - reduced form of glutathione at a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml, and / or

[0457] - ascorbic acid in a concentration of less than 20 mg / ml, for example 1 mg / ml to 15 mg / ml, 0.2 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml,

[0458] - B vitamins, preferably vitamin B6, in a concentration of 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 14 mg / ml, 0.1 mg / ml to 11 mg / ml, 0.2 mg / ml to 7.5 mg / ml, or 0.2 mg / ml to 2 mg / ml,

[0459] The composition optionally further comprises one or more components selected from the following:

[0460] c) at least one poly(oxyethylene) polymer as a stabilizer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol,

[0461] d) at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide, and

[0462] e) Anticoagulants and / or chelating agents, preferably EDTA.

[0463] In an embodiment, the at least one poly(ethylene oxide) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(ethylene oxide) polymers, preferably low molecular weight poly(ethylene oxide) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(ethylene oxide) polymers having a molecular weight of at least 1500.

[0464] In another embodiment, the collection device comprises a stabilizing composition, which is preferably liquid, more preferably aqueous, and which comprises:

[0465] a) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide. The caspase-specific peptide can be modified with an aldehyde, nitrile or ketone compound. According to one embodiment, the caspase-specific peptide is modified with an O-phenoxy (OPh) or fluoromethylketone (FMK) group, preferably at the carboxyl terminus. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh, Boc-D-(OMe)-FMK and Z-VAD(OMe)-FMK. According to one embodiment, the caspase inhibitor is selected from Q-VD-OPh and Z-VAD(OMe)-FMK, most preferably Q-VD-OPh.

[0466] b) one or more of the following compounds:

[0467] - N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0468] - reduced form of glutathione at a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml, and / or

[0469] - ascorbic acid in a concentration of less than 20 mg / ml, for example 1 mg / ml to 15 mg / ml, 0.2 mg / ml to 14 mg / ml, 0.5 mg / ml to 13.5 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml,

[0470] The composition optionally further comprises one or more components selected from the following:

[0471] c) at least one high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500, preferably in the range of 2000 to 40000, more preferably 2500 to 30000, 2500 to 25000 or 3000 to 20000;

[0472] d) at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (for example N,N-dimethylpropionamide) and / or butanamide,

[0473] e) at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer used, and wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) having a molecular weight of 1000 or less, preferably a molecular weight in the range of 200 to 800 or 200 to 600; and

[0474] f) anticoagulants and / or chelating agents, preferably EDTA.

[0475] In another embodiment, the collection device comprises a stabilizing composition, which is preferably liquid, more preferably aqueous, and which comprises:

[0476] a) at least one pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide as described above, most preferably Q-VD-OPh,

[0477] b) N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0478] The composition optionally further comprises:

[0479] c) at least one poly(oxyethylene) polymer as a stabilizer, wherein preferably the poly(oxyethylene) polymer is polyethylene glycol, more preferably unsubstituted polyethylene glycol,

[0480] d) at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (eg N,N-dimethylpropionamide) and / or butanamide, and

[0481] e) Anticoagulants and / or chelating agents, preferably EDTA.

[0482] In an embodiment, the at least one poly(ethylene oxide) polymer, preferably polyethylene glycol, is selected from low molecular weight poly(ethylene oxide) polymers, preferably low molecular weight poly(ethylene oxide) polymers having a molecular weight of 1000 or less, and / or high molecular weight poly(ethylene oxide) polymers having a molecular weight of at least 1500.

[0483] In another embodiment, the collection device comprises a stabilizing composition, which is preferably liquid, more preferably aqueous, and which comprises:

[0484] a) at least one pan-caspase inhibitor, more preferably a caspase inhibitor comprising a modified caspase-specific peptide as described above, most preferably Q-VD-OPh,

[0485] b) N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml,

[0486] The composition optionally further comprises:

[0487] c) at least one high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500, preferably in the range of 2000 to 40000, more preferably 2500 to 30000, 2500 to 25000 or 3000 to 20000;

[0488] d) at least one primary, secondary or tertiary amide, preferably N,N-dialkylpropionamide (for example N,N-dimethylpropionamide) and / or butanamide,

[0489] e) at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer used, and wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) having a molecular weight of 1000 or less, preferably a molecular weight in the range of 200 to 800 or 200 to 600; and

[0490] f) Optional anticoagulants and / or chelating agents, preferably EDTA.

[0491] The composition contained in the collecting device can be provided and e.g. prepared as described in step a) of the method according to the first aspect of the present invention, directly provided and e.g. prepared in the collecting device, or can be filled into the collecting device after preparation.

[0492] Providing a corresponding collection device, such as a container, such as a sample collection tube, preferably a blood collection tube, comprising the composition according to the sixth aspect of the present invention has the advantage that once the cell-containing biological sample is collected in the corresponding device, the sample is immediately stabilized.

[0493] The stabilizing composition and / or the individual compounds for stabilization contained in the collection container may be provided in liquid, semi-liquid or dry form. The compounds for stabilization may also be provided in the container as separate entities and may also be provided in the container in different forms. For example, one component may be provided in dry form, while another compound may be provided as a liquid. Other combinations are also feasible. Suitable formulation and manufacturing options are known to the skilled person. The advantage of using a solid stabilizing composition is that solids are generally chemically more stable than liquids. According to one embodiment, the inner wall of the container is treated / covered with the stabilizing composition of the present invention or with its individual components, such as the anticoagulant. The composition or component may be applied to the inner wall using, for example, a spray drying method. The stabilizing composition may be subjected to a liquid removal technique to obtain a substantially solid protective composition. Such liquid removal conditions and techniques are described in PCT / EP2015 / 055699, to which reference is made.

[0494] However, as explained above, it is preferred that the stabilizing composition is provided in liquid form, preferably in aqueous form. This has advantages in terms of irradiation sterilization and also has advantages for certain samples such as blood samples. Liquid compositions have the advantage that a mixture with the cell-containing biological sample to be stabilized can be obtained quickly, thereby providing a stabilizing effect essentially immediately once the sample comes into contact with the liquid stabilizing composition. In addition, liquid compositions are advantageous if a relatively large amount of stabilizing composition is used and therefore spray drying is not possible or difficult, or if the composition prevents the provision of a dry composition.

[0495] The amount of stabilizing composition contained in the container is effective to provide stabilization of the amount of sample to be collected in the container. According to one embodiment, the liquid stabilizing composition is contacted with the biological sample in a volume ratio selected from the following: 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10, 1:4 to 1:10 and 1:5 to 1:9, in particular about 1:6 to 1:8.

[0496] Preferably, the collection device is sterilizable, more preferably it can be sterilized using the method according to the first aspect of the invention. Again, for details, reference is made to the disclosure above in the first aspect of the invention, which also applies here. Therefore, it is highly preferred to irradiate the collection device comprising a composition in liquid, preferably aqueous form. If the collection device initially comprises a composition in solid form, the composition can be brought into liquid form before irradiation.

[0497] The device and the composition contained therein may be sterilized together, for example by the method according to the first aspect of the invention. This advantageously allows providing a sterilized end product ready for sample collection.

[0498] Therefore, preferably, the device is sterilized. More preferably, the device is a sterilized device obtainable and / or obtained by the method according to the first aspect of the present invention. Therefore, a device comprising a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample prepared using the method according to the first aspect of the present invention is considered and is preferred.

[0499] The collection device can be evacuated. The evacuation is preferably effective for drawing a specific volume of a cell-containing fluid biological sample into the interior. This ensures that the correct amount of sample is in contact with the pre-filled amount of stabilizing composition contained in the container, and therefore the stabilization is effective. According to one embodiment, the container comprises a tube with an open end sealed by a diaphragm. For example, the container is pre-filled with a limited amount of stabilizing composition in fixed or liquid form, and a limited vacuum is provided and sealed with a diaphragm. The diaphragm is configured to make it compatible with standard sampling accessories (such as cannulae, etc.). When in contact with, for example, a cannula, the sample amount predetermined by the vacuum is collected in the container. The corresponding embodiment is particularly advantageous for collecting blood. Suitable containers are, for example, disclosed in US 6,776,959.

[0500] The container may be made of glass, plastic or other suitable material. The plastic material may be an oxygen-impermeable material or may contain an oxygen-impermeable layer. Alternatively, the container may be made of a gas-permeable plastic material. The container of the present invention is preferably made of a transparent material. Examples of suitable transparent thermoplastic materials include polycarbonate, polyethylene, polypropylene and polyethylene terephthalate. The container may have a suitable size selected according to the volume of the biological sample to be collected. As described above, preferably, the container is evacuated to an internal pressure below atmospheric pressure. Such an embodiment is particularly suitable for collecting body fluids such as whole blood. The pressure is preferably selected to draw a predetermined volume of biological sample into the container. In addition to such vacuum tubes, non-vacuum tubes, mechanical separator tubes or gel barrier tubes can also be used as sample containers, particularly for collecting blood samples. Examples of suitable containers and capping devices are disclosed in US 5,860,397 and US 2004 / 0043505. As containers for collecting cell-containing samples, other collection devices, such as syringes, urine collection devices or other collection devices, can also be used. The type of container may also depend on the type of sample to be collected, and suitable containers are also available to the skilled artisan.

[0501] According to one embodiment, the container has an open top, a bottom and a side wall extending therebetween defining a chamber, wherein the chamber contains the composition according to the sixth aspect of the invention or the composition provided in step a) of the method according to the first aspect of the invention. It can be contained therein in liquid or solid form. According to one embodiment, it is a liquid, preferably an aqueous liquid. According to one embodiment, the container is a tube, the bottom is a closed bottom, the container also contains a closure in the open top, and the chamber is under reduced pressure. The advantages of the reduced pressure in the chamber are described above. Preferably, the closure can be pierced with a needle or a cannula, and the reduced pressure is selected to draw a specific volume of liquid sample into the chamber. According to one embodiment, the chamber is under a reduced pressure, the reduced pressure being selected to draw a specific volume of a liquid sample into the chamber, and the stabilizing composition is a liquid and is arranged in the chamber such that the volume ratio of the stabilizing composition to the specific volume of the cell-containing sample is selected from 10:1 to 1:20, 5:1 to 1:15 and 1:1 to 1:10, and preferably 1:5 to 1:10, more preferably 1:6 to 1:8. The related advantages are described above and are also discussed in PCT / EP2015 / 055699.

[0502] Preferably, the container is used to draw blood from a patient. According to one embodiment, it is used to draw 10 ml of blood from a patient. According to one embodiment, the stabilizing composition is a liquid and the volume is 2 ml or less and may be in the range of 0.5 ml to 2 ml, 0.75 ml to 1.75 ml and 1 ml to 1.5 ml.

[0503] There is also provided a method comprising the step of collecting, preferably drawing, a biological sample, preferably blood, directly from a patient into a chamber of a device or container according to the seventh aspect of the invention.

[0504] H. Kit

[0505] According to the eighth aspect, the present invention provides a kit comprising the composition according to the sixth aspect of the present invention or the collection device according to the seventh aspect of the present invention. The kit may also comprise other components, such as reagents for isolating extracellular nucleic acids.

[0506] I. Application

[0507] According to a ninth aspect, the present invention relates to the use of at least one compound selected from thiols (preferably N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives in a composition for protecting an extracellular nucleic acid population suitable for stabilizing a biological sample or its components during sterilization, particularly irradiation sterilization. These compounds are particularly suitable for protecting the contained caspase inhibitors from degradation during irradiation as described above. When one or more of these compounds are used, the degree of degradation is significantly reduced. The irradiation may preferably be ionizing irradiation, more preferably gamma irradiation, electron beam irradiation or X-rays, most preferably gamma irradiation. The irradiation and / or sterilization conditions applied may be as described above with respect to the first aspect of the present invention.

[0508] The disclosures about suitable compounds of the invention and combinations of compounds and suitable concentrations in the method according to the first aspect of the invention also apply to this aspect. The compound may, for example, be selected from N-acetylcysteine, glutathione (preferably reduced form), ascorbic acid and / or trolox. N-acetylcysteine ​​is highly preferred. Suitable concentrations are also described above, and the compound may, for example, but not limited to, be applied at concentrations indicated in Table A above. Suitable concentrations are also indicated in Table B above.

[0509] The caspase inhibitor may be selected from Q-VD-OPh, Boc-D-(OMe)-FMK and Z-VAD(OMe)-FMK. The caspase inhibitor may be selected from Q-VD-OPh and Z-VAD(OMe)-FMK, most preferably, the caspase inhibitor is Q-VD-OPh.

[0510] J. Other aspects

[0511] According to a tenth aspect, the present invention provides the use of at least one free radical scavenger for protecting an aqueous composition contained in a sample collection device, in particular a blood collection tube, during irradiation.

[0512] According to an eleventh aspect, there is provided a method for sterilizing an aqueous composition contained in a sample collection device, the method comprising:

[0513] a) preparing an aqueous composition comprising at least one free radical scavenger for inclusion in the sample collection device, and

[0514] b) sterilizing the composition by irradiation.

[0515] The aqueous composition of the tenth and eleventh aspects of the present invention may be a composition for stabilizing components contained in a biological sample, such as a cell-containing biological sample, preferably blood. Examples of target biological samples have been described in the first aspect of the present invention, and the sample stabilized by the aqueous composition may be such a sample. Preferably, the aqueous composition to be protected comprises a caspase inhibitor. The caspase inhibitor may be an inhibitor as disclosed in WO 2013 / 045457A1 or WO 2013 / 045458 A1. Suitable caspase inhibitors and concentrations have been disclosed in the first aspect of the present invention above. With reference to this disclosure, it also applies here. Moreover, suitable and preferred aqueous compositions have been disclosed in the first aspect of the present invention above. This disclosure also applies here, meaning that the aqueous composition according to the first aspect is an example of an aqueous composition that can be used in the tenth and eleventh aspects of the present invention. The aqueous composition may in particular be a composition for stabilizing circulating tumor cells, circulating cell-free RNA, circulating cell-free miRNA, viruses and / or exosomes. Stabilization may facilitate subsequent separation, analysis and / or diagnosis. Circulating tumor cells are of particular concern in mammals and humans suffering from cancer. Analysis of circulating tumor cells can, for example, provide information about tumor load, tumor staging and / or formation of distant metastasis. Circulating cell-free RNA and circulating cell-free miRNA are examples of extracellular nucleic acid populations. Viruses can be, for example, contained in biological samples from mammals or humans infected by viruses. Exosomes are cell-derived vesicles that are present in many biological fluids, including blood and urine. Exosomes, for example, participate in coagulation and intercellular signaling. Analysis of cell-free RNA and miRNA can be used to identify circulating biomarkers, detect somatic mutations and oncogene expression. In an embodiment, the sterilized aqueous composition is suitable for stabilizing intracellular nucleic acids (particularly intracellular RNA and / or intracellular DNA). In an embodiment, the sterilized aqueous composition is suitable for additional stable cell features, such as cell surface features and / or cell morphology. The details of such compositions and stabilization properties are described above, and reference is made to the disclosure that is also applicable to this above.

[0516] According to the tenth and eleventh aspects, at least one free radical scavenger can be a compound capable of reacting with free radicals in a biological system. It can be particularly at least one compound selected from thiols (such as N-acetylcysteine ​​or glutathione), water-soluble vitamins, and vitamin E or its derivatives, as described in the first aspect of the present invention. Suitable compounds and concentrations can be used as described in the first aspect of the present invention. Preferably, the at least one free radical scavenger has the activity of protecting the aqueous composition during irradiation, thereby reducing or eliminating the stabilization function of the aqueous composition caused by irradiation. The activity in reducing or eliminating the reduction of the stabilization function can be determined by comparing the stabilization function of the sterilized aqueous composition containing the at least one free radical scavenger with the composition to be tested, which is the same and preferably only different from the sterilized aqueous composition that does not contain the at least one free radical scavenger.

[0517] A suitable sample collection device that can be used in the tenth and eleventh aspects of the invention has been described above in the seventh aspect of the invention, and this description also applies here. Thus, the collection device can be evacuated. It can be made of glass, plastic or other suitable materials. According to one embodiment, the collection device or container may have an open top, a bottom, and side walls extending therebetween defining a chamber, wherein the aqueous composition is contained.

[0518] A method for isolating extracellular nucleic acids from a stabilized cell-containing biological sample, such as a blood sample, has been described above in conjunction with the third aspect. According to another aspect, a method for isolating nucleic acids from a stabilized cell-containing biological sample is provided, the method comprising:

[0519] a) stabilizing a cell-containing biological sample, wherein the stabilization comprises

[0520] i) obtaining a sterilized composition according to the method of the first aspect of the present invention; or

[0521] ii) obtaining the composition according to the sixth aspect of the present invention, wherein the composition is sterilized; or

[0522] iii) obtaining the sample collection device according to the seventh aspect of the present invention, wherein the composition contained in the sample collection device is sterilized;

[0523] and contacting the cell-containing biological sample with the sterilized composition; and

[0524] b) isolating nucleic acids from said stabilized sample.

[0525] Details about the sterilized stabilizing compositions and their stabilizing properties are described in detail above, and reference is made to the disclosures above that also apply hereto. In step b), intracellular nucleic acids and / or extracellular nucleic acids may be separated from the stabilized sample. Suitable separation methods are described above in conjunction with the third aspect of the invention, and reference is made to the corresponding disclosures that also apply hereto. In an embodiment, cells are removed from the cell-containing stabilized sample before separating the nucleic acids. In an embodiment, intracellular nucleic acids (e.g., DNA and / or RNA) are separated from the contained cells, for example, after the cells are separated from the remaining sample. For example, RNA can be isolated and used for gene expression analysis. Details are described above and reference is made to the corresponding disclosures.

[0526] The isolated nucleic acid can then be analyzed and / or further processed in step c) using suitable assays and / or analytical methods.

[0527] According to another aspect, there is provided a method for isolating cells from a stabilized cell-containing biological sample, the method comprising:

[0528] a) stabilizing a cell-containing biological sample, wherein the stabilization comprises

[0529] i) obtaining a sterilized composition according to the method of the first aspect of the present invention; or

[0530] ii) obtaining the composition according to the sixth aspect of the present invention, wherein the composition is sterilized; or

[0531] iii) obtaining the sample collection device according to the seventh aspect of the present invention, wherein the composition contained in the sample collection device is sterilized;

[0532] and contacting the cell-containing biological sample with the sterilized composition; and

[0533] b) isolating cells from said stabilized sample.

[0534] The isolated cells can be, for example, further analyzed. As discussed above, in embodiments, the sterilized stabilized composition also retains cell surface characteristics (e.g., cell surface epitopes or proteins) and / or cell morphology. Details are described above and reference is made to the disclosures above that also apply hereto.

[0535] According to another aspect, a method is provided, which comprises the step of collecting a sample from a patient directly into a chamber of a collection device according to the seventh aspect of the invention. Details about the collection device and the sample are described above. This refers to the corresponding disclosure. According to one embodiment, a blood sample is collected, preferably a blood sample is drawn from a patient.

[0536] The present invention is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to the methods and materials described herein can be used to practice or test embodiments of the present invention. Numerical ranges include numerical values ​​that limit the ranges. The titles provided herein do not limit the various aspects or embodiments of the present invention, and the present invention can be understood by referring to the specification as a whole.

[0537] As used in this specification and claims, references without indications of quantity include the plural unless the context clearly dictates otherwise. Thus, for example, reference to "a caspase inhibitor" includes a single type of caspase inhibitor as well as two or more caspase inhibitors. References to the "disclosure" and "invention" and the like include single or multiple aspects taught herein; and so forth. Aspects taught herein are encompassed by the term "invention".

[0538] As used in this specification, the expression "about" is intended to mean ±10% of a given value.

[0539] According to one embodiment, the subject matter described herein as comprising certain steps in the case of a method or comprising certain components in the case of a composition, solution and / or buffer refers to the subject matter consisting of the corresponding steps or components. The preferred embodiments described herein are preferably selected and combined, and the specific subject matter resulting from the corresponding combination of the preferred embodiments also belongs to the present disclosure.

[0540] The following items are also disclosed:

[0541] 1. A method for producing a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample, the method comprising:

[0542] a) providing a composition comprising:

[0543] i. at least one caspase inhibitor, and

[0544] ii. at least one compound selected from thiols and vitamins or their derivatives; and

[0545] b) irradiating the composition for sterilization.

[0546] 2. The method of item 1, comprising:

[0547] a) providing a composition comprising:

[0548] i. at least one caspase inhibitor, and

[0549] ii. at least one compound selected from thiols, water-soluble vitamins or their derivatives, and water-soluble derivatives of fat-soluble vitamins; and

[0550] b) irradiating the composition for sterilization.

[0551] 3. The method of item 1 or 2, comprising

[0552] a) providing a composition comprising:

[0553] i. at least one caspase inhibitor, and

[0554] ii. at least one compound selected from thiols, water-soluble vitamins or derivatives thereof, and water-soluble derivatives of fat-soluble vitamins, the thiol being N-acetylcysteine ​​or glutathione, optionally wherein the at least one compound is selected from thiols, water-soluble vitamins and water-soluble vitamin E derivatives, the thiol being N-acetylcysteine ​​or glutathione; and

[0555] b) irradiating the composition for sterilization.

[0556] 4. The method according to one or more of items 1 to 3, wherein irradiating the composition for sterilization has one or more of the following characteristics:

[0557] a) irradiating the composition by ionizing radiation;

[0558] b) irradiating the composition by gamma irradiation, electron beam irradiation or X-rays;

[0559] c) irradiating the composition by gamma irradiation or X-rays;

[0560] d) irradiating the composition by gamma irradiation;

[0561] e) irradiating the composition at an irradiation dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, about 8 kGy to about 25 kGy, or about 8 kGy to about 15 kGy;

[0562] f) irradiating the composition by gamma irradiation at an irradiation dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, about 8 kGy to about 25 kGy, or about 8 kGy to about 15 kGy;

[0563] g) irradiating the composition by gamma irradiation at an irradiation dose of 8 kGy to 25 kGy, or 8 kGy to 15 kGy; and / or

[0564] h) irradiation sterilization resulting in a sterility assurance level (SAL) of 10 -6 or lower.

[0565] 5. The method according to one or more of items 1 to 4, wherein the composition has one or more of the following characteristics:

[0566] a) the composition is prepared in solid form;

[0567] b) the composition is prepared in solid form and dissolved prior to irradiation to provide a liquid composition, which is preferably aqueous;

[0568] d) the composition is prepared in liquid form and irradiated;

[0569] e) the composition is aqueous;

[0570] f) the composition has an acidic pH;

[0571] g) the composition has a pH of 4.0 to 7.0, pH 4.1 to 6.9, pH 4.2 to 6.8, pH 4.3 to 6.6, pH 4.4 to 6.3, pH 4.5 to 6.0, or pH 4.5 to 5.5, and / or

[0572] h) the composition is buffered.

[0573] 6. The method according to one or more of items 1 to 5, comprising irradiating said composition in liquid form, preferably in aqueous form.

[0574] 7. The method according to one or more of items 1 to 6, wherein the composition comprises at least one compound selected from thiols, water-soluble vitamins or derivatives thereof, and water-soluble derivatives of fat-soluble vitamins at a concentration of less than 20 mg / ml, 15 mg / ml or less, 10 mg / ml or less, 7 mg / ml or less, 3 mg / ml or less, or 1.5 mg / ml or less, wherein the thiol is N-acetylcysteine ​​or glutathione.

[0575] 8. The method according to one or more of items 1 to 7, wherein the composition comprises

[0576] i. A compound selected from N-acetylcysteine, glutathione, B vitamins, ascorbic acid or a derivative thereof and trolox, wherein the glutathione is preferably in a reduced form, and the B vitamins are preferably vitamin B6;

[0577] ii. a compound selected from N-acetylcysteine, glutathione, ascorbic acid or a derivative thereof, and trolox, wherein the glutathione is preferably in a reduced form;

[0578] iii. a compound selected from the group consisting of N-acetylcysteine, glutathione, B vitamins, and ascorbic acid, wherein the glutathione is preferably in a reduced form and the B vitamins are preferably vitamin B6;

[0579] iv. a compound selected from N-acetylcysteine, glutathione, and ascorbic acid or a derivative thereof, wherein the glutathione is preferably in a reduced form; or

[0580] vN-Acetylcysteine.

[0581] 9. The method according to one or more of items 1 to 8, comprising:

[0582] a) providing a composition comprising:

[0583] i. at least one caspase inhibitor, preferably as defined in item 15, and

[0584] ii. At least one of the following

[0585] aa) N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml;

[0586] ab) glutathione at a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml, wherein the glutathione is preferably in reduced form;

[0587] ac) ascorbic acid or a derivative thereof at a concentration of less than 20 mg / ml, for example 0.1 mg / ml to 15 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml;

[0588] ad) B vitamins, preferably vitamin B6, in a concentration of 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 14 mg / ml, 0.1 mg / ml to 11 mg / ml, 0.2 mg / ml to 7.5 mg / ml, or 0.2 mg / ml to 2 mg / ml;

[0589] ae) Trolox at a concentration of 0.5 mg / ml to 5 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml, or 1 mg / ml to 1.4 mg / ml; and

[0590] b) irradiating the composition for sterilization,

[0591] Preferably, the composition comprises N-acetylcysteine ​​in a concentration as defined in aa).

[0592] 10. The method according to item 9, wherein the composition is irradiated in liquid form, preferably aqueous form and / or wherein the composition is irradiated sterilized by gamma irradiation.

[0593] 11. The method according to one or more of items 1 to 10, wherein the composition has one or more of the following characteristics:

[0594] a) the composition comprises N-acetylcysteine;

[0595] b) the composition comprises N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml;

[0596] c) the composition comprises N-acetylcysteine ​​and is sterilized by irradiation in liquid form, preferably aqueous form; and / or

[0597] d) The composition comprises N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml and is sterilized in an aqueous liquid form.

[0598] 12. The method according to one or more of items 1 to 11, wherein the composition has one or more of the following characteristics:

[0599] a) the composition comprises glutathione;

[0600] b) the composition comprises glutathione in reduced form;

[0601] c) the composition comprises glutathione at a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml, wherein the glutathione is preferably in reduced form;

[0602] d) the composition comprises glutathione and is sterilized in liquid form, preferably aqueous form, wherein the glutathione is preferably in reduced form; and / or

[0603] e) the composition comprises glutathione at a concentration of 0.03 mg / ml to 10 mg / ml, 0.04 mg / ml to 7.5 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, 0.3 mg / ml to 3 mg, 0.4 mg / ml to 2 mg / ml, or 0.4 mg / ml to 1.3 mg / ml and is sterilized in an aqueous liquid form, wherein the glutathione is preferably in a reduced form.

[0604] 13. The method according to one or more of items 1 to 12, wherein the composition has one or more of the following characteristics:

[0605] a) the composition comprises at least one water-soluble vitamin or a derivative thereof, wherein preferably the water-soluble vitamin is selected from ascorbic acid or a derivative thereof, vitamin B1, vitamin B2, vitamin B3, vitamin B6, folate or folic acid, vitamin B12, biotin and pantothenic acid, more preferably wherein the composition comprises ascorbic acid;

[0606] b) the composition comprises at least one water-soluble vitamin or a derivative thereof, preferably selected from ascorbic acid, vitamin B1, vitamin B2, vitamin B3, vitamin B6, folate or folic acid, vitamin B12, biotin and pantothenic acid, more preferably ascorbic acid or a derivative thereof, in a concentration of less than 20 mg / ml, for example 0.1 mg / ml to 15 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml;

[0607] c) the composition comprises at least one water-soluble vitamin or a derivative thereof and is sterilized in liquid form, preferably aqueous form;

[0608] d) the composition comprises ascorbic acid and is sterilized in liquid form, preferably aqueous form;

[0609] e) the composition comprises at least one water-soluble vitamin or derivative thereof in a concentration of less than 20 mg / ml, for example 0.1 mg / ml to 15 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml or 2 mg / ml to 11 mg / ml mg / ml, and is sterilized in liquid form, preferably aqueous form;

[0610] f) the composition comprises ascorbic acid or a derivative thereof in a concentration of less than 20 mg / ml, wherein the concentration may be selected from 0.1 mg / ml to 15 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml, and wherein the composition is sterilized by irradiation in liquid form, preferably aqueous form;

[0611] g) the composition comprises B vitamins, preferably vitamin B6, at a concentration of 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 14 mg / ml, 0.1 mg / ml to 11 mg / ml, 0.2 mg / ml to 7.5 mg / ml, or 0.2 mg / ml to 2 mg / ml; and / or

[0612] h) the composition comprises B vitamins, preferably vitamin B6, at a concentration of 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 14 mg / ml, 0.1 mg / ml to 11 mg / ml, 0.2 mg / ml to 7.5 mg / ml, or 0.2 mg / ml to 2 mg / ml, and wherein the composition is sterilized by irradiation in liquid form, preferably aqueous form.

[0613] 14. The method according to one or more of items 1 to 13, wherein the composition has one or more of the following characteristics:

[0614] a) the composition comprises a water-soluble derivative of a fat-soluble vitamin, wherein optionally the fat-soluble vitamin is selected from vitamin E, vitamin D, vitamin A and vitamin K;

[0615] b) the composition comprises a water-soluble vitamin E derivative, wherein preferably the water-soluble vitamin E derivative is trolox;

[0616] c) the composition comprises trolox;

[0617] d) the composition comprises a water-soluble derivative of a fat-soluble vitamin, preferably a water-soluble vitamin E derivative, more preferably trolox, at a concentration of 0.5 mg / ml to 5 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml, or 1 mg / ml to 1.4 mg / ml;

[0618] e) the composition comprises a water-soluble derivative of a fat-soluble vitamin and is sterilized by irradiation in liquid form, preferably aqueous form;

[0619] f) the composition comprises a water-soluble vitamin E derivative, preferably trolox, and is sterilized in liquid form, preferably aqueous form;

[0620] g) the composition comprises a water-soluble derivative of a fat-soluble vitamin at a concentration of 0.5 mg / ml to 5 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml, or 1 mg / ml to 1.4 mg / ml and is sterilized in liquid form, preferably aqueous form; and / or

[0621] h) The composition comprises a water-soluble vitamin E derivative, optionally trolox, at a concentration of 0.5 mg / ml to 5 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml, or 1 mg / ml to 1.4 mg / ml, and is sterilized in liquid form, preferably aqueous form.

[0622] 15. The method according to one or more of items 1 to 14, wherein the caspase inhibitor has one or more of the following characteristics:

[0623] a) the caspase inhibitor is a pan-caspase inhibitor;

[0624] b) the caspase inhibitor comprises a modified caspase-specific peptide;

[0625] c) the caspase inhibitor comprises a modified caspase-specific peptide, which is modified with an O-phenoxy (OPh) group, preferably at the carboxyl terminus;

[0626] d) the caspase inhibitor comprises a modified caspase-specific peptide, the peptide being modified with a glutamine (Q) group, preferably at the N-terminus;

[0627] e) the caspase inhibitor is selected from Q-VD-OPh, Boc-D-(OMe)-FMK and Z-Val-Ala-Asp(OMe)-FMK;

[0628] f) the caspase inhibitor is selected from Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK; and / or

[0629] g) The caspase inhibitor is Q-VD-OPh.

[0630] 16. The method according to one or more of items 1 to 15, wherein the composition further comprises one or more of the following:

[0631] a) an anticoagulant and / or a chelating agent, preferably EDTA;

[0632] b) poly(ethylene oxide) polymers as stabilizers; and / or

[0633] c) at least one primary, secondary or tertiary amide.

[0634] 17. The method according to one or more of items 1 to 16, wherein the composition further comprises at least one poly(ethylene oxide) polymer, optionally wherein the composition has one or more of the following characteristics:

[0635] a) the poly(oxyethylene) polymer is polyethylene glycol, preferably unsubstituted polyethylene glycol;

[0636] b) the composition comprises a poly(ethylene oxide) polymer which is a high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500;

[0637] c) the composition comprises at least one poly(oxyethylene) polymer having a molecular weight of less than 1500, preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less, more preferably the molecular weight is within the range selected from 100 to 800, 150 to 700, 200 to 600 and 200 to 500;

[0638] d) the composition comprises a poly(oxyethylene) polymer which is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500, and at least one other poly(oxyethylene) polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight poly(oxyethylene) polymer, wherein the other poly(oxyethylene) polymer is preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less; and / or

[0639] e) the composition comprises a poly(oxyethylene) polymer as a high molecular weight poly(oxyethylene) polymer and a poly(oxyethylene) polymer as a low molecular weight poly(oxyethylene) polymer, wherein the molecular weight of the high molecular weight poly(oxyethylene) polymer is within the range selected from 1500-50000, 1500 to 40000, 2000 to 30000, 2500 to 25000, 3000 to 20000, 3500 to 15000 and 4000 to 12500, and / or wherein the molecular weight of the low molecular weight poly(oxyethylene) polymer is 1000 or less and wherein preferably the molecular weight is within the range selected from 100 to 1000, 150 to 800, 150 to 700, 200 to 600 and 200 to 500.

[0640] 18. The method according to one or more of items 1 to 17, wherein the composition further comprises at least one primary, secondary or tertiary amide, optionally wherein the composition has one or more of the following characteristics:

[0641] a) The composition comprises at least one primary, secondary or tertiary amide of formula 1

[0642]

[0643] wherein R1 is a hydrogen group or an alkyl group, preferably a C1-C5 alkyl group, a C1-C4 alkyl group or a C1-C3 alkyl group, more preferably a C1-C2 alkyl group, R2 and R3 are identical or different and are selected from a hydrogen group and a hydrocarbon group having a carbon chain length of 1 to 20 atoms arranged in a straight or branched manner, preferably an alkyl group, and R4 is an oxygen, sulfur or selenium group, preferably R4 is oxygen; and / or

[0644] b) The composition comprises N,N-dialkylpropionamide, preferably N,N-dimethylpropionamide, and / or butanamide.

[0645] 19. The method according to one or more of items 1 to 18, wherein the composition comprises at least one compound of formula 1

[0646]

[0647] Wherein R1 is a hydrogen group or an alkyl group, preferably a C1-C5 alkyl group, a C1-C4 alkyl group or a C1-C3 alkyl group, more preferably a C1-C2 alkyl group, R2 and R3 are the same or different and are selected from a hydrogen group and a hydrocarbon group arranged in a straight chain or branched manner with a carbon chain length of 1 to 20 atoms, preferably an alkyl group, and R4 is an oxygen, sulfur or selenium group, preferably R4 is oxygen.

[0648] 20. The process according to item 19, wherein the at least one compound of formula 1 is a primary, secondary or tertiary carboxylic acid amide.

[0649] 21. The method according to one or more of items 1 to 20, wherein the sterilized composition has one or more of the following characteristics:

[0650] i) it is suitable for stabilizing intracellular nucleic acids contained in a cell-containing sample, wherein preferably stabilizing intracellular RNA and / or intracellular DNA;

[0651] ii) due to said stabilization it reduces the degradation of nucleic acids present in the cell-containing sample;

[0652] iii) it is suitable for stabilizing the transcriptome and / or transcript levels in the cells contained in the sample, wherein preferably, it is suitable for stabilizing the transcript levels of one or more marker genes selected from c-fos, IL-1β, IL-8 and p53, which are stabilized for at least 12h, at least 24h, preferably at least 48h after stabilization.

[0653] 22. The method according to one or more of items 1 to 21, wherein the sterilized composition does not induce lysis of nucleated cells or total cells.

[0654] 23. The method according to one or more of items 1 to 22, wherein the sterilized composition does not contain a cross-linking agent that induces nucleic acid-nucleic acid, protein-nucleic acid and / or protein-protein cross-links and does not involve the use of formaldehyde releasers.

[0655] 24. The method according to one or more of items 1 to 23, wherein the sterilized composition has the following stabilization characteristics:

[0656] a) said stabilization allows the separation of cells from the stabilized cell-containing sample;

[0657] b) the cell-containing sample is a blood sample, and wherein the cells contained in the blood sample are stabilized;

[0658] c) the cell-containing sample is a blood sample, and the leukocytes therein are stabilized;

[0659] d) preserve cell morphology;

[0660] e) retain the morphology of nucleated cells;

[0661] f) the sample is a blood sample and the lymphocytes and / or monocytes contained therein are stabilized;

[0662] g) retaining cell surface epitopes; and / or

[0663] h) Retention of cell surface proteins.

[0664] 25. A sterilizable composition, wherein the composition in sterilized form is suitable for stabilizing the extracellular nucleic acid population of a biological sample, wherein the composition is a composition as provided in step a) of one or more of items 1 to 18 or one or more of items 19 to 24, and wherein preferably the composition is sterilized.

[0665] 26. A method for stabilizing an extracellular nucleic acid population contained in a cell-containing biological sample, the method comprising:

[0666] a) obtaining a sterilized composition suitable for stabilizing the extracellular nucleic acid population of a biological sample according to the method defined in one or more of items 1 to 18 or one or more of items 19 to 24, or obtaining a composition according to item 25 in sterilized form; and

[0667] b) contacting the cell-containing biological sample with the sterilized composition for stabilization.

[0668] 27. A method for isolating extracellular nucleic acids from a stabilized cell-containing biological sample, the method comprising:

[0669] a) stabilizing the cell-containing biological sample according to the method of item 26; and

[0670] b) Isolation of extracellular nucleic acids.

[0671] 28. A method for processing and / or analyzing extracellular nucleic acids, the method comprising:

[0672] a) isolating extracellular nucleic acids from a stabilized cell-containing biological sample according to the method of claim 27; and

[0673] b) processing and / or analyzing the isolated extracellular nucleic acids.

[0674] 29. Use of at least one compound selected from the group consisting of thiols and vitamins or derivatives thereof in a composition suitable for stabilizing the extracellular nucleic acid population of a biological sample or a component thereof during radiation sterilization.

[0675] 30. The use of item 29, wherein the at least one compound is selected from thiols, water-soluble vitamins or derivatives thereof, and water-soluble derivatives of fat-soluble vitamins; or

[0676] wherein the at least one compound is selected from thiols, water-soluble vitamins or derivatives thereof, and water-soluble derivatives of fat-soluble vitamins, and the thiol is N-acetylcysteine ​​or glutathione; or

[0677] wherein the at least one compound is selected from thiols, water-soluble vitamins, and water-soluble vitamin E derivatives, and the thiol is N-acetylcysteine ​​or glutathione; or

[0678] The at least one compound is selected from thiol, ascorbic acid or its derivatives, and water-soluble vitamin E derivatives, and the thiol is N-acetylcysteine ​​or glutathione.

[0679] 31. A method for making a sterilizable composition, wherein the composition in sterilized form is suitable for stabilizing the extracellular nucleic acid population of a biological sample, the method comprising:

[0680] a) preparing a composition comprising:

[0681] i. at least one caspase inhibitor, and

[0682] ii. at least one compound selected from thiols and vitamin E or a derivative thereof; and optionally

[0683] b) sterilizing the composition.

[0684] 32. The method of item 31, wherein the composition prepared in step a) of item 31 is the composition prepared in step a) of one or more items 1-18 or one or more items 19-24.

[0685] 33. A sample collection device, such as a container, preferably a sample collection tube, comprising the sterilizable composition according to item 25.

[0686] 34. A kit comprising the sterilizable composition according to item 25 or the sample collection device according to item 33.

[0687] 35. A method for isolating nucleic acids from a stabilized cell-containing biological sample, the method comprising:

[0688] a) stabilizing a cell-containing biological sample, wherein the stabilization comprises

[0689] i. obtaining a sterilized composition according to the method defined in one or more of items 1 to 24; or

[0690] ii. obtaining a composition according to item 25, wherein the composition is sterilized; or

[0691] iii. Obtaining a sample collection device according to item 33, wherein the composition contained in the sample collection device is sterilized;

[0692] and contacting the cell-containing biological sample with the sterilized composition; and

[0693] b) isolating nucleic acids from said stabilized sample.

[0694] 36. The method according to item 35, wherein step b) comprises isolating intracellular nucleic acids, preferably intracellular RNA and / or intracellular DNA.

[0695] 37. The method according to item 35 or 36, comprising removing cells from the stabilized sample and isolating nucleic acid from the removed cells.

[0696] 38. A method for isolating cells from a stabilized cell-containing biological sample, the method comprising:

[0697] a) stabilizing a cell-containing biological sample, wherein the stabilization comprises

[0698] i. obtaining a sterilized composition according to the method defined in one or more of items 1 to 24; or

[0699] ii. obtaining a composition according to item 25, wherein the composition is sterilized; or

[0700] iii. Obtaining a sample collection device according to item 33, wherein the composition contained in the sample collection device is sterilized;

[0701] and contacting the cell-containing biological sample with the sterilized composition; and

[0702] b) isolating cells from said stabilized sample.

[0703] 39. A sterilized composition obtainable and / or obtained by a method according to one or more of items 1 to 24.

[0704] 40. A sterilized sample collection device, such as a container, preferably a sample collection tube, comprising the sterilized composition according to item 39, wherein the sterilized sample collection device is obtainable and / or obtained by the method according to one or more of items 1-24.

[0705] This application claims priority to EP 15 195 656.2 filed on November 20, 2015 and EP 16 163 863.0 filed on April 5, 2016. The contents of EP 15 195 656.2 and EP 16 163 863.0 are incorporated herein by reference in their entirety.

[0706] Example

[0707] The examples are for illustrative purposes only and are not to be construed as limiting the present invention in any way.

[0708] Abbreviations used:

[0709] ccfDNA: circulating cell-free DNA

[0710] PEG: polyethylene glycol

[0711] EDTA: Ethylenediaminetetraacetic acid

[0712] DMPA: dimethylpropionamide

[0713] HPLC: High Performance Liquid Chromatography

[0714] BCT: Blood Collection Tubes

[0715] kGy: kilogray

[0716] DMSO: dimethyl sulfoxide

[0717] I. Experimental Procedure

[0718] The effect of sterilization, in particular gamma irradiation sterilization, on a composition for stabilizing an extracellular nucleic acid population (hereinafter also referred to as a "stabilizing agent" or "stabilizing composition") was determined by two different means. The chemical degradation profile of the stabilizing composition was evaluated by high performance liquid chromatography (HPLC). A functional assay was performed to determine the effect of the sterilization on the activity of the composition to stabilize extracellular nucleic acids. In this assay, ccfDNA was analyzed as a preferred embodiment of the extracellular nucleic acid population.

[0719] The inventors were able to identify compounds that unexpectedly are able to mitigate or even eliminate the chemical degradation that occurs during sterilization of the stabilized composition. Importantly, these compounds simultaneously allow for optimal functioning of the stabilized composition once sterilized.

[0720] 1. HPLC test of stabilization reagents:

[0721] The caspase inhibitor (Q-VD-OPh) was dissolved in DMSO and diluted to generate a standard curve. Concentrations of 7.5, 15, 30 and 60 μg / ml were used to generate the standard curve.

[0722] Stabilization reagents containing a mixture of caspase inhibitors, EDTA, DMPA, and PEG were tested before and after gamma irradiation. Unique peaks of caspase inhibitors were identified and compared to standard curves for total quantification of caspase inhibitors. Concentrations of caspase inhibitors were determined before and after irradiation and these concentrations were compared for relative quantification.

[0723] 2. Manufacturing of PAXgene Blood ccfDNA Alpha Tube:

[0724] To make the PAXgene blood cfDNA Alpha blood collection tube (Alpha BCT), a BD standard test tube for a 10 ml blood draw was filled with 1.3–1.7 ml ccfDNA stabilization reagent. According to PCT / EP2015 / 055699, the exemplary stabilization reagent tested included a mixture of a caspase inhibitor (Q-VD-OPh), K2EDTA, DMPA, PEG300, and PEG10000. A vacuum was applied to the tube to allow 10.25 ml of blood to be drawn. The tube was sterilized by gamma irradiation using cobalt 60 as a high energy gamma ray source in the range of 5 to 35 kGy.

[0725] 3. Stabilization of whole blood:

[0726] Blood was drawn into Alpha BCT to stabilize ccfDNA levels. 10 ml of spray-dried EDTA tubes (BD) with 1.8 mg K2EDTA / ml whole blood were used as unstabilized negative controls. The tubes were inverted 10 times to mix blood and reagents. Stabilized and unstabilized blood samples were stored in an upright position at room temperature.

[0727] 4. Preparation of plasma:

[0728] Whole blood samples were centrifuged at 1.900 x g for 15 min at ambient temperature. The clarified plasma fraction was transferred to a new centrifuge tube. In the second round, plasma samples were centrifuged at 16.000 x g for 10 min in the case of unstabilized EDTA blood or at 1.900 x g for 10 min in the case of stabilized Alpha BCT blood. The supernatant was transferred to a new tube and used directly for purification of ccfDNA or stored at -20 ° C until use.

[0729] 5. Manual purification of ccfDNA using the QIAamp Circulating Nucleic Acid Kit:

[0730] DNA was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit (QIAGEN GmbH) using the protocol for purification of circulating nucleic acids from 1 ml, 2 ml or 3 ml serum or plasma. If not otherwise specified, 2 ml of plasma were mixed with proteinase K and lysis buffer ACL according to the manufacturer's recommendations, incubated at 60° C. for 30 min, mixed with buffer ACB, bound to a QIAamp Mini column with the aid of a QIAvac24Plus vacuum manifold, washed and eluted with 60 μl elution buffer AVE.

[0731] 6. Automated purification of ccfDNA on QIAsymphony:

[0732] CcfDNA was isolated from 2 ml of plasma using a preliminary version of the QIAsymphony circulating DNA protocol and kit (QIAGEN). If not otherwise specified, the QIAsymphony robot mixed 2 ml of plasma with proteinase K, binding buffer and magnetic beads. The beads with bound DNA were washed three times and the DNA was eluted with 60 μl elution buffer.

[0733] 7. Quantitative Real-time PCR Assay and Calculation of ccfDNA Copies:

[0734] To measure the amount of ccfDNA, real-time PCR assays were performed on Abi Prism HT7900 (Life Technologies) with 3 μl of eluate. Two fragments of the human 18S rDNA gene were amplified with multiple PCR using QuantiTect multiplex PCR kit reagents (QIAGENGmbH) in a 20 μl assay volume. Total quantification was achieved by comparing with a standard curve generated from 3000 diluted human genomic DNA to 0.3 genome equivalents (1 genome equivalent equals approximately 6.6 pg of human genomic DNA).

[0735]

[0736] Table 1: DNA target sequences detected by real-time PCR

[0737] Quantification of 66bp fragments is used to reflect the total amount of 18S rDNA copies in plasma. Quantification of 500bp is used to determine the amount of 18S rDNA copies from leukocytes derived from apoptosis or mechanical lysis in whole blood. Cell-free DNA has a typical length of 140–170bp (Forshew et al. (2012) Cancer Genomics 4 (136) 136ra68). Therefore, the 500bp fragment is derived from apoptotic, lysed or otherwise damaged blood cells. The increase in the copy number of the 500bp fragment between 0 and 3 or 6 days of storage is used as a measure of stability efficiency.

[0738] II. Embodiment

[0739] 1. Example 1: Effect of γ-irradiation on ccfDNA stabilization

[0740] PAXgene blood ccfDNA Alpha tubes were manufactured and sterilized by gamma irradiation at different energy levels. 10 ml whole blood samples from 8 donors were collected in 10 ml spray-dried K2EDTA tubes (unstabilized controls) and PAXgene blood ccfDNA Alpha tubes. Plasma was generated from 5 ml stabilized or unstabilized blood samples immediately after blood draw. The remaining blood was stored at room temperature for another 6 days before plasma was generated. ccfDNA was manually purified from 2 ml plasma and the copy number of the 18SrDNA gene was determined in triplicate by real-time PCR.

[0741] The stabilizing reagent mixture according to PCT / EP2015 / 055699 (for one Alpha tube) is: 11.45 μl of caspase inhibitor (Q-VD-OPh; 1 mg dissolved in 388 μl DMSO); the reagent also contains PEG10.000, PEG300, EDTA and DMPA, and water is added to 1.7 ml. After adding whole blood, the Alpha tube contains Q-VD-OPh at a final concentration of about 5 μM.

[0742] The unstabilized control contained K2EDTA at a final concentration of 1.8 mg / ml in whole blood.

[0743] Figure 1 The effect of gamma irradiation on ccfDNA levels in plasma is shown. The figure shows the average change and standard deviation (x-fold change) in the copy number of 66bp and 500bp fragments of the 18S rDNA gene in unstabilized EDTA blood (left) and stabilized blood samples drawn into Alpha tubes (non-sterile or sterilized with different doses of gamma irradiation). The samples were from 8 donors and stored at room temperature for 6 days.

[0744] It can be seen that the ratio of copy numbers (day 6 / day 0) is lower in the stabilized samples (Alpha tubes) compared to the unstabilized control (EDTA blood, left), demonstrating that stabilization of extracellular nucleic acids occurred in the stabilized samples. However, when comparing the stabilized samples, it can be seen that the efficiency of ccfDNA stabilization decreases with increasing gamma irradiation doses used to sterilize the stabilization reagent prior to adding the blood sample to be stabilized. The decrease in stabilization efficiency associated with increasing gamma irradiation doses can be seen from the increase in the copy number ratios (day 6 / day 0) obtained when the radiation dose increases.

[0745] in conclusion:

[0746] When applied to stabilization reagents containing caspase inhibitors, gamma irradiation used for sterilization has a dose-dependent effect on the subsequent stabilization of ccfDNA by the stabilization reagent. The higher the irradiation dose used for sterilization, the lower the stabilization in subsequent applications.

[0747] 2. Example 2: Degradation of caspase inhibitors by gamma irradiation

[0748] PAXgene blood ccfDNA Alpha tubes were manufactured and sterilized by gamma irradiation at different energy levels. For quantification by HPLC, a standard curve was generated with only the caspase inhibitor (Q-VD-OPh) dissolved in DMSO. The standard curve was generated in Figure 2 Table 2 (below) indicates the concentration of the caspase inhibitor and the peak area and retention time of the caspase inhibitor at different concentrations.

[0749]

[0750] Table 2: HPLC analysis results; preparation of standard curve

[0751] As a next step, the stabilization reagent from the Alpha tubes was measured before and after irradiation to determine the concentration of the caspase inhibitor. The unique peak of the caspase inhibitor was compared to the standard curve for total quantification. The percentage of caspase inhibitor that was not degraded after sterilization was calculated by comparison with the non-sterilized Alpha tubes.

[0752] The results are shown in Table 3 (below). The table shows the peak areas and retention times of the different samples tested as determined by HPLC, and the inhibitor concentrations calculated based on the standard curve.

[0753]

[0754] Table 3: Results of HPLC analysis; determination of % of caspase inhibitor remaining after irradiation.

[0755] The peaks caused by the other components in the stabilizing reagent according to PCT / EP2015 / 055699 appear earlier in the process than the peak of the caspase inhibitor. Although the caspase inhibitor appears at 26 min, the other components are closer to the range of 16-22 min. Therefore, the HPLC results of the stabilizing reagent are compared with the HPLC results of only the caspase inhibitor (used to generate the standard curve), proving that there is no interference in the detection of the caspase inhibitor caused by other components (EDTA, PEG and DMPA) in the stabilizing reagent.

[0756] in conclusion:

[0757] In conclusion, HPLC is a useful technique for quantifying the extent of caspase inhibitor degradation caused by gamma irradiation. Degradation of caspase inhibitors occurs during irradiation and stabilization of ccDNA levels is reduced after sterilization (see Figure 1) correlates with a decrease in the concentration of intact, undegraded caspase inhibitors.

[0758] To further examine the effects of different doses of gamma irradiation on caspase inhibitors, caspase inhibitors from different batches of stabilization reagents sterilized with different doses of gamma irradiation were quantified in PAXgene blood ccfDNA Alpha tubes. The Alpha tubes contained stabilization reagents as detailed in Table 3 above.

[0759] The results are shown in Table 4 (below). As can be seen, gamma irradiation has a dose-dependent effect on caspase inhibitor degradation.

[0760]

[0761] Table 4: Quantification of caspase inhibitors in stabilization reagents from different batches sterilized with different doses of gamma irradiation in PAXgene blood ccfDNA Alpha tubes.

[0762] in conclusion:

[0763] Gamma irradiation has a dose-dependent effect on the degradation of caspase inhibitors in stabilization reagents. The higher the irradiation dose, the more degradation occurs. HPLC can be used to quantify the degradation effect caused by gamma irradiation on caspase inhibitors.

[0764] 3. Example 3: Effect of adding scavengers on ccfDNA stabilization

[0765] To prevent degradation of the caspase inhibitor during sterilization by gamma irradiation, the ability of the components to protect the caspase inhibitor from degradation during irradiation was tested. To exclude that the additional components themselves had a negative impact on extracellular nucleic acid levels, these compounds were added to a stabilizing agent comprising a caspase inhibitor (Q-VD-OPh) and also comprising PEG, EDTA and DMPA and tested in a functional assay.

[0766] 10 ml whole blood samples from 8 donors were collected in 10 ml spray-dried K2EDTA tubes. Immediately after blood draw, the unstabilized blood samples were stabilized by adding 1.7 ml of the ccfDNA stabilization reagent according to PCT / EP2015 / 055699 with or without the addition of different compounds to test their effect on extracellular nucleic acid stabilization.

[0767] The stabilization reagent contains (for 10 ml K2EDTA whole blood):

[0768] 11.45 μl caspase inhibitor (Q-VD-OPh; 1 mg dissolved in 388 μl DMSO), the reagent also contains PEG 10.000, PEG 300, EDTA and DMPA, water to 1.7 ml. Scavengers are added as detailed further below.

[0769] After addition of whole blood, the Alpha tube contained Q-VD-OPh at a final concentration of 5 μM.

[0770] Immediately after blood draw (day 0), plasma was generated from 5 ml of stabilized blood samples. The remaining blood was stored at room temperature for another 6 days before plasma generation (day 6). ccfDNA was purified from 2 ml of plasma and the copy number of the 18SrDNA gene was determined in triplicate by real-time PCR.

[0771] a) Add N-acetylcysteine, ascorbic acid, gallic acid and tannic acid

[0772] N-acetylcysteine, ascorbic acid, gallic acid or tannic acid are added to the stabilizing agent at a concentration of Figure 3 As shown: N-acetylcysteine, 2 mg / ml; ascorbic acid, 5 mg / ml; gallic acid, 2 mg / ml; tannic acid, 1 mg / ml.

[0773] The final concentrations in whole blood were: 1.7 mM N-acetylcysteine ​​or 4.0 mM ascorbic acid or 1.7 mM gallic acid or 0.09 mM tannic acid.

[0774] The results are Figure 3 The figure shows the average change (x-fold change) in the copy number of the 66 bp and 500 bp fragments of the 18b rDNA gene in stabilized blood after storage with stabilization reagent alone (left) and with the addition of N-acetylcysteine, ascorbic acid, gallic acid, or tannic acid. The results are from blood stored at room temperature for 6 days from 8 donors.

[0775] in conclusion:

[0776] Certain free radical scavengers such as ascorbic acid and N-acetylcysteine ​​can be added to the stabilization reagent containing the caspase inhibitor without affecting the ccfDNA stabilization properties.

[0777] However, it was surprisingly found that the addition of certain free radical scavengers such as tannic acid or gallic acid reported in the prior art to be effective against gamma radiation-induced free radicals resulted in an increased release of DNA stabilized with the ccfDNA stabilization reagent in the blood, which can be Figure 3This is seen by the increase in the ratio of 500 bp copy numbers between day 6 and day 0 and the high standard deviation of the two fragments 66 and 500 bp. These compounds are therefore not suitable for use in a composition for stabilizing the extracellular nucleic acid population.

[0778] b) Addition of different concentrations of ascorbic acid, N-acetylcysteine ​​and glutathione (reduced form).

[0779] In another experiment, ascorbic acid, N-acetylcysteine ​​or glutathione (reduced form) were added to the stabilizing agent at concentrations such as Figure 4 As indicated in: ascorbic acid, 5 mg / ml, 10 mg / ml or 20 mg / ml; N-acetylcysteine, 2 mg / ml, 4 mg / ml or 10 mg / ml; glutathione (reduced), 0.1 mg / ml, 0.5 mg / ml or 1 mg / ml.

[0780] The final concentrations in whole blood are: 4.0 mM or 8.0 mM or 16.0 mM ascorbic acid; 1.7 mM or 3.5 mM or 8.7 mM N-acetylcysteine; or 0.05 mM or 0.23 mM or 0.47 mM glutathione (reduced).

[0781] The results are Figure 4 The figure shows the average change (x-fold change) in the copy number of the 66 bp and 500 bp fragments of the 18b rDNA gene in stabilized blood after storage with stabilization reagent alone (left) and after addition of different concentrations of ascorbic acid, N-acetylcysteine ​​or glutathione (reduced form). The blood was obtained from 8 donors and stored at room temperature for 6 days.

[0782] Figure 5 Shown are the average total copy numbers of a 500 bp fragment of the 18S rDNA gene in stabilized blood when measured immediately after blood collection.

[0783] in conclusion:

[0784] Stabilizing agents containing ascorbic acid, N-acetylcysteine ​​or glutathione (reduced form) showed good stabilizing properties at the concentrations tested ( Figure 4 ).from Figure 5 It can be seen that the addition of ascorbic acid at a concentration of 20 mg / ml, a concentration reported in the art to be (radio)protective against gamma radiation induced free radicals, leads to a decrease in the absolute copy number of ccfDNA.

[0785] c) Addition of different combinations of ascorbic acid, N-acetylcysteine ​​and glutathione

[0786] In another experiment, the effects of different combinations of ascorbic acid, N-acetylcysteine ​​and glutathione on the ccfDNA stabilizing properties of the stabilizing reagent were tested.

[0787] For this purpose, ascorbic acid, N-acetylcysteine ​​and glutathione (reduced form) were mixed as follows Figure 6 The indicated combinations and concentrations were added to the stabilizing reagent: ascorbic acid (2 mg / ml) and glutathione (0.2 mg / ml); N-acetylcysteine ​​(1 mg / ml) and glutathione (0.2 mg / ml); ascorbic acid (2 mg / ml) and N-acetylcysteine ​​(1 mg / ml); ascorbic acid (2 mg / ml), N-acetylcysteine ​​(1 mg / ml) and glutathione (0.2 mg / ml).

[0788] The final concentrations in whole blood were: 1.65 mM ascorbic acid and 0.1 mM glutathione (reduced); 0.9 mM N-acetylcysteine ​​and 0.1 mM glutathione (reduced); 1.65 mM ascorbic acid and 0.9 mM N-acetylcysteine; 1.65 mM ascorbic acid and 0.9 mM N-acetylcysteine ​​and 0.1 mM glutathione (reduced).

[0789] The results are Figure 6 The graph shows the average change (x-fold change) in the copy number of the 66 bp and 500 bp fragments of the 18b rDNA gene in stabilized blood from 8 donors and stored at room temperature for 6 days.

[0790] from Figure 6 It can be seen that good ccfDNA stabilization properties can be obtained with the tested clearing agent combinations.

[0791] in conclusion:

[0792] A free radical scavenger of the present invention, such as a combination of ascorbic acid, N-acetylcysteine ​​and glutathione (reduced form), may be added to a stabilization reagent comprising a caspase inhibitor at a suitable concentration without affecting the ccfDNA stabilization properties.

[0793] 4. Example 4: Effect of Addition of Scavengers on Caspase Inhibitor Concentration and CcfDNA Stabilization after γ-Irradiation

[0794] The effect of the addition of scavengers on the degradation of caspase inhibitors during gamma irradiation sterilization was analyzed. Also, the effect of the addition of scavengers (before sterilization in order to protect the caspase inhibitors) on the ccfDNA stabilization properties of the (sterilized) stabilization reagents was evaluated.

[0795] Stabilized reagents were prepared with or without the addition of free radical scavengers and sterilized in batches by gamma irradiation. Caspase inhibitor concentrations were measured by HPLC before and after irradiation to determine the effect of the addition of scavengers on the degradation of caspase inhibitors during gamma irradiation sterilization.

[0796] The stabilization reagent contains (for 10 ml K2EDTA whole blood):

[0797] 11.45 μl caspase inhibitor (Q-VD-OPh; 1 mg dissolved in 388 μl DMSO), the reagent also contains PEG 10.000, PEG 300, EDTA and DMPA, water is added to 1.7 ml. Scavengers are added as further detailed below. After the addition of whole blood, the Alpha tube contains Q-VD-OPh at a final concentration of 5 μM.

[0798] Scavengers were added to the stabilizing reagents at the concentrations shown in Table 5 (below).

[0799]

[0800] Table 5: Quantification of caspase inhibitors in stabilization reagents with or without scavengers before and after gamma irradiation

[0801] In order to analyze the effect of adding a scavenger before sterilization on the ccfDNA stabilization properties of the sterilized stabilization reagent, 10ml whole blood samples from 8 donors were collected in 10ml spray-dried K2EDTA tubes and stabilized with 1.7ml of each ccfDNA stabilization reagent with or without a scavenger. Plasma was generated from 5ml of stabilized blood samples immediately after blood was drawn. The remaining blood was stored at room temperature for another 6 days and then plasma was generated. CcfDNA was purified from 2ml plasma and the copy number of the 18S rDNA gene was determined in triplicate by real-time PCR.

[0802] The scavengers added and the concentrations used were Figure 7 Final concentrations in whole blood were 0.42 mM gallic acid, 4.0 mM ascorbic acid, 12 mM N-acetylcysteine, 0.05 mM glutathione, or 0.73 mM Trolox.

[0803] The results are Figure 7 Shown in Figure 2 are the average changes (x-fold changes) in the copy number of the 66 bp and 500 bp fragments of the 18S rDNA gene in blood samples stabilized with irradiated stabilization reagents containing a caspase inhibitor and with different scavengers. Average values ​​from 8 donors stored at room temperature for 6 days.

[0804] Gallic acid failed to prevent the reduction of ccfDNA stabilization induced by gamma irradiation, as can be seen from the higher ratio of the total copy number of 66 and 500 bp fragments between the 6th day after blood draw and the 0th day after blood draw, as well as the large standard deviation. This is consistent with the high degradation rate measured by HPLC (Table 5). In contrast, scavengers such as ascorbic acid, N-acetylcysteine, glutathione stabilized the level of ccfDNA, as measured by the copy number ratio between the 6th day and the 0th day. Trolox also showed a stabilizing effect.

[0805] in conclusion:

[0806] Surprisingly, only the addition of selected scavengers such as ascorbic acid, N-acetylcysteine, glutathione and Trolox in a stabilizing agent comprising a caspase inhibitor such as the stabilizing agent according to PCT / EP2015 / 055699 prevents the degradation of the caspase inhibitor caused by gamma irradiation.

[0807] It is also surprising that the addition of certain scavengers such as ascorbic acid, N-acetylcysteine, glutathione and Trolox prevented the negative effects of gamma irradiation on the stability levels of ccfDNA in blood stabilized with a stabilization agent comprising a caspase inhibitor.

[0808] In contrast, many scavengers that are reported to be able to dispose of free radicals cannot prevent the degradation of caspase inhibitors caused by irradiation and / or cause an increase in ccfDNA levels after blood storage and thus interfere with stabilization. This applies in particular to gallic acid.

[0809] 5. Example 5: Overview of Scavenger Activity

[0810] In Table 6 an overview of the tested scavengers showing protection against gamma irradiation on the one hand and effect on ccfDNA stability on the other hand is shown.

[0811]

[0812] Table 6: Summary - Tested scavengers and concentrations for protection of stabilization reagents containing caspase inhibitors against gamma irradiation. Concentration 1 indicates the concentration of the tested compound in mg / ml. Concentration 2 indicates the concentration of the tested compound in weight / volume (w / v) or volume / volume (v / v), depending on whether the compound is a liquid or solid.

[0813] in conclusion:

[0814] Only the addition of specific free radical scavengers such as ascorbic acid, N-acetylcysteine, glutathione and Trolox, in particular ascorbic acid, N-acetylcysteine ​​and glutathione, prevented or effectively reduced the degradation of caspase inhibitors due to gamma irradiation sterilization. Surprisingly, the addition of these free radical scavengers preserved the quality of ccfDNA stabilization achieved with the stabilization composition comprising caspase inhibitors.

[0815] The effective scavenging compounds and their effective concentrations differ significantly from previously published radioprotective excipients. For example, gallic acid, tannic acid, but also mannitol, isopropanol and Tween-80 have been previously described as scavengers. However, they do not provide gamma irradiation protection for stabilizing agents comprising caspase inhibitors. Furthermore, previously described scavengers such as tannic acid and gallic acid interfere with the stabilizing effect of the stabilizing agents.

[0816] 6. Example 6: Effects of scavengers and caspase inhibitor concentrations on caspase inhibitor protection during gamma irradiation

[0817] Stabilizing agents according to PCT / EP2015 / 055699 were manufactured with or without the addition of free radical scavengers at different concentrations and with different concentrations of caspase inhibitors. PAXgene blood ccfDNA Alpha tubes were manufactured by filling 1.7 ml of the corresponding stabilizing agent into a standard Vacutainer. The tubes were sterilized by gamma irradiation at different energy levels. Caspase inhibitor concentrations were measured by HPLC before and after irradiation (see Table 7 below).

[0818] Each tube of the stabilization reagent contains:

[0819] 3.3 μl or 6.6 μl or 9.8 μl of caspase inhibitor (Q-VD-OPh; 18 mg dissolved in 2 ml DMSO; final blood concentration 5, 10 or 15 μM, respectively), the reagent also contains PEG10.000, PEG300, EDTA and DMPA, water is added to 1.7 ml.

[0820] As shown in Table 7, scavengers were added to the stabilizing reagent in various concentrations and combinations.

[0821]

[0822]

[0823]

[0824]

[0825]

[0826] Table 7: Quantification of caspase inhibitors in stabilization reagents with or without different concentrations of scavengers and with different concentrations of caspase inhibitors before and after gamma irradiation.

[0827] in conclusion:

[0828] The free radical scavengers that can be used in conjunction with the present invention are effective over a wide range of concentrations. For the concentration range tested (scavenger and caspase inhibitor), the level of protection conferred by the scavenger is largely independent of the caspase inhibitor concentration used. Free radical scavengers can be combined to further improve protection.

[0829] 7. Example 7: Effect of scavenger concentration on ccfDNA stabilization in sterilized Alpha tubes

[0830] PAXgene blood ccfDNA Alpha tubes from Example 6 were selected for functional testing to determine the ccfDNA stabilization properties. N-acetylcysteine ​​was added to the stabilizing agent at different concentrations. Alpha tubes with 0.5, 1.0, 2.0 and 4.0 mg / ml N-acetylcysteine ​​were prepared as shown in Figure 8. The tubes used were non-sterile or sterilized with different doses of gamma irradiation before use. 10 ml whole blood samples from four donors were collected in 10 ml spray-dried K2EDTA tubes (unstabilized controls) and Alpha tubes (sterilized and unsterilized as controls) respectively. The final concentration of N-acetylcysteine ​​in whole blood was 0.44, 0.88, 1.75 and 3.5 mM. The unstabilized control tubes contained 1.8 mg / ml K2EDTA (final concentration in whole blood). Plasma was generated from 5 ml stabilized or unstabilized blood samples immediately after blood was drawn. The remaining blood was stored at room temperature for another 6 days, and then plasma was generated. ccfDNA was purified from 2 ml of plasma on a QIASYMPHONY and the copy number of the 18S rDNA gene was determined in triplicate by real-time PCR.

[0831] Each tube of the stabilization reagent contains:

[0832] 3.3 μl of caspase inhibitor (Q-VD-OPh; 18 mg dissolved in 2 ml DMSO; final blood concentration 5 μM), the reagent also contains PEG10.000, PEG300, EDTA and DMPA, water is added to 1.7 ml.

[0833] The stabilization of ccfDNA was analyzed by quantitative real-time PCR. The results are shown in Fig. 8A, B. The mean change and standard deviation (x-fold change) of the copy number of the 66 bp and 500 bp fragments of the 18S rDNA gene were compared between unstabilized EDTA blood and stabilized blood samples drawn into Alpha tubes (non-sterile or sterilized with different doses of gamma irradiation) containing 0.5, 1.0, 2.0, and 4.0 mg / ml N-acetylcysteine.

[0834] from Fig. 8A , B It can be seen that N-acetylcysteine ​​provides excellent protection over a wide range of irradiation doses, even at a high dose of 30 kGy, at all tested concentrations. Moreover, it can be seen from the non-irradiated (0 Gy) stabilizing agent containing N-acetyl-L-cysteine ​​that the addition of N-acetylcysteine ​​does not interfere with the stabilization of extracellular nucleic acids by the stabilizing agent.

[0835] in conclusion:

[0836] N-acetylcysteine ​​acts to protect against gamma irradiation over a wide range of concentrations. With the addition of free radical scavengers to the stabilizing agent, the dose-dependent effect of gamma irradiation on ccfDNA stability is eliminated. N-acetylcysteine ​​does not interfere with the stabilization of extracellular nucleic acids by the stabilizing agent, and it is preferably used as a protective agent to prevent degradation of caspase inhibitors during irradiation.

[0837] 8. Example 8: Effect of adding water-soluble vitamin B6 as a scavenger on the stabilization of ccfDNA levels after gamma irradiation

[0838] The effect of adding vitamin B6 as a scavenger to prevent degradation of caspase inhibitors during gamma irradiation sterilization was analyzed. Stabilized reagents were prepared with or without added vitamin B6 and sterilized in batches by gamma irradiation at 30 KGy.

[0839] 10ml whole blood samples from eight donors were collected in 10ml spray-dried K2EDTA tubes and stabilized by adding 1.7ml sterilized ccfDNA stabilizing reagent with or without scavenger immediately after blood drawing. The blood samples were stored at room temperature for 5 days and then plasma was generated. Using QIAsymphony PAXgene blood ccfDNA test kit and PAXcircDNA_2400_LAF (PreAnalytiX) scheme, the blood samples stabilized were used to automatically purify ccfDNA from 2.4ml plasma on QIAsymphony. As a control, using QIAsymphony circulating DNA scheme and test kit (QIAGEN), unstabilized blood samples were used to purify ccfDNA from 2.4ml plasma. The experiment also includes the addition of vitamin B6 but unsterilized stabilizing reagents. The total copy number of 18S rDNA gene (500bp fragment) was measured in triplicate by real-time PCR.

[0840] The stabilization reagent contains (for 10 ml K2EDTA whole blood):

[0841] 3.28 μl caspase inhibitor (Q-VD-OPh; 1 mg dissolved in 111 μl DMSO), the reagent also containing PEG, EDTA and DMPA, water added to 1.7 ml. Suitable polyethylene glycols are described above, such as PEG 300 and PEG 10.000. For stabilizing reagents containing the scavenger vitamin B6 (pyridoxine), vitamin B6 (pyridoxine) is added to the stabilizing reagent at a concentration of 2 mg / ml. The final concentration of the caspase inhibitor (Q-VD-OPh) in the stabilized blood is about 5 μM. The final concentration of vitamin B6 (pyridoxine) in whole blood is about 1.7 mM.

[0842] Fig. 9 The average total copy number of a 500 bp fragment of the 18S rDNA gene in blood samples from 8 donors stored at room temperature for 5 days is shown in Figure . The blood samples were stabilized using stabilizing agents containing (bars on the left) or not containing (third bar from the left) vitamin B6 as a scavenger. These stabilizing agents have been sterilized (30 kGy). As controls, stabilizing agents with vitamin B6 but not sterilized (second bar from the left) and unstabilized EDTA blood (fourth bar from the left) were included.

[0843] In blood samples stabilized with a stabilizing agent (irradiated and non-irradiated) containing vitamin B6 as a scavenger, the number of 500bp fragments of the 18S rDNA gene was only half that of a stabilizing agent (irradiated) without a scavenger. Since naturally occurring ccfDNA has a size of approximately 150bp, longer fragments originate from lysed or apoptotic leukocytes. Therefore, the low number of 500bp fragments of the 18SrDNA gene observed in blood samples stabilized with a stabilizing agent containing vitamin B6 as a scavenger indicates that the blood is effectively stabilized. In particular, the stabilization observed with an irradiated stabilizing agent containing vitamin B6 is superior to the stabilization observed with an irradiated stabilizing agent without a scavenger. This indicates that the stabilizing properties of the stabilizing agent are effectively protected by vitamin B6 during irradiation. In a negative control using unstabilized blood, the 500bp fragment copy number is even 25–50 times higher than that of a stabilized blood sample.

[0844] in conclusion:

[0845] Like the scavengers ascorbic acid, N-acetyl-L-cysteine, glutathione and Trolox, water-soluble vitamin B6 also prevents the negative effects of gamma irradiation on the stability levels of ccfDNA in blood stabilized with a stabilization agent containing a caspase inhibitor. Thus, the addition of vitamin B6 protects the stabilization composition during irradiation and allows the preparation of a sterilized stabilization composition with good stabilization properties.

[0846] 9. Example 9: Effects of different caspase inhibitors on stabilization of ccfDNA levels and effective protection of different caspase inhibitors during sterilization

[0847] The effect of adding different caspase inhibitors (Boc-D-(OMe)-FMK and Z-VAD(OMe)-FMK) on stabilizing ccfDNA levels in whole blood was tested. Furthermore, the protection of these different caspase inhibitors from degradation during irradiation sterilization was analyzed.

[0848] Stabilization reagents were made by adding Boc-D-(OMe)-FMK or Z-VAD(OMe)-FMK as caspase inhibitors with or without the scavenger N-acetyl-L-cysteine ​​(NAC) as sterilization protectant. In the case of the addition of NAC, the reagents were sterilized in batches by gamma irradiation at 30 kGy. Stabilization of ccfDNA levels was compared with PAXgene blood ccfDNA tubes (PreAnalytiX) and unstabilized EDTA blood.

[0849] 10ml whole blood samples from 6 donors are collected in 10ml spray-dried K2EDTA tubes, and stabilized by adding 1.7ml stabilizing agent, and collected in PAXgene blood ccfDNA tubes or remain unstable. Blood samples are stored at room temperature for 5 days, and then plasma is generated. Utilizing QIAsymphony PAXgene blood ccfDNA test kit and PAXcircDNA_2400_LAF (PreAnalytiX) scheme, stabilized blood samples are used to automatically purify ccfDNA from 2.4ml plasma on QIAsymphony. As a control, QIAsymphony circulating DNA scheme and test kit (QIAGEN) are used to purify ccfDNA from 2.0ml plasma using unstabilized blood samples. The total copy number of 18S rDNA gene (500bp fragment) is measured in triplicate by real-time PCR.

[0850] The stabilization reagent contains (for 10 ml K2EDTA whole blood):

[0851] 3.28 μl of caspase inhibitor (Boc-D-(OMe)-FMK dissolved as 1 mg in 217 μl DMSO, or Z-VAD(OMe)-FMK dissolved as 1 mg in 122 μl DMSO), the reagent also comprising PEG, EDTA and DMPA, water to 1.7 ml. Suitable polyethylene glycols, such as PEG 300 and PEG 10.000, are described above. For stabilizing reagents comprising N-acetyl-L-cysteine ​​(NAC) as a scavenger, N-acetyl-L-cysteine ​​is added to the stabilizing reagent at a concentration of 0.5 mg / ml. The final concentration of the caspase inhibitor in the stabilized blood is about 5 μM. The final concentration of the scavenger in whole blood is about 0.44 mM.

[0852] Fig.10The average total copy number of a 500 bp fragment of the 18S rDNA gene in blood samples from 6 donors stored at room temperature for 5 days is shown in Figure . The blood samples were stabilized using stabilization reagents containing different caspase inhibitors. Left bar: stabilization reagent containing the caspase inhibitor Boc-D-(OMe)-FMK (non-sterile); second bar from the left: stabilization reagent containing the caspase inhibitor Boc-D-(OMe)-FMK and N-acetylcysteine ​​as a scavenger (sterile); third bar from the left: stabilization reagent containing the caspase inhibitor Z-VAD(OMe)-FMK (non-sterile); fourth bar from the left: stabilization reagent containing the caspase inhibitor Z-VAD(OMe)-FMK and N-acetylcysteine ​​as a scavenger (sterile); fifth bar from the left: PAXgene blood ccfDNA tube; sixth bar from the left: EDTA blood that has not been stabilized.

[0853] from Fig.10 It can be seen that in blood samples stabilized with the caspase inhibitors Boc-D-(OMe)-FMK or Z-VAD(OMe)-FMK, the copy number of the 500bp fragment, which can be considered to be from apoptotic cells, is comparable to the number of fragments in blood stabilized with the PAXgene blood ccfDNA tube. Therefore, the stabilization reagent containing the caspase inhibitors Boc-D-(OMe)-FMK and Z-VAD(OMe)-FMK achieves good stabilization properties. The addition of the scavenger N-acetyl-L-cysteine ​​(NAC) effectively protects the caspase inhibitors Boc-D-(OMe)-FMK and Z-VAD(OMe)-FMK from γ-irradiation. In the negative control using unstabilized blood, the copy number of the 500bp fragment was 50-100 times higher than that of the stabilized blood sample.

[0854] in conclusion:

[0855] Like stabilizing agents including the caspase inhibitor Q-VD-OPh (see e.g. Fig. 9 ), stabilizing agents comprising other caspase inhibitors such as Boc-D-(OMe)-FMK or Z-VAD(OMe)-FMK also prevent the release of genomic DNA into the plasma and thus stabilize ccfDNA levels. The compounds used according to the invention, such as, in particular, N-acetylcysteine, protect stabilizing agents comprising different caspase inhibitors (such as Q-VD-OPh, Boc-D-(OMe)-FMK or Z-VAD(OMe)-FMK) during gamma irradiation and can prevent the negative effects of gamma-irradiation by adding free radical scavengers such as N-acetyl-L-cysteine. Sequence Listing <110> Qiagen Co., Ltd. <120> Method for preparing sterilized composition for stabilizing extracellular nucleic acid <130> 57 538 K <150> EP 15 195 656.2 <151> 2015-11-20 <150> EP 16 163 863.0 <151> 2016-04-05 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Human RNA forward primer <400> 1 gccgctagag gtgaaattct tg 22 <210> 2 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Human RNA reverse primer <400> 2 cattcttggc aaatgctttc g 21 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Human ribosomal DNA probe <400> 3 accggcgcaa gacggaccag a 21 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Human RNA forward primer <400> 4 gtcgctcgct cctctcctac tt 22 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Human RNA reverse primer <400> 5 ggctgctggc accagactt 19 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Human ribosomal DNA probe <400> 6 ctaatacatg ccgacgggcg ctgac 25

Claims

1. A sterilizable collection device suitable for collecting a cell-containing biological sample, wherein the collection device comprises a sterilized liquid composition, wherein the sterilized liquid composition is suitable for stabilizing an extracellular nucleic acid population of the biological sample, wherein the composition comprises: i. at least one caspase inhibitor, and ii. at least one compound selected from the group consisting of a thiol, a water-soluble vitamin, and a water-soluble vitamin E derivative, wherein the thiol is N-acetylcysteine ​​or glutathione, wherein the water-soluble vitamin is ascorbic acid or vitamin B6, and wherein the water-soluble vitamin E derivative is trolox; The composition is sterilized by irradiation in liquid form.

2. The collection device according to claim 1, wherein the collection device is sterilized.

3. The collection device according to claim 2, wherein the collection device and the composition contained therein are sterilized together.

4. The collection device according to claim 1, wherein: a) irradiating the composition and / or the collection device by ionizing radiation to sterilize; b) irradiating the composition and / or the collection device for sterilization by gamma irradiation, electron beam irradiation or X-rays; c) irradiating the composition and / or the collection device to sterilize at a radiation dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, about 8 kGy to about 25 kGy, or about 8 kGy to about 15 kGy, wherein the expression "about" means ±10% of a given value; d) irradiating the composition and / or the collection device by gamma irradiation at an irradiation dose of 5 kGy to 35 kGy, 6 kGy to 30 kGy, 7 kGy to 26 kGy, about 8 kGy to about 25 kGy, or about 8 kGy to about 15 kGy for sterilization, wherein the expression "about" means ±10% of a given value; and / or e) irradiation sterilization resulting in a sterility assurance level (SAL) of 10 -6 or lower.

5. The collection device according to claim 1, wherein the composition has one or more of the following characteristics: a) the composition is aqueous; b) the composition has an acidic pH; c) the composition has a pH of 4.0 to 7.0, pH 4.1 to 6.9, pH 4.2 to 6.8, pH 4.3 to 6.6, pH 4.4 to 6.3, pH 4.5 to 6.0, or pH 4.5 to 5.5; d) the composition is buffered; and / or e) the composition is sterilized by irradiation in aqueous form.

6. The collection device of claim 1, wherein the glutathione is in a reduced form.

7. The collection device of claim 1, wherein the composition comprises at least one compound selected from the group consisting of a thiol, ascorbic acid, vitamin B6, and trolox at a concentration of less than 20 mg / ml, 15 mg / ml or less, 10 mg / ml or less, 7 mg / ml or less, 3 mg / ml or less, or 1.5 mg / ml or less, wherein the thiol is N-acetylcysteine ​​or glutathione.

8. The collection device according to claim 1, wherein the composition comprises: i. at least one caspase inhibitor, and ii. At least one of the following aa) N-acetylcysteine ​​at a concentration of 0.05 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 7.5 mg / ml, 0.1 mg / ml to 5 mg / ml, 0.1 mg / ml to 2 mg / ml, 0.2 mg / ml to 1 mg / ml, or 0.3 mg / ml to 0.8 mg / ml; ab) glutathione at a concentration of 0.03 mg / ml to 10 mg / ml, 0.075 mg / ml to 5 mg / ml, 0.15 mg / ml to 4 mg / ml, or 0.4 mg / ml to 1.3 mg / ml; ac) ascorbic acid at a concentration of less than 20 mg / ml, 0.1 mg / ml to 15 mg / ml, 1 mg / ml to 13 mg / ml, 1.5 mg / ml to 12 mg / ml, or 2 mg / ml to 11 mg / ml; ad) vitamin B6 at a concentration of 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 14 mg / ml, 0.1 mg / ml to 11 mg / ml, 0.2 mg / ml to 7.5 mg / ml, or 0.2 mg / ml to 2 mg / ml; ae) Trolox at a concentration of 0.5 mg / ml to 5 mg / ml, 0.75 mg / ml to 3 mg / ml, 0.9 mg / ml to 2 mg / ml, or 1 mg / ml to 1.4 mg / ml.

9. The collection device of claim 8, wherein the glutathione is in a reduced form.

10. The collection device of claim 8, wherein the composition is sterilized in aqueous form.

11. The collection device according to any one of claims 1 to 10, wherein the caspase inhibitor has one or more of the following features: a) the caspase inhibitor is a pan-caspase inhibitor; b) the caspase inhibitor comprises a modified caspase-specific peptide; c) the caspase inhibitor comprises a modified caspase-specific peptide, the peptide being modified with an O-phenoxy (OPh) group and / or with a glutamine (Q) group; d) the caspase inhibitor comprises a modified caspase-specific peptide, the peptide being modified at the carboxyl terminus with an O-phenoxy (OPh) group and / or at the N-terminus with a glutamine (Q) group; e) the caspase inhibitor is selected from Q-VD-OPh, Boc-D-(OMe)-FMK and Z-Val-Ala-Asp(OMe)-FMK; f) the caspase inhibitor is selected from Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK; and / or g) The caspase inhibitor is Q-VD-OPh.

12. The collection device according to any one of claims 1 to 10, wherein the composition further comprises one or more of the following: a) anticoagulants and / or chelating agents; b) poly(ethylene oxide) polymers as stabilizers; and / or c) at least one primary, secondary or tertiary amide.

13. The collection device according to claim 12, wherein the anticoagulant and / or chelating agent is EDTA.

14. The collection device of any one of claims 1 to 10, wherein the composition further comprises at least one poly(ethylene oxide) polymer.

15. The collection device according to claim 14, wherein the composition has one or more of the following characteristics: a) the poly(ethylene oxide) polymer is polyethylene glycol; b) the composition comprises a poly(ethylene oxide) polymer which is a high molecular weight poly(ethylene oxide) polymer having a molecular weight of at least 1500; c) the composition comprises at least one low molecular weight poly(ethylene oxide) polymer having a molecular weight of 1000 or less or a molecular weight in a range selected from 100 to 800, 150 to 700, 200 to 600 and 200 to 500; d) the composition comprises a poly(oxyethylene) polymer which is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500 and at least one other poly(oxyethylene) polymer which is a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less; and / or e) the composition comprises a poly(oxyethylene) polymer as a high molecular weight poly(oxyethylene) polymer and a poly(oxyethylene) polymer as a low molecular weight poly(oxyethylene) polymer, wherein the molecular weight of the high molecular weight poly(oxyethylene) polymer is within the range selected from 1500-50000, 1500 to 40000, 2000 to 30000, 2500 to 25000, 3000 to 20000, 3500 to 15000 and 4000 to 12500, and / or wherein the molecular weight of the low molecular weight poly(oxyethylene) polymer is 1000 or less or the molecular weight is within the range selected from 100 to 1000, 150 to 800, 150 to 700, 200 to 600 and 200 to 500.

16. The collection device of claim 15, wherein the poly(ethylene oxide) polymer is unsubstituted polyethylene glycol.

17. The collection device according to any one of claims 1 to 10, wherein the composition comprises at least one compound of formula 1 wherein R1 is a hydrogen group or an alkyl group, R2 and R3 are the same or different and are selected from hydrogen groups and hydrocarbon groups having a carbon chain length of 1 to 20 atoms arranged in a straight or branched manner, and R4 is an oxygen, sulfur or selenium group.

18. The collection device according to claim 17, wherein R1 is a C1-C5 alkyl group, a C1-C4 alkyl group, a C1-C3 alkyl group or a C1-C2 alkyl group.

19. The collection device of claim 17, wherein the hydrocarbon group is an alkyl group, and / or wherein R4 is oxygen.

20. The collection device of claim 17, wherein the compound of Formula 1 is a primary, secondary or tertiary amide.

21. The collection device of claim 17, wherein the compound of Formula 1 is a primary, secondary or tertiary carboxylic acid amide.

22. The collection device according to any one of claims 1 to 10, wherein the composition comprises N,N-dialkylpropionamide and / or butanamide.

23. The collection device according to any one of claims 1 to 10, wherein the composition comprises N,N-dimethylpropionamide.

24. The collection device according to any one of claims 1 to 10, wherein the sterilized composition has one or more of the following characteristics: i) it is suitable for stabilizing the extracellular nucleic acid population in cell-containing biological samples; ii) it is suitable for stabilizing intracellular nucleic acids contained in cell-containing samples; iii) due to said stabilization it reduces the degradation of nucleic acids present in the cell-containing sample; iv) it is suitable for stabilizing the transcriptome and / or transcript levels in cells contained in said sample; v) it does not induce lysis of nucleated cells or total cells; and / or vi) it does not contain a cross-linking agent that induces nucleic acid-nucleic acid, protein-nucleic acid and / or protein-protein cross-links and does not involve the use of formaldehyde-releasing agents; And / or wherein the sterilized composition has the following stabilization characteristics: a) said stabilization allows the separation of cells from the stabilized cell-containing sample; b) the cell-containing sample is a blood sample, and wherein the cells contained in the blood sample are stabilized; c) the cell-containing sample is a blood sample, and the leukocytes therein are stabilized; d) preserve cell morphology; e) retain the morphology of nucleated cells; f) the sample is a blood sample and the lymphocytes and / or monocytes contained therein are stabilized; g) retaining cell surface epitopes; and / or h) Retention of cell surface proteins.

25. The collection device according to claim 24, wherein in feature ii), the intracellular RNA and / or the intracellular DNA are stabilized.

26. The collection device according to claim 24, wherein in feature iv), the composition is suitable for stabilizing the transcript level of one or more marker genes selected from c-fos, IL-1β, IL-8 and p53, and is stabilized for at least 12 h, at least 24 h or at least 48 h after stabilization.

27. The collection device according to any one of claims 1 to 10, wherein: a) the collection device is a collection device suitable for collecting blood, plasma and / or serum; b) the collection device is suitable for drawing blood from a patient; and / or c) The cell-containing biological sample is a blood sample.

28. The collection device of any one of claims 1 to 10, wherein the composition is contained in the collection device in an amount effective to provide stabilization of the amount of sample to be collected in the collection device.

29. A collection device according to claim 28, wherein the stabilizing composition is a liquid and is contacted with the biological sample in a volume ratio selected from the following: 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10, 1:4 to 1:10, 1:5 to 1:9, and about 1:6 to 1:8, wherein the expression "about" means a given value ±10%.

30. The collection device according to any one of claims 1 to 10, wherein the collection device is evacuated.

31. A collection device according to any one of claims 1 to 10, wherein the collection device is pre-filled with a limited amount of a stabilizing composition in liquid form, provided with a defined vacuum and sealed with a septum.

32. The collection device of any one of claims 1 to 10, wherein the collection device has an open top, a bottom, and sidewalls extending therebetween defining a chamber, wherein the composition is contained in the chamber.

33. The collection device of claim 32, wherein the composition is an aqueous liquid.

34. The collection device of claim 32, wherein the collection device is a tube, the bottom is a closed bottom, the collection device further comprises a closure in the open top, and the chamber is under reduced pressure.

35. A collection device according to claim 34, wherein the closure is capable of being pierced with a needle or cannula, and the reduced pressure is selected to draw a specific volume of liquid sample into the chamber.

36. A collection device according to claim 34, wherein the chamber is under a reduced pressure, the reduced pressure is selected to draw a specific volume of liquid sample into the chamber, and the composition is liquid and is arranged in the chamber so that the volume ratio of the stabilizing composition to the specific volume of the cell-containing sample is selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10, 1:5 to 1:10, and 1:6 to 1:

8.

37. The collection device according to any one of claims 1 to 10, wherein the collection device is sterilized and comprises a sterilized composition, wherein the sterilized collection device is obtainable by a method for producing a sterilized composition suitable for stabilizing an extracellular nucleic acid population of a biological sample, the method comprising: a) providing a composition as defined in claim 1 or any one of claims 5 to 26 in the collecting device; and b) irradiating the composition to sterilize it.

38. The collection device of any one of claims 1 to 10, wherein the composition is mixed with a cell-containing biological sample.

39. A collection device according to claim 38, wherein the cell-containing biological sample is whole blood and / or contains extracellular nucleic acids.

40. A method comprising the step of collecting a biological sample directly from a patient into a chamber of a collection device according to any one of claims 1 to 39.

41. The method of claim 40, wherein the biological sample is drawn from the patient and the biological sample is blood.

Images