Use of ultrafiltration for producing a purified allergenic extract

Abstract

A process for generating a final product comprising or consisting of a purified allergenic extract, comprising: (A) providing a crude allergenic extract, wherein the crude allergenic extract is obtained by isolation from a biological sample; (B) optionally, purifying the crude extract to obtain a purified crude extract, wherein the purifying step comprises: (B1) filtering the crude product to remove the natural microbial load with respect to the biological sample; (C) optionally, purifying the crude extract or the purified crude extract to obtain a purified extract, wherein the purifying step comprises: (C1) filtering the crude extract or the purified crude extract to remove impurities having a molecular weight of less than 5,000 Da. The process is characterized in that it comprises an ultrafiltration step for viruses which is functional for obtaining a final product which comprises or consists of a purified extract of allergens and which is substantially free of viruses and / or mycoplasmas. The present application also relates to a product for medical or diagnostic use, comprising one or more allergen extracts which are natural with respect to the starting biological sample or which are substantially free of viruses and / or mycoplasmas resulting from any external contamination.

Description

Technical Field

[0001] The present invention is in the technical field of a production process of a product containing an allergen extract useful for the therapeutic use in specific immunotherapy in the treatment of patients suffering from allergy or allergic symptoms; and the diagnostic use in the detection of allergy.

Background Art

[0002] Specific immunotherapy (ITS) is the first-choice therapy for the treatment of patients suffering from clinical symptoms of respiratory allergy or allergic species. In addition to reducing symptoms, ITS fights against the cause of allergy.

[0003] The WHO (World Health Organization) recognizes this as the only treatment that can "bring about a cure for the patient's allergy and change the quality of life".

[0004] Desensitization is based on the principle of administering to the patient a gradually increasing amount of a small amount of an allergen that causes an allergic reaction.

[0005] Currently, there are two forms of desensitization therapy: a sublingual form in which the allergen is left under the tongue for several minutes and an injectable form in which the doctor injects a gradually increasing amount of the allergen until the maximum dose is achieved.

[0006] The allergens that cause allergic reactions are generally extracted from samples or biological substrates of animal (mites; bee venom; pet epithelial derivatives), mitochondrial (mold / yeast cultures) origin or plant origin (herbs and pollen from plants). The nature of the starting materials particularly complicates the allergen extract from a qualitative point of view because they contain both the molecules of the reference allergen and the molecules belonging to the microbiome associated with the starting materials. For example, mite cultures from which antigens causing respiratory allergy to arthropods are extracted contain immunologically active bacterial endotoxins, such as lipopolysaccharide (LPS), the presence of which is systematically controlled in the production steps of pharmaceuticals in order to obtain the certifications required for marketing approval.

[0007] Over the past few decades, the metagenomics of viruses and the development of new technologies available for their use have revealed the remarkable diversity and ubiquity of viruses in the biosphere. Thus, it has begun to be investigated whether the biological materials used for allergen extraction can also be a source of immunogenic viral components.

[0008] The presence of viruses in allergen extracts can actually affect not only the production steps of pharmaceutical products (e.g., by affecting the increased efficiency of biological samples for industrial extraction purposes), but also the immune and inflammatory responses induced in patients, since viral proteins are known to have antigenic and immunogenic properties.

[0009] Vidal-Quist et al (Jose Cristian Vidal-Quist, Carmen Vidal, Fernando Escolar, Bart N. Lambrecht, Stephane Rombauts, Pedro Hernandez-Crespo. RNA viruses in the house dust mite Dermatophagoides pteronyssinus, detection in environmental samples and in commercial allergen extracts used for in vivo diagnosis. Allergy. 2021; 00:1-12) have shown that viral infections in mites of the Dermatophagoides pteronyssinus species are very common regardless of the origin of the mites (grown in industrial cultures or recovered from natural colonies). The authors found that there are seven different viral RNAs present in D. pteronyssinus.

[0010] Similar considerations have also been made for Mycoplasma, bacteria characterized by the absence of a cell wall, which are factors conferring resistance to many antibiotic treatments (which are precisely active against the synthesis of components forming the bacterial cell wall). Many Mycoplasma are naturally parasitic, but they can also attack and invade other cells. Mycoplasma can infect humans, animals, and plants, and they can be potential contaminants in biological starting materials from which allergens are extracted. Similar to viruses, their presence can also cause difficulties in production steps, for example, delaying and invalidating the production operations of allergenic extracts at the industrial level (https: / / www.citeqbiologics.com / mycoplasma-and-house-dust-mite-extracts / ).

[0011] Problems of the background art The chapter "Virus safety" of the European Pharmacopoeia (European Pharmacopoeia, 5.1.7) provides the requirements for virus safety regarding pharmaceuticals assuming the use of materials of human or animal origin.

[0012] The use of virus inactivation steps is being used in state-of-the-art technology in drugs that directly use biological origin materials (such as blood preparations, etc.).

[0013] To date, the risk of virus and / or Mycoplasma infection in products obtained by extraction containing allergenic proteins from pet starting materials has always been considered low or non-existent.

[0014] For this reason, there is no state-of-the-art evidence regarding the implementation of procedures suitable for reducing the virus and / or Mycoplasma load in products intended for specific immunotherapy (ITS).

[0015] The body responsible for the approval of the sale of this type of product does not actually require any certification proving the viral and / or mycoplasma content of the allergenic extract.

[0016] Nevertheless, the harvesting and / or propagation processes for the preparation of the raw materials, the protein extraction process, and the filtration and purification processes are carried out in systems that are potentially exposed to contact with personnel and are thus potentially exposed to viruses and bacteria.

[0017] Viral filtration to remove viruses based on a dimensional exclusion mechanism is widely used in the purification systems of biopharmaceuticals derived from mammalian cell cultures. Viral filtration is generally carried out through the use of filters with a mesh size in the nanometer range, and in this sense, nanofilters or, in some cases, ultrafiltration membranes for viruses, also known as virus filters. As a result, viral filtration is called nanofiltration or, in some cases, virus ultrafiltration.

[0018] However, virus filters are not easy to implement in sample purification systems because they tend to cause packing / clogging (also known as fouling) that contributes to a decrease in the flow of the filtrate and, as a result, a reduction in virus retention.

[0019] In the typical dimensional characteristics of a virus ultrafiltration system related to the surface of the filter in square centimeters and the filter material, the elements that can most frequently contribute to the clogging of the system are components or protein aggregates with a molecular weight greater than 150 kDa.

[0020] The differences in molecular weight among the target allergen proteins (1 - 150 kDa), viruses (150 - 7200 kDa), and mycoplasmas (0.2 - 0.3 μm) do not, in this sense, guarantee the easy removal of viruses by dimensional exclusion without, for example, entailing the risk of compromising the yield of the extract from the perspective of protein content.

[0021] Furthermore, allergenic proteins are unstable in aqueous solutions (both at room temperature and under refrigerated storage conditions) unless they rely on the addition of stabilizing excipients or technologies to improve their stability (e.g., lyophilization); thus, it should be noted that the application of the viral ultrafiltration step can lead to a reduction or loss of protein content due to the degradation of the molecule of interest.

[0022] In addition to degradation, the protein content can also be affected by the adsorption phenomenon of individual proteins of interest on the filter membrane.

[0023] In addition to the above problems, the implementation of viral ultrafiltration in the production system should be considered not only depending on the species contained in the sample to be filtered, but also usually requires adapting the working conditions to the production process implemented even among different batches of the same product. In other words, the viral ultrafiltration process is somewhat variable and can hardly be standardized. SUMMARY OF THE INVENTION

[0024] To reduce the risk of contamination by microorganisms, viruses, and / or mycoplasmas, the Applicant has developed a process for producing a final product comprising or consisting of a purified allergenic extract substantially free of viruses and / or mycoplasmas; in particular, the process of the present invention aims to investigate the implementation of a viral ultrafiltration step specialized in the removal of viruses and / or mycoplasmas.

[0025] Therefore, the object of the present invention is a process for producing a final product comprising or consisting of a purified allergenic extract, A) providing a crude allergenic extract in which the allergen is a natural origin allergen and can be obtained by isolation from a biological sample; B) optionally, purifying the crude extract to obtain a purified crude extract, wherein the purification step comprises at least Sub-step of filtering the crude product to remove the microbial load of the biological sample comprising the step of, and C) Optionally, a step of purifying the crude extract or the purified crude extract to obtain a purified extract, wherein the purification step comprises at least C1) Sub-step of filtering the crude extract or the purified crude extract to remove impurities having a molecular weight of 5,000 Da or less comprising the step of and comprising, characterized in that the process comprises ultrafiltration of one or more viruses, said ultrafiltration being functional for obtaining a final product substantially free of viruses and / or mycoplasmas, comprising or consisting of a purified allergenic extract is a process.

[0026] A further object of the present invention is a composition substantially free of viruses and / or mycoplasmas, comprising one or more allergen extracts, obtainable by the process described above. Said composition is preferably intended for medical use, for example in specific immunotherapy of subjects of allergic nature, or for diagnostic use for the detection of allergies.

[0027] Advantages of the present invention The development of the process object of the present invention has overcome the problems of the known art and achieved the following advantages and objectives: Obtaining a final product or an allergenic extract substantially free of microorganisms, viruses and / or mycoplasmas that are natural to the biological starting material or derived from external contamination: thus, the characterized product is safe for the end user since it does not contain antigenic substances that could interfere with immunospecific therapy. Implementation of the ultrafiltration step of viruses (see Example 2), which has been found to be functional not only for reducing the content of viruses and / or mycoplasmas in the final product comprising or consisting of an allergenic extract, but also for influencing the control of the content of bacterial endotoxins. Implementation of an efficient virus ultrafiltration step without fouling problems. This objective was specifically achieved through the use of a filter assembly including a prefilter upstream of the main filter. The implementation of this ultrafiltration step is further improved by the selection of a prefilter and subsequent filter of appropriate dimensions, which enables the system to obtain rapid ultrafiltration at a sustainable pressure (0.5 - 5 bar), specifically retain viruses and / or mycoplasmas, and instead allow allergenic proteins to pass through. Implementation of a virus ultrafiltration step that does not affect the product yield from the perspective of the protein content of the target allergen; the applicant has specifically experimented that the pore size of the filter does not negatively affect the protein and allergen content of the active substance being tested. Definition of the operating pressure that enables the virus ultrafiltration step to be carried out for 2 - 24 hours. Obtaining a production process that enables the generation of a purified allergen extract substantially free of viruses and / or mycoplasmas within 42 hours from the provision of the crude allergenic extract. Standardization of the virus ultrafiltration process and the entire production process, independent of the types of proteins contained in the extract and different batches. This process can actually be used for the production of products containing or consisting of allergen extracts of different natures.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0029] Definition "Allergen" means an antigen or set of antigens that induces an unwanted immune hypersensitivity reaction (or allergic reaction). Thus, "allergenicity" is related to allergens so defined. This definition applies throughout the following description, whether reference is made to a singular or plural allergen.

[0030] "Allergen of natural origin" means an allergen that occurs naturally and has not been manipulated or modified by genetic engineering or molecular biology techniques.

[0031] The "crude extract" (or raw extract) of an allergen means an extract obtained by isolating and separating the allergen originally contained in the starting material (or biological sample) (i.e., obtained by directly applying an extraction process to the allergen source). In other words, a crude allergen extract contains proteins that have been isolated and separated from the source material by themselves, regardless of their degree of purity. Extracts that may possibly be obtained by subsequent manipulation of the crude extract (or raw extract) are considered purified extracts (no longer crude or raw extracts).

[0032] "Purified, substantially free of virus and / or mycoplasma" means a product that has undergone a purification process adapted to exclude molecules that do not belong to allergens, a process to reduce the microbial load from the starting biological sample or external contaminants, and also a specific process for the removal of virus and / or mycoplasma. The viruses and mycoplasmas to be removed can be both those that are native to the starting biological sample and those resulting from any external contamination. The purified product substantially free of virus and / or mycoplasma complies with industrial regulations (ICH Harmonized Tripartite Guideline Q5A (R1), 1999: Viral Safety evaluation of biotechnology products derived from cell lines of human or animal origin. https: / / www.ema.europa.eu / en / ich-q5a-r1-quality-biotechnological-products-viral-safety-evaluation-biotechnology-products-derived; EMEA / CPMP / BWP / 268 / 95 / 3AB8A, 1996: Note for guidance on virus validation studies: The design, contribution and interpretation of studies validating the inactivation and removal of viruses) from the quantitative / qualitative point of view.

[0033] "Final product" means a product suitable for distribution and sale, taking advantage of its own biological activity. Thus, the final product either constitutes a product that can be used for therapeutic or diagnostic purposes in itself; or it can constitute a raw material suitable for further manipulation or mixing with other components for the preparation of a product that can be used for therapeutic or diagnostic purposes.

[0034] "Protein dosage" means the quantitative content of protein in a sample; for the purposes of the present invention, the allergen content present in a reference extract can be determined using relative specificity based on the protein dosage.

[0035] "Lowry method or assay", which is known in the art (LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265-75. PMID: 14907713), means a method for quantifying the protein dosage of a sample. The Lowry assay is based on a two-step procedure: (1) The biuret reaction: By adding a copper solution to a protein solution in a basic environment, a purple-violet color tone with a maximum absorption at 540 nm is obtained. (2) After adding the Folin reagent (phosphomolybdic acid-phosphotungstic acid reagent) that reacts with the tyrosine and tryptophan of the protein, a blue color tone (maximum 750 nm) is expected due to the formation of tungsten blue and molybdenum blue by reduction activated by the copper-protein complex.

[0036] "Native to the biological sample" means a material (microorganism, bacterium, virus, mycoplasma material) derived from or contained in the starting biological sample from which the allergen is extracted.

[0037] "Filtrate" means the fraction of the product subjected to filtration that passes through the membrane of the filtration system, i.e., is not retained by the membrane. "Filtrate" can also be referred to by the name "permeate".

[0038] "Residue" means the fraction of the product subjected to filtration that does not pass through the membrane of the filtration system, i.e., is retained by the membrane.

[0039] In the following of this specification, the present invention will be described in more detail. With reference to the process, the steps and sub-steps constituting the process will be described.

[0040] A - Provision of a crude allergen extract As already mentioned above, the allergens contained in the crude extract are natural allergens and are obtainable from biological samples.

[0041] Preferably, the allergens contained in the crude extract for the purposes of the present invention are allergens of animal origin obtainable from biological samples containing or consisting of materials classified as belonging to the animal kingdom.

[0042] More preferably, the allergens contained in the crude extract for the purposes of the present invention are allergens of Hymenoptera or mites or mammals (preferably dogs or cats).

[0043] According to a preferred embodiment, the isolation of the crude allergen extract from the biological sample can be carried out separately at a location different from the location where the process of the present invention is carried out; or this can constitute an integrated step of the production process.

[0044] When the isolation of the crude extract from the biological sample constitutes an integrated step of the production process of the present invention, the step (A) of providing the crude allergen extract preferably comprises or consists of at least a sub-step (A1) of isolating the allergen from the biological sample (or starting material).

[0045] Isolation for the purposes of the present invention means a set of techniques used for the separation of the crude allergen extract from the starting biological sample; thus isolation can optionally include solvent extraction and / or crude extract separation techniques.

[0046] The biological sample (or starting material) can be selected from the following: Preferably, mites in the form of dried and frozen mite cultures; the cultures may contain different mite components (e.g., feces, bodies, parts of mites, eggs). A mammalian material, preferably selected from epithelial material, mammalian hair or dandruff, preferably selected from mammalian hair and dandruff. Preferably, a venom bag from an entire insect, untreated venom in liquid form, treated venom in lyophilic form, and Hymenoptera selected from untreated venom.

[0047] According to a first preferred embodiment, the biological sample (or starting material) consists of mites. Preferably, the mite species selected for the purposes of the present invention are Dermatophagoides farinae, Dermatophagoides pteronyssinus, or Euroglyphus maynei.

[0048] In the case of mites, crude allergen extracts can be obtained by methods known to those skilled in the art (Prester, Ljerka, Kovacic, Jelena and Macan, Jelena. “Comparison of buffers for extraction of mite allergen der p 1 from dust” Archives of Industrial Hygiene and Toxicology, vol.63, no.3, 2012, pp.293 - 300). For example, mite cultures may be mixed with an aqueous solvent of ionic nature. The crude extract is isolated from the extraction medium by conventional separation techniques (centrifugation, filtration).

[0049] According to a second preferred embodiment, the mammalian biological sample (or starting material) consists of a sample of material derived from a dog (Canis lupus) or a cat (Felis catus), preferably a cat.

[0050] In the case of dogs or cats, crude extracts of the pet's epithelial material, hair, or dander can be obtained using methods known to those skilled in the art. Generally, this material is collected at animal protection facilities under the supervision of veterinarians (Fernandez-Caldas E, Cases B, El-Qutob D, Cantillo JF. Mammalian raw materials used to produce allergen extracts. Ann Allergy Asthma Immunol. 2017 Jul;119(1):1-8).

[0051] According to the third preferred embodiment, the biological sample (or starting material) consists of bee venom. Preferably, the bee species selected for the purposes of the present invention are Apis mellifera, Vespa Crabro, Vespa velutina, Vespa mandarinia, Vespula germanica, Vespula vulgaris, Polistes dominula, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, Dolichovespula, Vespula alascensis, Vespula maculifrons, Vespula flavopilosa, Vespula pensylvanica, and Vespula Squamosa; more preferably, they are selected from Apis mellifera, Vespa Crabro, Vespula germanica, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, Polybia paulista.

[0052] In the case of bee venom, the crude extract can be obtained using methodologies known to those skilled in the art (Greg Plunkett, PhD; Robert S. Jacobson, MS; David B.K. Golden, MD. Hymenoptera venoms used to produce allergen extracts. Ann Allergy Asthma Immunol 118 (2017) 649-654). For example, the venom sac is recovered from the insect (captured or from a living body obtained through breeding) by dissection, and the venom is recovered by appropriate recovery techniques. The venom can be extracted by rupturing it in an appropriate buffer solution from the sac to release the venom. The residue of the sac is removed by centrifugation and filtration. Thus, the obtained venom constitutes a crude allergen extract derived from Hymenoptera for the purposes of the present invention. Venom in the form of a liquid that has not been subjected to treatment also constitutes a crude extract of Hymenoptera; however, due to its high instability, mainly having an enzyme composition, this is not very preferable for subsequent treatment. A further example of a crude extract of Hymenoptera allergens is the venom of a liquid that has been extracted from the sac and treated by operations such as dissolution, filtration, and / or lyophilization by a method that ensures stability of the extracted venom over time.

[0053] Regardless of the source of the allergen, it should be noted that the crude composition has a total microbial load ≤ 1 × 10 12 CFU / g.

[0054] In the most preferred embodiment of the present invention, the starting crude allergenic extract (provided in step (A)) is a crude allergenic extract of mites, preferably Dermatophagoides farinae, or Dermatophagoides pteronyssinus, or Euroglyphus maynei; or a crude allergenic extract of the dander and / or hair of cats (Felis catus); or Bee venom, preferably from Apis mellifera, or Vespa Crabro, or Vespa velutina, or Vespa mandarinia, or Vespula germanica, or Vespula vulgaris, or Polistes dominula, or Polistes exclamans, or Bombus terrestris, or Bombus agrorum, or Bombus medics, or Dolichovespula, or Vespula alascensis, or Vespula maculifrons, or Vespula flavopilosa, or Vespula pensylvanica, or a crude allergenic extract of the bee venom of Vespula Squamosa; more preferably, a crude allergenic extract selected from the group consisting of Apis mellifera, Vespa Crabro, Vespula germanica, or Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, Polybia paulista selected from the group consisting of.

[0055] According to this most preferred embodiment, the step of providing the crude allergenic extract is performed in an environment of grade C and / or D according to ISO 14644-1.

[0056] When the biological sample consists of a mite culture, it should be noted that for the purpose of obtaining a crude extract, the sample is preferably mixed with a phosphate buffer as the extraction solvent. Preferably, the phosphate buffer has a pH in the range of 7.5 to 8.5, preferably equal to about 8.0.

[0057] The mixture is mixed in a manner convenient for the mixing step. At the end of stirring, the pH of the mixture can be adjusted with an alkaline reagent, an acid, or another buffer so that the pH of the mixture is in the range of 7.5 to 8.5, preferably equal to about 8.0.

[0058] Next, the mixture is left to stand for a period preferably in the range of 12 to 72 hours for extraction. For the purpose of extracting mite allergens, a cold extraction carried out at a temperature in the range of 2 to 8 °C is preferred.

[0059] For the purpose of isolating the extract, common separation techniques can be used. Preferably, the mixture is subjected to centrifugation, recovery of the supernatant (which constitutes the crude extract of the mite allergen), and removal of the precipitate.

[0060] It should be noted that when the starting material consists of cat (Felis catus) hair or dandruff, the extraction of the crude extract is carried out in the same manner as described above for the house dust mite.

[0061] Purification of the crude extract to obtain a B - purified crude extract According to a preferred embodiment, the process subject of the present invention includes a step (B) of purifying the crude allergen extract. The step (B) of purifying the crude allergen extract comprises or consists of at least a sub - step (B1) of filtering the crude extract to remove the microbial load of the biological sample.

[0062] The purification step (B) is not necessary in this sense if the starting crude extract already does not contain sufficient bacterial contaminants, and preferably, the purification step (B) can be avoided as long as the starting crude allergen extract is characterized by a microbial load < 100,000 CFU / mL, preferably ≦ 50,000 CFU / ml.

[0063] According to a preferred embodiment, the sub-step (B1) of filtering the microorganisms of the crude extract comprises or consists of a sub-step of performing a first-dimensional filtration using a first filter having a porosity in the range of 8 μm to 20 μm, preferably in the range of 8 μm to 10 μm, preferably having a dimension equal to 8 μm, and a sub-step of performing a second-dimensional filtration after the first-dimensional filtration using a second filter having a porosity in the range of 0.1 μm to 0.2 μm, preferably having a dimension equal to 0.2 μm. comprises or consists of.

[0064] It should be noted that the residue obtained from the sub-step of filtering the crude extract constitutes the microbial load of the biological sample and is thus discarded for the purpose of subsequent steps of the method according to the present invention; the permeate (or filtrate) obtained from the sub-step of filtering the crude extract constitutes the purified crude extract or a precursor of the purified crude extract.

[0065] Preferably, the sub-step (B1) of filtering the microorganisms of the crude extract is a conventional filtration or "dead-end" filtration, that is, filtration performed by applying pressure perpendicular to the membrane from the supply side of the sample to be filtered.

[0066] Preferably, the first filter used in the sub-step (B1) of filtering the microorganisms of the crude extract is a polypropylene (PP) filter.

[0067] Preferably, the second filter comprises a pre-filter having a porosity in the range of 0.45 μm to 0.65 μm, preferably in the range of 0.45 μm to 0.50 μm, preferably having a dimension equal to 0.45 μm.

[0068] Preferably, the pre-filter is structurally coupled to the filter to define a filter assembly for resolving continuity. The presence of the pre-filter preferably contributes to the definition of the production process such that it ends within or at 42 hours from the start of step (A) of providing the crude extract at a pressure in the range of preferably 0.5 bar to 5 bar, preferably 1.5 bar to 4 bar, preferably 1.8 bar to 3 bar, preferably equal to about 2 bar, enabling avoidance of clogging of the second filter.

[0069] Preferably, both the second filter and, if present, the relative pre-filter are made of polyethersulfone (PES).

[0070] Note that the purified crude extract preferably features a microbial load < 100,000 CFU / mL, preferably ≦ 50,000 CFU / ml.

[0071] Note that the purified crude extract preferably features a protein dosage (Lowry assay) of the final purified extract in the range of 150 to 250 wt% (the dosage of the final purified extract corresponds to 100%).

[0072] Preferably, the protein dosage of the purified crude extract of the mite allergen is in the range of about 3 mg / mL to 9 mg / mL, and preferably, the mite is Dermatophagoides farinae, or Dermatophagoides pteronyssinus, or Euroglyphus maynei.

[0073] Preferably, the protein dosage of the purified crude extract of the pet animal allergen is in the range of about 0.4 mg / mL to 9 mg / mL, and preferably, the pet animal is selected from Canis lupus and Felis catus.

[0074] Preferably, the protein dosage of the purified crude extract of bee venom allergen is in the range of about 0.3 mg / mL to 9 mg / mL. Preferably, the bee species are selected from Apis mellifera, Vespa Crabro, Vespa velutina, Vespa mandarinia, Vespula germanica, Vespula vulgaris, Polistes dominula, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, the genus Dolichovespula, Vespula alascensis, Vespula maculifrons, Vespula flavopilosa, Vespula pensylvanica, Vespula Squamosa, and Polybia paulista.

[0075] Preferably, the purified crude extract of the above bee venom allergen is in a lyophilized form; thus, the protein dosage stated represents the dosage of the extract before or after lyophilization when the lyophilized product is reconstituted to 1 mg / ml with physiological saline.

[0076] Particularly preferred embodiments of the bee species include Apis mellifera, Vespa Crabro, Vespula germanica, Polistes exclamans, Bombus terrestris, Bombus agrorum, and Bombus medics.

[0077] According to a preferred embodiment, the step of purifying the crude extract to reduce the microbial content of the extract is carried out in an environment of grade D according to ISO 14644-1.

[0078] Purification of the purified crude extract to obtain the C-purified extract According to a preferred embodiment, the process subject of the present invention includes a step of purifying the crude extract (when the step of purifying the crude allergen extract (step B) has not been previously performed) or a step of purifying the purified crude extract (when instead the crude extract must have previously undergone the purification step (B)).

[0079] The step (C) of purifying the crude extract or the purified crude extract comprises or consists of at least a sub-step (C1) of filtering the crude extract or the purified crude extract to remove impurities having a molecular weight ≦ 5,000 Da (or the "sub-step of removing impurities from the purified crude extract").

[0080] These impurities are molecules that do not belong to allergens and are salts and / or proteins that are natural to the starting biological sample, or salts and / or proteins introduced in previous steps of the process (for example, salts and / or proteins of the solvent solution that may be used during the extraction step). It is important to remove these impurities from the sample in order to control the isotonicity of the final extract and to prevent the specific immunotherapy product from causing a sensitization reaction by other protein species.

[0081] Thus, in this sense, the step of purifying the crude extract or the purified crude extract is not necessary when the isotonicity of the sample is already suitable for the preparation of the final product. As an example, in the production of a final product containing or consisting of bee venom allergen, there is a mechanical method of extracting the venom that does not require the use of a solvent that can affect the isotonicity of the crude extract, so it is not always necessary to perform the step of purifying the crude extract.

[0082] Preferably, the sub-step (C1) of removing impurities from the crude extract or the purified crude extract is carried out by diafiltration, preferably tangential flow mode diafiltration (tangential flow diafiltration), using a filter having a cut-off on the order of 5,000 Da.

[0083] The advantage of using diafiltration, preferably tangential flow mode diafiltration (tangential flow diafiltration), at this step of the present production process is that it has a molecular weight significantly lower than that of the allergenic protein of interest and for this very reason there is a possibility of removing molecules that could not be removed in any of the previous filtration steps (such as purification step (B) etc.). During diafiltration, the impurities pass through the membrane and are not retained (constituting the "permeate" or "filtrate"). The remaining part of the diafiltration extract retained by the membrane and containing proteins and molecules with a molecular weight above 5,000 daltons constitutes the "retentate".

[0084] More preferably, the sub-step (C1) of removing impurities from the purified crude extract comprises the following steps carried out using the same filter (or membrane): A first passage of the purified crude extract through a membrane having a cut-off of 5,000 Da to obtain a concentrated form (retentate) of the purified crude extract; At least 4 additional passages through the same membrane having a cut-off of 5,000 Da to wash the retentate obtained from each of the previous passages with physiological saline.

[0085] According to a preferred embodiment, the extract from which impurities have been removed in step (C1) can be further filtered using a filter having a porosity in the range of 0.1 μm to 0.2 μm, preferably equal to 0.1 μm.

[0086] The extract obtained from the sub-step (C1) of removing impurities from the purified crude extract, which is inclusive or without further downstream filtration (membrane 0.1 - 0.2 μm), constitutes the purified extract.

[0087] It should be noted that such purified extracts preferably have a microbial load < 100,000 CFU / mL, preferably ≦ 50,000 CFU / mL.

[0088] It should be noted that the purified extract preferably has a protein dosage of 100 - 300% by weight of the final purified extract (where the dosage of the final purified extract corresponds to 100%).

[0089] Preferably, the protein dosage of the purified extract of the mite allergen (the mite is preferably Dermatophagoides farinae, or Dermatophagoides pteronyssinus, or Euroglyphus maynei) is in the range of 1.9 mg / mL to 6.0 mg / mL.

[0090] Preferably, the protein dosage of the purified allergen extract from pet animals is in the range of about 0.25 mg / mL to 6.0 mg / mL, and the pet animal is preferably Canis lupus or Felis catus.

[0091] Preferably, the protein dosage of the purified extract of bee venom allergen is in the range of about 1.0 mg / mL to 3.0 mg / mL, and the order Hymenoptera preferably includes Apis mellifera, Vespa Crabro, Vespa velutina, Vespa mandarinia, Vespula germanica, Vespula vulgaris, Polistes dominula, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, the genus Dolichovespula, Vespula alascensis, Vespula maculifrons, Vespula flavopilosa, Vespula pensylvanica, and Vespula Squamosa. More preferably, the order Hymenoptera includes Apis mellifera, Vespa Crabro, Vespula germanica, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, Polybia paulista, and the protein dosage is in the range of about 0.2 mg / mL to 6.0 mg / mL.

[0092] According to a preferred embodiment, the step of purifying the purified crude extract is performed in an environment of grade D according to ISO 14644-1.

[0093] D, Provision and Packaging of Extracts for E-Packaging According to a preferred embodiment, the production process of the present invention includes other steps (D, E) aimed at packaging the purified extract after the purification step (C).

[0094] Preferably, the purified allergen extract is adjusted back to the desired titer (100%) by adding physiological saline to the purified allergen extract from the perspective of protein dosage (D).

[0095] The extract obtained from this back-titration step constitutes the purified and titrated allergenic extract.

[0096] Preferably, the protein dosage of the purified and titrated extract of mite allergens is in the range of about 1.9 mg / mL to 2.4 mg / mL, and the mite is preferably Dermatophagoides farinae or Dermatophagoides pteronyssinus, or Euroglyphus maynei.

[0097] Preferably, the protein dosage of the purified and titrated extract of pet animal allergens is in the range of about 0.25 mg / mL to 2.0 mg / mL, and the pet animal is preferably Canis lupus or Felis catus.

[0098] Preferably, the protein dosage of the purified and titrated extract of bee venom allergen is in the range of about 0.2 mg / mL to 2.0 mg / mL, and the order Hymenoptera preferably includes Apis mellifera, Vespa Crabro, Vespa velutina, Vespa mandarinia, Vespula germanica, Vespula vulgaris, Polistes dominula, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, the genus Dolichovespula, Vespula alascensis, Vespula maculifrons, Vespula flavopilosa, Vespula pensylvanica, and Vespula Squamosa, more preferably selected from Apis mellifera, Vespa Crabro, Vespula germanica, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, Polybia paulista.

[0099] Preferably, step (D) of the back-titration is carried out in a grade C room according to ISO 14644-1.

[0100] According to a preferred embodiment, before packaging, the purified and titrated allergenic extract is subjected to a final purification step (E) to remove any additional microbial load accumulated in previous processing steps. In other words, such additional microbial load should not be considered natural to the starting biological sample.

[0101] According to a preferred embodiment, the microbial filtration step (E) of the purified and titrated extract comprises or consists of a sub-step of performing dimensional filtration with a filter having a porosity of dimensions in the range of 0.1 μm to 0.2 μm, preferably equal to 0.1 μm. including or consisting of.

[0102] The extract obtained from the microbial filtration step (E) of the purified and titrated extract constitutes an embodiment of the final product according to the present invention.

[0103] It should be noted that the filtrate or permeate obtained from the microbial filtration step (E) of the purified and titrated extract contains the allergenic protein of interest and, in this sense, may constitute the final product according to the present invention; on the other hand, the residue obtained from the microbial filtration step (E) of the purified and titrated extract is discarded because it contains a microbial load that is not natural to the biological sample (if present).

[0104] Preferably, the step (E) of filtering the purified and titrated extract is conventional filtration or "dead-end" filtration, i.e., filtration performed by applying pressure perpendicular to the membrane from the supply side of the sample being filtered. The force inducing filtration is the pressure difference (differential pressure) between the inlet side and the outlet side of the filtrate.

[0105] Preferably, the filter comprises a pre-filter having a porosity with dimensions in the range of 0.45 μm to 0.65 μm, preferably in the range of 0.45 μm to 0.50 μm, preferably equal to 0.45 μm. The pre-filter is preferably structurally coupled to the filter in order to define a filter assembly for resolving continuity.

[0106] Preferably, the second filter and, if present, the relative pre-filter are made of polyethersulfone (PES).

[0107] It should be noted that the purified and titrated crude extract preferably features a microbial load < 100,000 CFU / mL, preferably ≤ 50,000 CFU / ml.

[0108] Ultrafiltration (or nanofiltration) of viruses According to a preferred embodiment of the present invention, the production process includes an ultrafiltration step of viruses that is functional to obtain a product that substantially does not contain viruses and / or mycoplasmas and that contains or consists of a purified extract of allergens.

[0109] The ultrafiltration step of viruses preferably uses a filter assembly including a dimensional filter including a filter having a porosity with dimensions in the range of about 20 nm to 100 nm, preferably 20 nm to 80 nm, preferably 20 nm to 50 nm, preferably 20 nm to 27 nm, preferably equal to 20 μm; a pre-filter coupled in front of (or upstream of) the filter and having a porosity with dimensions preferably in the range of about 100 nm to 900 nm, preferably in the range of 100 nm to 600 nm, preferably in the range of 100 nm to 200 nm, preferably equal to 100 nm is performed.

[0110] In the case of the ultrafiltration step of the virus, the residue is discarded because it contains the virus and / or mycoplasma, and probably a small protein fraction in the case of an allergenic protein having a dimension close to 150 kDa; on the other hand, it should be noted that the permeate obtained from the ultrafiltration of the virus does not contain the virus and / or mycoplasma, but constitutes a purified extract of the allergen within the scope of the present invention.

[0111] Preferably, the filters and prefilters that can be used in the ultrafiltration step of the virus are polyethersulfone (PES) or polyamide (PA) filters.

[0112] Preferably, the prefilter is structurally coupled to the filter to form a filter assembly for resolving continuity together with the filter.

[0113] More preferably, the ultrafiltration step is carried out at a constant pressure in the range of 0.5 to 5 bar, preferably about 1 to 5 bar, preferably about 1.5 to 4 bar, preferably about 1.8 to 3 bar, preferably equal to about 2 bar.

[0114] Thus, advantageously, the ultrafiltration carried out in this way can be completed in 2 to 24 hours, preferably 2 to 12 hours, preferably 2 to 6 hours, preferably 2 to 4 hours, preferably 2 to 3 hours, preferably 2 hours; more preferably, such an ultrafiltration time is in the range of 13 L.m -2 .h -1 or more, preferably 13 L.m -2 .h -1 ~30 L.m -2 .h -1 , preferably 20 to 30 L.m -2 .h -1 , preferably 25 L.m -2 .h -1 ~30 L.m -2 .h -1 and it should be noted that it is possible with a flow rate in this range.

[0115] Figure 1 schematically depicts a virus ultrafiltration system that can be used within the scope of the present invention. The depiction of the virus ultrafiltration system is intended to explain the manner of implementation of the ultrafiltration step in the process of generating a final product that contains or consists of an allergenic extract, but is not intended to limit the manner of utilization of the teachings of the present invention.

[0116] This system preferably includes a pressurizable container 1 (or "tank") made of stainless steel; the container is connected upstream to a compressed air line 2 and downstream to a filter assembly 3 in accordance with the present invention. According to a preferred embodiment, the filter assembly includes a filter 31 for ultrafiltration. A pre-filter 32 is coupled in front of the filter according to the present invention.

[0117] Container 1 is preferably removably connected upstream to a compressed air line 2 and downstream to a filter assembly 3 so that it can be assembled and disassembled in the step of washing or filling the sample. According to an alternative embodiment, the container is configured to be connected to a line for supplying a sample to be subjected to virus ultrafiltration (not shown).

[0118] The compressed air line 2 preferably includes a pressure controller 21 located upstream of the container and an introduction valve 22 (or "in-valve") that controls the entry of compressed air from the compressed air line into the container.

[0119] The virus ultrafiltration system also includes a discharge valve 23 (or "out-valve") located downstream of the container and upstream of the filter assembly, and this discharge valve controls the flow of material exiting the container 1 and entering the filter assembly 3.

[0120] The compressed air line 2 preferably includes a pressure sensor 23 for reading the pressure within the virus ultrafiltration system.

[0121] The compressed air line is connected to another system (not shown) that generates air under pressure.

[0122] The "driving" force of the virus ultrafiltration system is the pressure difference (or differential pressure) between the sample inlet and the filtrate outlet in the ultrafiltration membrane. This pressure is applied perpendicular to the membrane of the ultrafiltration membrane from the supply side. The induction force is the pressure difference (differential pressure) between the inlet side and the outlet side of the filtrate.

[0123] According to a preferred embodiment, the system includes an instrument for measuring the volume or weight of the filtrate 4 (scale as an example in the drawing), which is arranged downstream of the filter assembly 3 and communicates with the filter assembly 3 through a discharge line. Controlling the volume or weight of the filtrate is useful for monitoring the consistency and accuracy of the present production process.

[0124] More preferably, the virus ultrafiltration system includes a programmable logic controller 6 (PLC) for controlling the steps of the process. The PLC can be connected to the introduction valve and the discharge valve (22, 23); an instrument for measuring volume or weight as a function of the steps of the present process, or a production management system (not shown).

[0125] Preferably, the process for generating the purified extract according to the present invention can be carried out in 42 hours or less than 42 hours.

[0126] The reduction in the amount of virus / mycoplasma is usually the log reduction factor (LRF; in some cases, simply the logarithmic scale or "log" 10expressed in terms of the ratio between the virus / mycoplasma titers of the samples before and after filtration (ICH Harmonized Tripartite Guideline Q5A (R1), 1999: Viral Safety evaluation of biotechnology products derived from cell lines of human or animal origin. EMEA / CPMP / BWP / 268 / 95 / 3AB8A, 1996: Note for guidance on virus validation studies: The design, contribution and interpretation of studies validating the inactivation and removal of viruses.).

[0127] Preferably, the virus ultrafiltration step (and thus the entire production process) reduces the amount of virus / mycoplasma by at least 4 log 10 (LRF ≥ 4 log 10 ), preferably at least 6 log 10 (LRF ≥ 6 log 10 ), preferably at least 8 log 10 (LRF ≥ 8 log 10 ).

[0128] More preferably, the virus ultrafiltration step (and thus the entire production process) reduces the amount of virus / mycoplasma with a LRF in the range of 4 - 10 (including the extreme values), preferably in the range of 4 - 8 (including the extreme values).

[0129] The amount of virus in the sample may be determined, for example, by counting the virus particles present in the sample; this counting may be done by methodologies known to those skilled in the art such as the TCID 50 assay, plate assay, PCR assay, or electron microscopy.

[0130] The amount of mycoplasma in the sample can be determined, for example, by counting colony forming units (CFU), which can be performed under an optical or an electron microscope, or by calculating the copy of the mycoplasma genome by qPCR (quantitative real-time polymerase chain reaction).

[0131] Preferably, the product resulting from the virus ultrafiltration step contains ≦ 10 6 , preferably ≦ 10 4 , preferably ≦ 10 3 , preferably ≦ 10 2 , preferably ≦ 10, preferably ≦ 1, preferably ≦ 0.1, preferably ≦ 0.01 virus particles.

[0132] As an example, the viruses excluded by the implementation of the virus ultrafiltration step can be of the parvovirus, retrovirus, picornavirus types.

[0133] Preferably, the product resulting from the virus ultrafiltration step has a mycoplasma content of ≦ 50,000 CFU / mL ≦ 10,000 CFU / mL, preferably ≦ 1,000 CFU / mL, preferably ≦ 100 CFU / ml, ≦ 10 CFU / ml.

[0134] As an example, the mycoplasmas excluded by the implementation of the virus ultrafiltration step belong to the class Mollicutes (Spiroplasma, Mycoplasma, and Acholeplasma).

[0135] As already mentioned, virus ultrafiltration has also been proven to be effective in reducing the content of bacterial endotoxins; thus, the final product obtainable by the process subject of the present invention is not only substantially free of viruses and / or mycoplasmas by the implementation of the virus ultrafiltration step, but also substantially free of bacterial pathogens, preferably bacterial endotoxins. In any case, it should be noted that the step (B) of purifying the crude extract, when applied, contributes to reducing the bacterial load of the crude extract.

[0136] Preferably, the viral ultrafiltration step reduces the content of bacterial endotoxin by more than 90% or more than 1 log10, preferably in the range of about 90% to 99.9% or about 1 log 10 ~3 log 10 in the range, more preferably in the range of about 90% to 99.7% or about 1 log 10 ~2.5 log 10 in the range of values, and it is possible to reduce them.

[0137] The content of bacterial endotoxin can be determined according to state-of-the-art known methods (such as kinetic turbidimetry, chromogenic rate theory method, chromogenic or endpoint method, turbidimetric endpoint method), preferably according to the chromogen (or chromogenic) method.

[0138] The ultrafiltration step can be carried out one or more times (at one or more moments) in the production process object of the present invention. At least three preferred embodiments of the implementation of the viral ultrafiltration step in the process for producing a final product comprising or consisting of a purified extract of an allergen are described below; those skilled in the art will understand that such preferred embodiments are illustrative and do not limit the embodiments of the present invention, and similarly, other methods can also be carried out based on the teachings included in this application.

[0139] First Embodiment According to the first preferred embodiment of the production process of the present invention, the viral ultrafiltration step is carried out before the step (C) of filtering the purified crude extract.

[0140] More preferably, the viral ultrafiltration step is carried out on the crude extract of the allergen.

[0141] The ultrafiltration step for viruses can also be carried out after step (B) of purifying the crude allergenic extract, and more preferably after sub-step (B1) of filtering the crude extract to remove the microbial load of the biological sample. The ultrafiltration step for viruses is preferably carried out on the purified crude extract.

[0142] Second Embodiment According to a second preferred embodiment of the production process of the present invention, the ultrafiltration step for viruses is carried out after step (C) of filtering the purified crude extract, and more preferably after a sub-step of removing impurities from the purified crude extract. The ultrafiltration step for viruses is preferably carried out on the purified extract.

[0143] Such consecutive steps are particularly suitable for solving state-of-the-art known problems such as filter packing and variations in the virus ultrafiltration process.

[0144] It should be noted that the purified allergenic extract subjected to the ultrafiltration step for viruses and thus substantially free of viruses and / or mycoplasmas is preferably characterized by a protein dosage in the range of 100 to 300% by weight of the predicted protein dosage.

[0145] The purified allergenic extract subjected to the ultrafiltration step for viruses and thus substantially free of viruses and / or mycoplasmas is preferably characterized by a microbial load of <100,000 CFU / mL, preferably ≦50,000 CFU / ml.

[0146] Third Embodiment According to a third preferred embodiment of the present production process, the ultrafiltration step for viruses is carried out before packaging.

[0147] More preferably, the ultrafiltration step for viruses is carried out on the final product, understood as the product obtained from step (E) of filtering the microorganisms of the purified and titrated extract.

[0148] In this second embodiment, the final product subjected to virus ultrafiltration may contain one or more allergens among those that can be used for the purposes of the present invention.

[0149] It should be noted that, optionally, the above-described first, second, and third embodiments can be implemented simultaneously in the same process for generating a final product comprising or consisting of a purified extract of an allergen.

[0150] Allergens and products containing them For the purposes of the present invention, the allergen is preferably a mite allergen, the mite being preferably Dermatophagoides farinae or Dermatophagoides pteronyssinus, or Euroglyphus maynei; Bee venom allergen, the order Hymenoptera, preferably Apis mellifera, Vespa Crabro, Vespa velutina, Vespa mandarinia, Vespa velutina, Vespula germanica, Vespula vulgaris, Polistes dominula, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, the genus Dolichovespula, Vespula alascensis, Vespula maculifrons, Vespula flavopilosa, Vespula pensylvanica, and Vespula Squamosa, more preferably selected from Apis mellifera, Vespa Crabro, Vespula germanica, Polistes exclamans, Bombus terrestris, Bombus agrorum, Bombus medics, Polybia paulista; Pet animal allergen, the pet animal is preferably Canis lupus or Felis catus selected from.

[0151] In a preferred embodiment, the allergen for the purpose of the embodiments of the present invention is a mite allergen (Dermatophagoides farinae or Dermatophagoides pteronyssinus, or Euroglyphus maynei), and an allergen derived from cats (Felis catus).

[0152] The allergen for the purpose within the scope of the present invention is preferably characterized by a molecular weight in the range of 1 kDa to 150 kDa.

[0153] More preferably, the mite allergen has a molecular weight in the range of 10 kDa to 150 kDa.

[0154] More preferably, the bee venom allergen has a molecular weight in the range of 1 kDa to 150 kDa, preferably 1 kDa to 100 kDa.

[0155] A further object of the present invention is a final product for medical use, or for diagnostic use in vivo, or for diagnostic use in vitro, which contains one or more allergenic extracts and is substantially free of viruses and / or mycoplasmas.

[0156] Such a final product is preferably obtained by the process subject to the claims.

[0157] According to a preferred embodiment, the final product obtainable by the process of the present invention is a pharmaceutical product for medical use, which itself can be used in the treatment or prevention of allergies, a raw material that can be used to provide a pharmaceutical composition for medical use in the treatment or prevention of allergies constitutes.

[0158] Preferably, the final product according to the present invention is intended for medical use in specific, preventive, or therapeutic immunotherapy of allergic subjects. More preferably, it is intended for medical use in the treatment of clinical symptoms of allergies selected from the group consisting of mild and / or moderate atopic dermatitis, rhinitis, or rhinoconjunctivitis; mild and / or moderate asthma.

[0159] More preferably, the pharmaceutical composition according to the present invention is targeted at medical use in the prevention of the allergy march.

[0160] It should be noted that preferably, the final product according to the present invention can be used in both the introduction step and the maintenance step of specific immunotherapy treatment.

[0161] In the introduction step, the dose of the allergen is gradually increased until the maintenance dose is achieved.

[0162] Preferably, the pharmaceutical composition according to the present invention is in a sublingual or injectable form, preferably a sublingual composition.

[0163] In the case of an injectable, preferably subcutaneous injection composition, the purified allergen extract may be further manipulated and modified before being included in the final pharmaceutical composition. In particular, the allergen can be adsorbed onto L-tyrosine crystals by state-of-the-art known techniques to make it suitable for the intended route of administration; this can be subjected to a further filtration process for microorganisms, viruses, and / or mycoplasmas to produce a final product that is substantially sterile and suitable for subcutaneous administration.

[0164] These further filtration steps may require working in a sterile environment with a higher classification than that of grade C according to ISO 14644-1, such as an environment of grade B and / or A according to ISO 14644-1.

[0165] Alternatively, the end product according to the present invention is intended for use in the in vivo or in vitro diagnosis of allergies, preferably in the in vivo or in vitro diagnosis of respiratory allergies or contact allergies.

[0166] According to a preferred embodiment, the end product obtainable by the process of the present invention is a medicament for use in an in vivo or in vitro diagnosis that can itself be used for the detection of allergies a raw material that can be used to provide a pharmaceutical composition for in vivo or in vitro diagnostic use in the detection of allergies constitutes.

[0167] Examples of in vivo applications of the end product for diagnostic use are as follows: Patch test, i.e., a diagnostic test useful for the determination of contact allergy (DAC) consisting of applying a suspect substance (hapten) contained in a cell fixed with a patch to the back of the subject being tested. The patch is generally removed after being held at the site for 48 hours, and the appearance of any erythema, edema, and vesicles that may define an allergen positive is evaluated. Prick test, i.e., a skin test used to diagnose allergies, preferably respiratory allergies, which involves applying an allergen at a predetermined concentration to the skin and lightly puncturing the skin with a special lance to facilitate the penetration of this substance into the superficial layer of the skin. Alternatively, it is possible to perform an "intradermal test" which assumes the use of a larger needle that is convenient for subcutaneous injection of the allergen.

[0168] The end product according to the present invention can also be used in performing in vitro diagnostic tests that may be accompanied by or replace the use of in vivo diagnostic tests. Examples of in vitro applications of the end product for diagnostic use are for example, specific IgE dosages using an ELISA assay Administration of molecules involved in basophil activation (histamine, heparin, neutral protease, acid hydrolase, and other chemotactic factors), for example using a CAST-ELISA assay (measuring sulfidoleukotrienes produced in vitro after basophil stimulation by an allergen).

[0169] Preferably, the final product of the present invention may contain one or more of the above-mentioned allergens, regardless of whether it is intended for medical use or diagnostic use.

[0170] According to a preferred embodiment, the final product substantially free of virus and / or mycoplasma according to the present invention contains a purified allergen extract from Dermatophagoides farinae or Dermatophagoides pteronyssinus, or Euroglyphus maynei. More preferably, the pharmaceutical composition contains the above-mentioned mite-derived extract in a weight ratio of 1:1:1.

[0171] According to an alternative embodiment, the final product according to the present invention contains a purified extract of hair and dandruff allergens derived from Felis catus, substantially free of virus and / or mycoplasma.

Example

[0172] For illustrative and non-limiting purposes, the process of generating a purified extract substantially free of virus and / or mycoplasma according to the present invention is described below.

[0173] The following example represents a process for producing a purified extract of Euroglyphus maynei, which is also applicable per se to the production of purified extracts of D. pteronyssinus and D. farinae.

[0174] 1. Process for manufacturing a purified extract of Euroglyphus maynei Glossary SM: Source material, raw material EM1 API: Active substance, standardized origin extract in total protein with unique formulation Extraction ratio (w / V): Ratio between the amount of source material (grams) weighed in the weighing chamber and the volume of phosphate buffer pH 8.0 (mL) required to perform the steps of the cooling extraction process

[0175] Introduction The settings of the EM1 API detailed below were made within 42 hours and at the latest 42 hours from the start of the process (recovery of the source material from the freezer), and the setting time of the active substance was confirmed during the process validation. There was no interruption time during the manufacturing process.

[0176] All treatments are carried out at a controlled temperature (room temperature: 18 - 22 °C), except for the centrifugation step of the suspension by the extraction step and the separation step carried out at low temperature.

[0177] Table 2 shows the composition of the EM1 API of Euroglyphus maynei. [Table 1]

[0178] The formulation of the extract of the EM1 API of Euroglyphus maynei is shown in Table 3 below. [Table 2]

[0179] Principles for setting the EM1 API of Euroglyphus maynei This manufacturing process assumes the following main process steps: 1) Collection of source materials and solutions (Step 1A); transportation of materials to the weighing chamber, thawing and weighing of SM (Step 1B). 2) Cooling extraction of SM of Euroglyphus maynei in phosphate buffer pH 8.0; 3) Phase separation: centrifugation of the extract by separating the solid phase from the liquid phase (supernatant and precipitate); 4) Prefiltration and filtration: prefiltration (8 μm) and filtration (0.45 μm + 0.2 μm) of the extract; 5) Purification of the extract (nominal cut-off 5000 kDa): diafiltration and subsequent filtration (0.2 μm + 0.1 μm or.2 μm) of the diafiltered EM1; 6) Ultrafiltration (0.1 μm prefilter + 20 nm ultrafiltration membrane); 7) Return to protein titer: sampling of the diafiltered and ultrafiltered extract (untitrated EM1) for measuring the protein dosage in the CQ laboratory. Based on this, back-titration is performed using physiological saline (specification of protein dosage by the Lowry method: 1.9 - 2.4 mg / mL). 8) Filtration of the titrated EM1 (0.2 μm) and filling of containers: sampling of the titrated EM1 for measuring the microbial load and protein dosage with simultaneous filling of sterile containers and subsequent filtration at 0.2 μm; sampling for release analysis. 9) Transportation and storage in a dedicated quarantine area.

[0180] Description of the manufacturing process A flowchart of the process for manufacturing EM1 API of Euroglyphus maynei is shown in Figure 2.

[0181] Step 1 - Collection and thawing of source materials and transportation of materials from the storage chamber to the weighing chamber (Grade C room) 1) Take an aliquot of SM from the freezer. 2) Weigh the SM after thawing. 3) In the draft, weigh the desired aliquot of SM on a dedicated balance in a dedicated plastic container and seal the container. 4) Transport the materials to the room of Grade D specialized for extraction.

[0182] Step 2 - Cooling extraction in phosphate buffer pH 8.0 in the extraction / regulation room (room of Grade D) 1) Take the bottle containing phosphate buffer pH 8.0 from the refrigerator and put all the materials into the chemical draft. 2) Put the SM into a sterile bottle equipped with a stirring bar at the bottom, add phosphate buffer pH 8.0, close the bottle, and manually stir to completely wet the SM. 3) Transfer the remaining amount of phosphate buffer pH 8.0 into the extraction bottle and stir. 4) Place the extraction bottle on a stirrer and stir constantly for convenient phase mixing. At the end of stirring, confirm that the pH has not changed and correct it if necessary. 5) Close the bottle and weigh it with a dedicated balance. 6) Transfer the bottle to the refrigerator for the cooling extraction step.

[0183] Step 3 - Separation of phases (supernatant / precipitate) in the extraction / regulation room (room of Grade D) 1) Check the cooling extraction step. 2) Take the extraction bottle from the refrigerator. 3) Place the extraction bottle under stirring. 4) While stirring constantly, transfer the suspension to a tube for centrifugation. 5) At the end of centrifugation, transfer the supernatant (extract) into a sterile bottle. 6) Sample the supernatant for protein dosage control and for performing the microbial load (protein dosage: 3.0 - 9.0 mg / mL; total microbial load before filtration: less than or equal to 1×10 10 CFU / mL or the bioburden equal to it)

[0184] Step 4 - Prefiltration and filtration in the extraction / regulation room (Grade D) 1) Prefiltration of the extract with a PP filter having a porosity of 8 μm: Used to collect the extract to be pre-filtered, place the bottle closed with a screw cap on the balance and weigh it. Open the bottle intended to collect the filtrate and attach a filter system having a filter with a porosity of 8 μm. This system consists of a silicone tube (priming) with a filter attached at one end, which is housed in the filtrate bottle in the "in" → "out" direction from the bottle of the supernatant to be filtered to the bottle of the filtered supernatant. Operate the peristaltic pump and wait for the full volume to be filtered.

[0185] 2) Filtration of the extract through PES filters with porosities of 0.45 μm and 0.2 μm Open the bottle intended to collect the filtrate and attach a filter system having filters with porosities of 0.45 μm and 0.2 μm. This system consists of a silicone tube (priming) with a filter attached at one end, which is housed in the filtrate bottle in the "in" → "out" direction from the bottle of the pre-filtrate to be filtered to the bottle of the filtrate. Operate the peristaltic pump and wait for the full volume to be filtered. Collect samples and perform an analytical panel (protein dosage: 3.0 - 9.0 mg / mL; total microbial load before purification: bioburden lower than or equal to 50,000 CFU / mL; protein profile: the trace must include bands at 26.4 kDa and 18.0 kDa).

[0186] Step 5 - Purification of the extract in the diafiltration / dialysis / ultrafiltration room (Grade D) 1) Purification by diafiltration technology with volume reduction: The filtered extract is diafiltered using a diafiltration membrane with a nominal cut-off of 5,000 Daltons. The latter consists of a series of selective membranes with variable porosity and has the task of completely removing or reducing the concentration of salts, proteins, and molecules with a molecular weight of less than 5,000 Daltons that pass through the membrane and are not retained (this amount of waste is called the permeate or filtrate). The remaining part of the extract in diafiltration, which is retained by the membrane and contains proteins and molecules with a molecular weight of more than 5,000 Daltons, is called the residue.

[0187] Diafiltration with volume reduction assumes the following processing steps (1 concentration of the extract and 4 washes with physiological saline, V1 - V4). In each flow, a sample is taken for an analysis panel to evaluate that the diafiltered protein profile follows the standard profile (protein dosage for each diafiltration step: residue protein dosage V1: 3.0 - 9.0 mg / mL; V2: 2.5 - 7.5 mg / mL; V3 - V4: 2.0 - 6.0 mg / mL). Each diafiltration flow is carried out at a constant pressure of 2 bar until all the extract is within the system. The extract passes through the diafiltration membrane and is purified and concentrated from salts, proteins, and molecules with a molecular weight of less than 5,000 Daltons. Stop the peristaltic pump. Open the valve of the residue and collect the full volume into the cylinder. Confirm that the pH has not changed and correct it if necessary. Take a sample of EM1 using a sterile pipette of appropriate volume for an analysis panel (protein dosage: 2.0 - 6.0 mg / mL; protein profile: traces must not contain bands at 26.4 kDa and 18.0 kDa; microbial load before ultrafiltration: bioburden lower than or equal to 50,000 CFU / mL).

[0188] Filtration of the extract through a PES filter with a porosity of 0.2 μm or porosities of 0.2 μm and 0.1 μm Open the bottle intended to collect the filtrate and attach a filtration system with a filter having a porosity of 0.2 μm or porosities of 0.2 μm and 0.1 μm. This system consists of a silicone tube (priming) with a filter attached at one end, which is housed in the filtrate bottle in the "in" → "out" direction from the bottle of the purified substance to be filtered to the filtrate bottle. Operate the peristaltic pump and wait for the full volume to be filtered. 2) Perform the subsequent steps in the same room.

[0189] Step 6 - Ultrafiltration of dialysis filtrate EM1 in the diafiltration / dialysis room (Grade D) The ultrafiltration step uses two filters in a single process step: a prefilter with a porosity of 0.1 μm continuously connected to an ultrafiltration membrane with virus retention capacity having a porosity of 20 μm. The ultrafiltration membrane is connected to a vent filter (0.2 μm).

[0190] Perform the filtration under a constant pressure of 2 bar. 1) This system consists of a steel container for placing the extract to be ultrafiltered, with the following connections: "In" - connection: Inlet to a compressed air container containing no filtered oil at a constant pressure of 2 bar during use and having a porosity of 0.2 μm. "Out" - connection: Connection of the prefilter and the ultrafiltration membrane to the container. The ultrafiltration membrane has two outlets sequentially connected to two silicone tubes housed in the ultrafiltered extract recovery cylinder.

[0191] 2) Perform the following operations Conditioning of the filter with physiological saline Place the extract to be ultrafiltered in the container. Set the pressure to a constant pressure of 2 bar and wait for the full volume to pass completely into the collection cylinder.

[0192] 3) Use a sterile pipette of appropriate volume to collect a sample of EM1 and perform protein dosage (measurement) (protein dosage: 2.0 - 6.0 mg / mL).

[0193] 4) Transfer the non - titrated EM1 to a Grade C room specialized for non - sterile preparations for subsequent steps.

[0194] Step 7 - Back - titration of non - titrated EM1 in the non - sterile preparation room (Grade C) 1) Place a bottle containing both physiological saline for back - titration and non - titrated EMI on the working surface. 2) Based on the results of protein dosage (step 6.3), place the non - titrated EM1 at room temperature under constant magnetic stirring, and then back - titrate it with physiological saline (titration concentration of titrated EM1: 1.9 - 2.4 mg / mL). 3) Continue magnetic stirring. 4) Collect a sample to perform protein dosage (measurement) before the filtration step (protein dosage: 1.9 - 2.4 mg / mL). 5) Before moving to the subsequent steps, use a sterile pipette to sample the titrated EM1 for performing microbial load before filtration (total microbial load before filtration: bioburden lower than or equal to 50,000 CFU / mL).

[0195] Step 8 - 0.45 + 0.2 μm filtration of titrated EM1 in the non - sterile preparation room (Grade C) 1) Based on the weight of the obtained titrated EM1, set the number of bottles for collecting the filtrate. 2) On the LAF working surface, perform the following operations for each container. Place the bottle closed with a screw cap on the balance and press the "tare" key. Open the bottle intended to collect the filtrate and attach a filter system with a PES filter having a porosity of 0.45 + 0.2 μm. The system consists of a silicone tube (priming) with a filter attached at one end, which is housed in the filtrate bottle in the "in" → "out" direction from the bottle of titrated EM1 to be filtered to the filtrate bottle. Operate the peristaltic pump and wait for the full volume to be filtered.

[0196] 2. Analysis of the content of bacterial endotoxin after nanofiltration Glossary LAL: Lysate of amoebocytes of the American horseshoe crab (Limulus Polyphemus). These amoebocytes have the characteristics of reacting with endotoxin and causing blood coagulation of the American horseshoe crab in vivo. This property is utilized in tests for determining endotoxin. American horseshoe crab (Limulus Polyphemus): Ancient arachnids of the order Xiphosura that inhabit only the Atlantic coast of the United States Lipopolysaccharide (LPS): A pyrogenic component of the outer cell membrane of Gram-negative bacteria, consisting of a lipid part and a polysaccharide part, capable of inducing an animal's immune response. EM1: Origin extract EM1 API: Active substance, standardized origin extract in the total protein with a unique formulation R% = Recovery rate expressed as a percentage (range: 50 - 200%). It represents the degree of significance of the test. The % range of the recovery rate is set according to what is reported in the monograph of the current Ph.Eur. edition reference literature.

[0197] Introduction Bacterial endotoxins are lipopolysaccharides (LPS), which are components of the cell wall (pyrogens) of Gram-negative bacteria that can cause fever when injected into the bloodstream. These are constantly released into the environment where live bacteria are present and are completely released by the death of the bacteria themselves. Although there are a few pyrogens with structures other than LPS, it can be reasonably stated that the absence of bacterial endotoxins in the product implies the absence of pyrogenic components.

[0198] Bacterial endotoxins are resistant to high temperatures (up to 200 - 250 °C), pass through the sterilization filters commonly used in pharmaceutical preparations, are water-soluble and non-volatile. Thus, while it is not impossible to remove them from injectable preparations, it is difficult.

[0199] Method The Kinetic Chromogenic LAL method is an analytical method for determining the presence of bacterial endotoxins in a sample and quantifying them. The principle of this method involves measuring the reaction time required for the development of the color intensity after the reaction that occurs between LAL and bacterial endotoxins.

[0200] For the purposes of this test, Endosafe® Nexgen-PTS™ was used, which is based on the measurement of the color intensity of a parameter that is directly related to the concentration of endotoxins in the sample. The intensity of the color formed by the release of the chromophore is measured by a spectrophotometer set at an appropriate wavelength. The chromophore that causes the appearance of yellow is measured photometrically by the reader at 385 - 410 nm.

[0201] The time required for the appearance of the color varies depending on the amount of endotoxin present: if a large amount of endotoxin is present, the reaction occurs rapidly; in contrast, if the amount of endotoxin is small, the reaction occurs slowly.

[0202] The cartridge system of the Endosafe-PTS machine contains an accurate amount of LAL (approved by the FDA), a chromogenic substrate, and a standard endotoxin control (CSE). Each cartridge has four channels to which the LAL reagent and the chromogenic substrate are added. Additionally, the second and fourth channels also contain an endotoxin spike for the positive control.

[0203] Results

Table 3

[0204]

Table 4

Claims

1. A process for producing a final product containing or consisting of a purified extract of an allergen, A) A step of providing a crude extract of an allergen, wherein the allergen is of natural origin and can be obtained by isolation from a biological sample. B) A step of optionally purifying the crude extract to obtain a purified crude extract, wherein the purification step is at least B1) Substep of filtering the crude product to remove the microbial load from the biological sample. Steps including, C) Optionally, a step of purifying the crude extract or the purified crude extract to obtain a purified extract, wherein the purification step is at least C1) Substep of filtering the crude extract or the purified crude extract to remove impurities having a molecular weight of 5,000 Da or less. Steps and Includes, The process is characterized by comprising an ultrafiltration step of one or more viruses. process.

2. The ultrafiltration step of one or more viruses, A filter having a porosity of dimensions in the range of approximately 20 nm to 100 nm, Preferably, a pre-filter coupled before the filter and having a porosity in the range of approximately 100 nm to 900 nm, The process according to claim 1, carried out in a filter assembly including the following:

3. The process according to claim 1, wherein the ultrafiltration step of one or more viruses is performed at a constant pressure in the range of 0.5 bar to 5 bar.

4. The process according to claim 3, wherein the ultrafiltration step of one or more viruses is performed at time intervals ranging from 2 to 24 hours.

5. The substep (B1) of filtering the crude extract is A substep of performing first-dimensional filtration using a first filter having porosity in the range of 8 μm to 20 μm, A substep is performed after the first dimensional filtration using a second filter having a porosity in the range of 0.1 μm to 0.2 μm, and The process according to claim 1, including the process described in claim 1.

6. The process according to claim 1, wherein the substep (C1) of filtering the purified crude extract is carried out by dialysis filtration, preferably by tangential flow dialysis filtration.

7. The process according to claim 1, wherein the purified crude extract is characterized by a microbial load of <100,000 CFU / ml.

8. The process according to claim 1, wherein the allergen is selected from the group consisting of dust mite allergens, pet allergens, and bee venom allergens.

9. The process according to claim 1, wherein the allergen has a molecular weight in the range of 1 kDa to 150 kDa.

10. The process according to claim 1, wherein at least the steps of providing the crude extract (A), purifying the extract (B), and refining the extract (C) are carried out in a sterile environment of grade C and / or D according to ISO 14644-1.

11. The process according to claim 1, wherein the entire purification process is carried out within 42 hours or within 48 hours.

12. A final product for medical use or for in vivo diagnostic use, comprising one or more allergen extracts, which can be obtained by the production process described in claim 1.

13. In specific, prophylactic, or therapeutic immunotherapy for allergenic targets, or In the in vivo diagnosis of allergies, preferably respiratory or contact allergies, The final product for use according to claim 12.

14. The final product for medical use according to claim 12, in the form of a sublingual preparation or an injectable preparation.

15. Use of the final product according to claim 12 for in vitro diagnosis of allergies, preferably respiratory allergies or contact allergies.

Images