Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/94—Pancreatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/05—Living organisms or biological materials

Definitions

• the invention relates to a method for reducing the viral and microbial load of solids-containing biological extracts, a solid-containing biological extract produced by this method, as well as uses of such a solid-containing biological extract.
• Extracts derived from biological source material can be highly virulent.
• Viruses are nucleic acids that are surrounded by a protein shell. With enveloped viruses, an outer lipid envelope is added. Because viruses can not replicate independently, they rely on hosts. Accordingly, they occur in virtually all living things on earth. Few of the known viruses are pathogenic to humans because they have a high host specificity.
• extracts which are intended for human consumption or which are used as an active ingredient in medicaments should generally have as little or no viral load as possible. The actual production process does not always lead to a significant inactivation or removal of the viruses present, so that, in particular in the production of pharmaceutical active ingredients, additional abatement or inactivation steps must be integrated into the process.
• a particular challenge is the inactivation or removal of viruses from complex biological extracts whose active ingredients are enzyme mixtures, without destroying or altering the enzymatic activity of the proteins.
• pancreatin which is obtained as an extract from the porcine pancreas and is used in dried form as an oral therapeutic, as described in DE 3248588 A1.
• pancreas glands 1 originating from domestic pigs are first comminuted 2 and subjected to autolysis 3.
• the sieve filtrate is recovered by filtration 4 of the intermediate product thus obtained. 5.
• the enzymes present in the sieve filtrate are then precipitated 6, the mixture is filtered 7 and the filter cake is recovered 8.
• the filter cake obtained is finally ground 9, vacuum-dried 10 and ground again, whereby the pancreatin is obtained.
• the denoted by the reference numerals 2 to 10 characterized process steps each lead to intermediates, which are referred to below as intermediates.
• pancreatin The active substances in pancreatin are u. a. various polymer degrading enzymes, such as lipases, amylases and proteases.
• a prerequisite for the effectiveness of the therapeutic is that all enzymes are present in a particular ratio and in active form in the active ingredient.
• a peculiarity of the pancreatin is that the enzymes contained are partly in solution and partially bound to particles and thus it is a suspension.
• the invention relates to the treatment of blood plasma, that is a solution of biological active substances in water.
• Bradley DW et al. also describe a process for the treatment of blood plasma in "Pressure cycling technology: a novel approach to virus inactivation in plasma” (Transfusion, 40, 2000, 193).
• the high-pressure treatment of food has been described variously.
• mussels may be treated at high pressure to inactivate noroviruses or hepatitis A viruses (Kingsley et al., Inactivation of a Norovirus by High-Pressure Processing, Appl. Environ. Microbiol., 2007, 581, Calci et al. High-Pressure Inactivation of Hepatitis A Virus within Oysters, Appl. Environ. Microbiol., 2005, 339).
• the treatment should preserve the organoleptic properties of the food.
• the biological activity of enzymes or other drugs after high pressure treatment has not been studied. In addition, solids were tested in both cases.
• the high-pressure treatment can lead to a change in the food.
• fruits will turn brown when undergoing such treatment. This color change is on one due to an enzymatic reaction, indicating a change in the biological activity of these enzymes.
• a particular difficulty is in biological samples that are not present in a substantially homogeneous form.
• parts of the biologically active substances are in the liquid phase, other parts in the dispersed solid particles. Inactivation of viruses in the solid particles may therefore be associated with destruction of the active substances which are in the liquid phase, since the compressibility of the liquid phase is higher than that of the solid particles.
• pancreatin or the intermediates produced in the preparation represent exemplary solids-containing biological extracts.
• the corresponding substance is spiked with various laboratory strains and the titre determined before and after the treatment.
• the object of the invention is to eliminate the disadvantages of the prior art. It is intended in particular to provide a method for reducing the viral and microbial load of solids-containing biological extracts which is suitable for solids and suspensions, does not substantially reduce the activity of the enzymes contained in the biological extract, does not deteriorate the pharmacologically desired properties and does not produce toxic chemical compounds. Further, extracts and uses of such extracts prepared by the method should be indicated.
• a method for reducing the viral and microbial load of solids-containing biological extracts comprising the steps
• the biological activity of the solids-containing biological extract after high-pressure treatment corresponds to at least 50% of the biological activity of the solids-containing biological extract before high-pressure treatment
• the solids-containing biological extract is provided as a suspension comprising a liquid phase and solid particles dispersed therein, the mixture of biologically active agents being partially dissolved in the liquid phase and bound to another portion of the solid particles.
• the solids-containing biological extract may be provided in solid form.
• solids-containing biological extract is to be understood here as meaning an extract which comprises a biologically active substance selected from enzymes, proteins and peptides, or a mixture of such active substances. It is preferred that the solids-containing biological extract comprises a mixture of said biologically active agents. In the following, the solids-containing biological extract is also referred to as extract for the sake of simplicity.
• the solids-containing biological extract is preferably an extract obtained as an alcoholic or aqueous extract from animal organs.
• the contained biologically active agent or drug mixture may be present in the extract in both solution and solids.
• Such a solids-containing biological extract is a complex extract.
• the biologically active agent or mixture of such agents preferably has pharmaceutical activity.
• biologically active ingredient is understood to mean active pharmaceutical ingredients and therapeutic agents.
• Biologically active substances within the meaning of this invention are, for example, enzymes, proteins and peptides.
• Exemplary animal organs from which the extract can be obtained are the liver, the pancreas and the gastric mucosa.
• Examples of solids-containing biological extracts according to the invention are extracts which are obtained from the pancreas, in particular the pancreas of the pig. Specific examples are pancreatin as well as the intermediates obtained in its preparation. These intermediate stages are indicated in FIG. 2 by reference numerals 2 to 10. Preferred examples of these intermediates are the sieve filtrate (reference numeral 5 in Fig. 2) and the filter cake (reference numeral 8 in Fig. 2), after the recovery of which a high-pressure treatment can be carried out, i. H. the sieve filtrate and / or the filter cake can be subjected to a high-pressure treatment (reference numerals 13 and 14 in FIG. 2). Additionally or alternatively, a high-pressure treatment can also be carried out with each of the intermediate stages with the reference symbols 2, 3, 4, 6, 7, 9, 10 and 11.
• pancreas pancreatin is obtained as a pharmaceutical active substance.
• Pancreatin and the intermediates obtained in the preparation contain u. a. the enzymes lipase, amylase and protease. They are thus solid-containing biological extracts in the context of the present invention.
• the extracts are preferably provided as aqueous-alcoholic extracts in step (a) of the process according to the invention.
• the method according to the invention is applicable to all forms of viruses, such as DNA and RNA viruses, enveloped and non-enveloped viruses, furthermore to virions and prions or similar biological systems as well as bacteria and fungi.
• the method is preferably used to reduce the burden of non-enveloped viruses in pancreatin from porcine pancreas or in corresponding intermediates produced in the preparation (see Figure 2).
• the method according to the invention allows the reduction of viral and microorganism contamination of solid-containing biological extracts, without significantly reducing their enzymatic activity, deteriorating the pharmacologically intended properties or producing toxic chemical compounds.
• the biological activity of the solids-containing biological extract after high-pressure treatment should be at least 50% of the biological activity of the solids-containing biological extract before the high-pressure treatment, preferably at least 80%, more preferably at least 90%.
• the enzymatic activity of the solids-containing biological extract should be at least 50% of the enzymatic activity of the solids-containing biological extract after the high-pressure treatment before the high-pressure treatment, preferably at least 80%, more preferably at least 90%.
• its lipase activity, its amylase activity and / or its protease activity after high pressure treatment should be at least 50%, preferably 80%, more preferably 90% of its lipase activity, amylase activity and / or protease Activity before high pressure treatment.
• the viral infectivity of the solid-containing biological extract after high pressure treatment should have been reduced by a factor of greater than 1 log, preferably greater than 3 log, more preferably greater than 4 log, compared to viral infectivity prior to high pressure treatment.
• infectivity of non-enveloped viruses in pancreatin should be greater by a factor greater than that after treatment 1 logio, preferably greater than 3 log-m, more preferably greater than 4 log-io, be reduced compared to its infectivity before treatment.
• Viral infectivity is calculated by endpoint titration and subsequent calculation of the tissue culture infectious dose half-value (TCID 50 ) according to the Spearman-Kärber formula (Bundesan Attacher, No. 84, May 4, 1994). The virus titres calculated are reported as log TM TCID 50 per ml with confidence intervals of 95%.
• the reduction in viral load is given in accordance with EC Directive CMMP / BWP / 268/95, Annex II) as a logarithmic reduction factor, which is the difference in virus titers between a control sample and the pressure-treated samples.
• pancreatin The enzymatic activities of pancreatin are determined according to the monograph "Pancreas powder” published in Pharmacopoea Europaea 6.2, 01/2008: 0350.
• Step (b) of the process according to the invention is preferably carried out using pressures in the range from 1000 to 8000 bar. Preferably in the range of 2000 to 7000 bar, more preferably in the range of 3000 to 6000 bar.
• the high-pressure treatment can be carried out according to the invention at constant pressure or by pressure pulses.
• the high-pressure treatment can take place in a flow-through process or in a batch process in appropriate apparatuses.
• the exposure time of the high pressure treatment to the extract provided in step (a) is preferably 1 to 60 minutes.
• the high pressure treatment is preferred Temperatures between -20 0 C and 30 0 C, more preferably carried out between -10 0 C and 20 0 C.
• the liquid phase of the extract which is subjected to high-pressure treatment in step (b) may contain as solvent water or a mixture of water and an organic solvent.
• the proportion of solvent may range between 1 and 90% by weight, based on the weight of the suspension.
• the solvent used is preferably an alcohol, particularly preferably isopropanol.
• the proportion of isopropanol is typically between 5 and 85% by weight, based on the weight of the suspension.
• a solids-containing biological extract is further provided, which was obtained by the method according to the invention.
• This extract is characterized by low viral and microbial burden.
• its biological, and in particular its enzymatic activity is not substantially reduced, its pharmacologically intended properties are not impaired, and it has no toxic chemical compounds added during the treatment.
• the extract according to the invention obtained by high-pressure treatment or the biological active ingredient produced therefrom can be used for the production of medicaments, in particular in the context of oral enzyme therapy, and as food or food or dietary supplements.
• Fig. 2 is a flowchart of a method according to the invention for
• pancreatin Production of pancreatin from porcine pancreatic glands
• Fig. 3 is a graph showing the relative enzymatic activities after treatment of sieve filtrate from the pancreatin production process at different pressures in percent, based on the starting activity of the untreated sample.
• Fig. 4 is a graph showing the relative enzymatic activities after treatment of filter cake from the pancreatin production process at different pressures in percent, based on the starting activity of the untreated sample.
• pancreas glands 1 derived from domestic swine are the first crushed 2 and subjected to autolysis 3.
• the sieve filtrate can be recovered by filtration 4 of the intermediate product thus obtained. 5.
• the sieve filtrate can be subjected to a high-pressure treatment before further treatment.
• the enzymes present in the sieve filtrate are precipitated 6, the mixture is filtered 7 and the filter cake is recovered 8.
• the filter cake obtained can be subjected to a high-pressure treatment 14.
• the filter cake is finally ground 9, vacuum-dried 10 and ground again, whereby the pancreatin is obtained 12.
• high-pressure treatment of the screen filtrate 13 or (ii) high-pressure treatment of the filter cake 14 or (iii) high-pressure treatment of the screen filtrate 13 and high-pressure treatment of the filter cake 14 may be provided.
• the high-pressure treatment can also be performed on all other intermediates of the production process.
• the following example describes the treatment of intermediates from the pancreatin production process as solids-containing biological extracts derived from porcine pancreas at high pressure.
• the treatment was carried out in a batch process in a high-pressure apparatus.
• FCV Feiines Calicivirus
• the treatment was carried out in a batch process in a high-pressure apparatus.
• FCV stock solution in culture medium was subjected to high pressure treatment at different pressures and temperatures for different times.
• the virus titer was determined in the original sample and in the treated samples.
• Table 1 Virus titer (TCDI 50 in log 10 ) after high pressure treatment of an FCV solution

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• 2009
  • 2009-06-12 EP EP09007797A patent/EP2165717A1/de not_active Withdrawn
  • 2009-06-16 WO PCT/EP2009/004318 patent/WO2010022808A1/de not_active Ceased
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