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WO2012062841A1 - Indicateur d'humidité - Google Patents

Indicateur d'humidité Download PDF

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Publication number
WO2012062841A1
WO2012062841A1 PCT/EP2011/069813 EP2011069813W WO2012062841A1 WO 2012062841 A1 WO2012062841 A1 WO 2012062841A1 EP 2011069813 W EP2011069813 W EP 2011069813W WO 2012062841 A1 WO2012062841 A1 WO 2012062841A1
Authority
WO
WIPO (PCT)
Prior art keywords
moisture
lyophilisate
pama
drying
indicator
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2011/069813
Other languages
German (de)
English (en)
Inventor
Andy Wende
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Priority to CN201180054548.9A priority Critical patent/CN103261874B/zh
Publication of WO2012062841A1 publication Critical patent/WO2012062841A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • G01N31/222—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators for investigating moisture content
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N19/00—Investigating materials by mechanical methods
    • G01N19/10—Measuring moisture content, e.g. by measuring change in length of hygroscopic filament; Hygrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N25/00—Investigating or analyzing materials by the use of thermal means
    • G01N25/56—Investigating or analyzing materials by the use of thermal means by investigating moisture content
    • G01N25/62—Investigating or analyzing materials by the use of thermal means by investigating moisture content by psychrometric means, e.g. wet-and-dry bulb thermometers

Definitions

  • the present invention relates to a lyophilisate which has at least one moisture-indicating compound.
  • Another object of the present invention is the use of at least one compound selected from the group consisting of poly (acrylic acid-co-maleic acid), polyacrylic acid and / or a salt thereof, for moisture indication and a method for monitoring the moisture content of a lyophilisate.
  • Freeze-drying also known as lyophilization or sublimation drying, is a process for the gentle production of high-quality products. In freeze-drying, the ice crystals evaporate directly without the transition to the liquid state. The end product of freeze-drying is called lyophilisate Freeze-drying of the stabilization and preservation of liquid reagent formulations, such as pharmaceuticals and enzyme solutions.
  • the handling of lyophilizates is facilitated by the presence of moisture indicators indicating the level of moisture in the lyophilizate.
  • the moisture indicators available on the market are based essentially on a color reaction of dried salts during the rewetting of the lyophilisate.
  • the indicator system can be applied directly to a drying agent or to a carrier material, for example paper. In general, this application is followed by drying of the resulting indicator system so that it is "dry” in a starting color state, and when the indicator system then comes into contact with moisture, the system changes color. In this way, the exact humidity or material moisture content of the carrier materials can be determined.
  • drying agents are silica gels, alumina or Molekularsie- be.
  • 6,655,315 would not be added to any biological reaction because it consists essentially of carrier material and usually divalent salts (Co 2+ , Mg 2+ , Cr 2+, etc.) The latter are often highly inhibitory to enzyme reactions such as PCR.
  • US Pat. No. 6,665,315 does not mention the use of moisture indicators in lyophilizates at all.
  • WO 92/02804 A discloses a moisture indicator comprising cobalt thiocyanate as an inner mold material and a hygroscopic salt.
  • the hygroscopic salt is selected from the group consisting of copper chloride, sodium chloride and potassium chloride. This indicator system is not suitable for use because of the components used in sensitive biological lyophilizates.
  • WO 92/02804 A does not mention that the moisture indicator is suitable at all for use in lyophilisates.
  • the object of the present invention is to provide an indicator system for moisture in lyophilisates, which in particular does not have the disadvantages described above,
  • the present invention has the object to provide a moisture indicator which can be used in combination with lyophilizates of biological samples, but from this point of view is not understood that the biological sample is stored directly together with the moisture indicator, but in particular in that the moisture indicator is stored spatially separate from the biological sample but in the same atmosphere as the biological sample. Should it come in such an arrangement, comprising - spatially separated, but in the same atmosphere - at least one biological sample and at least one moisture indicator to a moisture absorption, this should be displayed by the separately present moisture indicator.
  • the moisture indicator should preferably be easy to produce,
  • a substantial absence of water means a relative humidity of 0%
  • a moisture indicator in combination with a lyophilizate, which has a volume reduction in comparison to the substantial absence of water of at least 30%, more preferably 60%, particularly preferably 90% Presence of a defined relative air humidity of the surrounding air of at least 70%, more preferably 50%, even more preferably 30%, particularly preferably 1 0%,
  • the moisture indicator itself is a lyophilisate. This can considerably simplify the production of arrangements comprising at least one biological sample and at least one moisture indicator since, for example, it is possible to lyophilize the at least one biological sample and the moisture indicator at the same time,
  • the solution of the underlying problem according to the invention does not require the use of a carrier material on which the humidity-indicating system (compound, complex, composite material, etc.) is applied, since it itself is present as an independent lyophilisate.
  • the moisture indicator according to the invention is itself a lyophilisate and thus suitable for monitoring the moisture state of lyophilized samples present, for example, in adjacent cavities.
  • the moisture indicator after taking up a defined amount of at least 60% by weight of water, based on the initial weight of the dry moisture indicator, the moisture indicator completely dissolves.
  • the object underlying the present invention is further solved by the use of a moisture indicator selected from the group consisting of poly (acrylic acid-co-maleic acid), polyacrylic acid and / or a salt thereof in a lyophilizate.
  • the present invention initially relates to a lyophilizate comprising at least one moisture indicator which has a volume reduction compared to the substantial absence of water of at least 30%, more preferably 60%, most preferably 90%, in the presence of a defined relative humidity of ambient air of at least 70%, more preferably 50%, even more preferably 30%, most preferably 10%.
  • a moisture indicator is understood as meaning a single compound, but also a composition of several individual compounds.
  • the present invention relates to a lyophilizate comprising at least one moisture indicator selected from the group consisting of poly (acrylic acid-co-maleic acid), polyacrylic acid and / or a salt thereof.
  • the compound poly (acrylic acid-co-maleic acid) (CAS number 52255-49-9) provided according to the invention is preferably a copolymer having an average molecular weight of about 50,000; Further, the molar ratio of acrylic acid and maleic acid monomers is preferably about 1: 1. In particular, in the context of the present invention, the use of the sodium salt of poly (acrylic acid-co-maleic acid) is suitable.
  • the moisture indicator according to the invention itself preferably represents a lyophilizate
  • the content of moisture-indicating compound, based in each case on the lyophilisate is preferably 30 to 100% by weight, preferably 40 to 100% by weight, particularly preferably 50 to 100% by weight -%.
  • the moisture indicator provided according to the invention is preferably used as lyophilizate in combination with at least one further biologically active constituent, wherein the moisture indicator and the at least one biologically active constituent are spatially separated, but at the same time in the same atmosphere.
  • This further biologically active ingredient may, for example, be selected from the group consisting of blood, its constituents; Proteins, in particular enzymes; viruses; Bacteria; hormones; antibiotics; fatty acids; lipids; Carbohydrates of any chain length; vaccines; Low molecular weight chemical compounds, their salts and organics.
  • the moisture-indicating compound and / or the at least one biologically active constituent may, for example, be incorporated in a cavity on a PCR strip or in a well on a microtiter plate, a (possibly not) standardized reaction vessel, reaction spaces, microfluidic environments or consumables .
  • the lyophilisate of the moisture-indicating compound may generally be obtained from at least one aqueous solution of the moisture-indicating compound.
  • the aqueous solution may have a concentration of moisture-fixing compound, in each case based on the aqueous solution, of 0.01 to 20% by weight, preferably 0.1 to 5% by weight, particularly preferably 0.25 to 2, 5 wt .-%, exhibit.
  • the aqueous solution may further comprise at least one further biologically active ingredient as defined above,
  • the lyophilizate according to the invention can be prepared, for example, according to the following procedure:
  • the water is sublimated, in which the product to be dried is dissolved. This is done at a vacuum that corresponds to the vapor pressure of the water at the temperature at which drying is to be carried out (eg: the product must be at least -40 ° C cold so that it does not change its structure during drying, ie lyophilized during the main drying with 0.1 2 mbar, this is the vapor pressure of the water at -40 ° C, so that the temperature to be dried product will be as long as -40 ° C, as water is still present).
  • the post-drying serves to remove the water molecules which are bound directly to the product to be dried. For this, a high vacuum of, for example, 0.001 mbar must be applied.
  • incubation means the time in which the mixture to be dried is frozen, the length of the incubation at a given glass transition temperature having an influence on the properties of the lyophilisate (especially on the appearance and structure of the freeze-drying cake) and can be suitably adapted by the skilled person.
  • the freezing provided in process step (ii) may be carried out at a temperature of -10 to -200 ° C, preferably -20 to -80 ° C, more preferably -30 to -50 ° C.
  • the duration of freezing may be 0.5 to 24 hours, preferably 0.5 to 8 hours, more preferably 1 to 3 hours,
  • the temperature can be raised to room temperature. Drying preferably takes place at at least 20 ° C. and a vacuum of 0.1 to 0.00001 mbar, more preferably 0.01 to 0.0001 mbar, particularly preferably 0.01 to 0.001 mbar, for a period of preferably 0 to 72 hours, more preferably 1 to 24 hours , more preferably 3 to 10 hours.
  • the lyophilisate according to the invention is preferably provided in a PCR vessel, in particular a PCR tube.
  • hygroscopic substances are added to the indicator solution to be lyophilized.
  • the lyophilisate changes its volume even at lower humidities than would be the case without addition.
  • the present invention relates to the use of a humectant-indicating compound which has a volume reduction compared to the substantial absence of water of at least 30%, more preferably 60%, most preferably 90%, in the presence of a defined relative humidity of the surrounding at least 70%, more preferably 50%, even more preferably 30%, more preferably 10%, of the moisture content,
  • Another object of the present invention is the use of a moisture keitsindigoingden compound selected from the group consisting of poly (acrylic acid-co-maleic acid), polyacrylic acid and / or a Saiz thereof, in a lyophilisate, for moisture indication.
  • the moisture indication is in a lyophilizate.
  • Another object of the present invention is a method for monitoring the moisture content of a lyophilisate, characterized in that (i) a lyophilisate comprising at least one moisture-proofing compound according to the invention is provided; and
  • the volume of the moisture-indicating compound can be monitored visually, and it can further be provided that the monitoring of the volume of the moisture-indicating compound takes place by comparison observation with an equivalent blank.
  • LoC lab-on-a-chip
  • PAMA poly (acrylic acid-co-maleic acid), Na salt
  • PEG polyethylene glycol
  • Lyophilisaten synonym for freeze-drying
  • MTP microtiter plate
  • a freeze-drying PAMA solution forms a dense network of PAMA as a solid. This is to be recognized by the fact that PAMA lyophilisates have a white body, which the person skilled in the art calls "cake". This is not unusual in itself. Surprising, however, was the observation that this cake disappears on exposure to moist air, as follows 50 ⁇ of a 0.5% or 5% PAMA solution was filled into PCR strips and freeze-dried (freezing at -80 ° C., incubation at -50 ° C.
  • the right four tubes contain the corresponding lyophilisates, starting from the 0.5% PAMA solution (1, and 2 nd tube from the left) and twice from the 5% PAMA solution (3 and 4, tubes from the left).
  • tube 2 (from the left) on both images with the inscription "0.5" contains the lyophilisate which was obtained starting from the 0.5% PAMA solution (left) (FIG.
  • FIGS. 2 a and 2 b A comparison of FIGS. 2 a and 2 b (same arrangement of the tubes as in FIGS. 1 a and b) shows, as already mentioned, that the 5% PAMA lyophilisate shows no change in its morphology even after incubation for 1 5 min, Trial 2:
  • FIG. 3 shows a time series of the humidification of PAMA lyophilizates (0 hours to 10 days).
  • MTP wells were described externally with a red permanent marker, resulting in a red area.
  • 2 MTP wells each were filled with 200 ⁇ 0, 1% / 0.5% / 2% / 5% PAMA incl. 2 ⁇ malachite green solution each. Thereafter, the filled MTP was freeze-dried as described in experiment 1,
  • the thus prepared MTP was successively 1 4 d on the laboratory bench (20 ° C, 35% rel, LF), 1 0 d in a desiccator (22 ° C, 45% rel, LF) and 2 d in a desiccator (22 ° C, 75% rel, LF). After 2 days on the bench, there was no further change in the PAMA cake at 35% rel. LF more recognizable ( Figure 5a). After 10 days at 45% rel, LF, the 0.1% and 0.5% PAMA cakes had shrunk (Figure 5b). After 2 days at 75% rel. Finally, all PAMA cakes had shrunken (Figure 5c).
  • FIG. 6 shows the freeze-dried cakes of 0.5% solutions of PAA, PEG8000, PEG35000, PEG3000 in the stated sequence from left to right.
  • Experiment 6 Quantification of the Water Consumption by the PAMA Lyophilisate
  • Table 1 Weight gain of PAMA lyophilisates (prepared according to approach a)) when incubated at different humidities
  • Table 2 Weight gain of PAMA lyophilisates (prepared according to approach b)) when incubated at different humidities
  • 11a and 11b show tubes from left to right with the following CsF & PAMA concentrations: OmM & 0.5%, 20mM & 0.5%, 30mM & 0.5%, 40mM & 0 , 5%, 50mM & 1%, 60mM & 1%, 70m & 1%, 80mM & 1%.
  • CsF & PAMA concentrations OmM & 0.5%, 20mM & 0.5%, 30mM & 0.5%, 40mM & 0 , 5%, 50mM & 1%, 60mM & 1%, 70m & 1%, 80mM & 1%.
  • Figure 1a shows the batches immediately after lyophilization: first row from above: 2x each 25 ⁇ 0.25%, 0.5%, 1%, 2.5% PAMA; second row from the top: 2x 25 ⁇ each 5% PAMA, 2x each 50 ⁇ 0.25%, 0.5%, 1% PAMA; third row from the top: 2x each 50 ⁇ 2.5%, 5% PAMA; 2x each ⁇ ⁇ 0.25%, 0.5% PAMA; bottom row: 2x each ⁇ ⁇ 1%, 2.5%, 5% PAMA,
  • FIG. 1b shows the same approaches as FIG. however, after the one-day incubation at 45% relative humidity

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Pendant l'incubation, le PAMA absorbe comparativement de grandes quantités d'eau (% en poids de matière solide) dans l'air humide. Il se produit ainsi une forte modification du lyophilisat de PAMA séché et réticulé. Cette modification se manifeste par un rétrécissement du lyophilisat originel. On peut supposer que les deux observations (absorption d'eau et rétrécissement) ont des origines communes.
PCT/EP2011/069813 2010-11-12 2011-11-10 Indicateur d'humidité Ceased WO2012062841A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201180054548.9A CN103261874B (zh) 2010-11-12 2011-11-10 湿度指示剂

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP10191025 2010-11-12
EP10191025.5 2010-11-12

Publications (1)

Publication Number Publication Date
WO2012062841A1 true WO2012062841A1 (fr) 2012-05-18

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ID=43827958

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PCT/EP2011/069813 Ceased WO2012062841A1 (fr) 2010-11-12 2011-11-10 Indicateur d'humidité

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Country Link
CN (1) CN103261874B (fr)
WO (1) WO2012062841A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4175207A (en) * 1975-02-07 1979-11-20 Elliott Stanley B Visual-type hygrometer
US4990284A (en) * 1989-10-11 1991-02-05 Brown & Williamson Tobacco Corporation Moisture indicating ink and package having same
WO1992002804A1 (fr) 1990-07-31 1992-02-20 Dansk Teknologisk Institut Indicateur d'humidite avec detecteur enveloppe
DE19508029A1 (de) * 1995-03-07 1996-09-12 Richard Prof Dr Sueverkruep Darreichungsform zur äußerlichen Applikation von ophthalmischen Arzneistoffen und Verfahren zur Herstellung derselben
US6655315B1 (en) 1999-07-30 2003-12-02 Levosil S.P.A. Moisture indicators for the absorbent capacity of a desiccant
US6665315B1 (en) 1998-11-27 2003-12-16 Oki Electric Industry Co., Ltd. Transmission apparatus automatically acquiring identifying information and independently measuring propagation delay
WO2007006682A1 (fr) * 2005-07-14 2007-01-18 Agfa Graphics Nv Derives phenylazo-acetoacetanilide comportant un groupe fonctionnel polymerisable et composes associes en tant que monomeres pour dispersants polymeriques de pigments pour encres d'impression jet d'encre
EP2253665A1 (fr) * 2009-04-22 2010-11-24 Dr. Suwelack Skin & Health Care AG Composition lyophilisée

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101556265A (zh) * 2009-05-18 2009-10-14 杨德成 一种湿度指示剂及其制造方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4175207A (en) * 1975-02-07 1979-11-20 Elliott Stanley B Visual-type hygrometer
US4990284A (en) * 1989-10-11 1991-02-05 Brown & Williamson Tobacco Corporation Moisture indicating ink and package having same
WO1992002804A1 (fr) 1990-07-31 1992-02-20 Dansk Teknologisk Institut Indicateur d'humidite avec detecteur enveloppe
DE19508029A1 (de) * 1995-03-07 1996-09-12 Richard Prof Dr Sueverkruep Darreichungsform zur äußerlichen Applikation von ophthalmischen Arzneistoffen und Verfahren zur Herstellung derselben
US6665315B1 (en) 1998-11-27 2003-12-16 Oki Electric Industry Co., Ltd. Transmission apparatus automatically acquiring identifying information and independently measuring propagation delay
US6655315B1 (en) 1999-07-30 2003-12-02 Levosil S.P.A. Moisture indicators for the absorbent capacity of a desiccant
WO2007006682A1 (fr) * 2005-07-14 2007-01-18 Agfa Graphics Nv Derives phenylazo-acetoacetanilide comportant un groupe fonctionnel polymerisable et composes associes en tant que monomeres pour dispersants polymeriques de pigments pour encres d'impression jet d'encre
EP2253665A1 (fr) * 2009-04-22 2010-11-24 Dr. Suwelack Skin & Health Care AG Composition lyophilisée

Also Published As

Publication number Publication date
CN103261874A (zh) 2013-08-21
CN103261874B (zh) 2016-05-11

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