WO2012062841A1 - Humidity indicator - Google Patents

Abstract

During incubation in humid air, PAMA absorbs comparatively large quantities of water (% by solid weight). This is accompanied by a significant change in the lyophilisate from dry and cross-linked PAMA. This change manifests itself in a shrinking of the initial lyophilisate. It can be assumed that both observations (water absorption and shrinkage) are causally linked.

Description

 MOISTURE INDICATOR

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. With this method, it is possible, for example, to maintain the activity of sensitive enzymes at room temperature for a long time to use the contents of the dried sample (as Pharmakum or biological Reacti Ones, such as the PCR (Polymerase Chain Reaction (PCR)), the lyophilisate is mixed with an adequate solvent, which is usually water. This leads to an immediate re-dissolution of the dried ingredients, which then re-unfold their original effect in solution.

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.

Commonly used drying agents are silica gels, alumina or Molekularsie- be.

An example in which a color change moisture indicator can be made is disclosed in US Pat. No. 6,655,315. This US patent describes a moisture indicator comprising one with copper chloride or with one or more to release Cu ²⁺ ions capable salts is colored, addition of the humidity indicator comprises one or more metal chlorides, a disadvantage of this therapeutic system is essentially that it is a composite of several components which must be mixed prior to use of the humidity indicator only in specific ratios, in In addition, it is undesirable for some biological samples if colored moisture indicators are used, since the color of the indicator or the color change occurring can cause optical impairment of the sample. In addition, the moisture indicator described in US Pat. No. 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. In addition, 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. In addition, WO 92/02804 A does not mention that the moisture indicator is suitable at all for use in lyophilisates.

Accordingly, 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, In particular, 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,

In the context of the present invention, investigations have been carried out on different indicator systems for use in lyophilisates and it has been found that compounds are generally suitable as indicator system in lyophilisates which have a volume reduction in comparison to the substantial absence of water of at least 30%, more preferably 60% %, particularly preferably 90%, in the presence of a defined relative air humidity of the surrounding air of at least 70%, more preferably 50%, even more preferably 30%, particularly preferably 10%,

In the context of the present invention, a substantial absence of water means a relative humidity of 0%,

The problem underlying the present invention is therefore initially solved by the use of 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%,

In this case, it is particularly preferred if 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,

In addition, 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.

Thus, 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.

In a particularly preferred embodiment of the present invention, 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.

Accordingly, 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%. In the context of the present invention, a moisture indicator is understood as meaning a single compound, but also a composition of several individual compounds, In particular, 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.

Since 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:

Preparing an aqueous solution comprising at least one moisture-indicating compound according to the invention;

 Introducing or applying the prepared mixture into a cavity or other surface;

 Freezing and incubating the mixture resulting from process step (ii); and

 Drying the mixture resulting from process step (iii) in a suitable vacuum, which drying phase may comprise main drying and post-drying at suitable depressions.

During the main drying, 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.

In the context of the present invention, 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,

During this drying step, 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.

In a further embodiment of the present invention, hygroscopic substances are added to the indicator solution to be lyophilized. As a result, the lyophilisate changes its volume even at lower humidities than would be the case without addition.

For water absorption and thus weight gain, essentially the absolute amount of moisture-indicating compound in the lyophilisate is crucial. For volume reduction, however, especially the percentage of moisture-indicating compound in the original solution (ie before lyophilization) is of crucial importance. That is, the higher the initial concentration in the solution before drying, the higher the humidity must be to allow the lyophilizate to take its shape changed.

Moreover, 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 keitsindizierenden 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.

As explained above, 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

 (ii) the volume of the moisture-indicating compound is observed

In the context of the present invention, 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.

The present invention will be explained in more detail with reference to the following examples,

Terms and abbreviations used in the following:

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

rel. LF = relative humidity Experiment 1: Change in the shape of freeze-dried PAMA by exposure to moisture

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 μΙ of a 0.5% or 5% PAMA solution was filled into PCR strips and freeze-dried (freezing at -80 ° C., incubation at -50 ° C. for 2 h, drying in vacuo (0, 2 mbar) at increasing temperatures (-40 ° C to 20 ° C), post-drying at 20 ° C and 0.002 mbar.) In the lid of the lyophilisate-equipped PCR tubes 1 0 μΙ water were pipetted and the tube with the appropriate lid In the case of the 0.5% PAMA lyophilisate, the cake began to "melt away" after 2 to 4 minutes. It reduced its volume and the surface looked increasingly glassy (Figure 1), the 5% - PAMA lyophilizate, however, showed no change in its morphology after 1 5 min incubation (Figure 2). The results of experiment 1 are shown in FIGS. 1 a and 1 b. 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). In FIGS. 1 a and 1 b, 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. before humidity incubation, right (Figure 1 b): wet incubation for 1 5 min), It can be seen that the lyophilisate has dissolved in tube 2 (from left) after the wet incubation, while the lyophilisate, which starting from the 5% PAMA solution was obtained, has not yet dissolved after the same wet incubation (on the right image (Figure 1 b) are the lyophilizates in the cover of Tubes 3 and 4),

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:

The following is an experiment in which 0.5% / 1% / 5% / 10% PAMA solutions were freeze-dried and then exposed to relative air humidities of 43-59%. The various PAMA cakes were prepared in PCR tubes as described above. Then, the PCR tubes were incubated in a desiccator in the presence of a CaCl ₂ solution (open, without lid) and photoprocessed at intervals. The relative humidity in the desiccator was thereby 43% (at the beginning) to 59% (at the end of the experiment ). Figure 3 shows that the 0.5% PAMA cake has already shrunk dramatically after less than 2 hours. For the same effect, the 1% approach took about 3 h, the 5% approach more than 5 h. The 10% -approach showed a completely shrunk lyophilizate after 10 days on the final photo documentation. In general, the final size of the wet PAMA cake was proportional to the amount of PAMA present,

The results are shown in FIG. 3, which shows a time series of the humidification of PAMA lyophilizates (0 hours to 10 days).

Trial 3:

The following two experiments show with the simplest means how to use the effect of "melting" PAMA on a moisture indicator.

A parafilm was described below from the English word "humid", after which a 0.5% PAMA solution was applied to the top of the parafilm and freeze-dried as described in experiment 1,

The prepared PAMA cake on parafilm was placed in a semi-open plastic box containing 4 ml of water. The changes in the PAMA cake were photographically documented over the next 1.5 h (Fig. 4). You can clearly see how the PAMA cake is almost invisible and the underlying lettering "humid" so "wet" reveals. Figure 4α shows on the left the freeze-dried PAMA cake before dampening, Figure 4b shows the PAMA cake after 45min of moist air and Figure 4c shows the PAMA cake after 1 h 45min of humid air. Trial 4:

The MTP wells were described externally with a red permanent marker, resulting in a red area. 2 MTP wells each were filled with 200 μΙ 0, 1% / 0.5% / 2% / 5% PAMA incl. 2 μΙ 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).

Trial 5:

This experiment shows that not every polymer substance shows a volume reduction on humidification. Polyacrylic acid (PAA, molecular weight 250,000), PEG3000, PEG8000 and PEG35000 (PEG = polyethylene glycol) were compared. 1 00 μΙ of these substances were, as already described several times, filled in PCR tubes and freeze-dried, then the lyophilizates were exposed to rising air humidities, Figure 6 shows how the lyophilizate cakes of the individual chemicals after 75 days incubation at 75 It can be seen clearly that the volume of the cake made of polyacrylic acid has greatly reduced, the PAA thus behaves similar to PAMA, the three PEG-cake, however, show no change and are therefore not as a basis for substance a moisture indicator in the context of the present invention suitable.

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

In order to better understand the phenomenon of extreme volume reduction of the PAMA cake in damp conditions, it was determined at which air humidity levels the morphology changes and how much water is absorbed.

Solutions with different PAMA concentrations (1-8% w / v) as well as different volumes of a 10% PAMA solution were pipetted into PCR strips and freeze-dried as described in the first experiment. The PCR tube strips were cut and the individual tubes were weighed with the lyophilizates. These were then exposed for 4 days to a relative humidity of approx. 60% at approx. 20 ° C. After this incubation time, the PCR tubes were weighed again If the quantity of water taken up (indicated by the weight difference between dry and moist state) is plotted against the initial amount of PAMA, a very clear proportionality between the amount of water absorbed and the originally dried PAMA solution volume (FIG. 7a) or , the PAMA concentration of the dried PAMA solution (Figure 7b). Both observations suggest that although the amount of water absorbed depends on the initial amount of PAMA, it does not depend on the original PAMA concentration.

Trial 7:

With this experiment, it can be clearly demonstrated that in fact only the absolute amount of PAMA present and not the concentration of the original solution determines how much water is taken up by a PAMA freeze-drying cake.

For this purpose, a) 20-100 μl of 0.1% PAMA solutions and b) 80 μl each of 1-8% PAMA solutions were filled into PCR tube strips (as triplicate) and freeze-dried as described at the beginning, The weights of the dry PAMA solutions Cakes were determined with a fine scale. Subsequently, the open tubes were exposed to increasing humidity (9%, 33%, 45%, 75%). exposed, in each case about 1 week at a certain humidity was incubated. Before each change to the next higher LF, the weight of the PAMA cake was redetermined.

From Figures 8a and 8b and tables _. 1 and 2, both approaches, both a) and b), show the same weight gain per milligram of PAMA starting weight,

Table 1: Weight gain of PAMA lyophilisates (prepared according to approach a)) when incubated at different humidities

 Mean weight gain [wt%]: 22 28 67

Table 2: Weight gain of PAMA lyophilisates (prepared according to approach b)) when incubated at different humidities

 Mean weight gain [% by weight]:

Experiment 8: In this experiment, it was determined how much water PEG lyophilizates (PEG = polyethylene glycol) take up in comparison to PAMA lyophilizates. In addition, 5 × PAMA or PEG8000 solutions in PC tubes were used for each 4x Ι ΟΟμΙ Freeze-dried (Figure 9a: 4 Tubes left with PAMA and 4 Tubes right with PEG8000), The weights of the dry substances were determined, Then the lyophilizates were one for 4 days exposed to relative humidity of 75% and weighed again. Figures 9a and 9b show photos of the approaches before and after the dampening. It can be clearly seen that the PEG8000 lyophilizates (4 tubes from the right) had no volume reduction, while the PAMA lyophilizates (4 tubes from the left) shrank very strongly (FIG. 8b). The weight determination revealed that the PAMA approaches were at the same weight of solids with 3.35 mg ca, had taken three times more water than the PEG8000 approaches (1, 05 mg). PAMA thus absorbs about 67% of its own solids weight in water (measured by several tests confirmed), while PEG8000 only 21%. Presumably, this high water absorption capacity is the reason for the changes in lyophilizate in PAMA.

Trial 9:

Solutions with different PAMA concentrations (0.01-1% w / v) were pipetted into duplicate PCR strips and lyophilized as described in the first experiment. The result is shown in Figure 1 0. The experiment showed that at least 0.25% PAMA must be in solution for freeze-drying to produce a good quality cake, Figure 10 shows freeze-dried cakes of solutions with increasing PAMA concentrations (from top left to bottom right: (2x each ) 0.01% / 0.025% / 0.05% / 0, 1% / 0.25% / 0.5% / 1%),

Trial 1 0:

Solutions with different PAMA (0.5, 1% w / v) and cesium fluoride concentrations (0/20/30 / 40mM or, 50/60/70/80 mM CsF) were pipetted into PCR strips and as described in the first experiment freeze-dried (Figure 1 la). Subsequently, the open tubes were successively exposed to rising humidities (7 days 9%, 14 days 24%, 1 day 34%), After 1 day at 34% rel, LF most approaches shrank sharply (Figure II b), you can see clearly recognize that the extent of shrinkage correlates with the concentration of CsF in the batch, FIGS. 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%. Experiment 1 1:

Solutions with different volumes (25-100 μΙ) of different PA-A concentrations (0.25-5% w / v) were freeze-dried as described in the first experiment. Figure 1a shows the batches immediately after lyophilization: first row from above: 2x each 25 μΙ 0.25%, 0.5%, 1%, 2.5% PAMA; second row from the top: 2x 25 μΙ each 5% PAMA, 2x each 50 μΙ 0.25%, 0.5%, 1% PAMA; third row from the top: 2x each 50μΙ 2.5%, 5% PAMA; 2x each Ι ΟΟμΙ 0.25%, 0.5% PAMA; bottom row: 2x each Ι ΟΟμΙ 1%, 2.5%, 5% PAMA,

 After 3 h, all lyophilizates shrank with 0.25% PAMA independently of the dried starting volume. After 1 day, all latches with less than or equal to 1% PAMA exhibited a severe shrinkage regardless of the starting volume , All 2.5% PAMA formulations shrank very little, irrespective of the starting volume. Only the 5% PAMA preparations did not show any shrinkage, again independently of the starting volume. FIG. 1b shows the same approaches as FIG. however, after the one-day incubation at 45% relative humidity,

Claims

claims 1 . A lyophilisate comprising at least one moisture indicator having a volume reduction compared to the substantial absence of water of at least 30% in the presence of a defined relative humidity of ambient air of at least 10%. 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. Lyophilisate according to claim 2, characterized in that lyophilisate comprises the sodium salt of poly (acrylic acid-co-maleic acid). Lyophilisate according to one of claims 1 to 3, characterized in that the content of moisture indicator, in each case based on the lyophilisate, 30 to 1 00 wt .-%, preferably 40 to 1 00 wt .-%, particularly preferably 50 to 1 00 wt .-%, is. Lyophilisate according to one of claims 1 to 4, characterized in that the lyophilizate is prepared according to the following procedure:  (i) preparing an aqueous solution comprising at least one moisture-indicating compound according to claim 1 or 2;  (ii) introducing or applying the prepared mixture into a cavity or other surface;  (iii) freezing and incubating the mixture resulting from step (ii); and  (iv) drying the mixture resulting from process step (iii) in a suitable vacuum, which drying phase may comprise main drying and post-drying at suitable depressions. 6. Lyophilisate according to one of claims 1 to 5, characterized in that the lyophilisate is provided in a PC vessel, in particular a PCR tube. , Arrangement comprising at least one lyophilisate according to any one of claims 1 to 6 and additionally at least one biologically active ingredient, characterized in that the lyophilisate and the at least one biologically active ingredient are spatially separated, but in contact with the same atmosphere An assembly according to claim 7, characterized in that said at least one biologically active ingredient is 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 chemical compounds, their salts and organics,, Arrangement according to claim 7 or 8, characterized in that the moisture indicator and / or the at least one biologically active ingredient is introduced separately from each other in a cavity on a PCR strip or in a cavity 10, use of at least one moisture-indicating compound which has a volume reduction of at least 30% in the presence of a defined relative air humidity of the surrounding air of at least 10% . 1 1, 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, 1 2, use according to claim 1 0 or 1 1, characterized in that the moisture indication is carried out in a lyophilisate, 1 3, A method for monitoring the moisture content of a lyophilisate, characterized in that (i) providing a lyophilisate comprising at least one moisture-indicating compound according to any one of claims 1 to 6; and (ii) the volume of the moisture-indicating compound is observed 1 4. The method of claim 1 3, characterized in that the volume of the moisture-indicating compound is visually monitored. A method according to claim 1 3 or 1 4, characterized in that the observation of the volume of the moisture-indicating compound is carried out by comparison observation with an equivalent blank,