EP1215135A1 - Bottle for photosensitive dyes - Google Patents

Abstract

Package that comprises a light-sensitive fluorochrome (I) is a bottle, at least the sides of which form a screen against light of wavelength 200-900 nm.

Description

The present invention relates to new packaging for photosensitive dyes and a new method of packaging fluorochromatic reagents for cytometry.

Flow cytometry is a very specific cell analysis technique used for decades. This technique involves at least one fluorochrome.

It is a powerful tool for cellular antigen analysis. This technique allows to analyze several antigenic targets at the same time thanks to combinations of antibodies conjugated to fluorochromes. The main fluorochromes used in cytometry are fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), PercP, tandems phycoerythrin-cyanine 5 (PC5), phycoerythrin-cyanine 7 (PC7) and phycoerythrin-texas red (ECD or PETR). These fluorochromes absorb light radiation particularly at 488 nm and 633 nm which are the main emission wavelengths of the lasers used on the cytometers. The tandems PC5, PC7, ECD use the principle of transfer of energy, i.e. the laser of the cytometer excites the molecule in the area spectral of phycoerythrin which restores the energy absorbed to the molecule acceptor (cyanine 5, cyanine 7 or Texas red) which will return it itself in radiative form at its emission wavelength.

Tandem dyes nevertheless absorb light throughout spectral zone and particularly in the visible zone (400-800 nm) and the Applicant has realized that these fluorochromes, alone or in tandem, were subject to significant degradation during their conservation.

• Apparition de cellules faussement positives
• Nécessité d'augmenter les compensations afin de retrouver les valeurs d'intensité de fluorescence initiales.

These degradations induce a deterioration in the efficiency of the energy transfer which leads to an increase in the fluorescence intensity of phycoerythrin at 580 nm. This phenomenon is expressed in cytometry by an increase in fluorescence (called leakage) in the phycoerythrin channel (FL2) which can lead to various problems:

• Appearance of false positive cells
• Need to increase the compensations in order to find the initial fluorescence intensity values.

The last point is particularly crucial in the case of automatic compensation computer systems.

The tandem phycoerythrin-allophycocyanin has been described for some time 20 years (Glazer et al. Biophys. J. (1983) 83, 383-386 and the tandems phycoerythrin-cyanine 5 for almost 10 years Lanier et al. Methods (1991) Vol 2 N ° 3 192-199. Since then, this kind of product has been marketed in bottles of brown or amber glass by BECTON DICKINSON Companies, PHARMINGEN, DAKO, IQP, or CALTAG.

It would therefore be desirable to have a packaging of fluorochromes ensuring the conservation of said fluorochromes during a extended duration.

After a number of tests, the applicant hypothesized that these fluorochromes could be sensitive to visible light. She then covered the side walls of the fluorochromes bottles with a sheath heat shrinkable absorbing visible light. A very noticeable improvement has then observed, however not yet giving complete satisfaction. She has then carried out other tests which did not bring any further progress.

While continuing her research, the plaintiff discovered with astonishment that the fact that the bottom of the sheathed bottles is not protected sufficient to degrade fluorochromes, when the bottles were handled or overturned flat on their side instead of being placed on their bottom. She then understood that these fluorochromes were very sensitive to wavelengths ranging from 200 to 900 nanometers, especially in visible light, particularly at wavelengths ranging from 400 to 700 nanometers and particularly at wavelengths ranging from 400 to 600 nanometers.

• par transfert de l'énergie lumineuse absorbée avec génération d'oxygène singulet (mécanisme dit de type II),
• par transfert d'électron à l'état excité avec génération de radicaux oxygénés (mécanisme dit de type I).

According to the applicant, the degradation of the tandems originates from a photo-induced oxidation phenomenon in which the molecules play the role of photo-sensitizer, that is to say that they are capable of activating oxygen:

• by transfer of the absorbed light energy with generation of singlet oxygen (so-called type II mechanism),
• by electron transfer in the excited state with generation of oxygen radicals (so-called type I mechanism).

The invention is based on the use of bottles opaque to radiation bright tandem exciters, i.e. between 200 and 900 nm and more particularly between 400 and 800 nm.

This is why the present request relates to packaging. comprising a fluorochrome sensitive to wavelengths ranging from 200 to 900 nanometers and particularly at wavelengths ranging from 400 to 600 nanometers placed in a bottle of which at least the side walls form a screen effective at radiations of the light spectrum between 200 and 900 nm, particularly at wavelengths from 400 to 800 nanometers, more particularly from 400 to 700 nanometers and particularly between 400 and 600 nm.

In the present application and in the following, the terms "Fluorochrome sensitive to visible light" means a fluorochrome whose structure is degraded by emission at the indicated wavelengths, by example fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), PercP, phycoerythrin-cyanine tandems 7 (PC7), phycoerythrin-cyanine 5 (PC5) and phycoerythrin-texas red (ECD or PETR), preferably the latter two. The terms "form an effective screen radiation "means that at least 95%, preferably at least 98%, particularly at least 99% and especially 100% of radiation from visible are stopped by the side walls of the bottle due to their structure (nature, treatment or sheathing in particular). The term "bottle" refers to preferably a small bottle preferably with a screw, fitted with a cap with or without septum. The bottle can be made of polyethylene, polypropylene, polycarbonate, but preferably glass. Its capacity can preferably range from 50 µl to 50 ml, especially from 100 µl to 20 ml, especially from 500 µl to 10 ml and all especially from 1 ml to 10 ml. For example, a 5 ml bottle can be filled per 1 to 2 ml of solution.

Particularly preferred are glass vials of about 5 ml.

Under preferential conditions for implementing the invention, the bottom of the bottle also forms an effective radiation shield visible.

Sheathing techniques are already known in perfumery or coating of vials forming an effective shield against spectrum radiation visible, used for aesthetic purposes.

Examples include powder coating electrostatic which consists in surrounding the bottles with a septum which is pierced by an electrode which is installed in the bottle to be covered. The vials are placed in an enclosure containing a powder sprayed, charged with a sign opposite to that of the bottle. The powder is deposited then on the bottle on which it is fixed for example by cooking in an oven at around 180 ° C. In the context of the invention, it is possible, for example, to use a epoxy resin powder preferably absorbing in the wavelengths above indicated, particularly 600 to 800 nanometers, black or blue in particular.

You can also sheath the bottle with a sheath advantageously heat shrink preferably also covering the bottom of the bottle.

We can still irradiate certain types of glasses by radiation gamma.

It is just as possible to make a molding using a film plastic, which has the advantage of being able to cover the bottom of the bottles.

The packaging object of the present invention have very interesting properties. The use of covered or treated bottles as described above eliminates the photoinduced oxidation phenomenon and provides excellent stability to fluorochromes-antibody conjugates or Tandem-antibodies.

These properties are illustrated below in the experimental part.

The present invention also applies to other conjugates antibody-tandems than those mentioned above, as well as all fluorochromes used in flow cytometry.

The present application also relates to a method for protecting fluorochromes sensitive to visible light and particularly to lengths wave ranging from 400 to 600 nanometers during their conservation, in which installs said fluorochromes in a bottle with at least the side walls have a structure that forms an effective screen for radiation from the light spectrum between 200 and 800 nm, particularly at wavelengths ranging from 400 to 800 nanometers, more particularly from 400 to 700 nanometers and particularly between 400 and 600 nm.

Preferential conditions for the implementation of packaging described above also apply to the other objects of the invention referred to above, in particular to the process for protecting fluorochromes.

• les figures 1A, B, C, D et E représentent les résultats de fluorescence obtenus avec des échantillons de conjugués Anticorps anti-CD3-Phycoérythrine-cyanine 5 contenus dans leur flacon d'origine ou d'un flacon selon l'invention, et soumis à l'action de la lumière, et
• les figures 2A, B, C et D représentent les résultats de fluorescence obtenus avec des échantillons de conjugués Anticorps anti-CD19-Phycoérythrine-texas red contenus dans leur flacon d'origine ou d'un flacon selon l'invention, et soumis à l'action de la lumière.

The following examples illustrate the present application.

• FIGS. 1A, B, C, D and E represent the fluorescence results obtained with samples of anti-CD3-Phycoerythrin-cyanine antibody conjugates contained in their original bottle or in a bottle according to the invention, and subjected to the action of light, and
• FIGS. 2A, B, C and D represent the fluorescence results obtained with samples of anti-CD19-Phycoerythrin-texas red antibody conjugates contained in their original bottle or in a bottle according to the invention, and subjected to l action of light.

• les figures 1A et 2A concernent un conjugué protégé de la lumière, servant de témoin,
• les figures 1B et 2B concernent un conjugué conditionné dans un flacon de verre commercial BeckmanCoulter Immunotech de la gamme IOTEST,
• les figures 1C et 2C concernent un conjugué conditionné dans un flacon de verre commercial BeckmanCoulter Immunotech de la gamme Cytostat,
• la figure 1D concerne un conjugué conditionné dans un flacon de verre commercial BECTON DICKINSON Pharmingen, et
• les figures 1E et 2D concernent un conjugué conditionné dans un flacon de verre commercial BeckmanCoulter Immunotech de la gamme IOTEST recouvert d'une couche de vernis époxy selon le procédé de poudrage électrostatique de l'exemple 1.

More precisely,

• FIGS. 1A and 2A relate to a conjugate protected from light, serving as a control,
• FIGS. 1B and 2B relate to a conjugate packaged in a BeckmanCoulter Immunotech commercial glass bottle from the IOTEST range,
• FIGS. 1C and 2C relate to a conjugate packaged in a BeckmanCoulter Immunotech commercial glass bottle from the Cytostat range,
• FIG. 1D relates to a conjugate packaged in a BECTON DICKINSON Pharmingen commercial glass bottle, and
• FIGS. 1E and 2D relate to a conjugate packaged in a BeckmanCoulter Immunotech commercial glass bottle from the IOTEST range covered with a layer of epoxy varnish according to the electrostatic powdering process of Example 1.

Example 1 : Coating of a glass bottle with epoxy resin

On lave les flacons.

On les passe dans un four de séchage.

On les passe ensuite dans une cabine dans laquelle les flacons sont recouverts d'un septum transpercé par une électrode.

La poudre de résine époxy Noir mat AKZO référence NOIR MAT 1P200 8391 (nouvelle référence AN052F) de charge opposée à celle de l'électrode pulvérisée dans la cabine s'applique par phénomène électrostatique sur la surface des flacons que l'on fait alors passer dans un four de cuisson à 180°C.

Commercial glass bottles are placed intended to receive anti-CD3-Phycoerythrin-cyanine 5 antibody conjugates from BeckmanCoulter Immunotech of the IOTEST range in a chain implementing the following steps:

We wash the bottles.

They are passed through a drying oven.

They are then passed through a cabin in which the bottles are covered with a septum pierced by an electrode.

AKZO matt black epoxy resin powder reference BLACK MAT 1P200 8391 (new reference AN052F) with a charge opposite to that of the electrode sprayed in the booth is applied by electrostatic phenomenon to the surface of the bottles which are then passed through a baking oven at 180 ° C.

EXAMPLE 2 Coating of a Glass Vial Using Polyethylene Heat Shrink Tubing

We covered the sides and the neck with 6ml commercial bottles in glass intended to receive anti-CD3-Phycoerythrin-cyanine antibody conjugates 5 of Beckman Coulter Immunotech from the IOTEST range using polyethylene heat-shrink tubing sold by the company SLEEVER INTERNATIONAL.

Example 3 : Manufacture of a glass vial covered with epoxy resin filled with a solution of anti-CD3-Phycoerythrin-cyanine antibody conjugates in PBS, BSA 2 mg / ml, sodium azide 0.1%.

Vials made according to Example 1 were filled with each 2 ml solution of anti-CD3-Phycoerythrin-cyanine antibody conjugates 5 in phosphate buffer / bovine serum albumin (2 mg / ml) / azide 0.1% sodium.

Example 4 : Preservation test of anti-CD3-Phycoerythrin-cyanine antibody conjugates 5

In order to demonstrate the effectiveness of the process of the invention, flasks commercial and modified products containing anti-CD3 antibodies conjugated to PC5 and ECD fluorochromes underwent a forced degradation test according to the method described in good manufacturing practices (Good Manufacturing Practice (GMP)) of the US Food and Drug Administration.

• Flacon commercial BeckmanCoulter Immunotech de la gamme IOTEST
• Flacon commercial BeckmanCoulter Immunotech de la gamme Cytostat
• Flacon commercial BeckmanCoulter Immunotech de la gamme IOTEST recouvert d'une couche de vernis époxy selon le procédé de poudrage électrostatique
• Flacon commercial BECTON DICKINSON Pharmingen.

Different packaging has been tested:

• BeckmanCoulter Immunotech commercial bottle from the IOTEST range
• BeckmanCoulter Immunotech commercial bottle from the Cytostat range
• BeckmanCoulter Immunotech commercial bottle from the IOTEST range covered with a layer of epoxy varnish using the electrostatic powdering process
• BECTON DICKINSON Pharmingen commercial bottle.

These bottles containing the antibody-tandem conjugates were introduced into a chamber equipped with fluorescent tubes and were subjected to a total light dose of 1.2 × 10 ⁶ lux.h.

Figure 1 shows the excellent protection of the contents of the vials in implementing the invention. Indeed the tandems introduced in the bottle BeckmanCoulter Immunotech from the IOTEST range covered with a layer of epoxy varnish according to the electrostatic powdering process have a autofluorescence in FL2 identical to that of the non-irradiated control. On the other hand all the other types of bottles show a light permeability which is translated by a "leak" in FL2, ie an increase in autofluorescence with the appearance of false positive cells.

EXAMPLE 5 Preservation Test for Anti-CD19-phycoerythrin-texas Red Antibody Conjugates

We operated as in Example 4, but with conjugates Anti-CD19-phycoerythrin-texas red antibodies. The results are given to the figure 2.

Analysis of the results leads to the same conclusions as Example 4.

Claims (7)

A package comprising a set consisting of a fluorochrome sensitive to visible light and a bottle in which it is placed, at least the side walls of said bottle forming an effective radiation shield of the light spectrum between 200 and 900 nm. A package according to claim 1, characterized in that the bottom of the bottle also forms an effective screen for radiation from the visible spectrum. A packaging according to claim 1 or 2, characterized in that the effective radiation shielding effect is obtained by coating by electrostatic powdering, by sheathing the bottle of a sheath, by irradiation of certain types of glasses by gamma radiation, or by overmolding. A packaging according to one of claims 1 to 3, characterized in that the fluorochrome is chosen from fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), PercP, or is one of the phycoerythrin tandems -cyanine 7 (PC7), phycoerythrin-cyanine 5 (PC5) and phycoerythrin-texas red (ECD or PETR). A package according to one of claims 1 to 4, characterized in that the side walls of the bottle have a structure which stops at least 95% of visible radiation. A process for the protection of light-sensitive fluorochromes visible during storage, in which said fluorochromes are installed in a bottle of which at least the side walls form an effective shield against radiations of the light spectrum between 200 and 900 nm. A method according to claim 6, characterized in that the side walls of the bottle have a structure which stops at least 95% of visible radiation.

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