MX2013004002A - Formulation for the lyophilization of the monoclonal anti a gal antibody for being used as a diagnosis agent. - Google Patents

Abstract

The present invention describes a composition for the lyophilization of the monoclonal anti a-Gal antibody, containing a polyol, a polymer, albumin as a cryoprotector, potassium chloride and a carbohydrate, using as a base a buffer of phosphates and proClin 300 as preservers. The present invention also describes the methodology for carrying out the composition of lyophilized anti a-Gal antibody. According to the present invention, the preparation is stable at room temperature where the formation of aggregates and the denaturalization of the monoclonal antibody are prevented during the shelf life of the product. The preparation resulting from the present invention may be used for the formulation of the anti a-Gal antibody for being used as a diagnosis agent in in-vitro testing.

Description

FORMULATION FOR THE LIOFILIZATION OF THE MONOCLONAL ANTIBODY ANTI a-GAL FOR USE AS A DIAGNOSTIC AGENT FIELD OF THE INVENTION The present invention is directed to the preparation of a composition for lyophilization of the anti-a-Gal monoclonal antibody. The lyophilized anti-Gal monoclonal antibody is used as a diagnostic reagent in in vitro assays.

BACKGROUND OF THE INVENTION A monoclonal antibody is a globulin (protein) that reacts specifically with an epitope. They are essential tools in; the clinical and biotechnological field, and have proven useful in the diagnosis and treatment of diseases, as well as in the study of pathogen-host interactions and the labeling, detection and quantification of various molecules.

The lyophilization process is widely used in the pharmaceutical industry to improve the stability and storage time (expiration) of thermolabile compounds, especially proteins for therapeutic and diagnostic uses. The lyophilized preparations not only have the advantage of greater stability, they also provide a better and easier handling, both for transport purposes and for storage purposes of a commercial product. The stability of a compound during the lyophilization process and its subsequent storage depend to a large extent on the formulation of the preparation. The lyophilization process consists of two broad steps: on the one hand the freezing of the preparation and on the other hand, the drying of the frozen solid, by means of a vacuum sublimation process.

The critical properties of a formulation include the collapse temperature, the stability of the compound and the properties of the excipients used. In the formulations of lyophilized preparations, the elimination of water minimizes the formation of degradation products, for example deamination or degradation by hydrolysis, as well as the formation of aggregates. (Andya et al, AAPS PharmSCi 2003; 5 (2), article 10) However, water plays a critical role in the formation of the native structure of the protein, which could represent a serious problem in the lyophilization process of a monoclonal antibody. In an aqueous environment, the hydrophobic interaction and hydrogen bonding forces determine the folding of the protein. The dominant force is the hydrophobic effect, because it is the factor that influences that during the folding, the non-polar amino acids are directed towards the center of the molecule to avoid contact with the solvent and vice versa, the residues that contain the polar structures are predominantly exposed to the surface. The hydrogen bonds formed between the water molecules and the polar parts of the molecule contribute to the stabilization of the tertiary structure of the protein. (Ohtake et al., Advanced Drug delivery reviews, vol 63, issue 13, Oct 2011, pp1053-1073), (Chang et al, Journal of Pharmaceutical sciences vol 98 No. 9, 2009, pp 2886-2908.

On the other hand, another drawback is that proteins are particularly : sensitive to the stress generated by a lyophilization process and are easily degraded during the process. (Dráber et al., Journal of immunological Methods 181 (1995) 37-43), (Wang et al. Journal of Pharmaceutical Sciences, vol. 96, No. 1, 2007). The low temperature at which the process is carried out is not a guarantee of the stability of the final product because many proteins undergo cold denaturation (cold denaturation) or a denaturation at the interface (protein-air and protein-ice).

The formulation of freeze-dried preparations with carbohydrates as excipients allows the formation of amorphous and high viscosity environments under dehydration conditions, forming what is known as a matrix 'i vitrea. (Kaushik et al., Journal of Biological Chemistry, vol 278, Issue of July 18 pp26458-26465,2003) The suspension of the protein in this vitreous form reduces the protein-protein contact that can lead to aggregation processes. In addition, carbohydrates contain multiple hydroxyl groups that can form hydrogen bonds with the polar groups present on the surface of the protein, similar to those formed with water molecules, which facilitates the preservation of the tertiary structure of said macromolecules. (Chang et al, Journal of Pharnaceutical sciences vol 98 No. 9, 2009, pp 2886-2908.

; ' On the other hand, the use of any carbohydrate during a lyophilization process is not possible, since some of them generate Maillard reactions, also called non-enzymatic glycosylation of the proteins. During these reactions, a carbohydrate of the so-called "reducing sugar" reacts with protein residues containing lysine, arginine, asparagine and glutamine, resulting in a condensation reaction between the carbonyl group of the carbohydrate and the amino group of the aforementioned amino acids. previously, resulting in a Shiff base and a water molecule. This causes the vitreous transition temperature (Tg) to be reduced and may lead to the formation of aggregates and the appearance of compounds that can provide a brown color to the final product, with the consequent loss of activity (Chang et al, Journal of Pharmaceutical sciences vol 98 No. 9, 2009, pp 2886-2908). There is also another series of excipients that, in isolation, contribute little to the stabilization of a protein during the lyophilization process, but in the right proportions can provide a stability to the preparation that allows it even be stored at room temperature. Examples of these are polyols such as sorbitol and polymers such as polyethylene glycol (PEG), which interact with proteins and the environment. Therefore, the presence of salts and a buffered solution that allows the regulation of the pH is also necessary. (Wang et al Journal of pharmaceutical Sciences, vpl96, No. 1, 2007).

Once a formulation has been developed that allows the protein of interest to successfully undergo a lyophilization process, different tests of its stability are performed to check its behavior under different environmental conditions, since this type of product is routinely used. they should be stored at low temperatures to preserve their immunogenic properties, (Dráber et al., Journal of immunological Methods 181 (1995) 37-43), (Kaushik et al., Journal of Biological Chemistry, Vol 278, issue of july 18, pp26458- 26465, 2003), however, the conservation of these products in cold chain can represent serious problems both for their transportation and for their use in areas where the required refrigeration systems are not available, therefore, a preparation that allows transportation and storage for a certain time of the freeze-dried product without losing its characteristics allows a better handling and conservation of the product.

; OBJECT OF THE INVENTION It is an object of the present invention to provide a composition for the preparation of lyophilized anti-Gal antibody. Said composition minimizes the stress of the molecule during the lyophilization process, as well as decreases the rate of degradation, in order to provide the product with an adequate shelf life.

A second object is to provide a methodology for the preparation of the composition of the lyophilized anti-Gal antibody, from the formation of a system formed by a carbohydrate, a polymer and a polyol that together form a matrix that allows the stable preservation to ambient temperature of said macromolecule.

A third object is to provide a composition of the monoclonal anti-Gal antibody useful as a diagnostic reagent in in vitro assays.

BRIEF DESCRIPTION OF THE DRAWINGS Figure No.1 Structure of the trehalose Figure No. 2 Phase diagram of trehalose Figure No. 3 Vitreous transition temperature of trehalose Figure No. 4 Comparison of the stability of the lyophilisate at different storage temperatures. (20 ° C and 4-8 ° C) Figure No. 5 Stability study of the lyophilized preparation at 50 ° C.

Figure No. 6. Activity / storage time ratio at 50 ° C Figure No. 7 Data on the percentage of activity of reconstituted preparations stored at different temperatures.

Figure No. 8 Stability study of reconstituted preparations stored at different temperatures DESCRIPTION OF THE INVENTION | The present invention describes the methodology for carrying out a lyophilized preparation, having the anti-a-Gal antibody as the main ingredient. The formulation of the present invention retains the activity and prevents two different excipients from interfering with the characteristics of the final test, such as sensitivity, specificity and reproducibility, mainly when the final use of the antibody is as a diagnostic agent.

Based on the foregoing, a composition has been developed that forms a stable lyophilisate through the formation of a matrix. The components are 1) a monoclonal antibody, 2) trehalose as the first stabilizer and matrix shaper, 3) sorbitol as the second stabilizer, 4) bovine serum albumin as the first I í cryoprotectant, 5) polyethylene glycol (PEG) 8000 as the second cryoprotectant and matrix shaper 6) Potassium Chloride 7) a buffer to adjust the pH value and finally 8) Proclin 300 as a preservative.

Monoclonal antibodies commonly undergo aggregation in solution when they are subjected to storage at 4 ° C for long periods and many are denatured by lyophilization processes. The antibody to lyophilize can first be contained in a phosphate buffer solution (without sodium chloride) or in a supernatant obtained from the cell culture of the hybridoma producer, always in the smallest possible volume, at most in a proportion of 1% with respect to the volume end of the preparation to lyophilize. The concentration of the monoclonal antibody i It can be variable up to a maximum of 30pg / ml in the final solution.

The use of the phosphate buffer as the base of the composition makes it possible to avoid formation of aggregates at the time of reconstitution. The molecular structure of trehalose (FIG. 1) inhibits the process of protein aggregation and unfolding. This effect is produced by producing a crystalline matrix that restricts the mobility of the antibody and also acts as a substitute for water molecules, forming with the macromolecule (antibody) hydrogen bonds that allow the folding (tertiary structure) of said molecule to be conserved . Trehalose is highly soluble, non-reducing and non-hygroscopic. In the phase diagram of the trehalose (Figure No. 2), it can be observed that as the concentration of the same increases (in this case due to the effect of sublimation that is part of the process of 1 lyophilization), the vitreous body is formed. Albumin is used as a cryoprotectant during the lyophilization process and to prevent the adsorption of proteins to the walls of the container. It contributes to increase the solubility of the antibody in solution. Albumin contains 7 disulfide bridges, which contribute to form a structure and to carry out a process of spherical impediment between protein-protein interactions. In addition, it helps to increase the viscosity of the solution by limiting the movement of the antibody.

The addition of sorbitol in the presence of trehalose results in an increase in the stability of the monoclonal antibody by molecularly carrying out the formation of crystals and thermodynamically an increase in the vitreous transition temperature (Figure No. 3). i i Potassium chloride potentiates the formation of the polymer solution, as well as helping to prevent the formation of aggregates. It allows the vitreous body to be formed stably during the freezing process and contributes to the protection of the antibody during the reconstitution process. In addition to the formation of the polymer solution, the PEG 8000 is a cryoprotectant that helps to preserve the secondary structure. It works as a co-solvent and helps the formation of the tablet during the process of sublimation, resulting in a complete pill. It works as a stabilizer and inhibits possible antibody interactions • I with the buffer components. PEG 8000 also contributes to the solubilization of both bovine serum albumin and monoclonal antibody.

The Ploclin 300 is a condom with biocidal components used for the control of microorganisms in reagents and diagnostic agents that are used in vitro form. It has a broad spectrum, compatibility with the components of the formula, stability and low toxicity, thus increasing the shelf life and product. This condom does not affect the functionality of the antibody and does not interfere with the colorful reactions of the different enzymes that could be used for the development of an analysis. This type of preservatives are added mainly for the preservation of the product once it has been reconstituted according to the instructions for use and in the period of life stipulated for said product.

The process of preparing the composition comprises the following steps: first, a base buffer solution is prepared with potassium monobasic phosphate and potassium di basic phosphate, both at a concentration of 100 mM, adjusting a pH of 7.2. The pH value is adjusted by the addition of sodium hydroxide or hydrochloric acid solutions. In the base solution, the dissolution of sorbitol and trehalose is carried out together with the monoclonal antibody and the bovine serum albumin. The dissolution process is carried out at a temperature of 15-20 ° C, with a slow dissolution. The order of addition of these components is important. As a first step, all sorbitol must be dissolved slowly and in half of the expected volume (the concentration should be between 16 and 20%, by weight of the total volume of the formulation), preferably between 17 and 19% by weight of the total volume of the formulation) and half of the amount of trehalose to be dissolved (from 4% to 6% by weight of the total volume of the formulation, preferably between 4.5-5.5%, by weight of the total volume of the formulation). Once these reagents have dissolved, the monoclonal antibody must be added adjusted to the required concentration and previously dissolved or conditioned in the same phosphate buffer, (taking care that this operation is carried out in the smallest possible volume) and just afterwards, add bovine serum albumin (0.03 to 0.07% by weight of the total volume of the formulation, preferably 0.04 to 0.06% by weight of the total volume of the formulation). Stir until total dissolution. This solution should be left at rest at least 2 minutes After said interval of time, the rest of the trehalose should be added and a slow dissolution should be performed. The final solution is stirred until the formation of a crystalline solution and then left to stand for 2 more minutes. Once the above time has elapsed, PEG 8000 is added, the concentration of the PEG 8000 is between 0.5% and 1% by weight of the total volume of the formulation, preferably between 0.7 and 0.9% by weight of the total volume of the formulation and in an intercalated manner, potassium chloride is added in the same manner, at a final concentration of between 0.1 to 0.5% by weight of the total volume of the formulation, preferably between 0.2 to 0.3% by weight of the total volume of the formulation. The stirring at this point is carried out very slowly as well as the addition of the reagents. The sorbitol in the presence of the PEG 8000 forms a liquid polymeric solution (gel), which will be noticed when observing a change in the viscosity of the solution, but said solution should still be fluid.

Once the total dissolution of the components has been carried out, Proclin is added 300, at a final concentration of between 0.07 to 1.3% Volume / total volume of the solution, preferably between 0.09 and 1.1%, Volume / total volume of the solution adjust the pH of the solution in a range of between 7.2 to 7.4 and finally, to estimate the solution to the final volume that has been previously determined using the phosphate buffer (the preparation can be applied to any total volume, while the proportions of the components are saved). When finished, let the solution stand for 10 seconds. minutes or until the presence disappears; of bubbles in the solution. The solution already prepared should remain at rest with the aim that the formation of the cross-links is completed and the gel formed acquires the final consistency. From this moment, the preparation must be protected from light. The resulting solution is fractionated and placed in amber flasks, which are frozen at -70 ° C. When reaching -70 ° C, the viscosity of the solution is much larger and the vitreous state of trehalose is potentiated. These jars, once frozen, are ready for the lyophilization process.

The lyophilization process can be carried out at a condenser temperature of -50 ° C, for 24 hours and K mTorr of vacuum.

RESULTS The final lyophilized preparation has the appearance of a compact white tablet. The reconstitution time of the tablet is 3-5 seconds and the appearance of the resulting solution is crystalline, without the presence of turbidity or precipitation.

Lyophilization of the anti-alpha-gal antibody The anti-a-Gal monoclonal antibody was produced through the culture of its producing hybridoma and then conditioned in phosphate buffer (100mM) and carried to a concentration of 1.43 mg with a final volume of 130 ml already formulated according to the present invention. Once the preparation was ready, it was fractionated in amber glass jars of type 1 borosilicate glass of 1 ml capacity and lyophilized for 24 hours under the aforementioned conditions. An immediate analysis after the lyophilization process, using a colorful test, verifies that the i Monoclonal antibody has not lost avidity for its antigen and that the formulation does not interfere with the performance of an immunological assay.

Stability y In order to make a comparative analysis and measure the efficiency of the lyophilization process, a series of samples of lyophilized preparations were stored under defined temperature conditions: -20 ° C, 4 ° C, 20 ° C and 50 ° C, the latter as extreme temperature. Every so often, samples of the lyophilized preparations that were stored in each condition, were reconstituted with ultra pure water to determine by means of an immunological assay (ELISA) the presence and activity of the antibody. The method of evaluation is analogous to the method in which said monoclonal antibody will be used as a diagnostic reagent.

Monoclonal antibodies are thermolabile substances, that is to say, at temperatures above 30 ° C they suffer a degradation or denaturation process. The denaturation by heat is accompanied by a decrease in solubility, segregation of protein molecules, loss of activity, loss of affinity for their antigen, among others and therefore are routinely stored at refrigeration temperatures (4 to 8 ° C) in order to minimize these effects. Although a temperature of 20 ° C may not cause an immediate denaturation process, the exposure time of the molecule may cause a gradual degradation process until the total loss of activity, resulting in a downward tendency of the activity of the molecule. antibody over the storage time. Therefore, the fact of conducting a y of the effect of a 1 temperature of 50 ° C allows to verify the behavior of the lyophilized preparation in i critical conditions.

By monitoring this y for 6 months, we observed a sustained activity at different storage temperatures (Figure No. 4). The initial activity of the composition is 1.5 units and throughout the y an average loss of activity is obtained for the samples stored at 20 ° C of practically zero and 26% for the samples stored in refrigeration (2 to 8 ° C). The variations in the different measurements observed in figure 4 are attributed to the method, however all the readings are within statistical control, since they are within two standard deviations with respect to the general average of the same. It is marked inside the same figure the detection limit of the method (0.5 units).

In the case of a critical temperature (50 ° C), a prevalence of the activity is observed up to a period of 14 days, retaining a remaining activity at the end of said period. (Figure 5). Figure 6 shows the loss of activity with respect to the storage time at this same temperature, observing that at 30 days only retains 3% of the initial activity.

In none of the cases was precipitation or turbidity found when the preparations were reconstituted.

Verifying the behavior of the preparation in! Freezing conditions, a study was carried out storing freeze-dried preparations at -20 ° C. In the results of this study it is observed that during 14 days of storage, practically the activity is conserved at 100%.

Reconstitution study In order to verify the preservation that the formulation confers to the monoclonal antibody when the tablet formed is again liquid, a series of lyophilized preparations were reconstituted, which were placed at 4 ° C (refrigeration) and at -20 ° C (freezing), Figures 7 and 8. In these figures it can be seen that at the end of the study (30 days of storage in the respective conditions), for the temperature of -20 ° C there is an activity of 91.7% with respect to the initial activity, while in the case of the reconstituted and stored compounds from 2 to 8 ° C, the reconstituted preparation conserves 58.3% of its activity with respect to the initial activity, for which it is concluded that the components of the formulation provide adequate protection, still reconstituted . It should be noted that normally an antibody stored in refrigeration (2 to 8 eC) retains its activity for an average of 7 days.

Claims (19)

1. - Formulation for lyophilization of the anti-GAL monoclonal antibody for use as a diagnostic agent, characterized in that it comprises: to. Anti-GAL monoclonal antibody; b. Trehalose; c. Sorbitol; d. Bovine serum albumin; and. Polyethylene glycol (PEG) 8000; F. Potassium chloride; g. Proclin 300; Y h. A pH buffer solution

2. - The formulation according to claim 1 characterized in that it contains a maximum of 30pg / ml of anti-GAL monoclonal antibody; about 5% by weight of trehalose, about 18% by weight of sorbitol, about 0.05% by weight of serum albumin, about 0.8% by weight of polyethylene glycol (PEG) 8000, about 0.3% by weight of potassium chloride and about 0.1% by weight of Proclin 300, all of the above having as a diluent 100mM phosphate buffer

3. - The formulation according to claim 1, characterized in that the pH buffer is a phosphate buffer and the pH is between 7.2 and 7.4.

4. The formulation according to claim 1, characterized in that the anti-a-Gal monoclonal antibody can be contained in a phosphate buffer solution or in a supernatant obtained from the cell culture of the producing hybridoma.

5. - A process for the preparation of the formulation according to claim 1, characterized said process because it comprises the following steps: to. Prepare a base buffer solution with potassium monobasic phosphate and di-basic potassium phosphate, at a concentration of 100 mM, adjusting the pH to 7.2; b. Dissolve slowly at a temperature between 15 and 20 ° C, the total amount of sorbitol and 50% of the total amount of trehalose; in half the volume planned for the final solution. c. Once these reagents have dissolved, add the adjusted monoclonal antibody to the required concentration; and immediately add the bovine serum albumin and stir until total dissolution; d. Leave the solution at rest for at least 2 minutes, after said interval of time, add the rest of the trehalose and dissolve slowly and. Stir until the formation of a crystalline solution and then let it rest for 2 more minutes F. After the time, add to the previous solution PEG 8000, and so i; intercalated, potassium chloride is added. Add Proclin 300 and adjust the pH of the solution in a range of between 7.2 to 7.4 g. Emphasize the solution to the final volume that has been determined with the buffer Í phosphates h. At the end, let the solution stand for 10 minutes or until the presence of bubbles disappears in the solution, protect from light and place in amber bottles, which are frozen at -70 ° C.

6. - The process according to claim 5, characterized in that in step b) the order of addition comprises; first dissolve slowly and in half the volume of the base solution, the sorbitol, whose concentration is between 16 to 20% by weight of the total volume of the formulation, preferably between 17 and 19%; then dissolve half of the total amount of trehalose, at a concentration of 4% to 6%, preferably between 4.5-5.5% by weight of the total volume of the formulation.

7. The process according to claim 5, characterized in that in step c) the bovine serum albumin is present in 0.03 to 0.07% by weight of the total volume of the formulation, preferably between 0.04 to 0.06% by weight of the total volume of the formulation. i the formulation.

8. The process according to claim 5, characterized in that in step f) where the concentration of the PEG 8000 is between 0.5% and 1%, by weight of the total volume of the formulation preferably between 0.7 and 0.9% by weight of the total volume of the formulation.

9. The process according to claim 5, characterized in that in step f) the concentration of potassium chloride is between 0.1 to 0.5% by weight of the total volume of the formulation, preferably between 0.2 to 0.3% by weight of the total volume of the formulation.

10. The process according to claim 5, characterized in that once the r i total dissolution of the components in step f), Proclin 300 is added, at a final concentration of between 0.07 to 1.3% by weight of the total volume of the formulation, preferably between 0.09 and 1.1% by weight of the total volume of the formulation ,

The process according to claim 5, characterized in that in step f) the stirring is carried out very slowly as well as the addition of the reactants.

12. The process according to claim 5, characterized in that the mixture of sorbitol and PEG 8000 forms a liquid polymer solution (gel) fluid.

13. The process according to claim 5, characterized in that the resulting product is stable at room temperature.

14. The process according to claim 5, characterized in that the final lyophilized preparation has the appearance of a compact white tablet.

15. The process according to claim 5, characterized in that: the reconstitution time of the tablet is 3-5 seconds and the appearance of the resulting solution is crystalline, without the presence of turbidity or precipitation.

16. The process according to claim 5, characterized in that the product obtained has a prevalence of activity at a critical temperature of 50 ° C for a period of 14 days, retaining a remaining activity at the end of said period.

17. The process according to claim 5, characterized in that the lyophilized preparations stored at -20 ° C, during 14 days of storage the activity is conserved up to approximately 100%.

18. The process according to claim 5, characterized in that the obtained product which is reconstituted and stored at -20 ° C, retains an activity of 91.7% with respect to the initial activity during a storage period of 30 days.

19. The process according to claim 5, characterized in that the reconstituted product preserves 58.3% of its activity with respect to the initial activity at 30 days of its reconstitution, when it is stored at temperatures between 2 and 8 ° C.