RU2788164C2 - Process for reduction in endotoxin content in gelatin - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: group of inventions relates to the pharmaceutical industry, namely to a method for reduction in an endotoxin level in gelatin, as well as production of gelatin with a low endotoxin content, and its use. The method for reduction in an endotoxin level in gelatin includes: dissolution of salt in a gelatin solution containing gelatin and a solvent to form a gelatin-salt solution, wherein an endotoxin content in gelatin is at least 6000 EU/g, and a salt concentration in the gelatin-salt solution is 125-300 mM, and salt is sodium chloride; filtration of the gelatin-salt solution by means of an anion-exchange adsorber, as a result of which the endotoxin content in filtrate of the gelatin-salt solution is less than 2500 EU/g; and desalination of filtrate of the gelatin-salt solution to form a gelatin solution with a low endotoxin content, wherein the endotoxin content is less than 2500 EU/g. A gelatin solution with a low endotoxin content is also described. A method for the production of a lyophilized tablet for delivery of a pharmaceutically active ingredient is described. A lyophilized tablet for delivery of a pharmaceutically active ingredient is also described.

EFFECT: reduction in an endotoxin level in gelatin.

21 cl, 11 dwg, 6 tbl, 4 ex

Description

LINK TO RELATED APPLICATIONS

This application claims priority under U.S. Provisional Application No. 62/640394, filed March 8, 2018, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for reducing the content of endotoxin in gelatin. More specifically, the present invention relates to a method for reducing endotoxin in gelatin by filtering gelatin saline using anion exchange and desalting the filtered gelatin saline.

BACKGROUND OF THE INVENTION

Vaccines are usually delivered by parenteral route: intramuscular, intradermal, or subcutaneous. However, mucosal vaccine delivery (via the buccal mucosa, sublingual mucosa, nasal mucosa, oral mucosa, or vaginal mucosa) is of increasing interest as a means of inducing local and distant antibody-mediated immune responses as well as systemic immune responses. In addition, mucosal vaccine delivery via solid dosage forms (eg, buccal/sublingual tablet, oral tablet or capsules, vaginal swab) has a number of advantages, such as the possibility of mass immunization as well as independence from the cold chain. In addition, mucosal delivery of the vaccine may be suitable for patients with a fear of injections, whereby the patient can self-administer the vaccine. The buccal-sublingual route of administration has been used for many years to deliver drugs and small molecules into the bloodstream, but its use as a means of mucosal vaccine delivery has received little attention.

One of the main criteria for mucosal delivery of a vaccine is the level of endotoxin present in the pharmaceutical composition. Endotoxin is a complex lipopolysaccharide (“LPS”) found in the outer membrane of Gram-negative bacteria that is released after cell death or lysis. Endotoxin can cause physiological changes in the human body and therefore can affect organ systems and disrupt human humoral and cellular communication systems. For parenteral drugs, there is a strict limitation on the content of endotoxin, since the human body can only tolerate a small amount of endotoxin in the blood.

However, there is usually little safety factor for oral buccal/sublingual medications that contain high levels of endotoxin because aerobic and anaerobic bacteria colonize in the oral cavity. For example, in a healthy adult with good oral hygiene, the average level of endotoxins in the oral microflora corresponds to approximately 2 endotoxin units (EU)/ml of saliva (1 ng=10 EU). Thus, the daily endotoxin production for a healthy adult with good oral hygiene exceeds 1000 EU/day. However, in terms of vaccine delivery, depending on the mucosal site, endotoxin can eliminate, stop, or enhance the immune response.

SUMMARY OF THE INVENTION

The level of endotoxin in buccal/sublingual drugs used for vaccination (particularly against infection) can positively or negatively influence the immune response. In addition, mucosal delivery adjuvants are often added to enhance the immune response. The presence of endotoxins may negatively or positively mask the beneficial effects of this excipient. In addition, it has been suggested that endotoxin induces tolerance to antigens at the sublingual level, which may neutralize the immunostimulatory function of the excipient. Accordingly, dosage forms such as buccal/sublingual tablets (used for mucosal delivery of a vaccine to treat an infection) should have a low endotoxin content.

Many dosage forms use matrix excipients to provide oral dispersible properties. Gelatin is one of the main matrix-forming substances. Gelatin is obtained from the hydrolysis of collagen under acidic or alkaline conditions. Because it is a product of natural origin, it contains varying amounts of endotoxin. For example, in fish gelatin, the level of endotoxin can range from 6,000 to 30,000 EU/g of gelatin. Other gelatins may contain endotoxin in an amount of at least about 3,000 EU/g, about 4,000 EU/g, about 5,000 EU/g, about 6,000 EU/g, about 10,000 EU/g, about 12,000 EU/g, about 14000 EU/g, about 15000 EU/g, about 20000 EU/g, about 25000 EU/g or about 30000 EU/g.

Accordingly, in order to obtain dosage forms made from gelatin with a low content of endotoxin, the inventors have found a method that reduces the amount of endotoxin in gelatin. The resultant low endotoxin gelatin can be further used as a matrix forming agent in various dosage forms. In addition, low endotoxin gelatin can be used in a variety of other applications such as granules in stick packs and softgels.

Provided herein are methods for reducing the level of endotoxin in gelatin and producing gelatin with a low endotoxin content. The process involves dissolving the salt in a gelatin solution and filtering the gelatin-salt solution through anion exchange to reduce endotoxin levels. After the level of endotoxins in the gelatin saline is reduced, the low endotoxin gelatin saline is desalted, thereby obtaining a low endotoxin gelatin solution. The low endotoxin gelatin solution can be used in various pharmaceutical compositions, including vaccines.

In some embodiments, the method of reducing the level of endotoxin in gelatin comprises dissolving a salt in a solution containing gelatin and a solvent to form a gelatin saline solution, wherein the endotoxin content of the gelatin is at least 6000 EU/g; filtering the gelatin saline solution with an anion exchange adsorber, whereby the content of endotoxin in the gelatin saline filtrate is less than 2500 EU/g; desalting the gelatin-brine filtrate to form a low endotoxin gelatin solution having an endotoxin content of less than 2500 EU/g. In some embodiments, the salt concentration in the gelatin saline solution is 75-300 mM. In some embodiments, the salt concentration of the gelatin saline is about 145-155 mM. In some embodiments, the gelatin solution is a 1-20% by weight gelatin solution. In some embodiments, the salt is sodium chloride. In some embodiments, the gelatin is fish gelatin. In some embodiments, dissolving the salt in the gelatin solution comprises heating the gelatin solution to 50-70°C. In some embodiments, the solvent is water. In some embodiments, the endotoxin content of the gelatin saline solution and the low endotoxin gelatin solution is less than 1000 EU/g. In some embodiments, filtering the gelatin saline solution through an anion exchange adsorber can reduce the endotoxin content of the solution by at least 95%. In some embodiments, at least about 85% of the amount of gelatin is found in the gelatin saline filtrate after the gelatin saline is filtered through an anion exchange adsorber. In some embodiments, the method further comprises removing the solvent from the low endotoxin gelatin solution to form low endotoxin gelatin that has an endotoxin content of less than 2500 EU/g.

In some embodiments, the desalting of the gelatin-brine filtrate is performed by diafiltration. In some embodiments, the diafiltration process includes diluting the gelatin saline filtrate with a second solvent and filtering the dilute gelatin saline filtrate to form a solution of the dilute gelatin filtrate. In some embodiments, the dilute gelatin-brine filtrate is filtered until the conductivity of the dilute gelatin filtrate solution is less than 25% of the conductivity of the gelatin solution. In some embodiments, the ratio of gelatin saline filtrate to second solvent is 1:1-1:4. In some embodiments, the second solvent is removed from the diluted gelatin solution in the filtrate to form a low endotoxin gelatin solution. In some embodiments, the second solvent is removed from the dilute gelatin filtrate solution until the weight of the dilute gelatin filtrate solution is less than 5% of the weight of the gelatin brine filtrate. In some embodiments, the implementation of the second solvent includes water.

In some embodiments, a low endotoxin gelatin solution is prepared by a process that includes dissolving a salt in a gelatin solution containing gelatin and solvent to form a gelatin saline solution, wherein the endotoxin content of the gelatin is at least 6000 EU/g; filtering the gelatin saline solution with an anion exchange adsorber, whereby the content of endotoxin in the gelatin saline filtrate is less than 2500 EU/g; desalting the gelatin-brine filtrate to form a low endotoxin gelatin solution having an endotoxin content of less than 2500 EU/g. In some embodiments, the low endotoxin gelatin solution comprises a solvent and gelatin, wherein the gelatin has an endotoxin content of less than 2500 EU/g.

In some embodiments, a method for preparing a dosage form for delivering a pharmaceutically active ingredient comprises dissolving a salt in a gelatin solution containing gelatin and a solvent to form a gelatin saline solution, wherein the endotoxin content of the gelatin is at least 6000 EU/g; filtering the gelatin saline solution with an anion exchange adsorber, whereby the content of endotoxin in the gelatin saline filtrate is less than 2500 EU/g; desalting the gelatin-brine filtrate to form a low endotoxin gelatin solution having an endotoxin content of less than 2500 EU/g; dispensing the composition containing the low endotoxin gelatin solution into a pre-made mould; and lyophilizing the low endotoxin gelatin solution to form a dosage form.

In some embodiments, a dosage form for delivering a pharmaceutically active ingredient is prepared by a method that includes dissolving a salt in a gelatin solution containing gelatin and a solvent to form a gelatin saline solution, wherein the endotoxin content of the gelatin is at least 6000 EU/g ; filtering the gelatin saline solution with an anion exchange adsorber, whereby the content of endotoxin in the gelatin saline filtrate is less than 2500 EU/g; desalting the gelatin saline filtrate to form a low endotoxin gelatin solution having an endotoxin content of less than 2500 EU/g; dispensing the composition containing the low endotoxin gelatin solution into a pre-made mould; and lyophilizing the low endotoxin gelatin solution to form a dosage form.

Additional advantages will be apparent to those skilled in the art from the following detailed description. The examples and descriptions provided herein are to be considered as illustrative and not as limiting of the scope of the present invention.

All publications, including patent documents, scientific articles and databases referred to in this application, are incorporated by reference in their entirety in all respects to the same extent as if each individual publication were individually incorporated by reference. If a definition given herein conflicts with or is otherwise inconsistent with the definition given in patents, applications, published applications and other publications, which are incorporated herein by reference, the definition given in this document takes precedence over the definition that is included in this document by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The exemplary embodiments are described with reference to the accompanying figures, in which:

Figure 1 illustrates a flowchart of the method for reducing the level of endotoxin in gelatin, described in this document.

Figure 2 illustrates the block diagram of the stage of obtaining gelatin saline.

Figure 3 illustrates a flowchart for the gelatin saline filtration step using anion exchange to remove endotoxin described herein.

Figure 4 is a graph illustrating the experimental results of the effect of salt concentration in gelatin saline on endotoxin removal.

Figure 5 illustrates a block diagram of the desalting step of the filtered gelatin brine described herein.

Figure 6 is a plot of endotoxin in sample fractions versus total volume of gelatin saline filtered in the manner described in Example 1.

Figure 7 is a plot of endotoxin in sample fractions versus total volume of gelatin saline filtered by the method described in Example 2.

Figure 8 is a plot of endotoxin in sample fractions versus total volume of gelatin saline filtered by the method described in Example 3.

Figure 9A is a schematic illustration with a photograph of a fully wetted tablet.

Figure 9B is a schematic photograph of a tablet with hard lumps.

Figure 9C is a schematic photograph of a tablet with a matrix film of disintegrated composition that forms on the surface of the lyophilized tablet (peel).

In the figures, like reference numerals refer to like components unless otherwise indicated. Also, the figures are not drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

Through the methods described herein, gelatin with a low level of endotoxin can be obtained. The low endotoxin gelatin can then be used in a variety of applications including, but not limited to, as a matrix forming agent in a pharmaceutical dosage form. Figure 1 shows a flow diagram of a method 100 for reducing the level of endotoxin in gelatin.

Preparation of gelatin saline solution

The first step 101 may include preparing a gelatin saline solution by dissolving the salt in the gelatin solution. Figure 2 shows a block diagram of the stage 101 of dissolving the salt in the gelatin solution. The content of endotoxin in the original gelatin solution may be equivalent to its content in the gelatin in solution. In addition, the content of endotoxin in the gelatin saline solution will also be comparable to the content of the original gelatin solution.

Step 101 may include dissolving the gelatin in a solvent to form a gelatin solution. Preferably, the gelatin is completely soluble in the solvent. The dissolution of the gelatin in the solvent can be facilitated by stirring and/or heating the solution. In some embodiments, the gelatin solution may be heated to about 40-80°C, about 50-70°C, about 55-65°C, about 58-62°C, or about 60°C to help dissolve the gelatin in the solvent. . After the gelatin has completely dissolved in the solvent, the gelatin solution can be cooled to room temperature.

In some embodiments, the gelatin may be non-gelatinous gelatin, gelatinous gelatin, including fish gelatin, bovine gelatin, porcine gelatin, chicken gelatin, or a combination thereof. Although the examples described below use fish gelatin as a typical gelatin, the principles established for the methods described herein apply to other types of gelatin and the corresponding grades of gelatin available in each type of gelatin. In some embodiments, the solvent may be water (including purified water).

The concentration of gelatin in the gelatin solution may vary depending on the end use of the gelatin. Thus, the specific amount of gelatin and the specific amount of solvent used may vary depending on the desired concentration of gelatin. In some embodiments, the concentration of gelatin in the gelatin solution may be about 1-20% by weight, about 2-15% by weight, about 3-12% by weight, about 5-12% by weight, about 8-12% by weight. by weight or about 10-12% by weight.

Before salt is added to the gelatin solution, the conductivity of the gelatin solution can be measured. This pre-salt conductivity measurement can be used when comparing the final low endotoxin gelatin solution after the desalting process. In addition, the conductivity of the gelatin saline solution can also be measured before the endotoxin reduction step.

As shown in Figure 2, the gelatin can be completely dissolved in the solvent to form a gelatin solution. The dissolution of the gelatin can be facilitated by stirring and heating the gelatin solution. The salt may be dissolved in the gelatin solution to form a gelatin saline solution.

In some embodiments, the salt may be sodium chloride, potassium chloride, lithium chloride, calcium chloride, other chloride salts, or combinations thereof.

As described in more detail below, the inventors have found that the salt concentration of the gelatin saline solution plays an important role in the anion exchange process in step 102 to remove endotoxin from gelatin. In particular, the inventors have found that performing anion exchange without the use of salt in a gelatin solution can cause the gelatin to plug/clog the filters used in the anion exchange process in step 102.

In addition, as shown below in the description of step 102, the inventors have found that a certain salt concentration has a direct effect on the endotoxin removal step. Thus, the salt concentration in gelatin saline may be about 50-500 mM, about 75-300 mM, about 100-200 mM, about 125-175 mM, about 140-160 mM, about 145-155 mM, or about 150 mm.

Endotoxin Reduction by Gelatin-Saline Filtration Using Anion Exchange

After preparation of the gelatin saline, endotoxin can be removed from the gelatin in step 102. FIG. 3 shows a block diagram of the step 102 for filtering the gelatin saline using anion exchange (“AEX”). The inventors have found that using the principles of anion exchange chromatography, endotoxin can be removed from a gelatin solution. Anion exchange chromatography refers to a method for separating molecules based on their charge. In particular, anion exchange chromatography uses a positively charged ion exchange resin with an affinity for molecules having net negative surface charges. Endotoxin molecules have net negative surface charges. Thus, endotoxin can bind to the positively charged ion exchange resin and be removed from the gelatin saline solution.

As noted above, the inventors have found that during the endotoxin reduction process, the gelatin solution without salt can clog/clog the filters of the anion exchanger. Without wishing to be bound by any theory, the inventors believe that the salt in the gelatin solution can modify the charge on the gelatin molecule to a sufficient level, thereby reducing the adhesion of the gelatin to the anion exchange device. Thus, the gelatin-saline solution can pass through the anion exchange device as a filtrate, while endotoxins are retained by the device.

The anion exchange filter may be in the form of a membrane adsorber (e.g. commercially available Sartobind Q from Sartorius Stedim, Mustang E from Pall Life Sciences) or resin (e.g. commercially available Fractogel EMD TMAE Hicap (M) Resin and Eshmuno Q Resin). from Merk Millipore). In addition, membrane adsorbers are available in various sizes. For example, Sartobind Q is available in several sizes including 1 ml, 7 ml, 75 ml, 159 ml, 1.2 L and 5 L filter capacities. The choice of adsorber size may be based on the volume to be filtered, the point of endotoxin penetration for the treated solution, and/or various processing conditions.

Although endotoxin can bind to a positively charged ion exchange resin in an anion exchange device, the inventors have found that the salt concentration of the gelatin solution can have a significant effect on the removal of endotoxin through the anion exchange device. To determine the effect of salt concentration on endotoxin removal, the inventors prepared a 10% by weight solution of fish gelatin in purified water with various concentrations of sodium chloride and filtered them using Nartobind Q 1 ml Nano with a filter flow of 1 ml. The inventors then determined the endotoxin content as well as the gelatin content of the gelatin saline before and after filtration. The results of the influence of salt concentration on the removal of endotoxin in gelatin saline solution are shown in the following table 1 and figure 4.

Table 1

Solution before/after filtration Endotoxin content (EU/g) Gelatin content (% by mass) (10% gelatin) 7000 10.5; 10.4 Before filtering
(10% Gelatin + 150 mM NaCl) 4140 10.3; 10.3 After filtering
(10% Gelatin + 150 mM NaCl) 191 10.1; 10.1 Before filtering
(10% Gelatin + 250 mM NaCl) 5760 10.0; 10.2 After filtering
(10% Gelatin + 250 mM NaCl) 464 10.6; 10.7 Before filtering
(10% Gelatin + 300 mM NaCl) 4610 10.3; 10.6 After filtering
(10% Gelatin + 300 mM NaCl) 441 10.5; 10.4

As shown in Table 1 above and Figure 4, the inventors have found that in the case where the salt concentration of the fish gelatin-NaCl solution is about 150 mM, the filter can achieve the greatest endotoxin reduction in the solution. In the case where the salt concentration is much higher than 150 mm, less endotoxin can be removed. Furthermore, in the case where the salt concentration is much lower than 150 mM, this may cause the gelatin to clog/block the anion exchange device.

After reducing the content of endotoxin, the conductivity of the gelatin-brine filtrate can be measured. In some embodiments, during the endotoxin reduction step, the gelatin solution may be reduced in endotoxin by at least about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%. %.

In some embodiments, the level of endotoxin in the gelatin saline filtrate may be less than about 3000 EU/g, about 2500 EU/g, about 2000 EU/g, about 1500 EU/g, about 1000 EU/g, about 750 EU/g, about 500 EU/g, about 250 EU/g, about 200 EU/g, about 150 EU/g or about 100 EU/g. In some embodiments, at least about 85%, about 90%, about 95%, about 98%, or about 99% of the original gelatin content is retained after the endotoxin reduction step. This can be assessed by comparing the concentration of gelatin before and after anion exchange filtration.

Desalting the filtered gelatin saline solution

After endotoxin has been reduced in the gelatin saline, the salt added in step 101 must be removed or reduced in step 103. FIG. . One method by which salt can be removed from a gelatin saline solution is by diafiltration. Diafiltration is a process that involves removing salt from a solution based on molecular size using filters that are permeable to micromolecules. To facilitate diafiltration, the gelatin saline solution can be further diluted with water to reduce its viscosity; in particular in the case where the solution has a high content of gelatin. The added water can then be removed in a concentration step. In some embodiments, the salt can be removed without diluting the gelatin saline solution with water, and therefore a subsequent concentration step is not required; for example, for a gelatin saline solution with a lower gelatin content.

The filtered gelatin-saline solution can be introduced into the diafiltration system. Examples of diafiltration systems include, but are not limited to, the Sartoflow Advanced crossflow filtration system from Sartorius Stedim, the KMPi TFF system from Spectrum Labs (Repligen), or the Cogent M1 TFF system from Merck. The weight of the filtered gelatin saline solution and the conductivity can be measured prior to diafiltration.

In some embodiments, the gelatin-brine filtrate from the endotoxin reduction step may be diluted with a solvent. In some embodiments, the solvent may be water (including purified water). The ratio of gelatin saline filtrate to solvent may be about 1:1-1:6, about 1:1-1:5, about 1:1-1:4, about 1:1-1:3, about 1:1 -1:2 or about 2:1-1:1. In some embodiments, the ratio of gelatin saline filtrate to solvent may depend on the concentration of gelatin in the gelatin saline filtrate. The mixture of gelatin-brine and solvent may be stirred and/or heated to ensure sufficient mixing.

At the beginning of the diafiltration, the conductivity of the mixture can be determined. In addition, the conductivity can be continuously monitored and the mixture can be continuously stirred during the entire diafiltration process. The diafiltration may be stopped when the conductivity of the mixture reaches a relatively similar or comparable conductivity value to the original gelatin solution prior to salt addition in step 101. The relatively same or comparable value may be when the value is less than about 25%, about 20 %, about 15%, about 10%, about 5%, about 2% or about 1% of the original value.

After removing the salt, the dilute gelatin solution can be concentrated. The concentration process may remove excess solvent added during dilution prior to diafiltration. The concentration process can be carried out by means of a system for concentration. Examples of downconcentration systems include, but are not limited to, the Sartoflow Advanced crossflow filtration system from Sartorius Stedim, the KMPi TFF systems from Spectrum Labs (Repligen), or the Cogent M1 TFF system from Merck. Concentration of the dilute gelatin solution can be stopped when the weight of the dilute gelatin solution reaches relatively the same or comparable weight of the pre-diluted gelatin-brine filtrate. Relatively the same or comparable value may be when the value is less than about 25%, about 20%, about 15%, about 10%, about 5%, about 2 or about 1% of the original value.

After diafiltration, a gelatin solution with a reduced content of endotoxin remains. In addition, the concentration or content of gelatin in this low endotoxin demineralised gelatin solution may have relatively the same or similar gelatin concentration/content as the filtered gelatin saline solution after the endotoxin reduction step. The relatively same or comparable value may be when the value is less than about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, or about 1% of the original value. The low endotoxin gelatin solution may have an endotoxin content of less than about 3000 EU/g, about 2500 EU/g, about 2000 EU/g, about 1500 EU/g, about 1000 EU/g, about 750 EU/g, about 500 EU/g, about 250 EU/g, about 200 EU/g, about 150 EU/g or about 100 EU/g. Thus, the content of endotoxin, after the stage of its removal, can be maintained in the process of diafiltration.

In some embodiments, at least about 85%, about 90%, about 95%, about 98%, or about 99% of the original gelatin content is retained after the desalting step. This can be assessed by comparing the concentration of gelatin before and after diafiltration. In some embodiments, at least about 85%, about 90%, about 95%, about 98%, or about 99% of the original gelatin is retained after two steps: endotoxin reduction and desalting.

In some embodiments, the solvent in the reduced endotoxin gelatin solution can be removed. For example, a solution of endotoxin-reduced gelatin can be subsequently dried to a low-endotoxin hard gelatin. This low endotoxin gelatin can be in the form of a powder and can be used as a starting material for a variety of applications including a pharmaceutical formulation. In some embodiments, the level of endotoxin in the low endotoxin gelatin may be less than about 3000 EU/g, about 2500 EU/g, about 2000 EU/g, about 1500 EU/g, about 1000 EU/g, about 750 EU/g, about 500 EU/g, about 250 EU/g, about 200 EU/g, about 150 EU/g or about 100 EU/g.

Use in pharmaceutical compositions

Gelatin solutions with a low content of endotoxin can be used in various pharmaceutical compositions. For example, the gelatin or low endotoxin gelatin solutions prepared herein can be used in the dosage forms described in US Pat. Nos. 4,371,516; 4305502; and 4,758,598 and UK Patent Nos. 1,548,022 and 211,423, which are hereby incorporated by reference.

In some embodiments, the process for manufacturing a dissolving dosage form for delivering a pharmaceutically active ingredient (“API”) may include the steps of: (a) dispensing a gelatin-containing composition into a preform mold; and (b) lyophilizing the composition to form a dissolving dosage form.

Used in this document, the term "dosed" refers to the placement of a predetermined aliquot of a solution or suspension. As used herein, the term "preformed mould" refers to any suitable container or cavity in which an aqueous solution or suspension can be placed, which is subsequently lyophilized; in some preferred embodiments of the present invention, the preform is a blister pack with one or more blister cells.

The composition in step (a) may include a matrix-forming agent. The matrix-forming agent may be any conventional non-gelling matrix-forming agent. Suitable non-gelling matrix forming agents include, but are not limited to, non-gelling gelatins (including low endotoxin gelatin prepared according to the process described above), modified starches, pullulan, non-gelling fish starch, maltodextrins, low molecular weight dextrans, starch esters, cellulose gums with low and average molecular weight and combinations thereof. The matrix-forming agent may also be any conventional gelling matrix-forming agent. Suitable gelling matrix-forming agents include, but are not limited to, gelling gelatin (including low endotoxin gelatin prepared as described above), carageenan gums, hyaluronic acid, pectins, starches, sodium carboxymethyl cellulose, agar, gellan gum, guar gum, tragacantan, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, carbomer, poloxamer, polyacrylic acid, polyvinyl alcohol, alginates, and poly(glycolic acid), and combinations thereof. In some embodiments, the matrix-forming agent may be a combination of a non-gelling matrix-forming agent and a gelling matrix-forming agent. A person skilled in the art, if necessary, can easily determine the appropriate amounts of these matrix-forming substances.

The composition in step (a) is usually in the form of a solution or suspension. Accordingly, the solvent is also present in the composition. A suitable solvent can be easily selected by one skilled in the art once the final composition of the composition is known, ie the pharmaceutically active ingredient, excipient, etc. to be present. Preferred solvents include ethanol, isopropanol, other lower aliphatic alcohols and water, and more preferably water.

The composition in step (a) may also contain an additional pharmaceutically acceptable substance or excipient. Such additional pharmaceutically acceptable substances or excipients include, without limitation, sugars such as mannitol, dextrose and lactose, inorganic salts such as sodium chloride and aluminum silicates, mammalian gelatins, fish gelatin, modified starches, preservatives, antioxidants, surfactants. thickening agents, coloring agents, flavoring agents, pH modifiers, sweeteners, flavoring agents, and combinations thereof. Suitable dyes include iron oxide red, black and yellow, and FD&C dyes such as FD&C blue #2 and FD&C red #40, and combinations thereof. Suitable flavors include mint, raspberry, licorice, orange, lemon, grapefruit, caramel, vanilla, cherry and grape flavors and flavors and combinations thereof. Suitable pH modifiers include citric acid, tartaric acid, phosphoric acid, hydrochloric acid, maleic acid, and sodium hydroxide, and combinations thereof. Suitable sweeteners include aspartame, acesulfame K and thaumatin and combinations thereof. Suitable flavoring agents include sodium bicarbonate, ion exchange resins, cyclodextrin inclusion compounds, adsorbates or microencapsulated actives, and combinations thereof. The person skilled in the art can easily determine the appropriate amounts of these various additional excipients, if necessary. Mannitol, which is an organic compound with the formula (C ₆ H ₈ (OH) ₆ ) and which is generally known to those skilled in the art, is the preferred additional pharmaceutically acceptable substance.

The composition of step (a) may also contain a pharmaceutically active ingredient. As used herein, the term “pharmaceutically active ingredient” refers to a drug that can be used in the diagnosis, elimination, amelioration, treatment, or prevention of a disease. Any pharmaceutically active ingredient can be used to achieve the objectives of the present invention. Obviously, a person skilled in the art will easily understand that some pharmaceutically active ingredients are more suitable for use with the non-gelling matrix forming agent of the composition of step (a) than, for example, with the gelling forming matrix substance of step (b). Suitable pharmaceutically active ingredients include, without limitation: analgesics and anti-inflammatory agents, antacids, anthelmintics, antiarrhythmic agents, antibacterial agents, anticoagulants, antidepressants, antidiabetic agents, antidiarrheal agents, antiepileptic agents, antifungal agents, antigout agents, antihypertensive agents, anti malaria, anti-migraine agents, antimuscarinics, antitumor agents and immunosuppressants, antiprotozoals, antirheumatic agents, antithyroid agents, antivirals, anxiolytics, sedatives, hypnotics and antipsychotics, beta-blockers, inotropic agents, corticosteroids, cough suppressants, cytotoxic agents, decongestants or decongestants, diuretics, enzymes, antiparkinsonian agents, gastrointestinal agents, histamine β antagonists receptors, lipid regulators, local anesthetics, neuromuscular agents, nitrates and antianginals, nutrients, opioid analgesics, oral vaccines, proteins, peptides and recombinant drugs, sex hormones and contraceptives, spermicides and stimulants; and their combinations. A list of specific examples of these active ingredients can be found in US Pat. No. 6,709,699, which is incorporated herein by reference. The pharmaceutically active ingredient, if present, is present in the composition of step (a) in an amount necessary to produce the desired physiological effect as determined by clinical studies. A person skilled in the art can easily determine the appropriate amount of active ingredient to include in a dosage form made in accordance with the present invention.

The composition of stage (a) can be obtained by any conventional method, which is incorporated herein by reference. Most typically, the matrix-forming agent, solvent, and optional ingredients may be mixed together at a given temperature to form a solution. In some embodiments, the matrix-forming agent and solvent may be a low endotoxin gelatin solution prepared in accordance with the process described above. Any optional ingredients may be mixed into the gelatin solution. The solution may then be cooled while the active ingredient may be added.

Likewise, the dosing described in step (a) may be carried out by any known method or device, including sequential dosing as described in WO 2011/115969.

In step (b), the compositions dosed in step (a) are freeze-dried to form a dissolving dosage form. Typically, pre-formulated dosage formulations are frozen by any means known in the art, such as by passing them through a liquid nitrogen tunnel, preferably within one to about ten minutes. A person skilled in the art will easily understand how fast they need to be passed through the tunnel. The dosage compositions in pre-formulated molds can then be lyophilized at low pressure (ie, under vacuum).

Dosage forms of the present invention are dissolving dosage forms and, accordingly, have the distinct advantage of faster disintegration. The route of administration may be oral, vaginal or nasal, although oral is preferred. After placement in the mouth and contact with saliva, the dosage form may disintegrate within 1 to 180 seconds, 1 to 120 seconds, 1 to 60 seconds, preferably 1 to 30 seconds, more preferably within about 1 to about 10 seconds and most preferably less than 5 seconds.

In some embodiments, dosage forms may have less than about 200 EU/dosage form, less than about 150 EU/dosage form; less than about 125 EU/dosage form; less than about 100 EU/dosage form; less than about 90 EU/dosage form; less than about 75 EU/dosage form; or less than about 50 EU/dosage form.

Examples

First of all, all vessels and laboratories used to prepare gelatin saline should be cleaned and disinfected as required to minimize bioburden and/or introduce additional endotoxin.

Example 1

An amount of fish gelatin containing about 7000 EU/g of gelatin was added to some purified water and completely dissolved (by heating to about 60° C. to facilitate the dissolution of the gelatin) to obtain a 10% by weight solution of fish gelatin. Then the solution was cooled to room temperature and a control sample was taken to determine the conductivity. Next, some sodium chloride (“NaCl”) was added to the gelatin solution to give a NaCl concentration of 150 mM in the gelatin-NaCl solution. Then the conductivity of the gelatin-NaCl solution was measured.

Before endotoxin removal, an anion exchange device (Sartobind Q Single Sep mini filter with a throughput of 7 ml) was preliminarily prepared. Preparation prior to use of the anion exchanger and associated tubing was performed according to the pretreatment procedure. A 1M sodium hydroxide solution (“NaOH”) was used to clean and disinfect the system, followed by flushing the system with a 1M NaCl solution to remove the NaOH solution. The adsorber was filled with a gelatin-NaCl solution to remove the 1M NaCl solution used to prepare for use. Using a peristaltic pump, the gelatin-NaCl solution was then loaded and passed through a 7 ml Sartobind Q Single Sep mini filter. The pump was set to maintain a constant pressure to feed the solution through the adsorber with minimal back pressure buildup. The filtered gelatin-NaCl solution was collected in a clean container and the conductivity of this filtered solution was measured.

Approximately 435 g of the gelatin-NaCl solution (in 30 g fractions) was filtered through a 7 ml Sartobind Q Single Sep mini filter. The binding capacity and the moment of penetration through the filter in relation to endotoxin were evaluated by collecting fractions of the filtrate at regular intervals and assessed the content of endotoxin and analytical gelatin content. The results are summarized in the following table 2 and in Fig.6.

TABLE 2

Total weight of filtered gelatin solution (g) Endotoxin content (EU/g) Content of gelatin in the sample (% by mass) Step 101: Preparation of gelatin solution. 10% gelatin solution 0 Not tested Not tested 10% gelatin solution with 150 mM NaCl 0 6440 10.3; 10.3 Step 102: Endotoxin Reduction filling thirty N/A N/A Filtered fraction 1 60 <100 10/5; 10.2 Filtered fraction 2 90 Not tested Not tested Filtered fraction 3 120 112 10.6; 10.4 Filtered fraction 4 150 Not tested Not tested Filtered fraction 5 180 122 10.4; 10.5 Filtered fraction 6 210 Not tested Not tested Filtered fraction 7 240 129 10.4; 10.4 Filtered fraction 8 270 Not tested Not tested Filtered fraction 9 300 144 10.3; 10.5 Filtered fraction 10 330 Not tested Not tested Filtered fraction 11 360 138 10.7; 10.5 Filtered fraction 12 390 Not tested Not tested Filtered fraction 13 420 Not tested Not tested Filtered fraction 14 435 149 10.6; 10.6

Table 2 shows that endotoxin content in gelatin is reduced with the Sartobind Q filter device. The data also shows endotoxin level plateauing/stabilizing after filtering 300 g (after fraction 9) of the solution, with the device eventually being blocked at fraction 14, when, due to adsorber blockage, only 15 ml were collected. This indicates that the test adsorber had reached equilibrium at the time when about 300 g of gelatin saline was filtered. Instead of endotoxin penetration, continued accumulation of endotoxin on the filter device resulted in blockage of the device.

A sample of the gelatin saline solution before and after filtration was also analyzed by UV spectroscopy, and the analysis showed a similar content in the sample, confirming that the gelatin was not removed during the filtration process.

Example 2

An amount of fish gelatin containing about 14,600 EU/g of gelatin was added to some purified water and completely dissolved (by heating to about 60° C. to facilitate the dissolution of the gelatin) to give a 12% by weight fish gelatin solution. Then the solution was cooled to room temperature and a control sample was taken to measure the conductivity. Next, some sodium chloride (“NaCl”) was added to the gelatin solution to give a NaCl concentration of 150 mM in the gelatin-NaCl solution. Then the conductivity of the gelatin-NaCl solution was determined.

Before endotoxin removal, an anion exchange device (75 ml Sartobind Q capsule) was preliminarily prepared. Preparation prior to use of the anion exchanger and associated tubing was performed according to the pretreatment procedure. 1M sodium hydroxide solution (“NaOH”) was used to clean and disinfect the system, followed by flushing the system with 1M NaCl solution to remove the NaOH solution. The adsorber was filled with a gelatin-NaCl solution to remove the 1M NaCl solution used for preparation prior to use. Using a peristaltic pump, the gelatin-NaCl solution was then loaded and passed through 75 ml Sartobind Q. The pump was set to maintain a constant pressure to drive the solution through the adsorber with minimal back pressure buildup. The filtered gelatin-NaCl solution was collected in a clean container and the conductivity of this filtered solution was measured.

Approximately 3000 g of the gelatin-NaCl solution was filtered through a 75 ml Sartobind Q filter. The binding capacity and endotoxin penetration point through the filter were determined by collecting fractions of the filtrate at regular intervals and determining the endotoxin content and the gelatin content of the sample. The results are summarized in the following table 3 and in Fig.7.

TABLE 3

Total weight of filtered gelatin solution (g) Conductivity (mS/cm) Endotoxin content (EU/g gelatin) Content of gelatin in the sample (% by mass) Step 101: Preparation of gelatin solution 12% gelatin solution 0 (0.8 -0.9)* 14600 Not tested 12% gelatin solution with 150 mM NaCl Before filtration 0 Not tested 13600 12.4; 12.2 Step 102: Endotoxin Reduction filling 224 - - - Before filtering fraction 1 312 - - - Filtered fraction 1* 335 - <100 12.1;12.3 Before filtering fraction 2 588 - - - Filtered fraction 2* 611 - Not tested Not tested Before filtering fraction 3 937 - - - Filtered fraction 3* 960 - 148 12.4; 12.6 Before filtering fraction 4 1230 - - - Filtered fraction 4* 1253 - Not tested Not tested Before filtering fraction 5 1623 - - - Filtered fraction 5* 1648 - 188 12.2; 12.2 Before filtering fraction 6 1905 - - - Filtered fraction 6* 1927 - Not tested Not tested Before filtering fraction 7 2184 - - - Filtered fraction 7* 2203 - 175 12.2; 12.3 Before filtering fraction 8 2419 Filtered fraction 8* 2437 - Not tested Not tested Before filtering fraction 9 2604 Filtered fraction 9* 2617 - 158 12.3; 12.3 The total fraction collected on the filter (fraction 1-9) 188 - - - Total filtered gelatin solution (excluding fractions 1-9) 2429 13.4 119; < 100 Not tested

*The sample fractions collected were approximately 20 g each.

Table 3 shows that 75 ml Sartobind Q has the ability to reduce endotoxin in a batch of gelatin from an initial endotoxin level equivalent to 14600 EU/g gelatin to less than 200 EU/g gelatin. Thus, about 2.5 kg of 12% by weight gelatin saline can be filtered using a 75 ml capsule, with endotoxin levels beginning to plateau/stabilize after about 1.6 kg has been filtered (after fraction 5 ) 12% by weight gelatin saline, assuming that the equilibrium position has been reached. The penetration point was not observed.

Analysis of the gelatin in solution showed no significant change in the content of the sample between the gelatin solutions before and after filtration, confirming that there was no loss of gelatin in the anion exchange membrane adsorber.

Example 3

An amount of fish gelatin containing approximately 14,600 EU/g of gelatin was added to the amount of purified water and completely dissolved (with heating to approximately 60° C. to facilitate the dissolution of the gelatin) to obtain a 12% by weight solution of fish gelatin. Then the solution was cooled to room temperature and a control sample was taken to measure the conductivity. Then, some sodium chloride (“NaCl”) was added to the gelatin solution to give a NaCl concentration of 150 mM in the gelatin-NaCl solution. Then the conductivity of the gelatin-NaCl solution was measured.

An anion exchange device (75 ml Sartobind Q capsule) was pre-prepared prior to endotoxin removal. According to the pretreatment procedures. 1M sodium hydroxide solution (“NaOH”) was used to clean and disinfect the system, followed by flushing the system with 1M NaCl solution to remove the NaOH solution. The adsorber was filled with a solution of gelatin-NaCl with the removal of 1M NaCl solution used for preparation before use. The gelatin-NaCl solution was then loaded using a peristaltic pump and passed through 75 ml Sartobind Q. The pump was set to maintain a constant pressure to drive the solution through the adsorber with minimal back pressure buildup. The filtered gelatin-NaCl solution was collected in a clean container and the conductivity of this filtered solution was measured.

Approximately 5000 g of the gelatin-NaCl solution was filtered through a 75 ml Sartobind Q filter. Fifteen (15) filtered fractions, each weighing approximately 20 g, were collected for analysis. Between successive fractions, approximately 300 g of the gelatin solution was filtered.

The first 2000 g of the filtered gelatin saline solution was transferred to a diafiltration system (Sartorius Sartoflow Advanced) for salt removal using diafiltration and concentration processes. The diafiltration system was set up in accordance with the operating instructions. Then, for cleaning and disinfection of the system, preparations were made before using the diafiltration system in accordance with the operating instructions. A 1M NaOH solution was used to clean and disinfect the system, followed by flushing the system with purified water to remove the NaOH solution. Before use, the diafiltration system was flushed with a stream of clean water to check that the cross-flow filter (cassette) was clean and compliant. Then, before starting diafiltration, the settings (pressure and flow) were optimized.

The filtered gelatin-saline solution was loaded into the tank of the diafiltration system. The weight of the solution that was loaded into the tank was recorded, and the conductivity of the loaded solution was measured using a conductivity sensor. The loaded filtered gelatin-brine solution was diluted with purified water (with a ratio of filtered gelatin-brine solution: water 2:1 or 1:1 or 1:2 or 1:3 or 1:4 depending on the concentration of gelatin). The mixture was stirred to check that the mixture was well mixed.

The diafiltration system was then adjusted to concentrate the dilute desalted gelatin solution by removing excess water added for dilution until the weight of the desalted gelatin solution was the same as that of the filtered gelatin saline before dilution. At the end, the diafiltration system was cleaned, and then flushed with pure water after being used to check the cleanliness of the cross-flow filter (cassette).

Filtration of the remaining gelatin-salt solution (about 3000 g) was continued to the end. The binding capacity and filter penetration point for endotoxin were determined by collecting fractions of the filtrate at regular intervals and the endotoxin content and analytical gelatin content were determined. The results are summarized in the following table 4 and in Fig.8.

TABLE 4

Total weight of filtered gelatin solution (g) Conductivity (mS/cm) Endotoxin content (EU/g gelatin) Content of gelatin in the sample (%) Step 101: Preparing the gelatin solution 12% gelatin solution 0 (~-0.9)* 14,600 12% gelatin solution with 150 mM NaCl 0 13.2 14000 12.4; 12.6 Step 202: Endotoxin Reduction Loading (224) - N/A N/A Before filtering fraction 1 524 - - - Filtered fraction 1* 540 134 12.6; 12.6 Before filtering fraction 2 840 - - - Filtered fraction 2* 857 - Not tested Not tested Before filtering fraction 3 1157 - - - Filtered fraction 3* 1177 - 269 Not tested Before filtering fraction 4 1476 - - - Filtered fraction 4* 1494 Not tested Not tested Before filtering fraction 5 1794 - - - Filtered fraction 5* 1810 - 697 12.4; 12.4 Before filtering fraction 6 2110 - - - Filtered fraction 6* 2127 - Not tested Not tested *The collected filtered gelatin solution was transferred for diafiltration (~ total volume 2L) Before filtering fraction 7 2427 - - - Filtered fraction 7 2449 - 3780 Not tested Before filtering fraction 8 2748 - - - Filtered fraction 8 2771 - Not tested Not tested Before filtering fraction 9 3071 - - - Filtered fraction 9 3092 - Not tested 12.3, 12.5 Before filtering fraction 10 3392 - - Filtered fraction 10 3412 - Not tested Not tested Before filtering fraction 11 3712 - Filtered fraction 11 3732 - 3480 Not tested Before filtering fraction 12 4032 Filtered fraction 12 4051 Not tested Not tested Before filtering fraction 13 4351 Filtered fraction 13 4366 2160 12.9; 12.4 Before filtering fraction 14 4666 Filtered fraction 14 4690 Not tested Not tested Before filtering fraction 15 4990 Filtered fraction 15 5002 1010 12.3; 12.4 Step 103: Removal of NaCl * harvested filtered gelatin/before diafiltration N/A - Not tested Not tested * collected filtered gelatin solution / after filtration N/A 1.2 405 11.6; 11.4

*Conductivity is determined as a separate reference sample.

Table 4 demonstrates that 75 ml Sartobind Q has the ability to reduce endotoxin in a batch of gelatin with an endotoxin level equivalent to 14/600 EU/g gelatin to less than 1000 EU/g gelatin for approximately the first 2 kg of 12 wt % gelatin - saline solution. Endotoxin levels were maintained as this material passed through the diafiltration system. For the next 3 kg of gelatin saline, which was filtered, the level of endotoxin continued to rise, followed by a decrease. This indicates endotoxin penetration after 2 kg was filtered using 75 ml Sartobind Q.

With regard to gelatin content, no significant differences were found in the amount of gelatin in the filtered samples with a reduced content of endotoxin compared to the samples before filtration. This confirmed (as in the previous examples) that the gelatin did not bind to the anion exchange membrane of the adsorber during filtration. For the sample after diafiltration, the sample showed a comparable content of gelatin in the sample. In addition, the conductivity measurements of the desalted sample showed a conductivity value comparable to the value of the sample before salt treatment, confirming the effectiveness of the desalting process.

Example 4

For example 4, a batch of lyophilized tablets based on an aqueous solution of fish gelatin with reduced endotoxin content was made using the three stages described in figures 2, 3 and 5. The purified water used in example 4 had an endotoxin level of less than 0.1 EU /ml

An aqueous solution of fish gelatin and sodium chloride, 3 kg, containing 9% by weight of fish gelatin and 150 mm NaCl, was obtained by the method shown in figure 2 and in the attached paragraphs. The fish gelatin was added to purified water and completely dissolved (by heating to approximately 60° C.) to obtain a fish gelatin solution. The solution was cooled to room temperature and NaCl was added to the fish gelatin solution. The level of endotoxin in the resulting gelatin-saline solution was measured and was 1100 EU/g.

To reduce the level of endotoxin in the gelatin saline solution, the solution was filtered through an anion exchange device (75 ml Sartobind Q capsule) as shown with reference to Figure 3 and the accompanying paragraphs. Prior to filtration, the Sartobind Q capsule and associated tubing was cleaned and disinfected by pumping 1M sodium hydroxide solution through the system using a peristaltic pump. Purified water was then pumped through the system to remove the sodium hydroxide solution. Next, the system was prepared by pumping 1M NaCl solution through the system, and then part of the gelatin-brine solution to remove the NaCl solution. This portion of the gelatin saline was discarded.

To reduce the endotoxin content, the remainder of the gelatin saline was filtered through a Sartobind Q filter. The resulting gelatin saline filtrate was approximately 2.3 kg. The gelatin-brine filtrate was then transferred to a diafiltration system (Sartorius Sartoflow Advanced) to remove the salt using the diafiltration and concentration processes described in FIG. 5 and the accompanying paragraphs.

Before starting the desalting/concentration process, the diafiltration system was cleaned and disinfected using 1M NaOH solution, followed by rinsing with purified water. Before use, the system was flushed with a stream of clean water. The system was then tuned for optimal pressure and flow rate using a cross-flow filter (cassette) with a cutoff of 10 kDa.

The diafiltration system was then set up for constant volume diafiltration. The conductivity of the solution was monitored and the diafiltration process was stopped when the conductivity reached the target value, which in this example was less than 0.5 mS/cm ^-1 . The diafiltration system was then adjusted to concentrate the filtered desalted gelatin solution to a target gelatin concentration of 12% by weight (range 10-14%). At the end, the resulting gelatin solution (filtered-desalted/concentrated) was measured for gelatin and endotoxin content. The resulting solution had a gelatin content of 11% by weight and an endotoxin level of 83 EU/g. Collected about 1.8 kg of the resulting gelatin solution.

The following Table 5 summarizes the production of low endotoxin gelatin lyophilized tablets from Example 5.

Table 5

Ingredient % by mass Amount in mg per 500 mg aliquot for dosing Amount in mg after freeze drying Buffer solution (to simulate a liquid vaccine composition) 25% 125 mg ~2.6 mg** Low endotoxin fish gelatin (in target 12% by weight fish gelatin solution) 50% 250 mg (contains 30 mg fish gelatin) 30 mg** mannitol 8% 40 mg 40 mg Trehalose 0.75% 3.75 mg 3.74 mg pH modifier in sufficient quantity to achieve a pH value in the range of 6.5-7.8 ~1.3 mg ~0.04 mg** Water (for preparing aqueous matrix mixture) enough to reach 100% ~73.5 mg Water removed

**after removing the water in the freeze drying process

In this example, a 1 kg simulated vaccine formulation was prepared for the manufacture of lyophilized tablets. The composition mixture contained 50% by weight filtered fish gelatin solution with a reduced content of endotoxins (ie, 0.5 kg of filtered fish gelatin solution obtained by the method described above was used in the manufacture). The composition was obtained by adding and dissolving mannitol and trehalose in a filtered solution of fish gelatin at a temperature of from 13 to 17ºC. The pH value was adjusted to the target value of 7.4 using a pH modifier (eg 3% NaOH solution). A simulated liquid vaccine composition (in this case a buffer solution) was then added and the pH value was checked to be still within the specified range. Water was then added to bring the volume up to batch size.

The composition was dosed by weight (wet dose) into the wells of preformed blister packs. In this case, a 500 mg aliquot was dosed into each well in a preformed blister. After dosing, the blister packs were passed through a liquid nitrogen freezing tunnel where the water in the mixture was frozen in the blister cells. Upon exiting the freezing tunnel, the product was stored frozen in refrigerated cabinets prior to lyophilization. The required freeze time (also referred to as annealing time) was applied to the frozen tablets to anneal the tablets. In this case, the exposure time was 6 hours. The frozen tablets were then placed on the freezer shelves where ice crystals were removed from the frozen tablets by low pressure sublimation. The resulting lyophilized tablets had a very high porosity, which ensures rapid disintegration. This disintegration is characterized by a dispersion time (wetting and disintegration time) determined by an in-house study. Samples of the five resulting lyophilized tablets were examined and found to be typically less than 10 seconds. The results are summarized in Table 6. In some embodiments, a dispersion time of less than 60 seconds is also considered acceptable for the product when a slower dispersion/disintegration profile of the tablet is desired. The resulting lyophilized tablets were also evaluated for endotoxin content. It has been found to average less than 75 EU per tablet.

Table 6

Tablet moisturizing time Tablet disintegration time Range (n=5)
seconds Average time (n=5) seconds Range (n=5)
seconds Average time (n=5) seconds < 2 - < 4 < 3 < 5 -< 7 < 6

Measurement methods

Conductivity Measurement : The conductivity of a solution is measured using a conductivity meter. The conductivity meter measures the number of charged particles or ions in a solution. Ions responsible for conductivity appear in electrolytes when dissolved in water. Salts (eg sodium chloride), acids and bases are electrolytes. The conductivity value is proportional to the concentration of electrolytes. The conductivity sensor is calibrated using a conductivity calibration solution. Then the conductivity of the solutions is measured. In the present invention, the salt is added to the gelatin solution in step 101 and then removed in step 103 by desalting. To complete the desalting process, the conductivity of the desalted gelatin solution must be comparable to that of the original gelatin solution (before salt addition). Gelatin has some residual ions, so the conductivity is measured with a solution of gelatin (without salt) and the original conductivity is determined. This conductivity value is then used to set up the diafiltration system. The solution is diafiltered until the conductivity reaches this initial value. When the value is reached, the concentration process begins and the conductivity is maintained.

Measurement of endotoxin content: The content of endotoxin in solution is determined in accordance with the pharmacopoeial method “EP2.6.14/USP<81> Bacterial Endotoxins - Method D Chromogenic Kinetic.” The Bacterial Endotoxin Test (BET) is used to detect or quantify endotoxins from Gram-negative bacteria using a lysate of horseshoe crab amoebocytes (Limulus polyphemus or Tachypleus tridentatus). The method used for this test is a chromogenic method based on the appearance of staining after cleavage of a synthetic peptide-chromogenic complex. In practice, a sample of the product is prepared to obtain a sample concentration of 10 mg/ml and using a predetermined dilution (1:1000) tested for endotoxin content using a standard curve for comparison purposes.

Sample Gelatin Measurement : UV spectroscopic analysis was used to determine the gelatin content of the solutions described herein. A calibration curve for the content of gelatin in the sample was built by measuring the UV absorption of the calibration samples (concentration range from 0.006 to 0.26 mg/ml) at a wavelength of 220 nm. An appropriate amount of test samples was measured, placed in a 100 ml volumetric vessel, and made up to volume with deionized water so that the theoretical concentration of these solutions was about 0.05 mg/ml. The UV absorption of the samples was measured at 220 nm, from which the actual concentration of gelatin in mg/ml was determined. Based on this value, the percentage of gelatin in the test samples was determined.

Salt Concentration Measurement: Salt concentration can be determined by conductivity (i.e. using a conductivity meter). A calibration curve can be obtained by measuring the conductivity of a saline solution over a range of salt concentrations. Gelatin may have some residual electrolytes; accordingly, the initial concentration may be, for example, ~0.8-1 mS/cm. By adding salt to a gelatin solution, the conductivity can be measured (which will be referred to electrolyte residue + added salt. For example, ~13 mS/cm). The removal of the added salt can be controlled during the diafiltration process until its amount again reaches the original value. At this point, it can be assumed that all added salt has been removed.

Salt concentration or conductivity can be measured when preparing a gelatin solution; after adding salt to the gelatin solution to form a gelatin saline solution; and throughout the diafiltration process.

Measurement of dispersion characteristics (study in vitro ): Characteristics of dispersion (studyin vitro): a minimum of 5 tablets are subjected to the study. First prepare a beaker containing about 200 ml of purified water at 20 ± 0.5°C. Each tablet is then removed from the blister pack and placed on the surface of the water. The time is taken as the period of time required for each tablet to completely moisten or disintegrate. Humidification corresponds to the period of time required for complete hydration of the tablet. Moistening of the tablet may occur in discrete areas that eventually fuse together, with the entire tablet becoming hydrated. The study of dispersion is considered complete when the central part of the tablet is a moistened mass. Thus, the moistening time is determined from the moment the central part of the tablet is moistened, since this is the thickest part of the tablet. Moisture time is recorded for each of the five tablets. The maximum time for each study is 60 seconds. Time exceeding this value may be written simply as “greater than 60 seconds”. Disintegration corresponds to the time required for the tablet to disintegrate. This time can be determined from the beginning of the disintegration of the tablet at the edges. The disintegration time is recorded for each of the five tablets. The maximum time for each study was 60 seconds. Time exceeding this value may be logged as “greater than 60 seconds”. In some cases, the tablet will not completely hydrate or completely disintegrate during this time. Sometimes hard lumps may be present in the tablet; in other cases, the surface of the tablet may not be wetted at all. In addition, the entire tablet may be covered with a hard crust. In case of occurrence of these phenomena, this is recorded in the description with an indication of “hard lumps” or “remaining crust”, as the case may be. The formation of "hard lumps" and/or "crust" may indicate the destruction of the microstructure during lyophilization. In FIG. 9A-C show a simplified side view and a top view of the three possible non-dispersed states of the tablets as they appear in water. The photographs, in figa-C, show some typical tablets in similar categories. The criterion for the study of dispersion characteristics is that 5 tablets can be completely moistened and/or disintegrate into a palpable mass without the presence of hard lumps and crust in 60 seconds or less. In some embodiments, the dosage forms described herein can completely hydrate and/or disintegrate into a palpable mass without the presence of hard lumps and/or crust in 60 seconds or less.

Definitions

Unless otherwise indicated, all technical terminology, designations and other technical and scientific terms or terminology used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms with common meanings are defined herein for clarity and/or reference purposes, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from what is commonly understood in the art.

The reference to “about”, when applied to values or parameters in this document, includes (and also describes) deviations that relate to that value or parameter as such. For example, a description referring to "about X" includes a description of "X". In addition, an indication of the phrases “less than”, “greater than”, “maximum”, “at least”, “less than or equal to”, “greater than or equal to”, or other similar phrases, followed by a list of values or parameters, is for applying a phrase to each value or parameter in a list of values or parameters. For example, a statement that a solution has a concentration of at least about 10 mM, about 15 mM, or about 20 mM means that the solution has a concentration of at least about 10 mM, at least about 15 mM, or at least about 20 mm.

As used herein, the singular forms are also intended to include plural forms, unless the context clearly indicates otherwise. It should also be appreciated that the term “and/or” as used herein refers to and encompasses all possible combinations of one or more of the associated listed elements. In addition, it should be appreciated that the terms "comprises", "comprising", "comprises" and/or "comprising", as used herein, refer to the presence of predetermined features, integers, steps, operations, elements, components, and /or units, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, units and/or groups thereof.

In this application, in the text and in the figures, several numerical ranges are described. The described numerical ranges essentially include any range or value within the described numerical ranges, including endpoints, even if the description does not specifically indicate the limitation of the range, since the present invention can be practiced with respect to all the described numerical ranges.

The foregoing description is provided to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, the present invention is not limited to the present embodiments, but should cover the widest scope in accordance with the principles and features disclosed herein.

Claims (37)

1. A method for reducing the level of endotoxin in gelatin, including: dissolving a salt in a gelatin solution containing gelatin and a solvent to form a gelatin saline solution, wherein the endotoxin content of the gelatin is at least 6000 EU/g, the salt concentration of the gelatin saline solution is 125-300 mM and the salt is sodium chloride ; filtering the gelatin saline solution with an anion exchange adsorber, whereby the content of endotoxin in the gelatin saline filtrate is less than 2500 EU/g; and desalting the gelatin-brine filtrate to form a low endotoxin gelatin solution, the endotoxin content being less than 2500 EU/g. 2. The method according to claim 1, characterized in that the salt concentration in the gelatin saline solution is 145-155 mm. 3. The method according to claim 1 or 2, characterized in that the gelatin solution is a 1-20% by weight solution of gelatin. 4. Process according to any one of claims 1 to 3, characterized in that the salt is sodium chloride. 5. The method according to any one of claims 1 to 4, characterized in that the gelatin is fish gelatin. 6. Method according to any one of claims 1 to 5, characterized in that the dissolution of the salt in the gelatin solution comprises heating the gelatin solution to 50-70°C. 7. Method according to any one of claims 1 to 6, characterized in that the solvent is water. 8. A method according to any one of claims 1 to 7, characterized in that the endotoxin content of the gelatin saline solution and the low endotoxin gelatin solution is less than 1000 EU/g. 9. A method according to any one of claims 1 to 8, wherein filtering the gelatin saline solution through an anion exchange adsorber can reduce the endotoxin content of the solution by at least 95%. 10. The method according to any one of claims 1 to 9, characterized in that at least 85% of the amount of gelatin remains in the gelatin saline filtrate after filtering the gelatin saline through an anion exchange adsorber. 11. The method according to any one of claims 1 to 10, characterized in that the desalting of the gelatin saline filtrate is carried out by diafiltration. 12. The method of claim 11 wherein the diafiltration process comprises diluting the gelatin saline filtrate with a second solvent and filtering the dilute gelatin saline filtrate to form a dilute gelatin solution filtrate. 13. The method of claim 12, wherein the dilute gelatin saline filtrate is filtered until the conductivity of the dilute gelatin solution filtrate is less than 25% of the gelatin solution conductivity. 14. The method according to claim 12 or 13, characterized in that the ratio of the gelatin saline filtrate and the second solvent is 1:1-1:4. 15. The method according to any one of claims 12-14, characterized in that the second solvent is removed from the dilute gelatin solution filtrate to form a low endotoxin gelatin solution. 16. The method of claim 15 wherein the second solvent is removed from the dilute gelatin solution filtrate until the weight of the dilute gelatin solution filtrate is less than 5% by weight of the gelatin saline filtrate. 17. The method according to any one of claims 12 to 16, characterized in that the second solvent contains water. 18. The method of any one of claims 1 to 17, further comprising removing the solvent from the low endotoxin gelatin solution to form low endotoxin gelatin that has an endotoxin content of less than 2500 EU/g. 19. Low endotoxin gelatin solution prepared by a method comprising: dissolving a salt in a gelatin solution containing gelatin and a solvent to form a gelatin saline solution, wherein the endotoxin content of the gelatin is at least 6000 EU/g of gelatin, the salt concentration of the gelatin saline solution is 125-300 mM, and the salt is chloride sodium; filtering the gelatin saline solution with an anion exchange adsorber, whereby the content of endotoxin in the gelatin saline filtrate is less than 2500 EU/g; desalting the gelatin-brine filtrate to form a low endotoxin gelatin solution having an endotoxin content of less than 2500 EU/g. 20. A method for producing a lyophilized tablet for delivery of a pharmaceutically active ingredient, comprising: dissolving a salt in a gelatin solution containing gelatin and a solvent to form a gelatin saline solution, wherein the endotoxin content of the gelatin is at least 6000 EU/g, the salt concentration of the gelatin saline solution is 125-300 mM, and the salt is sodium chloride ; filtering the gelatin saline solution with an anion exchange adsorber, whereby the content of endotoxin in the gelatin saline filtrate is less than 2500 EU/g; desalting the gelatin-brine filtrate to form a low endotoxin gelatin solution having an endotoxin content of less than 2500 EU/g; dispensing the composition containing the low endotoxin gelatin solution into a pre-made mould; and lyophilizing the low endotoxin gelatin solution to form a lyophilized tablet. 21. Lyophilized tablet for the delivery of a pharmaceutically active ingredient obtained by a method including: dissolving a salt in a gelatin solution containing gelatin and a solvent to form a gelatin saline solution, wherein the endotoxin content of the gelatin is at least 6000 EU/g, the salt concentration of the gelatin saline solution is 125-300 mM, and the salt is sodium chloride ; filtering the gelatin saline solution with an anion exchange adsorber, whereby the content of endotoxin in the gelatin saline filtrate is less than 2500 EU/g; desalting the gelatin-brine filtrate to form a low endotoxin gelatin solution having an endotoxin content of less than 2500 EU/g; dispensing the composition containing the low endotoxin gelatin solution into a pre-made mould; and lyophilizing the low endotoxin gelatin solution to form a lyophilized tablet.

Images