Spherical Protein Particles and Methods of Making and Using Them

Abstract

This invention relates to SPPs, spherical nanocrystalline composite particles or crystalline SPPs of biologically active proteins or compositions, including formulations, comprising such SPPs, spherical nanocrystalline composite particles or crystalline SPPs. More particularly, methods are provided for the production of SPPs, spherical nanocrystalline composite particles or crystalline SPPs of high concentrations of biologically active proteins, and for the preparation of stabilized SPPs, spherical nanocrystalline composite particles or crystalline SPPs for use alone, or in dry or slurry compositions. This invention also relates to methods for stabilization, storage and delivery of biologically active proteins using SPPs, spherical nanocrystalline composite particles or crystalline SPPs. The present invention further relates to methods using SPPs, spherical nanocrystalline composite particles or crystalline SPPs, or compositions or formulations comprising such SPPs, spherical nanocrystalline composite particles or crystalline SPPs, for biomedical applications, including biological delivery to humans and animals.

Description

TECHNICAL FIELD OF THE INVENTION[0001]This invention relates to spherical protein particles (“SPPs”), spherical nanocrystalline composite particles and crystalline SPPs, methods for producing them and methods and compositions, including formulations, for using them.[0002]More particularly, the present invention further relates to methods using SPPs, spherical nanocrystalline composite particles and crystalline SPPs for biological delivery to humans and animals. More specifically, the SPPs, spherical nanocrystalline composite particles and crystalline SPPs of this invention can be used to provide alternative dosage / delivery forms, e.g., aerosol, needleless injection, for delivery of biologically active pharmaceutical proteins.[0003]The present invention further relates to methods using SPPs, spherical nanocrystalline composite particles or crystalline SPPs, or compositions, including formulations, containing them, for biomedical applications, including more particularly, highly conce...

AI Technical Summary

Benefits of technology

[0204]Another advantage of the present invention is that SPPs, spherical nanocrystalline composite particles or crystalline SPPs of biologically active proteins, or compositions or formulations comprising them, can be dried by lyophilization (see Example 26, Method 3). Lyophilization, or freeze-drying allows water to be separated from the composition or formulation. The SPPs, spherical nanocrystalline composite particles or crystalline SPPs, or compositions or formulations comprising them, are first frozen and then placed in a high vacuum. In a vacuum, the crystalline H2O sublimes, leaving behind the intact SPP, spherical nanocrystalline composite particle or crystalline SPP, or composition or formulation thereof, containing only the tightly bound water. Such processing further stabilizes the SPP, spherical nanocrystalline composite particle or crystalline SPP, or composition or formulation thereof, and allows for easier storage and transportation at typically encountered ambient temperatures.

[0205]The SPPs, spherical nanocrystalline composite particles or crystalline SPPs of this invention may also be spray-dried (see Example 26, Method 6). Spray drying allows water to be separated from the SPP, spherical nanocrystalline composite particle or crystalline SPP, or composition or formulation thereof. It is highly suited for the continuous production of dry solids in either powder, granulate or agglomerate form from liquid feedstocks as solutions, emulsions, and pumpable suspensions. Spray drying involves the atomization of a liquid feedstock into a spray of droplets and contacting the droplets with hot air in a drying chamber. The sprays are produced by either rotary (wheel) or by nozzle atomizers. Evaporation of moisture from the droplets and formation of dry particles proceed under controlled temperature and airflow conditions. Relatively high temperatures are needed for spray drying operations. However, heat damage to products is generally only slight, because of an evaporative cooling effect during the critical drying period and because the subsequent time of exposure to high temperatures of the dry material may be vary short. Powder is discharged continuously from the drying chamber. Operating conditions and dryer design are selected according to the drying characteristics of the product and the powder specification. Spray drying is an ideal process where the end product must comply with precise quality standards regarding particle size distribution, residual moisture content, bulk density and particle shape.

[0206]This feature is especially desirable for therapeutic proteins and protein vaccines, including anti-idiotypic antibodies, which can be dispensed into single dose sterile containers (“ampules”) or alternatively, any desired increment of a single dose as a slurry, in a composition or formulation. The ampules containing the dispensed slurries or compositions or formulations can then be capped, batch frozen and lyophilized under sterile conditions. Such sterile containers can be transported throughout the world and stored at ambient temperatures. Such a system is useful for providing sterile vaccines and therapeutic proteins to remote and undeveloped parts of the world. At the point of use, the ampule is rehydrated with the sterile solvent or buffer of choice and dispensed. For such preparations, minimal or no refrigeration is required.

[0207]The SPPs, spherical nanocrystalline composite particles or crystalline SPPs of this invention may also be nitrogen-dried (see Example 26, Method 1), air-dried (see Example 26, Method 5), air-dried after addition of organic solvents (see Example 26, Method 4), or vacuum oven-dried (see Example 26, Method 2).

[0208]In another embodiment of this invention, SPPs, spherical nanocrystalline composite particles or crystalline SPPs of biologically active proteins according to this invention may be crosslinked for additional stability. This allows for the use of such SPPs, spherical nanocrystalline composite particles or crystalline SPPs, or compositions or formulations comprising them, in areas of pH extremes, such as the gastrointestinal tract of humans and animals. For example, SPPs, spherical nanocrystalline composite particles or crystalline SPPs of biologically active proteins or vaccines, e.g., monoclonal antibody or anti-idiotypic antibody SPPs, spherical nanocrystalline composite particles or crystalline SPPs, may be crosslinked using one of a variety of crosslinkers, including, but not limited to, Dimethyl 3,3′-dithiobispropionimidate.HCl (DTBP), Dithiobis (succinimidylpropionate) (DSP), Bis maleimido-hexane (BMH), Bis[Sulfosuccinimidyl]suberate (BS), 1,5-Difluoro-2,4-dinitrobenzene (DFDNB), Dimethylsuberimidate.2HCl (DMS), Disuccinimidyl glutarate (DSG), Disulfosuccinimidyl tartarate (Sulfo-DST), 1-Ethyl-3-[3-Dimethylaminopropyl]carbodiimide hydrochloride (EDC), Ethylene glycolbis[sulfosuccinimidylsuccinate] (Sulfo-EGS), N-[g-maleimidobutyryloxy]succinimide ester (GMBS), N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB), Sulfosuccinimidyl-6-[a-methyl-a-(2-pyridyldithio)toluamido]hexanoate (Sulfo-LC-SMPT), Bis-[b-(4-azidosalicylamido) ethyl]disulfide (BASED) and glutaraldehyde (GA).

[0209]In a further embodiment of this invention, SPPs, spherical nanocrystalline composite particles or crystalline SPPs of a protein, such as an intact antibody or scFv fragment of an antibody, may be radiolabelled to be used in antibody radiation therapies. In such a therapy, for example, an SPP, spherical nanocrystalline composite particle or crystalline SPP containing a radiolabelled anti-cancer antibody or scFv fragment, or a composition or formulations comprising them, can be delivered according to this invention, to the site of the cancer. After delivery, the released antibody or scFv fragment binds to its targeted cancer antigen and delivers the radioisotope directly to the cancerous cells or tumor. The release of the antibody may be timed according to this invention. Theoretically, useful radiolabels include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I. Practically, however, in vivo use in radiotherapies would limit the radiolabel to 131I, 90Y, or any other radiolabels defined by a short half-life. For example, the monoclonal antibody Rituximab has been labelled with 90Yttrium (90Y), in order to be used for radioimmunotherapy in patients with non-Hodgkin's lymphomas. This compound is commercially available as Zevalin™ (IDEC Pharmaceuticals, (San Diego, Calif.)).Encapsulation of SPPs, Spherical Nanocrystalline Composite Particles or Crystalline SPPs of Biologically Active Proteins in Polymeric Carriers:

Problems solved by technology

Protein drugs are generally formulated for parenteral administration, i.e., injection or infusion, because of their extremely poor bioavailability.

As a result, medical care for patients who require parenteral administration of protein drugs is often expensive and time-consuming.

Furthermore, patient compliance is often problematic, especially for those patients who require long-term treatment.

These methods are problematic because they typically denature proteins by heat and mechanical stress.

The method disclosed in the '910 patent requires suspending the protein of interest in 90% organic solvent, which is not suitable for a number of proteins.

Furthermore, the method disclosed in Morita requires the addition of organic solvents, e.g., methylene chloride, to remove the PEG used in a previous step, which, as stated above, is not suitable for a number of proteins.

Also, the method disclosed in Morita requires the use of PEG, which may or may not stabilize the protein being used.

Another limitation of the Morita method is that the disclosed technique involves rapid cooling of the material and can be applied only to freeze stable products.

Ice formation is usually destructive to the protein crystal lattice, which destabilizes the protein molecule, and sometimes leads to the formation of amorphous precipitate.

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