WO2016202713A1

WO2016202713A1 - Method of freezing protein solutions

Info

Publication number: WO2016202713A1
Application number: PCT/EP2016/063422
Authority: WO
Inventors: Martin List
Original assignee: F. Hoffmann-La Roche Ag; Hoffmann-La Roche Inc.
Priority date: 2015-06-15
Filing date: 2016-06-13
Publication date: 2016-12-22

Abstract

The present invention relates to a method for freezing a protein solution wherein the protein solution is frozen as scale ice and uses of the resulting scale ice.

Description

Method of freezing protein solutions

The present invention relates to a method for freezing liquid protein or peptide solutions in the form of scale ice and uses thereof.

Liquid protein or peptide solutions are widely used as therapeutics for the treatment of various diseases. An important class of therapeutic proteins marketed as liquid solutions are mono- clonal antibodies such as Rituxan, Herceptin, Avastin. The handling and processing of therapeutic protein or peptide solutions usually involves the freezing of the protein or peptide solutions for storage and transport. The freezing of therapeutic protein or peptide solutions often involves the filling of the protein or peptide solutions in Cryovessels and then deep-freezing the protein or peptide solution by a cooling media. The frozen protein or peptide solution is then stored and transported in the Cryovessels. Typical Cryovessels contain a few liters up to a few hundreds of liters.

A drawback of this method is that concentration gradients are formed in the solution, caused by freezing from the cold exchange surface towards the center of the container. The degree of gradient formation depends on the speed of freezing and the thickness of the ice to be formed. The thickness of the ice proportionally slows down freezing because of its insulation properties. Concentration gradients lead to inhomogeneity and may cause degradation of the protein. Furthermore the Cryovessels are costly and the handling of the empty Cryovessels is time intensive and expensive e.g. cleaning, sterilization and transport of the Cryovessels. Furthermore, the freezing of a protein solution in a Cryovessel or any other larger volume leads to an ice block which has to be thawed completely and homogenized in order to be able to take a sample of the protein solution or to process only part of the solution. The repetition of thaw freeze cycles leads to protein denaturation and the loss of protein activity.

Therefore, there is a need for a method for freezing proteins solutions which overcomes at least in part the drawbacks of the prior art methods. The present invention provides a method to freeze a protein or peptide solution with reduced formation of concentration gradients, by limiting the thickness of the resulting ice to < 5 mm, preferably 2 - 3- mm. The use of a continuous process to freeze protein or peptide solutions in above dimensions, making the process independent of the batch size, resulting in homogenous ice chips (scale ice), limiting formation of concentration gradients to the thickness of the chips.

SHORT DESCRIPTION OF THE FIGURES Fig. 1A and IB show a schematic of a state of the art protein solution freezing process taking place in a cryovessel. Fig. 1 A shows the protein solution at the start of the freezing process. At the start of the freezing process the protein molecules (API molecules) are evenly distributed in the solvent and the solution is homogenous. Fig. IB shows the protein solution during the freezing process during the freezing process ice crystals form at the edge of the cryovessel inte- rior pushing API molecules and other dissolved compounds towards the center. The solution becomes inhomogeneous.

Fig.2 shows a schematic of the inventive freezing method of a protein solution in thin layer. The temperature gradient in the freezing solution is reduced compared to the state of the art freezing methods and the resulting frozen solution (scale ice) is characterized by a homogenous concentration of API molecules in the frozen solution;

Fig. 3 shows a schematic view of a continuous ice maker.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention a scale ice machine can be used to perform the method of the present invention. The scale ice machine is self-emptying, applying only minimal mechanical stress to the ice, easy to clean, and performs in a continuous mode. Basic principle is a body in cylindrical shape. The body is double walled, allowing chilling it down to a temperature below about -7 °C. The protein solution is fed in a constant adjustable flow to run down the inner wall of the body.

In one embodiment of the present invention, the scale ice formed at the inner wall of the scale machine is removed by compressing the cylindrical wall slightly into an oval shape. The ice is brittle and will crack loose from the wall, subsequently falling down to the bottom.

In one embodiment of the present invention, the scale ice formed at the inner wall of the scale machine is removed by a flexible in-liner in the body of the ice machine. The in-liner is to be made from a suitable elastomer. Tight contact with the body of the ice machine may be achieved by applying under pressure between in-liner and body. Cracking of the ice is caused by applying overpressure between in-liner and body. In one embodiment of the present invention, the scale ice formed at the inner wall of the scale machine is removed by a rotating helical screw, moving along the body in a planetary type path. Distance to the body is adjustable. The helical screw touches the ice spot wise, causing it to crack and fall to the bottom. The term "scale ice" refers to ice mainly consisting of large, flat, thin pieces of ice (5-1 mm) having a temperature of about -7°C. Scale ice can be produced by freezing ice onto the inner wall of a large vertical drum. A rotating knife then goes around the surface, cracking the ice from the walls.

The term "protein" as used herein refers to an amino acid chain, where the chain has great- er than 50 amino acid residues, which can be obtained, for example, from either chemical synthesis or DNA-based recombinant methods.

The term "peptide" as used herein refers to an amino acid chain, where the chain has from 8 to 50 amino acid residues.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi- specific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')₂; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is di- rected against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

Pharmaceutical fformulations of a therapeutic protein, in particular a monoclonal antibody, can be prepared by mixing such protein having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohex- anol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically accepta- ble carriers herein further include insterstitial drug dispersion agents such as soluble neutral- active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluroni- dase glycoproteins, such as rHuPH20 (HYLENEX^®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Claims

1. A method for freezing a liquid protein or peptide solution with reduced formation of protein concentration gradients, wherein the protein solution is frozen as scale ice and the resulting scale ice has a thickness smaller than about 5 mm, preferably about 2 - 3 mm.

2. The method of claim 1, wherein the protein solution comprises an antibody, preferably a monoclonal antibody, more preferably a therapeutic antibody.

3. The method of claim 1 or 2, wherein the protein solution is a pharmaceutical formulation comprising a monoclonal antibody.

4. The method of claim 1 to 3, wherein the method is a continuous process.

5. The method of claim 1 to 4, wherein the scale ice is produced by an automatic scale ice machine.

6. A method of storing a frozen protein solution comprising the steps of: a) freezing a protein solution according to claims 1 to 5, b) filing the scale ice in bags for storage, preferably disposable bags, c) storing the scale ice bags at a suitable temperature, usually of about - 10°C to -70°C.

7. A method of transporting a frozen protein solution comprising the steps of: a) freezing a protein solution according to a method of claims 1 to 5, b) filling the scale ice in bags, c) transporting the scale ice bags to a location for storage or further processing.

8. A method of dispensing a protein solution comprising the steps of: a) dispensing the required amount of scale ice produced according a method of claims 1 to 5, b) thawing the frozen scale ice in a suitable container and c) dispensing the thawed solution of step b) in containers for patient application.

9. Use of a scale ice machine for the production of a frozen protein or peptide solution.