KR20190117631A - Excipient Compounds for Protein Processing Treatment - Google Patents

Abstract

The present invention provides a viscosity-lowering excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and low molecular weight aliphatic polyacids; And adding a viscosity-lowering excipient compound in a viscosity-lowering content to a carrier solution for a protein-related process comprising the protein of interest, and a method of improving the parameters of a protein-related process, A carrier solution is disclosed that includes a dissolved liquid medium and a viscosity-lowering excipient and is lower in viscosity than the control solution, which is substantially the same as the carrier solution except that a viscosity-lowering excipient is present.

Description

Excipient Compounds for Protein Processing Treatment

Related Applications

This application claims the benefit for US Provisional Application No. 62 / 459,893, dated February 16, 2017. The entire contents of these US provisional applications are incorporated herein by reference.

The present invention relates generally to formulations for the delivery and processing of biopolymers.

Biopolymers can be used for therapeutic or non-therapeutic purposes. Biopolymer-based therapeutics such as antibody or enzyme preparations are widely used to treat diseases. Non-therapeutic biopolymers, such as enzymes, peptides, and structural proteins, have utility in non-therapeutic uses such as home, nutrition, industrial, and industrial use.

Biopolymers used for therapeutic purposes must be formulated to allow introduction into the body to treat a disease. For example, in certain circumstances, antibody and protein / peptide biopolymer formulations are advantageously delivered by the subcutaneous (SC) or intramuscular (IM) route, rather than by intravenous (IV) infusion. In order to achieve better patient compliance and ease of SC or IM infusion, the liquid volume of the syringe is typically limited to 2 to 3 cc, and the viscosity of the formulation is typical so that the formulation can be delivered using conventional medical devices and small caliber needles. Should be less than about 20 centipoise (cP). These delivery parameters do not always meet the dosing requirements for the delivered agent.

Antibodies, for example, may have to be delivered at high dose levels to exert the intended therapeutic effect. To deliver antibodies at high dose levels using a limited liquid volume, the formulation may require high concentrations of the antibody in the delivery vehicle and may often exceed the 150 mg / mL level. At this dose level, the viscosity versus concentration curve of the protein solution goes through a linear-nonlinear transition, and as the concentration increases, the viscosity of the formulation becomes significantly higher. However, high viscosities are not suitable for standard SC or IM delivery systems. Solutions of biopolymer-based therapeutics are also susceptible to stability problems such as precipitation, turbidity, opalescence, denaturation and gel formation, reversible or irreversible aggregation. Stability issues either limit the shelf life of the solution or require special handling.

One method for preparing injectable protein formulations is to convert the therapeutic protein solution into a powder that can be reconstituted to produce a suspension suitable for SC or IM delivery. Lyophilization is the standard technique for preparing protein powders. Freeze-drying, spray drying and even super-critical-fluid extraction after precipitation have also been used to prepare protein powders for future reconstitution. Powder suspensions have a low pre-dissolution viscosity (relative to solutions of the same total volume) and may therefore be suitable for SC or IM injection as long as the particles are small enough to pass through the needle. However, there is an inherent risk that protein crystals present in powder form can trigger an immune response. Uncertain protein stability / activity after re-dissolution causes additional problems. There is still a need in the art for the preparation of low viscosity protein preparations for therapeutic purposes while avoiding the problems caused by protein powder suspensions.

In addition to the therapeutic uses of the aforementioned proteins, biopolymers such as enzymes, peptides and structural proteins can be used for non-therapeutic uses. Such non-therapeutic biopolymers can be produced in a number of different ways, for example, delivered from plant sources, animal sources or from cell culture.

Non-therapeutic proteins can be prepared, transported, stored and handled as granular or powdered substances or as solutions or typically as dispersions in water. Biopolymers for non-therapeutic use may be spherical or fibrous proteins, and certain forms of such materials may limit solubility or exhibit high viscosity upon dissolution. Such solution properties can cause problems in formulating, handling, storing, pumping and performance of non-therapeutic materials, and thus methods are needed to lower the viscosity and improve solubility and stability of non-therapeutic protein solutions.

Proteins are complex biopolymers, each with a uniquely folded 3D structure and surface energy map (hydrophobic / hydrophilic region and charge). In the case of concentrated protein solutions, these macromolecules can interact strongly and even inter-lock in a complex manner depending on their actual morphology and surface energy distribution. Strong and specifically interacting "hot spots" cause protein clustering, increasing the viscosity of the solution. To address this problem, several excipient compounds are used in biotherapeutic formulations to reduce the viscosity of the solution by delaying local interactions and clustering. These attempts are adjusted individually, often empirically, and sometimes through in silico simulation. Combinations of excipient compounds may be provided, but optimization of such combinations should again be done empirically, case by case.

There is still a need in the art for a truly universal approach to lowering the viscosity of protein preparations at certain concentrations under non-linear conditions. There is also a further need in the art to achieve this viscosity drop while maintaining protein activity. It would also be desirable to adapt the viscosity-lowering system for use in formulations with an adjustable sustained release profile and for formulations suitable for depot injection. There is also a need for improvements in the manufacturing process of proteins and other biopolymers.

The invention, in embodiments, a protein; And an excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and low molecular weight aliphatic polyacids. It relates to a liquid formulation, added in a viscosity-reducing amount. In an embodiment, the protein is a PEGylated protein and the excipient is a low molecular weight aliphatic polyacid. In an embodiment, the formulation is a pharmaceutical composition, the therapeutic formulation comprises a therapeutic protein, and the excipient compound is a pharmaceutically acceptable excipient compound. In an embodiment, the formulation is a non-therapeutic formulation and the non-therapeutic formulation comprises a non-therapeutic protein. In an embodiment, the viscosity-lowering content lowers the viscosity of the formulation below the viscosity of the control formulation. In an embodiment, the viscosity of the formulation is at least about 10% less than the viscosity of the control formulation, or at least about 30% less than the viscosity of the control formulation, or at least about 50% less than the viscosity of the control formulation, or At least about 70% or less than the viscosity of the control formulation or at least about 90% or less to the viscosity of the control formulation. In an embodiment, the viscosity is less than about 100 cP, or less than about 50 cP, or less than about 20 cP, or less than about 10 cP. In an embodiment, the excipient compound has a molecular weight of <5000 Da, or <1500 Da, or <500 Da. In an embodiment, the formulation comprises at least about 25 mg / mL protein, or comprises at least about 100 mg / mL protein, or comprises at least about 200 mg / mL protein, or at least about 300 protein. in mg / mL. In an embodiment, the formulation comprises about 5 mg / mL to about 300 mg / mL excipient compound, or about 10 mg / mL to about 200 mg / mL excipient compound, or about 20 mg excipient compound / mL to about 100 mg / mL, or the excipient compound at about 25 mg / mL to about 75 mg / mL. In an embodiment, the formulation has improved stability compared to the control formulation. In an embodiment, the excipient compound is a hindered amine. In an embodiment, the hindered amine is selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin and acesulfame potassium. In an embodiment, the hindered amine is caffeine. In an embodiment, the hindered amine is an anesthetic compound for topical injection. Hindered amines may have independent pharmacological properties, and hindered amines may be present in amounts with pharmacological effects independent of the formulation. In an embodiment, the hindered amine can be present in the formulation in an amount less than a therapeutically effective amount. Independent pharmacological activity may be local anesthetic activity. In an embodiment, the hindered amine with independent pharmacological activity is combined with a second excipient which further lowers the viscosity of the formulation. The second excipient compound may be selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin and acesulfame potassium. In an embodiment, the formulation can comprise an additional substance selected from the group consisting of preservatives, surfactants, sugars, polysaccharides, arginine, proline, hyaluronidase, stabilizers and buffers.

The invention also includes administering a liquid therapeutic agent to a mammal; The therapeutic agent comprises a therapeutically effective amount of the therapeutic protein; The formulation further comprises a pharmaceutically acceptable excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and low molecular weight aliphatic polyacids; A therapeutic agent relates to a method of treating a disease or disorder in a mammal, wherein the agent is effective for treating the disease or disorder. In an embodiment, the therapeutic protein is a PEGylated protein and the excipient compound is a low molecular weight aliphatic polyacid. In an embodiment, the excipient is a hindered amine. In an embodiment, the hindered amine is a local anesthetic compound. In an embodiment, the formulation is administered by subcutaneous infusion or intramuscular infusion or intravenous infusion. In an embodiment, the excipient compound is present in the therapeutic formulation in a viscosity-lowering content, wherein the viscosity-lowering content decreases the viscosity of the therapeutic agent below the viscosity of the control formulation. In an embodiment, the therapeutic formulation has improved stability compared to the control formulation. In an embodiment, the excipient compound is essentially a pure substance.

The invention also includes administering a liquid therapeutic formulation by infusion; The therapeutic agent comprises a therapeutically effective amount of the therapeutic protein; The formulation further comprises a pharmaceutically acceptable excipient compound selected from the group consisting of topical injectable anesthetic compounds; Pharmaceutically acceptable excipient compounds are added to the formulation in a viscosity-lowering content; Reduces the pain experienced when administering a therapeutic formulation comprising an excipient compound compared to when a control therapeutic formulation is administered in a mammal; A method of reducing pain at the site of infusion of a therapeutic protein in a mammal in need, wherein the control therapeutic formulation does not comprise an excipient compound but otherwise is identical to the therapeutic formulation.

The present invention, in embodiments, comprises a therapeutic protein; And preparing a protein liquid formulation comprising an excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and low molecular weight aliphatic polyacids; The protein liquid formulation shows improved stability compared to the control protein liquid formulation; A method for improving the stability of a protein liquid formulation, wherein the control protein liquid formulation contains no excipient compounds and otherwise is identical to the protein liquid formulation. The stability of the liquid formulation may be cold storage conditions stability, room temperature stability or elevated temperature stability.

In addition, the present invention, in embodiments, the protein; And an excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and low molecular weight aliphatic polyacids, wherein the presence of the excipient compound in the formulation is a protein. Features that improve protein-protein interactions as measured by diffusion interaction parameter kD or second virial coefficient B22. In an embodiment, the agent is a therapeutic agent, which comprises a therapeutic protein. In an embodiment, the formulation is a non-therapeutic formulation, which comprises a non-therapeutic protein.

The invention also relates to a method of improving a protein-related process comprising, in an embodiment, the steps of providing the above-described liquid formulation and using the same in a processing method. In an embodiment, the processing method comprises filtration, pumping, mixing, centrifugation, membrane separation, lyophilization or chromatography. In an embodiment, the processing method is selected from the group consisting of cell culture harvesting, chromatography, virus inactivation and filtration. In an embodiment, the processing method is a chromatography process or a filtration process. In an embodiment, the filtration process is a virus filtration process or an ultrafiltration / diafiltration process.

The present invention also provides a viscosity comprising one or more excipient compounds selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, and diones and sulfones. Providing a lowering excipient additive and adding one or more excipient compounds in a viscosity-lowering content to a carrier solution for a protein-related process comprising the protein of interest, whereby the parameters are improved. It relates to a method for improving the parameters of a related process. In an embodiment, the parameter can be selected from the group consisting of protein production cost, protein yield, protein yield and protein production efficiency. The parameter may be a proxy parameter. In an embodiment, the protein-related process is an upstream treatment process. The carrier solution for the upstream treatment process may be a cell culture medium. In an embodiment, when the carrier solution is a cell culture medium, adding the excipient additive to the carrier solution comprises the first substep of adding the excipient additive to the supplemental medium to prepare the excipient-containing supplement medium, and the excipient A second substep of adding the containing supplemental medium to the cell culture medium. In other embodiments, the protein-related process is a downstream treatment process. The downstream process may be a chromatography process, and the chromatography process may be a protein-A chromatography process. In an embodiment, the chromatography process recovers the protein of interest, wherein the protein of interest improves protein-related parameters selected from the group consisting of improved purity, improved yield, reduced particle size, reduced misfolding, or reduced aggregation compared to the control solution. It features. In an embodiment, the protein-related parameter to be improved is improved recovery of the subject protein from the chromatography process. In other embodiments, the protein-related process is selected from the group consisting of filtration, infusion, syringe, pumping, mixing, centrifugation, membrane separation, and lyophilization, wherein the process selected is characterized by force consumption may be low. In an embodiment, the protein-related process is selected from the group consisting of a cell culture process, a cell culture harvesting process, a chromatography process, a virus inactivation process and a filtration process. In an embodiment, the protein-related process is a virus inactivation process and the virus inactivation process is performed at a level of pH about 2.5 to about 5.0, or the virus inactivation process is performed at a higher pH than the control process. In other embodiments, the protein-related process is a filtration process. The filtration process can be a virus removal filtration process or an ultrafiltration / diafiltration process. The filtration process may be characterized by filtration-related parameter improvements. Improvements in filtration-related parameters can be faster filtration rates than control solutions. An improvement in the filtration-related parameters may be that the amount of aggregated protein is less compared to the amount of aggregated protein generated by the control filtration process. An improvement in the filtration-related parameters may be that the mass transfer efficiency is higher than the mass transfer efficiency of the control filtration process. An improvement in the filtration-related parameters may be that the concentration or yield of the target protein is higher than the concentration or yield of the target protein achieved by the control filtration process.

The present invention also relates to the aforementioned process, wherein the viscosity-lowering excipient additive comprises two or more excipient compounds. In an embodiment, the at least one excipient compound is a hindered amine. In an embodiment, the at least one excipient compound is a group consisting of caffeine, saccharin, acesulfame potassium, aspartame, theophylline, taurine, 1-methyl-2-pyrrolidone, 2-pyrrolidinone, niacinamide and imidazole Is selected from. In an embodiment, the at least one excipient compound is selected from the group consisting of caffeine, taurine, niacinamide and imidazole. In an embodiment, the at least one excipient compound is an anionic aromatic excipient, and in some embodiments, the anionic aromatic excipient can be 4-hydroxybenzenesulfonic acid. In embodiments, the viscosity-lowering content is about 1 mg / mL to about 100 mg / mL of one or more excipient compounds, or the viscosity-lowering content is about 1 mM to about 400 mM, or viscosity- The descent content is from about 2 mM to about 150 mM. In an embodiment, the carrier solution comprises an additional substance selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers and buffers. In an embodiment, the subject protein is a therapeutic protein and the therapeutic protein can be a recombinant protein or monoclonal antibody, polyclonal antibody, antibody fragment, fusion protein, PEGylated protein, antibody-drug conjugate, synthetic polypeptide, protein May be selected from the group consisting of fragments, lipoproteins, enzymes and structural peptides. In an embodiment, the method further comprises adding a second viscosity-lowering excipient to the carrier solution, wherein adding the second viscosity-lowering excipient further improves the parameters.

The present invention also relates to a carrier solution comprising a liquid medium in which a target protein is dissolved and a viscosity-lowering additive, wherein the carrier solution has a low viscosity compared to the control solution. The carrier solution may further comprise additional materials selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers and buffers.

1 is a block diagram illustrating the steps of a fermentation process (“upstream process”) for producing therapeutic proteins, eg monoclonal antibodies.
FIG. 2 is a block diagram showing the steps of a purification process (“downstream process”) to prepare a therapeutic protein, eg, a monoclonal antibody.

The present invention relates to a formulation capable of enabling delivery of a concentrated protein solution and a method for producing the same. In an embodiment, the methods described herein can make a therapeutic or non-therapeutic protein into a lower viscosity liquid formulation or a higher concentration liquid formulation as compared to traditional protein solutions. In an embodiment, the methods herein can produce liquid formulations with improved stability compared to traditional protein solutions. A stable formulation is one that substantially retains its physical or chemical stability and its therapeutic or non-therapeutic efficacy upon storage in a storage environment, whether in a cold storage environment, a room temperature environment or an elevated storage environment. Advantageously, a stable formulation may also provide protection against the aggregation or precipitation of the protein dissolved in the formulation. For example, a cold storage environment may involve storage in a refrigerator or freezer. In some examples, the cold storage environment may involve storage at temperatures of 10 ° C. or less. In a further example, the cold storage environment involves storage at a temperature of about 2 ° C to about 10 ° C. In another example, the cold storage environment involves storage at a temperature of about 4 ° C. In an additional example, the cold storage environment entails storage at freezing temperatures, such as about −20 ° C. or less. In another example, the cold storage environment entails storage at a temperature of about -20 ° C to about 0 ° C. Room temperature storage environments may involve storage at ambient temperatures, such as from about 10 ° C to about 30 ° C. An elevated storage environment may involve storage at temperatures above a certain temperature. Stability at elevated temperatures, eg, at about 30 ° C. to 50 ° C., can be used as part of accelerated aging experiments to predict long term storage at typical ambient (10-30 ° C.) conditions.

Proteins in solution tend to form entanglements, which can interfere with the therapeutic or non-therapeutic efficacy of the protein and limit the translational mobility of the entangled chains. Well known to those skilled in the art. In an embodiment, an excipient compound described herein can inhibit protein clustering due to specific interactions between the excipient compound and the target protein in solution. The excipient compounds described herein may be natural or synthetic, and are preferably substances recognized as generally safe substances ("GRAS") designated by the FDA.

One. Justice

For the purposes herein, the term “protein” refers to an amino acid sequence having a chain length sufficient to form an individual tertiary structure, typically having a 1-3000 kD molecular weight. In some embodiments, the molecular weight of the protein is about 50-200 kD; In other embodiments, the molecular weight of the protein is about 20-1000 kD or about 20-2000 kD. In contrast to the term “protein”, the term “peptide” refers to an amino acid sequence that does not have an individual tertiary structure. A wide variety of biopolymers are included within the term "protein". For example, the term “protein” refers to a therapeutic or non-therapeutic protein, including antibodies, aptamers, fusion proteins, PEGylated proteins, synthetic polypeptides, protein fragments, lipoproteins, enzymes, structural peptides, and the like. Can be.

As a non-limiting example, therapeutic proteins include hormones and prohormones (eg, insulin and proinsulin, glucagon, calcitonin, thyroid hormones (T3 or T4 or thyroid-stimulating hormones), parathyroid hormones, follicle-stimulating hormones, luteinizing hormones). , Growth hormone, growth hormone releasing factor, etc.); Coagulation and anti-coagulation factors (e.g., tissue factors, von Willebrand's factor, factor VIIIC, factor IX, protein C, plasminogen activators (eurokinase, tissue-type plasminogen activators), thrombin ); Cytokines, chemokines and inflammatory mediators; Interferon; Colony-stimulating factor; Interleukins (eg, IL-1 to IL-10); Growth factors (eg, vascular endothelial growth factor, fibroblast growth factor, platelet derived growth factor, transformation growth factor, neurotrophic growth factor, insulin-like growth factor, etc.); albumin; Collagen and elastin; Hematopoietic factors (eg, erythropoietin, thrombopoietin, etc.); Osteoinductive factor (eg, bone morphogenic proteins); Receptors (eg, integrins, cadherin, etc.); Surface membrane proteins; Transport protein; Regulatory proteins; Antigenic proteins (eg, viral components acting as antigens); And mammalian proteins, such as antibodies. The term "antibody" is used herein in its broadest sense and includes, but is not limited to, monoclonal antibodies (eg, full-length antibodies with immunoglobulin Fc regions), single chain molecules, bispecific and multispecific antibodies, diabodies. , Antibody-drug conjugates, antibody compositions with multiple epitope specificities, polyclonal antibodies (e.g., polyclonal immunoglobulins used as therapeutics in immunocompromised patients), antibody fragments (e.g., Fc, Fab, Fv and F (ab ') 2) is used to include. In addition, an antibody may be referred to as an "immunoglobulin". Antibodies are understood to target specific proteins or non-protein "antigens" that are biologically important substances; A therapeutically effective amount of an antibody administered to a patient may form a complex with the antigen to modify biological properties to exert a therapeutic effect in the patient.

In an embodiment, the protein is PEGylated, ie, comprising poly (ethylene glycol) (“PEG”) and / or poly (propylene glycol) (“PPG”) units. PEGylated proteins or PEG-protein conjugates have utility as therapeutic uses due to beneficial properties such as solubility, pharmacokinetics, pharmacodynamics, immunogenicity, kidney clearance and stability. Non-limiting examples of PEGylated proteins include PEGylated interferon (PEG-IFN), PEGylated anti-VEGF, PEG protein conjugate drugs, Adagen, Pegaspargase, Pegpill Pegfilgrastim, Pegloticase, Pegvisomant, Pegylated Epoetin-β and Cetolizumab pegol.

PEGylated proteins can be synthesized by various methods, such as the reaction of a protein with a PEG reagent having one or more reactive functional groups. Reactive functional groups on the PEG reagent may form linkages with proteins at target protein sites such as lysine, histidine, cysteine and N-terminus. Typical PEGylation reagents have reactive functional groups, such as aldehyde, maleimide or succinimide groups, which have specific reactivity to targeted amino acid residues on the protein. PEGylated reagents may have a PEG chain length of about 1 to about 1000 PEG and / or PPG repeat units. Another pegylation method involves glyco-pegylation, in which the protein is first glycosylated and then the glycated residues are PEGylated in a second step. Some PEGylation processes proceed with the help of enzymes such as sialyltransferase and transglutaminase.

PEGylated proteins may provide therapeutic advantages over natural non-pegylated proteins, but such materials may have physical or chemical properties that make purification, dissolution, filtration, concentration and administration difficult. PEGylation of proteins can result in higher viscosity of the solution compared to natural protein, so in general, PEGylated protein solution formulations should be at lower concentrations.

The protein therapeutic agent is preferably formulated in a stable, low viscosity solution for administration to the patient in a minimal injection volume. For example, subcutaneous (SC) or intramuscular (IM) injection of the drug should generally be a small infusion volume, preferably less than 2 mL. The SC and IM injection routes are well suited for self-administration administration, which is a less expensive and more accessible form of treatment compared to intravenous (IV) injections performed only under direct medical supervision. Formulations for SC or IM injections should have a low viscosity and generally less than 30 cP, preferably less than 20 cP, so that the therapeutic solution can easily pass through the gauge with a small needle. The combination of small injection volume and low viscosity poses a problem of using PEGylated protein therapeutics in the SC or IM injection route.

Proteins having therapeutic efficacy may be referred to as “therapeutic proteins”; An agent comprising a therapeutically effective amount in a therapeutic protein may be referred to as a "therapeutic agent." In addition, a therapeutic protein contained in a therapeutic formulation may be referred to as its "protein active ingredient." Typically, a therapeutic formulation will include a protein active ingredient in a therapeutically effective amount, and excipients, with or without other optional ingredients. As used herein, the term "treatment" includes both treatment of existing disorders and prevention of disorders. Therapeutic proteins include, for example, bevacizumab, trastuzumab, adalimumab, infliximab, etanercept, darbepoetin alfa, epoetin alfa proteins such as epoetin alfa, cetuximab, filgrastim and rituximab. Other therapeutic proteins will be familiar to those skilled in the art.

"Treatment" includes any measure intended to cure, treat, alleviate, ameliorate, remedy or otherwise benefit a disorder, including preventing or delaying the onset of the symptoms of the disorder and / or alleviating or ameliorating the symptoms of the disorder. Patients in need of treatment include both those already suffering from a particular disorder, and patients in need of preventing the disorder. A disorder is any condition that affects the homeostatic well-being of a mammal, such as an acute or chronic disease, or a pathological condition that makes a mammal vulnerable to an acute or chronic disease. Non-limiting examples of disorders include cancer, metabolic disorders (eg diabetes), allergic disorders (eg asthma), skin disorders, cardiovascular disorders, respiratory disorders, blood disorders, musculoskeletal disorders, inflammatory or rheumatic disorders, Autoimmune disorders, gastrointestinal disorders, urinary system disorders, sexual and reproductive disorders, and neurological disorders. For therapeutic purposes, the term "mammal" may refer to any animal classified as a mammal, including humans, livestock, pets, farm animals, sports animals, working animals, and the like. "Treatment" may therefore include both veterinary and human treatments. For convenience, a mammal in which such "treatment" is in progress may be referred to as a "patient." In certain embodiments, the patient can be a patient of any age, such as an intrauterine fetal animal.

In an embodiment, the treatment comprises providing a therapeutically effective amount of a therapeutic agent to the mammal in need. A “therapeutically effective amount” is at least the minimum concentration of therapeutic protein administered to a mammal in need thereof for achieving the prevention of a related disorder or for the treatment of an existing disorder, such treatment or prevention being a “therapeutic intervention”. Therapeutically effective amounts of various therapeutic proteins that may be contained as active ingredients in a therapeutic formulation may be familiar to those in the art; Or in the case of therapeutic proteins to be subsequently applied or developed for therapeutic intervention, the therapeutically effective amount can be determined by standard techniques performed by one of ordinary skill in the art within the scope of conventional experiments.

Proteins used for non-therapeutic purposes (ie, those that do not involve treatment), such as for home, nutritional, commercial, and industrial use, may be referred to as "non-therapeutic proteins." Formulations containing non-therapeutic proteins may be referred to as "non-therapeutic agents." Non-therapeutic proteins can be derived from plant sources, animal sources or produced from cell culture; It may also be an enzyme or structural protein. Non-therapeutic proteins can be used in home, nutritional, commercial, and industrial applications such as catalysts, human and animal nutrients, processing aids, cleaners, and waste disposal.

The main category of non-therapeutic biopolymers is enzymes. Enzymes have a number of non-therapeutic uses, for example as catalysts, as human and animal nutritional ingredients, as processing aids, as cleaners, and as waste treatments. Enzyme catalysts are used to accelerate various chemical reactions. Examples of enzyme catalysts for non-therapeutic use include catalase, oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase. Uses of enzymes in the nutritional aspects of humans and animals include nutraceuticals, nutrient sources of proteins, chelating or controlled delivery of micronutrients, digestive aids and supplements; It may be derived from amylases, proteases, trypsin, lactase and the like. Enzyme processing aids are used to improve the production of food and beverage products in processes such as baking, brewing, fermentation, juice processing and wine making. Examples of these food and beverage processing aids include amylase, cellulase, pectinase, glucanase, lipase and lactase. In addition, enzymes can be used to make biofuels. Ethanol for biofuels can be supported, for example, by enzymatic digestion of biomass feedstocks such as cellulosic materials and lignocellulosic materials. Treatment of cellulase and ligninase with these raw materials converts the biomass into fuel fermentable materials. In other commercial applications, enzymes are used as detergents, cleaners, stain lifting aids for laundry, dish washing, surface cleaning and equipment cleaning applications. Typical enzymes for this purpose include proteases, cellulases, amylases and lipases. In addition, non-therapeutic enzymes are used in a variety of commercial and industrial processes such as fabric softening with cellulase, leather processing, waste treatment, contaminated sediment treatment, water treatment, pulp bleaching and pulp softening and debonding. . Typical enzymes for this purpose are amylases, xylanases, cellulases and ligninases.

Other examples for non-therapeutic biopolymers include fibrous or structural proteins such as keratin, collagen, gelatin, elastin, fibroin, actin, tubulin or their hydrolyzed, degraded or derivatized forms. These materials are used in the manufacture and blending of food ingredients such as gelatin, ice cream, yoghurt and compaction; As a thickener, it is added to foods as a source of rheology modifiers, texture enhancers and nutritional proteins. In the cosmetic and personal care industry, collagen, elastin, keratin and hydrolyzed keratin are widely used as components in skin care and hair care products. Another example for a non-therapeutic biopolymer is whey protein such as beta-lactoglobulin, alpha-lactalbumin and serum albumin. These whey proteins are mass produced as a by-product of dairy operations and are used for a variety of non-therapeutic uses.

2. Measure

In an embodiment, the protein-containing formulations described herein exhibit resistance to monomer loss as measured by size exclusion chromatography (SEC) analysis. In the SEC assay of the present invention, the main analyte peak is generally due to the target protein contained in the formulation, which is called the monomer peak. The monomer peak represents the amount of the protein active ingredient in the target protein, eg, monomer state, as opposed to the aggregated state (dimer, trimer, oligomeric form, etc.) or the fragmented state. The monomer peak area can be compared to the total amount of peaks in monomer, aggregated and fragment form associated with the target protein. That is, the stability of the protein-containing preparation can be observed by the relative content of monomers after the elapsed time; The stability improvement of the protein-containing formulations of the invention can thus be measured as the higher monomer percentage after a certain elapsed time compared to the% monomer in the control formulation without excipients.

In an embodiment, the ideal stability is one having a monomer peak of 98-100% as measured by SEC analysis. In an embodiment, the stability improvement of the protein-containing formulation of the invention is measured as the higher monomer percentage after exposure to the stress condition, compared to the% monomer of the excipient-free control formulation when the control formulation is exposed to the same stress conditions. Can be. In embodiments, the stress condition may be cold storage, hot storage, air exposure, bubble exposure, shear condition exposure or freeze / thaw cycle exposure.

In an embodiment, the protein-containing formulations described herein exhibit resistance to increase in protein particle size as measured by dynamic light scattering (DLS) analysis. Herein, in the DLS analysis, the particle size of the protein in the protein-containing formulation can be observed by the median particle diameter. Ideally, the median particle diameter of the target protein represents the active ingredient in the monomeric state, rather than in the aggregated (dimer, trimer, oligomer, etc.) or fragmented state, and therefore does not vary relatively when performing DLS analysis. Should not. The increased median particle diameter may be indicative of aggregated protein. That is, the stability of the protein-containing formulation can be observed by the relative change in median particle diameter after elapsed time.

In an embodiment, the protein-containing formulations described herein are resistant to forming a polydisperse particle size distribution as measured in dynamic light scattering (DLS) analysis. In an embodiment, the protein-containing formulation can comprise a monodisperse particle size distribution of colloidal protein particles. In an embodiment, the ideal stability result is less than 10% change in median particle diameter compared to the initial median particle diameter of the formulation. In an embodiment, the stability improvement of the protein-containing formulations of the invention can be measured as a low percent change in median particle diameter after a certain elapsed time compared to the median particle diameter in an excipient-free control formulation. In an embodiment, the stability improvement of the protein-containing formulations of the invention is such that the percent change in median particle diameter after exposure to stress conditions is the percentage change in median particle diameter in an excipient-free control formulation exposed to the same stress conditions. Compared with%, it can be measured as small. In embodiments, the stress condition may be cold storage, high temperature storage, air exposure, bubble exposure, exposure to shear environment, or freeze / thaw cycle exposure. In an embodiment, the stability improvement of the protein-containing formulation therapeutic formulation of the present invention, when measured by DLS, results in a particle size distribution in an excipient-free control formulation when the polydisperse particle size distribution is exposed to the same stress conditions. Compared to the polydispersity of, it can be measured as low.

In an embodiment, the protein-containing formulation of the invention exhibits resistance to precipitation as measured by turbidity, light scattering and / or particle count analysis. In turbidity, light scattering or particle counting analysis, low values generally mean that the number of particles suspended in the formulation is smaller. An increase in turbidity, light scattering or particle counting may mean that the solution of the target protein is not stable. That is, the stability of the protein-containing formulation can be observed by the relative result of turbidity, light scattering or particle counting after elapsed time. In an embodiment, the ideal stability result is a low and relatively constant turbidity, light scattering or particle count value. In an embodiment, the stability improvement of the protein-containing formulation of the invention, after a certain period of time, results in more turbidity, light scattering or particle counting compared to the turbidity, light scattering or particle count values of the excipient-free control formulation. Can be measured as low. In an embodiment, improving the stability of the protein-containing formulations described herein includes turbidity of an excipient-free control formulation that is exposed to stress conditions and whose turbidity, light scattering or particle counts are exposed to the same stress conditions, As compared to light scattering or particle counting, it can be measured as being low. In embodiments, the stress condition may be cold storage, high temperature storage, air exposure, bubble exposure, exposure to shear environment, or freeze / thaw cycle exposure.

3. Therapeutic formulation

In one aspect, the formulations and methods described herein provide a stable liquid formulation with improved viscosity or reduced viscosity that comprises a therapeutic protein and an excipient compound in a therapeutically effective amount. In an embodiment, the formulation may improve the stability of the active ingredient at an acceptable concentration and at the same time providing an acceptable viscosity. In an embodiment, the formulation provides an improvement in stability compared to the control formulation; For the purposes herein, a control formulation is a formulation that contains the same protein active ingredient in all manners as the therapeutic formulation on a dry weight basis, except that the excipient compound is deficient. In an embodiment, the stability improvement of the protein containing formulation is when the percentage of soluble aggregates, microparticles, subvisible particles or gel form is low compared to the control formulation.

Viscosities of protein liquid formulations include, but are not limited to, the nature of the protein itself (eg, enzymes, antibodies, receptors, fusion proteins, etc.); Its size, three-dimensional structure, chemical composition and molecular weight; Its concentration in the formulation; Components other than the protein of the formulation; Preferred pH range; Storage conditions of the preparation; And various factors, such as the method of administering the formulation to the patient, can be affected. The most suitable therapeutic protein for use with the excipient compounds described herein is preferably essentially pure, i.e. free of contaminating proteins. In an embodiment, the “essentially pure” therapeutic protein comprises at least 90 wt%, or preferably at least 95 wt%, or more preferably, 99 wt% of the therapeutic protein, based on the total weight of therapeutic protein and contaminating protein in the composition. It is a protein composition containing at least by weight. For clarity, proteins added as excipients are not intended to be included in this definition. Therapeutic agents described herein are pharmaceutical-grade agents, ie, in a form in which the desired therapeutic effect of the protein active ingredient can be achieved, wherein the agent to be administered does not contain components that are toxic to the mammal and are used to treat the mammal. As a formulation for use, intended for use.

In an embodiment, the therapeutic formulation comprises at least 25 mg / mL protein active ingredient. In other embodiments, the therapeutic formulation comprises at least 100 mg / mL protein active ingredient. In other embodiments, the therapeutic formulation comprises at least 200 mg / mL protein active ingredient. The therapeutic formulation solution contains at least 300 mg / mL of the protein active ingredient. In general, the excipient compounds described herein may be added to the therapeutic formulation in an amount of about 5 to about 300 mg / mL. In an embodiment, an excipient compound can be added at an amount of about 10 to about 200 mg / mL. In an embodiment, an excipient compound can be added at an amount of about 20 to about 100 mg / mL. In embodiments, excipients may be added in an amount of about 25 to about 75 mg / mL.

Excipient compounds of various molecular weights are selected for particular beneficial properties when used in combination with a protein active ingredient in a formulation. Examples of therapeutic agents comprising excipient compounds are provided below. In an embodiment, the excipient compound has a molecular weight of <5000 Da. In an embodiment, the excipient compound has a molecular weight of <1000 Da. In an embodiment, the excipient compound has a molecular weight of <500 Da.

In an embodiment, the excipient compound described herein is added to the therapeutic formulation in a viscosity-lowering content. In an embodiment, the viscosity-lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 10% compared to the control formulation; For the purposes herein, a control formulation is a formulation containing the same protein active ingredient in all manners as the therapeutic formulation on a dry weight basis, except for the absence of excipient compounds. In an embodiment, the viscosity-lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 30% compared to the control formulation. In an embodiment, the viscosity-lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 50% compared to the control formulation. In an embodiment, the viscosity-lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 70% compared to the control formulation. In an embodiment, the viscosity-lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 90% compared to the control formulation.

In an embodiment, the viscosity-lowering content achieves a therapeutic formulation having a viscosity of less than 100 cP. In other embodiments, the therapeutic formulation has a viscosity of less than 50 cP. In other embodiments, the therapeutic formulation has a viscosity of less than 20 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term "viscosity" means a dynamic viscosity value as measured by the method described herein.

Therapeutic formulations according to the invention have particular beneficial properties. In an embodiment, the therapeutic agent is resistant to shear degradation, phase separation, clouding out, oxidation, deamidation, aggregation, precipitation and denaturation. In an embodiment, the therapeutic formulation is processed, purified, stored, syringed, filtered and centrifuged more efficiently than the control formulation. In an embodiment, the therapeutic agent is administered to the patient at a high concentration of therapeutic protein. In an embodiment, the therapeutic formulation is administered to the patient with little or no discomfort when encountering a similar formulation without a therapeutic excipient. In an embodiment, the therapeutic agent is administered as a depot injection. In an embodiment, the therapeutic agent extends the half-life of the therapeutic protein in the body.

These properties of the therapeutic formulations described herein include the use of small caliber needles for which clinical circumstances, that is, intramuscular injection tolerance of patients, and acceptable (eg 2-3 cc) injection volumes are typical for IM / SC purposes. In such circumstances, administration of the agent in an intravenous or subcutaneous injection will be permitted in situations in which the agent will be administered in an effective amount in one injection at one injection site under these conditions. In contrast, SC / IM injection of conventional formulations will not be suitable for the clinical situation, as injection of therapeutic proteins at similar doses using conventional formulation techniques will be limited due to the high viscosity of conventional formulations. Solutions with high concentrations of therapeutic proteins formulated with the addition of excipient compounds described herein can be administered to a patient using syringes or prefilled syringes.

In an embodiment, the therapeutic excipient has antioxidant properties that stabilize the therapeutic protein from oxidative damage. In an embodiment, the therapeutic formulation is stored at ambient temperature or in refrigerated conditions for an extended period of time without losing the appreciable potency of the therapeutic protein. In an embodiment, the therapeutic formulation is dried to store until needed; It is then reconstituted by addition of a suitable solvent, for example water. Advantageously, formulations prepared as described herein may be stable for extended periods of time, for months to years. If an exceptionally long storage period is desired, the formulation can be preserved in the freezer (and may be reactivated in the future) without fear of protein denaturation. In an embodiment, the formulation may be prepared for long term storage without cooling.

Methods of preparing the therapeutic formulations may be familiar to those skilled in the art. Therapeutic formulations of the invention can be prepared, for example, by adding an excipient compound to the formulation before or after the therapeutic protein is added to the solution. The therapeutic formulation can be processed, for example, by combining the therapeutic protein and excipient to a first (low) concentration and then filtering or centrifuging to produce a second (high) concentration of the therapeutic protein. Therapeutic formulations may be prepared using one or more excipient compounds having chaotrope, kosmotrope, hydrotrope and salts. Therapeutic formulations may be prepared with one or more excipient compounds using techniques such as encapsulation, dispersion, liposomes, vesicle formation, and the like. Methods of making a therapeutic formulation comprising an excipient compound described herein can include a combination of excipient compounds. In an embodiment, a combination of excipients may be useful for low viscosity, improved stability or pain reduction at the injection site. Other additives such as preservatives, surfactants, sugars, sucrose, trehalose, polysaccharides, arginine, proline, hyaluronidase, stabilizers, buffers and the like can be added to the therapeutic formulation during manufacture. As used herein, pharmaceutically acceptable excipient compounds are substances that are suitable and nontoxic for animal and / or human administration.

4. Non-Therapeutic Formulations

In one aspect, the formulations and methods described herein provide stable liquid formulations with improved or reduced viscosity, comprising an effective amount of a non-therapeutic protein and an excipient compound. In an embodiment, the formulation improves stability while providing an acceptable concentration and acceptable viscosity of the active ingredient. In an embodiment, the formulation provides an improvement in stability compared to the control formulation; For the purposes herein, a control formulation is a non-therapeutic formulation free of excipient compounds and a formulation containing the same protein active ingredient in all manners on a dry weight basis.

Viscosities of protein liquid formulations include, but are not limited to, the nature of the protein itself (eg, enzymes, structural proteins, degree of hydrolysis, etc.); Its size, tertiary structure, chemical composition and molecular weight; Its concentration in the formulation; Components of preparations other than proteins; Preferred pH range; And various storage factors such as storage conditions of the preparation.

In an embodiment, the non-therapeutic formulation comprises at least 25 mg / mL protein active ingredient. In other embodiments, the non-therapeutic formulation comprises at least 100 mg / mL protein active ingredient. In other embodiments, the non-therapeutic formulation comprises at least 200 mg / mL protein active ingredient. In another embodiment, the non-liquid therapeutic formulation comprises at least 300 mg / mL protein active ingredient. In general, the excipient compounds described herein are added to the non-therapeutic formulation in an amount of about 5 to about 300 mg / mL. In an embodiment, the excipient compound is added in an amount of about 10 to about 200 mg / mL. In an embodiment, the excipient compound is added in an amount of about 20 to about 100 mg / mL. In an embodiment, the excipient is added in an amount of about 25 to about 75 mg / mL.

Excipient compounds of various molecular weights are selected for particular beneficial properties when combined with the protein active ingredient in the formulation. Examples of non-therapeutic formulations comprising an excipient compound are provided below. In an embodiment, the excipient compound has a molecular weight of <5000 Da. In an embodiment, the excipient compound has a molecular weight of <1000 Da. In an embodiment, the excipient compound has a molecular weight of <500 Da.

In an embodiment, the excipient compound described herein is added to the non-therapeutic formulation in a viscosity-lowering content. In an embodiment, the viscosity-lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 10% compared to the control formulation; For the purposes herein, a control formulation is a formulation that contains the same protein active ingredient in all manners as the therapeutic formulation, except that there is no excipient compound on a dry weight basis. In an embodiment, the viscosity-lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 30% compared to the control formulation. In an embodiment, the lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 50% compared to the control formulation. In an embodiment, the lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 70% compared to the control formulation. In an embodiment, the lowering content is an amount of an excipient compound that lowers the viscosity of the formulation by at least 90% compared to the control formulation.

In an embodiment, the viscosity-lowering content makes a non-therapeutic formulation having a viscosity of less than 100 cP. In other embodiments, the non-therapeutic formulation has a viscosity of less than 50 cP. In other embodiments, the non-therapeutic formulation has a viscosity of less than 20 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term "viscosity" means a kinematic viscosity value.

Non-therapeutic formulations according to the invention may have certain beneficial properties. In an embodiment, the non-therapeutic agent exhibits resistance to shear degradation, phase separation, clouding out, oxidation, deamidation, aggregation, precipitation, and denaturation. In an embodiment, the therapeutic formulation can be effectively processed, purified, stored, pumped, filtered and centrifuged as compared to the control formulation.

In an embodiment, the non-therapeutic excipient has antioxidant properties that stabilize the non-therapeutic protein from oxidative damage. In an embodiment, the non-therapeutic formulation is stored at ambient temperature or in refrigerator conditions for an extended period of time without losing the appreciable potency of the non-therapeutic protein. In an embodiment, the non-therapeutic formulation is dried to store until needed; It can then be reconstituted by addition of a suitable solvent, for example water. Advantageously, formulations prepared as described herein are stable for extended periods of time, for months to years. If an exceptionally long storage period is desired, the formulation can be preserved in the freezer (and may be reactivated in the future) without fear of protein denaturation. In an embodiment, the formulation is prepared for long term storage without cooling.

Methods of preparing non-therapeutic formulations comprising the excipient compounds described herein may be familiar to those skilled in the art. For example, an excipient compound may be added to the formulation before or after the non-therapeutic protein is added to the solution. Non-therapeutic formulations may be prepared at a first (low) concentration and then advanced to a second (high) concentration by filtration or centrifugation. Non-therapeutic formulations may be prepared using one or more excipient compounds with chaotrope, kosmotrope, hydrotrope and salts. Non-therapeutic formulations may be prepared using one or more excipient compounds using techniques such as encapsulation, dispersion, liposomes, vehicle formation, and the like. Other additives can be added to the non-therapeutic formulations, including preservatives, surfactants, stabilizers and the like during manufacture.

5. Excipient compound

Reference is made herein to several excipient compound species, suitable for use with one or more therapeutic or non-therapeutic proteins, which may be configured to contain high concentrations of protein (s). Some of the excipient compound categories described below are as follows: (1) hindered amines; (2) anionic aromatics; (3) functionalized amino acids; (4) oligopeptides; (5) short-chain organic acids; (6) low molecular weight aliphatic polyacids; And (7) dions and sulfones. Without wishing to be bound by theory, excipient compounds referred to herein are those associated with specific fragments, sequences, structures, or sections of the therapeutic protein that will participate in interparticle (ie, protein-protein) interactions. I think. The association of these excipient compounds with a therapeutic or non-therapeutic protein can mask the interaction between proteins so that the protein can be formulated at high concentrations without excessively increasing the viscosity of the solution. The excipient compound may advantageously be water soluble and therefore suitable for use with an aqueous vehicle. In an embodiment, the excipient compound has a water solubility of> 10 mg / mL. In an embodiment, the excipient compound has a water solubility of> 100 mg / mL. In an embodiment, the excipient compound has a water solubility of> 500 mg / mL. Advantageously for therapeutic proteins, excipient compounds can be derived from biologically acceptable and non-immunogenic substances and are therefore suitable for pharmaceutical use. In therapeutic embodiments, the excipient compound may be metabolized in the body to produce byproducts that are biologically suitable and non-immunogenic.

a. Excipient Compound Category 1: Hindered Amine

High concentration solutions of therapeutic or non-therapeutic proteins can be prepared into formulations using hindered amine small molecules as excipient compounds. As used herein, the term “hindered amine” refers to a small molecule containing one or more bulky or sterically hindered groups consistent with the examples below. Hindered amines can be used in free base form, protonated form, or a combination thereof. In the protonated form, the hindered amine can be combined with anionic counterions such as chloride, hydroxide, bromide, iodide, fluoride, acetate, formate, phosphate, sulfate or carboxylate. Hindered amine compounds that can be used as excipient compounds include secondary amines, tertiary amines, quaternary ammonium, pyridinium, pyrrolidone, pyrrolidine, pyri, such that the excipient compound has a cationic charge in an aqueous solution at neutral pH. Ferridine, morpholine or guanidinium groups. Hindered amine compounds also include one or more bulky or sterically hindered groups, such as cyclic aromatic, cycloaliphatic, cyclohexyl, or alkyl groups. In an embodiment, the sterically hindered group can be basically an amine group such as dialkylamine, trialkylamine, guanidinium, pyridinium or quaternary ammonium. Without wishing to be bound by theory, it is believed that hindered amine compounds associate with aromatic moieties of proteins such as phenylalanine, tryptophan and tyrosine by cationic pi (pi) interactions. In an embodiment, the cationic group of the hindered amine can be affinity for the electron rich pi structure of the aromatic amino acid residue of the protein, so that it can mask this site of the protein, thereby lowering the tendency for the masked protein to associate and aggregate.

In an embodiment, the hindered amine excipient compound is imidazole, 1-methylimidazole, 4-methylimidazole, 1-hexyl-3-methylimidazolium chloride, histamine, 4-methylhistamine, alpha-methylhistamine , Betahistin, beta-alanine, 2-methyl-2-imidazoline, 1-butyl-3-methylimidazolium chloride, uric acid, potassium urate, betazole, carnosine, aspartame, It has a chemical structure comprising imidazole, imidazoline or imidazolidine groups or salts thereof, such as saccharin, acesulfame potassium, xanthine, theophylline, theobromine, caffeine and anserine. In an embodiment, the hindered amine excipient compound is dimethylethanolamine, dimethylaminopropylamine, triethanolamine, dimethylbenzylamine, dimethylcyclohexylamine, diethylcyclohexylamine, dicyclohexylmethylamine, hexamethylene acetabolite. Anide, poly (hexamethylene biguanide), imidazole, dimethylglycine, agmatine, diazabicyclo [2.2.2] octane, tetramethylethylenediamine, N, N-dimethylethanolamine, Ethanolamine phosphate, glucosamine, choline chloride, phosphocholine, niacinamide, isnicotinamide, N, N-diethyl nicotinamide, nicotinic acid sodium salt, tyramine, 3-aminopyridine, 2,4,6-trimethylpyridine, 3 Pyridine methanol, nicotinamide adenosine dinucleotide, biotin, morpholine, N-methylpyrrolidone, 2-pyrrolidinone, procaine, lidocaine, dicyandiamide-ta Lean adducts, 2-pyridylethylamine, dicyandiamide-benzyl amine adducts, dicyandiamide-alkylamine adducts, dicyandiamide-cycloalkylamine adducts and dicyandiamide-aminomethaneforces Selected from the group consisting of phonic acid adducts. In an embodiment, the hindered amine compound according to the present application is formulated as a protonated ammonium salt. In an embodiment, the hindered amine compound according to the invention is formulated as a salt with an inorganic anion or an organic anion as a counterion. In an embodiment, the high concentration solution of the therapeutic protein or non-therapeutic protein is formulated with a combination of caffeine and benzoic acid, hydroxybenzoic acid or benzenesulfonic acid as an excipient compound. In an embodiment, the hindered amine excipient compound is metabolized in the body to form a biologically friendly byproduct. In some embodiments, the hindered amine excipient compound is present in the formulation at a concentration of about 250 mg / ml or less. In additional embodiments, the hindered amine excipient compound is present in the formulation at a concentration of about 10 mg / ml to about 200 mg / ml. In yet another aspect, the hindered amine excipient compound is present in the formulation at a concentration of about 20 to about 120 mg / ml.

In an embodiment, certain hindered amine excipient compounds may have other pharmacological properties. For example, xanthine is one category of hindered amines that have independent pharmacological properties, such as stimulant properties and bronchodilator properties, upon systemic absorption. Representative xanthines include caffeine, aminophylline, 3-isobutyl-1-methylxanthine, paraxanthine, pentoxifylline, theobromine, theophylline and the like. Methylated xanthine is understood to affect cardiac contractility, heart rate and bronchial dilatation. In some embodiments, the xanthine excipient compound is present in the formulation at a concentration of about 30 mg / ml or less.

Another category of hindered amines with independent pharmacological properties is anesthetic compound for topical injection. Topical injectable anesthetic compounds have a three-component molecular structure of (a) a lipophilic aromatic ring, (b) an intermediate ester or amide linkage, and (c) a secondary or tertiary amine. Hindered amines. Hindered amines in this category are understood to interfere with nerve conduction by inhibiting the influx of sodium ions, leading to local anesthesia. The lipophilic aromatic ring of the local anesthetic compound may consist of carbon atoms (eg benzene ring) or may comprise heteroatoms (eg thiophene ring). Representative topical injectable anesthetic compounds include, but are not limited to, amylocaine, articaine, bupivicaine, butacaine, butanilicaine, chlorprocaine (chlorprocaine), cocaine, cyclomethycaine, dimethocaine, dimethocaine, editocaine, hexylcaine, isobucaine, levobupivacaine, Lidocaine, metabutethamine, metabutoxycaine, mepivacaine, meprylcaine, propoxycaine, prilocaine, procaine procaine, piperocaine, tetracaine, trimecaine, and the like. Topical injectable anesthetic compounds may have several advantages in protein therapeutic formulations, such as reduced viscosity, improved stability and lower pain upon injection. In some embodiments, the local anesthetic compound is present in the formulation at a concentration of about 50 mg / ml or less.

In an embodiment, hindered amines with independent pharmacological properties are used as excipient compounds according to the formulations and methods described herein. In some embodiments, an excipient compound with independent pharmacological properties is present in an amount that has no pharmacological effect or is not therapeutically effective. In other embodiments, an excipient compound with independent pharmacological properties is present in an amount that has a pharmacological effect and / or is therapeutically effective. In certain embodiments, hindered amines with independent pharmacological properties are used in combination with other excipient compounds selected to lower the viscosity of the formulation, wherein the hindered amines with independent pharmacological properties benefit from pharmacological activity. Used to provide. For example, topical injection anesthetic compounds can be used to lower the viscosity of the formulation and also to reduce pain upon injection of the formulation. Injection pain reduction can be caused by anesthetic characteristics; Or when a viscosity falls with an excipient | filler, a force may be low for injection. In other embodiments, topical injection anesthetic compounds can be used in combination with other excipient compounds that lower the viscosity of the formulation to provide the desired pharmacological benefit of reducing local sensation upon injection of the formulation.

b. Excipient Compound Category 2: Anionic Aromatic Compounds

High concentration solutions of therapeutic or non-therapeutic proteins can be formulated with anionic aromatic small molecule compounds as excipient compounds. Anionic aromatic excipient compounds may include aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, phenol, hydroxyaryl, heteroaromatic groups, or fused aromatic groups. Anionic aromatic excipient compounds may also include anionic functional groups such as carboxylates, oxides, phenoxides, sulfonates, sulfates, phosphonates, phosphates or sulfides. While anionic aromatic excipients may be described as acids, sodium salts, or the like, it is understood that excipients may be used in various salt forms. While not wishing to be bound by theory, anionic aromatic excipient compounds can associate with the cationic portion of the protein to mask this portion of the protein, thereby reducing the interaction between protein molecules causing the viscosity of the protein-containing formulation. It is regarded as a bulky steric hindrance molecule.

In embodiments, examples of the anionic aromatic excipient compounds include salicylic acid, aminosalicylic acid, hydroxybenzoic acid, aminobenzoic acid, para-aminobenzoic acid, benzenesulfonic acid, hydroxybenzenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, hydroquinone Sulfonic acid, sulfanilic acid, vanillic acid, vanillin, vanillin-taurine adducts, aminophenols, anthranilic acid, cinnamic acid, kumaric acid, adenosine monophosphate, indole acetic acid, potassium urate, furan dicarboxylic acid, furan-2-acrylic acid, Compounds such as 2-furanpropionic acid, sodium phenylpyruvate, sodium hydroxyphenylpyruvate, dihydroxybenzoic acid, trihydroxybenzoic acid, pyrogallol, benzoic acid and salts of the aforementioned acids. In an embodiment, the anionic aromatic excipient compound is formulated in an ionized form. In an embodiment, the anionic aromatic compound is formulated as a salt of a hindered amine, such as dimethylcyclohexylammonium hydroxybenzoate. In an embodiment, the anionic aromatic excipient compound is formulated with various counterions such as organic cations. In an embodiment, a high concentration solution of therapeutic or non-therapeutic protein is formulated with an anionic aromatic excipient compound and caffeine. In an embodiment, the anionic aromatic excipient compound is metabolized in the body to form a biologically suitable byproduct.

c. Excipient Compound Category 3: Functionalized Amino Acids

High concentration solutions of therapeutic or non-therapeutic proteins may be formulated with one or more functionalized amino acids, wherein oligopeptides comprising one functionalized amino acid or one or more functionalized amino acids may be used as excipient compounds. have. In an embodiment, the functionalized amino acid compound comprises a molecule ("amino acid precursor") that can be hydrolyzed or metabolized to become an amino acid. In an embodiment, the functionalized amino acid can comprise an aromatic functional group such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromatic group or fused aromatic group. In an embodiment, the functionalized amino acid compound may comprise esterified amino acids such as methyl, ethyl, propyl, butyl, benzyl, cycloalkyl, glyceryl, hydroxyethyl, hydroxypropyl, PEG and PPG esters. In an embodiment, the functionalized amino acid compound is selected from the group consisting of arginine ethyl ester, arginine methyl ester, arginine hydroxyethyl ester and arginine hydroxypropyl ester. In an embodiment, the functionalized amino acid compound is charged with the ionic compound at neutral pH in aqueous solution. For example, one amino acid can be derivatized by forming an ester such as acetate or benzoate and the hydrolysis product will be acetic acid or benzoic acid plus amino acid which is a natural substance. In an embodiment, the functionalized amino acid excipient compound is metabolized in the body to form a biologically suitable byproduct.

d. Excipient Compound Category 4: Oligopeptides

High concentration solutions of therapeutic or non-therapeutic proteins can be formulated with oligopeptides as excipient compounds. In an embodiment, the oligopeptide is designed so that the structure has a charged portion and a bulky portion. In an embodiment, the oligopeptide consists of 2-10 peptide subunits. Oligopeptides may be bifunctional, for example cationic amino acids coupled with non-polar amino acids, or anionic amino acids coupled with non-polar amino acids. In an embodiment, the oligopeptide consists of 2-5 peptide subunits. In an embodiment, the oligopeptide is a homopeptide such as polyglutamic acid, polyaspartic acid, poly-lysine, poly-arginine and poly-histidine. In an embodiment, the oligopeptide has a net cationic charge. In other embodiments, the oligopeptide is a heteropeptide such as Trp2Lys3. In an embodiment, the oligopeptide can have a cross structure such as an ABA repeating pattern. In an embodiment, the oligopeptide may comprise both anionic and cationic amino acids, for example Arg-Glu. Without wishing to be bound by theory, oligopeptides include structures capable of associating with proteins in a manner that reduces the intermolecular interactions that result in highly viscous solutions; For example, oligopeptide-protein associations can be charge-charge interactions that leave some non-polar amino acids to break the hydrogen bonds of the hydration layer around the protein, thereby reducing the viscosity. In some embodiments, oligopeptide excipients are present in the composition at up to about 50 mg / ml.

e. Excipient Compound Category 5: Short-chain Organic Acids

As used herein, the term "short chain organic acid" refers to a C2-C6 organic acid compound and salts, esters or lactones thereof. This category includes saturated and unsaturated carboxylic acids, hydroxy functionalized carboxylic acids and linear, branched or cyclic carboxylic acids. In an embodiment, the acid group of the short chain organic acid is carboxylic acid, sulfonic acid, phosphonic acid or salts thereof.

In addition to the four excipient categories described above, high concentration solutions of therapeutic or non-therapeutic proteins are, as excipient compounds, acidic or salt forms of short-chain organic acids such as sorbic acid, valeric acid, propionic acid, caproic acid and ascorbic acid. Can be formulated together. Examples of excipient compounds in this category include potassium sorbate, taurine, calcium propionate, magnesium propionate and sodium ascorbate.

f. Excipient Compound Category 6: Low Molecular Weight Aliphatic Polyacids

High concentration solutions of therapeutic or non-therapeutic PEGylated proteins can be formulated with certain excipient compounds that can lower the viscosity of the solution, such excipient compounds being low molecular weight aliphatic polyacids. As used herein, the term "low molecular weight aliphatic polyacid" is an organic aliphatic polyacid having a molecular weight <about 1500 and two or more acidic groups, wherein the acidic group is understood as a proton-donating moiety. Non-limiting examples of acidic groups include carboxylate, phosphonate, phosphate, sulfonate, sulfate, nitrate and nitrite groups and the like. Acidic groups of low molecular weight aliphatic polyacids may be in the form of anionic salts such as carboxylates, phosphonates, phosphates, sulfonates, sulfates, nitrates and nitrites; Its counterions can be sodium, potassium, lithium and ammonium. Specific examples of low molecular weight aliphatic polyacids useful for interacting with the PEGylated proteins described herein include maleic acid, tartaric acid, glutaric acid, malonic acid, citric acid, ethylenediaminetetraacetic acid (EDTA), aspartic acid, glutamic acid , Alendronic acid, etidronic acid and salts thereof. As the anionic salt form, a further example for a low molecular weight aliphatic acid phosphate (PO ₄ ^3-), hydrogen phosphate (HPO ₄ ^{3 -),} dimethyl hydrogen phosphate (H ₂ PO ₄ ^-), sulfate (SO ₄ ^{2 -),} bisulfate (HSO ₄ ^-), pyrophosphate ^{_{(P 2 O 7 4 -)}} , carbonate (CO ₃ ^2-) and bicarbonate (HCO ₃ ^-) and the like. The counterion of the anionic salt may be Na, Li, K or ammonium ion. In addition, these excipients may be used in combination with excipients. Herein, the low molecular weight aliphatic polyacids may also be alpha hydroxy acids, in which there is a hydroxy group adjacent to the first acidic groups such as glycolic acid, lactic acid and gluconic acid and salts thereof. In an embodiment, the low molecular weight aliphatic polyacid is in oligomeric form with more than two acidic groups, such as polyacrylic acid, polyphosphate, polypeptides and salts thereof. In some embodiments, the low molecular weight aliphatic polyacid excipient is present in the composition at a concentration of about 50 mg / ml or less.

g. Excipient Compound Category 7: Diones and Sulfones

Effective viscosity-lowering excipients include sulfone, sulfonamide or dione functional groups which are soluble in pure water up to 1 g / L or more at 298 K and have a net neutral charge at pH 7. It may be a molecule. Preferably, this molecule has a molecular weight of less than 1000 g / mol, more preferably less than 500 g / mol. Diones and sulfones effective in lowering viscosity have a plurality of hetero bonds, are water soluble, have a net charge of zero at pH 7, and are not strong hydrogen bond donors. Without wishing to be bound by theory, double binding characteristics may allow for weak pi-stacking interactions with proteins. In an embodiment, charged proteins are not effective because at high protein concentrations, and in proteins that produce high viscosity only at high concentrations, because electrostatic interaction is a longer range of interactions. The solvated protein surface is usually hydrophilic, making it water soluble. Hydrophobic regions of a protein are usually hidden within a three-dimensional structure, but the structure continues to unfold, unfolding and refolding (sometimes "breathing"), and the hydrophobic regions in contact with the hydrophobic regions of adjacent proteins May cause aggregation by interaction. The pi-stacking feature of diones and sulfone excipients can mask hydrophobic patches that may be exposed during this “breed”. Another important role of the excipient may be to break hydrophobic interactions and hydrogen bonds between closely located proteins, thereby effectively lowering the viscosity of the solution. Diones and sulfone compounds conforming to this content include dimethyl sulfone, ethyl methyl sulfone, ethyl methyl sulfonyl acetate, ethyl isopropyl sulfone, bis (methylsulfonyl) methane, methane sulfonamide, methionine sulfone, 1,2- Cyclopentanedione, 1,3-cyclopentanedione, 1,4-cyclopentanedione, butane-2,3-dione, and the like.

6. Protein / Excipient Solutions: Properties and Processes

In certain embodiments, hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, or short chain organic acids, low molecular weight aliphatic polyacids, and the aforementioned excipient compounds, such as diones and sulfones, or combinations thereof (hereinafter, Solutions of therapeutic or non-therapeutic proteins, formulated with "excipient additives") result in improving protein-protein interaction characteristics, as measured by protein diffusion interaction parameter kD or second viral coefficient B22. Bring it. As used herein, “improvement” of one or more protein-protein interaction parameters achieved by a test formulation using the excipient compound or combinations thereof described above is similar to a corresponding formulation that does not contain a particular excipient compound or excipient additive. As compared to the test agent in, it may mean a reduction in attracting protein-protein interactions. These improvements can be identified by measuring specific parameters that apply to the entire process or one aspect thereof, which parameters are any measurement parameters attached to the process that can quantify the strain and compare it to the previous state or control. The parameters may be related to the process itself, such as efficiency, unit cost, yield or speed.

In addition, the parameter may be a proxy parameter related to a feature aspect of a larger process. For example, parameters such as kD or B22 parameters may be referred to as proxy parameters. kD and B22 can be measured using standard techniques in the industry and can be indicators of process-related parameters such as protein stability in solution or improvement of solution properties. Without being bound by theory, it is understood that a high negative kD value may mean that the protein has a strong attracting interaction, which may cause aggregation, instability and rheological problems. When formulated in the presence of certain excipient compounds or combinations thereof described above, the same protein may have improved proxy parameters that are lower negative kD values or kD values close to or higher than zero, which improved proxy parameters Related to the improvement of process-related parameters.

In embodiments, certain excipient compounds described above or combinations thereof, such as hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, and / or diones and sulfones Silver, heat transfer, gas transfer, centrifugation, chromatography, membrane separation, concentration by centrifugation, tangential flow filtration, radial flow filtration, axial flow filtration, freeze drying and gel electrophoresis It is used to improve protein-related processes such as the preparation, processing, aseptic filling, purification and analysis of protein-containing solutions using processing methods such as filtration, syringe, transport, pumping, mixing, heating or cooling. In this process and related protein-related processes, the protein of interest is dissolved in a solution carried through the processing apparatus. This solution, referred to herein as a "carrier solution," refers to a cell culture medium (e.g., containing a secreted target protein), a cell lysate obtained after cytolyzing a host cell (if the target protein is present in the cytolysate), Eluents (containing the protein of interest after chromatographic separation), electrophoretic solutions, transport solutions for transporting the protein of interest through the tubes of the processing apparatus, and the like. Carrier solutions containing the protein of interest may also be referred to as protein-containing solutions or protein solutions. As described in more detail below, one or more viscosity-lowering excipients may be added to the protein-containing solution to improve various processing aspects. As used herein, the terms "improve", "improvement" and the like refer to a beneficial change in the subject parameter in the carrier solution when the parameter is compared with the same parameter measured in the control solution. As used herein, "control solution" means a solution substantially free of viscosity-enhancing excipients but substantially similar to a carrier solution. As used herein, a “control process”, such as a control filtration process, a control chromatography process, etc., is a protein-related process that is substantially similar to the subject protein-related process and is performed with a control solution instead of a carrier solution.

For example, in a process where a protein-containing solution is pumped through a conduit (e.g., a flow chamber, a pipe or a tubing), before or during the pumping process, a viscosity-lowering excipient is added to the protein solution as described above, It is possible to substantially reduce the forec and power required for pumping. Fluids generally exhibit flow resistance, i. The power required for pumping, P, is calculated as head H and capacity Q as shown in the following formula:

P to HQ (Equation 1)

Viscous fluids tend to increase the power required for the pump, lower pumping efficiency, reduce pump head and capacity, and increase frictional resistance in the pipe. The addition of the aforementioned viscosity-enhancing excipients to the protein solution before or during pumping can reduce the processing cost by substantially reducing the head (H, equation 1) or the capacity (Q, equation 1) or both. Viscosity lowering benefits can be seen, for example, by improved throughput, increased yield or reduced processing time. In addition, friction reduction from fluid transfer through conduits represents a significant proportion of the costs associated with such fluid transport. The addition of the viscosity-lowering excipients described above to the protein solution before or during pumping can substantially lower the processing cost by reducing the friction involved in the pumping process. Processing cost measurements are process parameters that can be improved by using viscosity-lowering excipients.

The process and method of processing such protein solutions may be improved in efficiency due to the low viscosity, improved solubility or improved stability of the protein in the solution during the preparation, processing, purification and analysis steps. Proxy parameter measurements or process efficiency measurements, such as viscosity, solubility or stability of proteins in solution, are process parameters that can be improved by using viscosity-lowering excipients. It is understood that several other factors negatively affect protein viscosity, solubility and stability during the process. For example, protein-containing solutions may be used during manufacture and purification, including, but not limited to, significant shear stresses generated by manipulating protein solutions through typical process operations such as pumping, mixing, centrifugation, and filtration. You will be exposed to a variety of physical stressors. In addition, during this process step, bubbles may be trapped in the fluid that the protein can adsorb. This interfacial tension, coupled with the typical shear stress encountered during the process, can unfold the adsorbed protein molecules and cause aggregation. In addition, significant protein unfolding may occur during pump cavitation events and upon exposure to solid surfaces such as ultrafiltration and diafiltration membranes during manufacture. This phenomenon can worsen protein folding and product quality.

For Newtonian fluids, the stress τ imposed by a given process is determined by the shear rate γ and the viscosity η of the fluid, as represented by the following equation:

τ = γη (Equation 2)

By formulating a protein solution together with one or more of the aforementioned excipient compounds or combinations thereof, the viscosity of the solution is reduced, ie the shear stress to which the protein solution is exposed is reduced. Reduced shear stress can improve the stability of the formulation under treatment, as evident, for example, by better or better preferred measurement results of the process parameters. Such improved process parameters can include metrics such as protein aggregation, reduced levels of particles or subvisibles (identified macroscopically as turbidity), reduced production loss or improved overall yield. . As another example of an improved process parameter, a decrease in viscosity of a protein-containing solution can reduce the processing time of the solution. The processing time of a given unit operation is generally inversely proportional to the shear rate. Thus, for certain specific stresses, decreasing the viscosity of the protein solution by adding the above excipient compounds or combinations thereof involves increasing the shear rate (γ, equation 2), i.e. shortening the process time.

While the process is in progress, it is understood that the protein in solution may be a desired protein active ingredient, eg, a therapeutic protein or a non-therapeutic protein. Using the excipients mentioned herein to facilitate the processing of the protein active ingredients can result in increased production rates or yields of the protein active ingredients, improved efficiency of certain processes, reduced energy use, and the like. This arbitrary performance is a process parameter that is improved by using viscosity-lowering excipients. It is also understood that protein contaminants may occur during certain processing techniques, such as during the fermentation and purification steps of bioprocessing. If the contaminants are removed faster, more thoroughly or more effectively, the processing of the desired protein, ie the protein active ingredient, can also be improved; This performance is a process parameter that is improved by using viscosity-lowering excipient compounds or additives. As mentioned herein, by lowering the viscosity of a solution, improving protein stability, and / or increasing protein solubility, certain excipients described herein may improve delivery of the desired protein active ingredient, and improper protein contamination Improve the removal of material; These two effects are process parameters that are improved by using viscosity-lowering excipients or additives, which show that these excipients or additives improve the overall process of protein production.

Special platform unit manipulations for therapeutic protein production and purification give other examples of the beneficial use of the viscosity-lowering excipients described herein, and yet other examples where these excipients or additives improve process parameters. For example, the introduction of one or more of the above-mentioned viscosity-enhancing excipients into such production and purification processes can provide substantial molecular stability and recovery improvements and reduced operating costs.

In the art, techniques widely used to produce and purify therapeutic proteins such as monoclonal antibodies are generally understood to consist of a fermentation process and a subsequent series of purification process steps. Fermentation or upstream processing (USP) involves culturing a therapeutic protein in a bioreactor, typically using a bacterial or mammalian cell line. The USP may, in embodiments, include steps such as those shown in FIG. 1. In an embodiment, the purification or downstream process (DSP) can comprise steps such as those shown in FIG. 2.

As shown in FIG. 1, USP may begin with thawing 102 a vial from a master cell bank (MCB). The MCB can be amplified as shown in step 104 to build a working cell bank (not shown), and / or to create a working stock for further production. As shown in steps 108 and 110, the cells are cultured in a series of seed and production bioreactors to produce the bioreactor product 112, from which desired, as shown in step 114, is obtained. Therapeutic proteins can be harvested. After the harvesting step 114, the products are further purified (ie, DSP, as described in detail in FIG. 2 and described below in detail), or these products are typically frozen and stored by storage at about −80 ° C. Bulk can be stored.

In an embodiment, protein production by cell culture techniques can be improved by using the aforementioned excipients, as confirmed by improvements in process-related parameters. In an embodiment, a preferred excipient can be added in USP in an amount effective to reduce the viscosity of the cell culture medium by at least 20%. In other embodiments, preferred excipients may be added in the USP in an amount effective to reduce the viscosity of the cell culture medium by at least 30%. In embodiments, preferred excipients may be added to the cell culture medium in an amount of about 1 mM to about 400 mM. In an embodiment, a preferred excipient can be added to the cell culture medium in an amount of about 20 mM to about 200 mM. In embodiments, preferred excipients may be added to the cell culture medium in an amount of about 25 mM to about 100 mM. Preferred excipients or combinations of excipients may be added directly to the cell culture medium, or it may be a constituent of a more complex supplemental medium, eg, a nutrient-containing solution or "feed," each formulated and added to the cell culture medium. Solution, "as a constituent of" solution ". In an embodiment, a second viscosity-lowering compound can be added to the carrier solution either directly or via a supplemental medium, wherein the second viscosity-lowering solution further improves specific subject parameters.

As discussed below, there are a number of process-related parameters that can be improved using one or more viscosity-lowering excipients in the USP. For example, in embodiments, the use of viscosity-lowering excipients may improve parameters such as cell proliferation rate and / or proliferation level at stages such as inoculum amplification 104 and cell culture 108 and 110, or And / or improve proxy parameters that correlate with improvements in various process parameters. For example, the aforementioned excipients may be added to the USP process at the same stage as the production bioreactor stage 110 to lower the viscosity of the cell culture medium, thus improving heat transfer efficiency and gas transfer efficiency. The cell culture process requires oxygen injection to allow the cells to express the protein, that is, the diffusion of oxygen into the cell may be a rate-limiting step, thus improving the oxygen uptake rate by improving the gas delivery efficiency due to the viscosity decrease of the solution. It can improve the rate or amount and / or efficiency of protein expression. In this regard, parameters such as oxygen uptake and gas delivery efficiency may be considered proxy parameters, where improvements are associated with process parameter improvements, ie, improved protein expression or improved process efficiency. In another example, the availability of viscosity-lowering excipients may improve the proxy parameters such as solubility of protein growth factors required for protein expression, eg, during inoculator amplification step 104 and cell culture steps 108 and 110. Through which the process can be improved; Due to the improved solubility of growth factors, cells can make greater use of these materials and thus promote cell proliferation.

In an embodiment, process parameters such as protein recovery or protein recovery in the USP can be improved by lowering the viscosity in the USP through various mechanisms. For example, by using the excipients described above, at the end of the cell lysis process in harvesting 114 from the finished cell culture, recovery of the therapeutic protein may be more efficient or improved. Without being bound by theory, such viscosity-lowering excipients can increase the efficiency of the therapeutic protein spreading away from other cytolytic components by lowering the viscosity of the expressed protein. In addition, separation of membranes and other cell debris from protein-containing supernatants can be achieved with faster separation rates or higher supernatant purity by using viscosity-lowering excipients, ie improving the USP efficiency parameters of the process. can do. In addition, the protein separation step using a centrifugation or filtration step can be more quickly achieved using a viscosity-lowering excipient because the excipient lowers the viscosity of the medium.

In an embodiment, as an additional advantage, the use of the viscosity-lowering excipients described above for cell culture reduces protein misfolding and aggregation, thereby increasing process parameters such as protein yield in USP. Since cell culture is optimized to achieve maximum yield of recombinant protein, the protein produced can be expressed in a high concentration manner, causing mis-folding; It is understood that the addition of viscosity-lowering excipients can reduce the attractant protein-protein interactions leading to mis-folding and aggregation, thereby increasing the amount of complete recombinant protein available at harvest 114.

The downstream process (DSP) shown in FIG. 2, in an exemplary embodiment, involves a series of steps to achieve recovery and purification of therapeutic proteins, eg monoclonal antibodies, biologics, vaccines and other biologics. At the end of USP, the therapeutic target protein can be secreted from the host cell and lysed in the cell culture medium. The therapeutic protein can also be lysed in fluid medium after cytolysis of the host cell at the end of the USP sequence. DSP recovers and purifies the protein of interest from the lysed solution (eg, culture medium or host cell lysis medium). During DSP, (i) various contaminants (eg, insoluble cell debris and particulates) are removed from the medium, and (ii) protein products are isolated through techniques such as extraction, precipitation, adsorption or ultrafiltration, and (iii) protein The product is purified through techniques such as affinity chromatography, precipitation or crystallization, and (iv) the product is further polished and virus removed.

As shown in FIG. 2, the feedstock from the cell culture harvest 200 (also shown in FIG. 1) is first subjected to affinity chromatography 204, typically involving Protein A chromatography or other similar chromatography steps. . Virus inactivation step 208 typically involves maintaining the feedstock at a low pH. One or more polishing chromatography steps 210 and 212 are performed to remove impurities such as host cell protein (HCP), DNA, charged variants and aggregates. Cation exchange (CEX) chromatography is typically used as the first polishing chromatography step 210, but a second chromatography step 212 can be preceded or followed. The second chromatography step 212 further removes product related impurities such as host-cell related impurities (eg HCP or DNA) or aggregates. Anion exchange (AEX) chromatography and hydrophobic interaction chromatography (HIC) can be used as the second chromatography step 212. Virus filtration 214 is performed to achieve virus removal. Final purification step 218 may include ultrafiltration and diafiltration, and preparation for formulation.

In general, as described above, the purification process or DSP after the fermentation process may comprise (1) cell culture harvesting, (2) chromatography (eg, Protein A chromatography and polishing chromatography steps, eg, ion exchange and hydrophobic interactions). Chromatography, (3) virus inactivation, and (4) filtration (eg, virus filtration, sterile filtration, dialysis, and ultrafiltration and diafiltration steps for concentrating the protein and exchanging the protein with the formulation buffer). can do. Examples that illustrate the advantages of using the viscosity-lowering excipients described herein to improve the process parameters associated with this purification process are described below. Viscosity-lowering excipients or combinations thereof may be introduced at any DSP step by adding it to a carrier solution or in any other way to achieve contact of the subject protein with the excipient, either in soluble or stabilized form. . In an embodiment, the second viscosity-lowering compound can be added to the carrier solution in the DSP, and the second viscosity-lowering compound further improves certain subject parameters.

(1) Cell Culture Harvesting: Cell culture harvesting generally involves centrifugation and deep filtration operations that physically remove cell debris from protein-containing solutions. The centrifugation step allows more complete separation of soluble proteins from cell debris thanks to the viscosity-lowering excipient. Whether by batch or continuous processing, separation by centrifugation is necessary to make the dense phase as hard as possible to maximize recovery of the target protein. In an embodiment, the addition of the aforementioned excipients or combinations thereof can increase the protein yield, which is a process parameter, for example by increasing the yield of protein-containing concentrate flowing out from the dense phase of the separation process by centrifugation. . The depth filtration step is a viscosity-limiting step, ie it can be made more efficient by using excipients that lower the solution viscosity. This process can also cause bubble entry into the protein solution, which can be coupled with shear-induced stress to destabilize the therapeutic protein molecule being purified. As mentioned above, the addition of a viscosity-lowering excipient to the protein-containing solution prior to and / or during harvesting of the cell culture can protect the protein from such stresses to improve the quantitative product recovery, a process parameter.

(2) Chromatography: After cell culture harvest by centrifugation or filtration, the therapeutic protein is typically separated from the fermentation broth using chromatography. If the therapeutic protein is an antibody, protein A chromatography is used: Since protein A is selective for IgG antibodies, it will bind kinetically at high flow rates and doses. Cation exchange (CEX) chromatography can be used as a cost-effective alternative to Protein A chromatography. When using CEX, the pH of the feed must be titrated to optimize the kinetic binding capacity before loading into the column, lowering its conductivity. In addition, mimetic resins can also be used as an alternative to protein A chromatography. These resins provide immunoglobulins, for example ligands for binding to Ig-binding proteins such as protein G or protein L, synthetic ligands, or protein A-like porous polymers.

Other chromatography processes can be used for the DSP. Ion exchange chromatography (IEC) can be used to remove impurities introduced during the previous process, such as leaked protein A, endotoxins or viruses from cell lines, residual host cell proteins or DNA, or media components. have. IEC can be used directly after Protein A chromatography, whether CEX or anion exchange chromatography. Hydrophobic interaction chromatography (HIC) can complement the IEC, which is generally used as a polishing step to remove aggregates. In an embodiment, the use of the excipients described above may increase the solubility of the host cell protein in the chromatography column loading step and lower its viscosity. In an embodiment, the use of the aforementioned excipients can increase the solubility of the therapeutic protein in the chromatographic column loading step and the elution step and lower its viscosity.

The chromatographic process in protein purification involves (a) a low pH environment during elution from the Protein A chromatography column, (b) an elevated local protein concentration in the pore-space of the chromatography resin (often 300-400). mg / mL levels), (c) elevated salt concentration in ion exchange chromatography, and (d) elevated salting-out agent concentration in the eluate from the HIC column. . The addition of a viscosity-lowering excipient to the protein-containing solution as described before and / or after chromatography passes through the chromatography column of the protein so that it is less exposed to the potentially harmful environment caused by the chromatography process steps. It can be made easy. In addition, increasing the concentration of local protein in the column pore-space may render the material in this space highly viscous, resulting in significant back pressure on the column. To relieve back pressure, media with relatively large pores are generally used. However, large pore media have lower resolving power than small pore. Incorporation of viscosity-modifying excipients, as described above, allows the pores to utilize smaller chromatography media. In an embodiment, the elution step of Protein A chromatography exposes the therapeutic protein to a low pH environment, which can lead to decreased solubility of the target protein and increased aggregation thereof: the addition of excipients can increase the solubility of the target protein. Thus, the recovery yield of the Protein A chromatography step is improved. In other embodiments, excipients can elute the target protein from the Protein A resin at higher pH, thereby reducing the chemical stress on the target protein, thereby reducing the amount of protein degradation that occurs during processing. It is possible to improve the protein yield which is a parameter.

(3) Virus Inactivation : The virus inactivation process typically involves long-term maintenance of the protein solution at low pH, for example below pH 4. However, this environment can destabilize the therapeutic protein. For example, before and / or during the virus inactivation process, formulating a protein in the presence of a viscosity-lowering excipient by adding a viscosity-lowering excipient can result in process parameters such as protein stability or solubility, or overall yield thereof. Can be improved.

(4) Filtration : The filtration process includes a virus filtration process (nanofiltration) to remove viral particles and an ultrafiltration / diafiltration process to concentrate the protein solution and exchange the buffer system.

(a) Virus filtration purifies the protein solution by removing viral particles, which may be twice the size of recombinant human monoclonal antibodies. Thus, the virus filtration membrane may be nano-sized pores. Because of the small size of the pores through which the protein must pass, the filtration step can cause stress on the protein and involve a significant level of membrane fouling from protein aggregated particles. The addition of a viscosity-lowering excipient, for example, before and / or during filtration, as described above, can reduce the measurable parameters such as back pressure in the filtration system by increasing the overall diffusivity. And can mitigate membrane fouling tendencies by mitigating protein-protein interactions that cause membrane fouling. The end result is an improvement in these parameters, which means an improvement in the performance of the virus filtration unit in protein purification.

(b) Ultrafiltration and diafiltration (UF / DF) processes concentrate the protein solution and replace the buffer system by passing the protein-containing solution through a filter membrane with a specific molecular weight cutoff smaller than the protein of interest. At this stage, the protein solution is exposed to high shear stresses in the filter unit, elevated protein concentrations and the adsorption of proteins to hydrophobic membranes typically used in UF / DF processes, all of which can increase protein aggregation. As mentioned above, the addition of viscosity-lowering excipients, for example, before and / or during the UF / DF process, increases the overall diffusion coefficient (as measured by increasing kD) to reduce back pressure in the filtration system. Can be. This not only reduces membrane overall shear stress but also promotes de-diffusion reduction from the filter membrane, lowering effective protein concentration at the membrane interface and increasing permeate flux. As a result, viscosity-lowering excipients can be used to improve the parameters associated with higher throughput, reduced production loss and increased overall yield in these filtration processes. In addition, passing viscous fluids through ultra- and diafilters can result in a significant pressure drop throughout the filter device, which can result in inefficient separation. Formulating a protein solution in the presence of a viscosity-lowering excipient, as described above, can substantially reduce the pressure drop across the filter device, thereby reducing both process parameters, operation cost and process time. This can be improved.

After adding the excipients to complete the upstream protein treatment or downstream purification, the excipients may remain as part of the drug substance mixture or may be separated from the protein active ingredient. To separate excipients from protein active ingredients, typical small molecule separation methods such as buffer exchange, ion exchange, ultrafiltration and dialysis can be used. The use of such excipients, in addition to the beneficial effects on the protein purification process as described above, can protect and maintain the devices used for protein preparation, processing and purification. For example, device-related processes such as cleaning, sterilization and maintenance of protein processing devices may be facilitated by the use of excipients described above, due to reduced fouling, lower denaturation, lower viscosity and improved solubility of the protein. And the parameters associated with such process improvements are likewise improved.

While the use of excipient compounds to improve the upstream and / or downstream processes has been described broadly herein, it is understood that combinations of excipients may be added together to achieve desirable effects such as improvement of the subject parameters. The term “excipient additive” may refer to one excipient compound that leads to a desired effect or parameter improvement, or a combination of excipient compounds that achieves a desired effect or parameter improvement.

Example

material:

· Bovine Gamma Globulin (BGG), Purity> 99%, Catalog # G5009, Sigma Aldrich

· Histidine, Sigma Aldrich

· Other materials mentioned in the Examples below are purchased from Sigma Aldrich unless otherwise stated.

EXAMPLE  1: Preparation of formulation containing excipient compound and test protein

The formulations were prepared using excipient compounds and test proteins, and the test proteins are intended to mimic the therapeutic proteins to be used in the therapeutic formulation or non-therapeutic proteins to be used in the non-therapeutic formulation. The formulations were prepared in 50 mM histidine hydrochloride to which various excipient compounds were added to determine the viscosity in the following manner. First, histidine hydrochloride is dissolved by dissolving 1.94 g of histidine in distilled water, titrating to pH 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO) and then diluting the final volume to 250 mL with distilled water in a flask. First prepared. The excipient compound was then dissolved in 50 mM histidine HCl. An excipient list is provided in Examples 4, 5, 6 and 7. In some cases, the excipient compound was titrated to pH 6 before dissolving in 50 mM histidine HCl. In this case, the excipient compound was first dissolved in deionized water at about 5 wt% and then titrated to pH 6.0 with hydrochloric acid or sodium hydroxide. The prepared salt solution was placed in a convection laboratory oven at about 65 ° C. to evaporate water and to isolate the solid excipient. Once the excipient solution in 50 mM histidine HCl was prepared, test protein bobbin gamma globulin (BGG) was dissolved at a rate of about 0.336 g of BGG per mL of excipient solution. This achieved a final protein concentration of about 280 mg / mL. BGG solution in 50 mM histidine HCl with excipients was prepared in a 20 mL vial and stirred at 100 rpm overnight on a rotary shaker table. The BGG solution was then placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove the collected air before viscosity measurement.

EXAMPLE  2: viscosity measurement

Viscosity measurements for the formulations prepared as mentioned in Example 1 were performed using a DV-IIT LV cone & plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature, and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of three collected data points was obtained and recorded as the viscosity of the sample.

EXAMPLE  3: protein concentration measurement

The absorbance of the protein solution was measured under a wavelength of 280 nm in a UV / VIS spectrometer (Perkin Elmer Lambda 35) to determine the concentration of protein in the experimental solution. First, the instrument was calibrated to zero absorbance using 50 mM histidine buffer at pH 6. The protein solution was then diluted 300-fold with the same histidine buffer and the absorbance was recorded at 280 nm. The final concentration of protein in solution was calculated using an extinction coefficient of 1.264 mL / (mg x cm).

Example  4: Hindered Amine  Formulations with added excipient compounds

280 mg / mL BGG formulations were prepared as in Example 1, with some samples added with excipient compounds. In the test, hydrochloride salts of dimethylcyclohexylamine (DMCHA), dicyclohexylmethylamine (DCHMA), dimethylaminopropylamine (DMAPA), triethanolamine (TEA), dimethylethanolamine (DMEA) and niacinamide Was investigated as an example of the hindered amine excipient compound. In addition, the taurine-dicyanidiamide adduct and the hydroxybenzoic acid salt of DMCHA were investigated as examples of hindered amine excipient compounds. The viscosity of each protein solution was measured as in Example 2, and the results are shown in Table 1 below, and the following table shows the viscosity lowering effect of the added excipient compound.

Table 1

Exam number Additive excipients Excipient Concentration (mg / mL) Viscosity (cP) Viscosity reduction rate 4.1 none 0 79 0% 4.2 DMCHA-HCl 28 50 37% 4.3 DMCHA-HCl 41 43 46% 4.4 DMCHA-HCl 50 45 43% 4.5 DMCHA-HCl 82 36 54% 4.6 DMCHA-HCl 123 35 56% 4.7 DMCHA-HCl 164 40 49% 4.8 DMAPA-HCl 87 57 28% 4.9 DMAPA-HCl 40 54 32% 4.10 DCHMA-HCl 29 51 35% 4.11 DCHMA-HCl 50 51 35% 4.14 TEA-HCl 97 51 35% 4.15 TEA-HCl 38 57 28% 4.16 DMEA-HCl 51 51 35% 4.17 DMEA-HCl 98 47 41% 4.20 DMCHA-hydroxybenzoate 67 46 42% 4.21 DMCHA-hydroxybenzoate 92 42 47% 4.22 Product of Example 8 26 58 27% 4.23 Product of Example 8 58 50 37% 4.24 Product of Example 8 76 49 38% 4.25 Product of Example 8 103 46 42% 4.26 Product of Example 8 129 47 41% 4.27 Product of Example 8 159 42 47% 4.28 Product of Example 8 163 42 47% 4.29 Niacinamide 48 39 51% 4.30 N-methyl-2-pyrrolidone 30 45 43% 4.31 N-methyl-2-pyrrolidone 52 52 34%

EXAMPLE  5: Anionic  Formulations with addition of aromatic excipient compounds

280 mg / mL BGG formulations, with excipient compounds added to some samples, were prepared as in Example 1. The viscosity of each solution was measured as in Example 2 and the results are shown in Table 2 below, and the table below shows the viscosity lowering effect of the added excipient compound.

TABLE 2

Exam number Additive excipients Excipient Concentration (mg / mL) Viscosity (cP) Viscosity reduction rate 5.1 none 0 79 0% 5.2 Sodium aminobenzoate 43 48 39% 5.3 Sodium hydroxybenzoate 26 50 37% 5.4 Sodium sulfanilate 44 49 38% 5.5 Sodium sulfanilate 96 42 47% 5.6 Sodium indole acetate 52 58 27% 5.7 Sodium indole acetate 27 78 One% 5.8 Vanillic acid, sodium salt 25 56 29% 5.9 Vanillic acid, sodium salt 50 50 37% 5.10 Sodium salicylate 25 57 28% 5.11 Sodium salicylate 50 52 34% 5.12 Adenosine monophosphate 26 47 41% 5.13 Adenosine monophosphate 50 66 16% 5.14 Sodium benzoate 31 61 23% 5.15 Sodium benzoate 56 62 22%

EXAMPLE  6: Oligopeptide  Formulations with added excipient compounds

Oligopeptides (n = 5) having the N-terminus of the free amine and the C-terminus of the free acid were prepared from NeoBioLab Inc. (Woburn, Mass.) Was synthesized with> 95% purity. Dipeptide (n = 2) was synthesized in 95% purity at LifeTein LLC (Somerset, NJ). 280 mg / mL BGG formulations, with excipient compounds added to some samples, were prepared as in Example 1. The viscosity of each solution was measured as in Example 2 and the results are shown in Table 3 below, and the table below shows the viscosity lowering effect of the added excipient compound.

TABLE 3

Exam number Additive excipients Excipient Concentration (mg / mL) Viscosity (cP) Viscosity reduction rate 6.1 none 0 79 0% 6.2 ArgX5 100 55 30% 6.3 ArgX5 50 54 32% 6.4 HisX5 100 62 22% 6.5 HisX5 50 51 35% 6.6 HisX5 25 60 24% 6.7 Trp2Lys3 100 59 25% 6.8 Trp2Lys3 50 60 24% 6.9 AspX5 100 102 -29% 6.10 AspX5 50 82 -4% 6.11 Dipeptide LE (Leu-Glu) 50 72 9% 6.12 Dipeptide YE (Tyr-Glu) 50 55 30% 6.13 Dipeptide RP (Arg-Pro) 50 51 35% 6.14 Dipeptide RK (Arg-Lys) 50 53 33% 6.15 Dipeptide RH (Arg-His) 50 52 34% 6.16 Dipeptide RR (Arg-Arg) 50 57 28% 6.17 Dipeptide RE (Arg-Glu) 50 50 37% 6.18 Dipeptide LE (Leu-Glu) 100 87 -10% 6.19 Dipeptide YE (Tyr-Glu) 100 68 14% 6.20 Dipeptide RP (Arg-Pro) 100 53 33% 6.21 Dipeptide RK (Arg-Lys) 100 64 19% 6.22 Dipeptide RH (Arg-His) 100 72 9% 6.23 Dipeptide RR (Arg-Arg) 100 62 22% 6.24 Dipeptide RE (Arg-Glu) 100 66 16%

EXAMPLE  8: Guanil  Taurine Excipient Synthesis

Guanyl taurine was prepared according to the method described in US Pat. No. 2,230,965. 3.53 parts of taurine (Sigma-Aldrich, St. Louis, MO) are mixed with 1.42 parts of dicyandiamide (Sigma-Aldrich, St. Louis, MO) and then ground until a homogeneous mixture is obtained with a mortar and pestle. It was. The mixture was then placed in a flask and heated at 200 ° C. for 4 hours. The product was used without further purification.

EXAMPLE  9: protein formulation containing excipient compound

The formulations were prepared using excipient compounds and test proteins, and the test proteins were intended to mimic the therapeutic protein to be used in the therapeutic formulation or the non-therapeutic protein to be used in the non-therapeutic formulation. These formulations were prepared in 50 mM histidine hydrochloride buffer aqueous solution with various excipient compounds added to determine the viscosity in the following manner. First, histidine hydrochloride buffer is dissolved by dissolving 1.94 g of histidine in distilled water, titrating to pH 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO), and then diluting to a final volume of 250 mL with distilled water in a flask. Was prepared first. The excipient compound was then dissolved in 50 mM histidine HCl buffer. An excipient list is shown in Example 4. In some cases, the excipient compound was dissolved in 50 mM histidine HCl buffer and the resulting solution was titrated with a small amount of sodium hydroxide or hydrochloric acid to pH 6 before dissolving the model protein. In some cases, the excipient compound was titrated to pH 6 before dissolving in 50 mM histidine HCl. In this case, the excipient compound was first dissolved in deionized water at about 5 wt% and then titrated to pH 6.0 with hydrochloric acid or sodium hydroxide. The prepared salt solution was placed in a convection laboratory oven at about 65 ° C. to evaporate water and to isolate the solid excipient. Once the excipient solution in 50 mM histidine HCl was prepared, test protein bobbin gamma globulin (BGG) was dissolved at a rate at which a final protein concentration of about 280 mg / mL was achieved. BGG solution in 50 mM histidine HCl with excipients was prepared in a 20 mL vial and stirred at 100 rpm overnight on a rotary shaker table. The BGG solution was then placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove the collected air before viscosity measurement.

Viscosity measurements for the formulations prepared as above were performed using a DV-IIT LV viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of three collected data points was obtained and recorded as the viscosity of the sample. The viscosity of the excipient containing solution was normalized to the viscosity of the excipient-free model protein solution. The normalized viscosity is the ratio of the viscosity of the excipient-containing model protein solution to the viscosity of the excipient-free model protein solution.

Table 4

Exam number Additive excipients Excipient Concentration (mg / mL) Normalized Viscosity (cP) Viscosity reduction rate 9.1 DMCHA-HCl 120 0.44 56% 9.2 Niacinamide 50 0.51 49% 9.3 Isonicotinamide 50 0.48 52% 9.4 Tyramine HCl 70 0.41 59% 9.5 Histamine HCl 50 0.41 59% 9.6 Imidazole HCl 100 0.43 57% 9.7 2-methyl-2-imidazoline HCl 60 0.43 57% 9.8 1-butyl-3-methylimidazolium chloride 100 0.48 52% 9.9 Procaine HCl 50 0.53 47% 9.10 3-aminopyridine 50 0.51 49% 9.11 2,4,6-trimethylpyridine 50 0.49 51% 9.12 3-pyridine methanol 50 0.53 47% 9.13 Nicotinamide Adenine Dinucleotide 20 0.56 44% 9.15 Sodium Phenylpyruvate 55 0.57 43% 9.16 2-pyrrolidinone 60 0.68 32% 9.17 Morpholine HCl 50 0.60 40% 9.18 Agmartin Sulfate 55 0.77 23% 9.19 1-butyl-3-methylimidazolium iodide 60 0.66 34% 9.21 L-Anserine Nitrate 50 0.79 21% 9.22 1-hexyl-3-methylimidazolium chloride 65 0.89 11% 9.23 N, N-diethyl nicotinamide 50 0.67 33% 9.24 Nicotinic acid, sodium salt 100 0.54 46% 9.25 Biotin 20 0.69 31%

EXAMPLE  10: excipient Combination and  Preparation of Formulations Containing Test Proteins

The formulations were prepared using the first excipient compound, the second excipient compound, and the test protein, and the test protein is intended to mimic the therapeutic protein to be used in the therapeutic formulation or the non-therapeutic protein to be used in the non-therapeutic formulation. The first excipient compound was selected from compounds having both anionic and aromatic functional groups as listed in Table 5 below. The second excipient compound was selected from compounds having a non-ionic or cationic charge and imidazoline or benzene ring at pH 6 as listed in Table 5 below. Formulations of these excipients were prepared in 50 mM histidine hydrochloride buffer to measure viscosity in the following manner. First, histidine hydrochloride is dissolved by dissolving 1.94 g of histidine in distilled water, titrating to pH 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO) and then diluting the final volume to 250 mL with distilled water in a flask. Prepared. Then, the first or second excipient compound was dissolved in 50 mM histidine HCl. The combination of the first and second excipients was dissolved in 50 mM histidine HCl and the resulting solution was titrated with a small amount of sodium hydroxide or hydrochloric acid to pH 6 before dissolving the model protein. After the excipient solution was prepared as above, test protein bobbin gamma globulin (BGG) was dissolved in each test solution at a rate at which a final protein concentration of about 280 mg / mL was achieved. BGG solution in 50 mM histidine HCl with excipients was prepared in a 20 mL vial and stirred at 100 rpm overnight on a rotary shaker table. The BGG solution was then placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove the collected air before viscosity measurement.

Viscosity measurements for the formulations prepared as above were performed using a DV-IIT LV viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of three collected data points was obtained and recorded as the viscosity of the sample. The viscosity of the excipient-containing solution was normalized to the viscosity of the excipient-free model protein solution and summarized in Table 5 below. The normalized viscosity is the ratio of the viscosity of the excipient-containing model protein solution to the viscosity of the excipient-free model protein solution. This example shows that a combination of the first and second excipients may provide better results than a single excipient.

Table 5

First excipient
Second excipient
Standardized viscosity Exam number designation Concentration (mg / mL) designation Concentration (mg / mL) 10.1 Salicylic acid 30 none 0 0.79 10.2 Salicylic acid 25 Imidazole 4 0.59 10.3 4-hydroxybenzoic acid 30 none 0 0.61 10.4 4-hydroxybenzoic acid 25 Imidazole 5 0.57 10.5 4-hydroxybenzene sulfonic acid 31 none 0 0.59 10.6 4-hydroxybenzene sulfonic acid 26 Imidazole 5 0.70 10.7 4-hydroxybenzene sulfonic acid 25 Caffeine 5 0.69 10.8 none 0 Caffeine 10 0.73 10.9 none 0 Imidazole 5 0.75

EXAMPLE  11: excipient Combination and  Preparation of Formulations Containing Test Proteins

The formulations were prepared using the first excipient compound, the second excipient compound, and the test protein, and the test protein is intended to mimic the therapeutic protein to be used in the therapeutic formulation or the non-therapeutic protein to be used in the non-therapeutic formulation. The first excipient compound was selected from compounds having both anionic and aromatic functional groups as listed in Table 6 below. The second excipient compound was selected from compounds having a non-ionic or cationic charge and imidazoline or benzene ring at pH 6 as listed in Table 6 below. Formulations of these excipients were prepared in distilled water to measure the viscosity in the following manner. The combination of the first and second excipients was dissolved in distilled water and the resulting solution was titrated to pH 6 with a small amount of sodium hydroxide or hydrochloric acid before dissolution of the model protein. After preparing an excipient solution in distilled water, test protein bobbin gamma globulin (BGG) was dissolved at a rate at which a final protein concentration of about 280 mg / mL was achieved. A BGG solution in distilled water with excipients was prepared in a 20 mL vial and stirred at 100 rpm overnight on a rotary shaker table. The BGG solution was then placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove the collected air before viscosity measurement.

Viscosity measurements for the formulations prepared as above were performed using a DV-IIT LV viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of three collected data points was obtained and recorded as the viscosity of the sample. The viscosity of the excipient-containing solution was normalized to the viscosity of the excipient-free model protein solution and summarized in Table 6 below. The normalized viscosity is the ratio of the viscosity of the excipient-containing model protein solution to the viscosity of the excipient-free model protein solution. This example shows that a combination of the first and second excipients may provide better results than a single excipient.

Table 6

First excipient Second excipient Standardized
Viscosity Exam number designation Concentration (mg / mL) designation Concentration (mg / mL) 11.1 Salicylic acid 20 none 0 0.96 11.2 Salicylic acid 20 Caffeine 5 0.71 11.3 Salicylic acid 20 Niacinamide 5 0.76 11.4 Salicylic acid 20 Imidazole 5 0.73

EXAMPLE  12: Preparation of a formulation containing an excipient compound and PEG

Materials: All materials were purchased from Sigma-Aldrich (St. Louis, Mo.). The formulations were prepared using excipient compounds and PEG, which PEG was intended to mimic the PEGylated therapeutic protein used in the therapeutic formulation. These formulations were prepared by mixing PEG solution in the same volume with excipient solution. These two solutions were prepared in Tris buffer (pH 7.3) consisting of 10 mM Tris, 135 mM NaCl, 1 mM trans-cinnamic acid.

PEG solutions were prepared by mixing 3 g of poly (ethylene oxide) (Aldrich Catalog # 372781) with an average Mw of 1,000,000 with 97 g of Tris buffer. The mixture was stirred overnight to dissolve completely.

An example of preparation of an excipient solution is as follows: 0.4 g of citric acid (Aldrich cat. # 251275) is dissolved in 5 mL of Tris buffer and titrated to pH 7.3 using a minimum amount of 10 M NaOH, about 80 mg / citric acid in Tris buffer. mL solution was prepared.

0.5 mL PEG solution and 0.5 mL excipient solution were mixed to prepare a PEG excipient solution, which was then mixed using vortex for several seconds. Control samples were prepared by mixing 0.5 mL of PEG solution with 0.5 mL of Tris buffer.

EXAMPLE  13: Determination of viscosity of excipient compound and PEG-containing formulation

Viscosity measurements for the prepared formulations were performed using a DV-IIT LV viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of three collected data points was obtained and recorded as the viscosity of the sample.

The results shown in Table 7 show the viscosity lowering effect of the added excipient compound.

TABLE 7

Exam number Excipient Excipient Concentration (mg / mL) Viscosity (cP) Viscosity reduction rate 13.1 none 0 104.8 0% 13.2 Citric Acid Na Salt 40 56.8 44% 13.3 Citric Acid Na Salt 20 73.3 28% 13.4 Glycerol Phosphate 40 71.7 30% 13.5 Glycerol Phosphate 20 83.9 18% 13.6 Ethylene diamine 40 84.7 17% 13.7 Ethylene diamine 20 83.9 15% 13.8 EDTA / K salt 40 67.1 36% 13.9 EDTA / K salt 20 76.9 27% 13.10 EDTA / Na salt 40 68.1 35% 13.11 EDTA / Na salt 20 77.4 26% 13.12 D-gluconic acid / K salt 40 80.32 23% 13.13 D-gluconic acid / K salt 20 88.4 16% 13.14 D-gluconic acid / Na salt 40 81.24 23% 13.15 D-gluconic acid / Na salt 20 86.6 17% 13.16 Lactic Acid / K Salt 40 80.42 23% 13.17 Lactic Acid / K Salt 20 85.1 19% 13.18 Lactic Acid / Na Salt 40 86.55 17% 13.19 Lactic Acid / Na Salt 20 87.2 17% 13.20 Ethidronic acid / K salt 24 71.91 31% 13.21 Ethidronic acid / K salt 12 80.5 23% 13.22 Ethidronic Acid / Na Salt 24 71.6 32% 13.23 Ethidronic Acid / Na Salt 12 79.4 24%

EXAMPLE  14: BSA PEG per molecule  With one chain Pegylated  BSA manufacturing

To the beaker, 200 mL of phosphate buffered saline (Aldrich Cat. # P4417) and 4 g of BSA (Aldrich Cat. # A7906) were added and mixed with a magnetic rod. Then 400 mg of methoxy polyethylene glycol maleimide, MW = 5,000, (Aldrich Cat. # 63187) were added. The reaction mixture was allowed to react overnight at room temperature. The next day, the reaction was stopped by adding 20 drops of 0.1 M HCl. The reaction product was characterized by SDS-PAGE and SEC, and PEGylated BSA was clearly observed. The reaction mixture was placed in an Amicon centrifuge tube of molecular weight cutoff (MWCO) 30,000 and concentrated to several mL. The sample was then diluted 20-fold with 50 mM histidine buffer of about pH 6 and then concentrated until a high viscosity fluid was obtained. The absorbance of the protein solution was measured at 280 nm and the final concentration of the protein solution was determined using the BSA excitation coefficient 0.6678. As a result, the final concentration of BSA in the solution was found to be 342 mg / mL.

EXAMPLE  15: BSA PEG per molecule  With several chains Pegylated  BSA manufacturing

0.5 g of BSA was mixed with 100 mL of buffer to prepare a 5 mg / mL BSA (Aldrich A7906) solution in 25 mM phosphate buffer at pH 7.2. Then 1 g of methoxy PEG propionaldehyde Mw = 20,000 (JenKem Technology, Plano, TX 75024) was added followed by 0.12 g of sodium cyanoborohydride (Aldrich 156159). The following day, the reaction mixture was diluted 13-fold with Tris buffer (10 mM Tris, 135 mM NaCl, pH = 7.3) and concentrated using an Amicon centrifuge tube of MWCO 30,000 until reaching a concentration of about 150 mg / mL.

EXAMPLE  16: Lysozyme PEG per molecule  With several chains Pegylated Lysozyme  Produce

0.5 g of lysozyme was mixed with 100 mL of buffer to prepare a 5 mg / mL lysozyme (Aldrich L6876) solution in 25 mM phosphate buffer at pH 7.2. Then 1 g of methoxy PEG propionaldehyde Mw = 5,000 (JenKem Technology, Plano, TX 75024) was added followed by 0.12 g of sodium cyanoborohydride (Aldrich 156159). The reaction proceeded overnight at room temperature. The next day, the reaction mixture was diluted 49-fold with 25 mM phosphate buffer, pH 7.2, and concentrated using an Amicon centrifuge tube at MWCO 30,000. The absorbance of the protein solution was measured at 280 nm and the final concentration of the protein solution was determined using the lysozyme excitation coefficient 2.63. As a result, the final concentration of lysozyme in the solution was found to be 140 mg / mL.

EXAMPLE  17: BSA PEG per molecule  With one chain Pegylated  Of BSA Effect of Excipients on Viscosity

6 or 12 mg of excipient salt was added to 0.3 mL of pegylated BAS solution to prepare pegylated BSA formulations (of Example 14) with excipient added. The solution was gently stirred and mixed and the viscosity was measured by RheoSense microVisc equipped with an A10 channel (100-micron depth) at a shear rate of 500 sec ⁻¹ . Viscometer measurements were completed at ambient temperature.

The results shown in Table 8 show the viscosity lowering effect of the added excipient compound.

Table 8

Exam number Excipient Excipient Concentration (mg / mL) Viscosity (cP) Viscosity reduction rate 17.1 none 0 228.6 0% 17.2 Alpha-cyclodextrin sulfate Na salt 20 151.5 34% 17.3 K acetate 40 89.5 60%

EXAMPLE  18: BSA PEG per molecule  With several chains Pegylated  Of BSA Effect of Excipients on Viscosity

8 mg of excipient salt was added to 0.2 mL of pegylated BAS solution to prepare pegylated BSA formulations (of Example 15) added with citric acid Na salt as excipients. The solution was gently stirred and mixed and the viscosity was measured by RheoSense microVisc equipped with an A10 channel (100-micron depth) at a shear rate of 500 sec ⁻¹ . Viscometer measurements were completed at ambient temperature. The results shown in Table 9 show the viscosity lowering effect of the added excipient compound.

Table 9

Exam number Additive excipients Excipient Concentration (mg / mL) Viscosity (cP) Viscosity reduction rate 18.1 none 0 56.8 0% 18.2 Citric Acid Na Salt 40 43.5 23%

EXAMPLE  19: Lysozyme PEG per molecule  With several chains Pegylated Lysozyme  Effect of Excipients on Viscosity

6 mg of excipient salt was added to 0.3 mL of pegylated lysozyme solution to prepare pegylated lysozyme formulations (of Example 16) added with potassium acetate as excipient. The solution was gently stirred and mixed and the viscosity was measured by RheoSense microVisc equipped with an A10 channel (100-micron depth) at a shear rate of 500 sec ⁻¹ . Viscometer measurements were completed at ambient temperature. The results shown in the table below show the viscosity lowering effect of the added excipient compound.

Table 10

Exam number Excipient Excipient Concentration (mg / mL) Viscosity (cP) Viscosity reduction rate 19.1 none 0 24.6 0% 19.2 K acetate 20 22.6 8%

EXAMPLE  20: excipient Combination  Protein preparations

Formulations were prepared using excipient compounds or two excipient compounds and test proteins, and the test proteins were intended to mimic the therapeutic proteins to be used in the therapeutic formulation. These formulations were prepared in 20 mM histidine buffer with the addition of various excipient compounds to determine the viscosity in the following manner. The excipient combination was dissolved in 20 mM histidine and the resulting solution was titrated to pH 6 with a small amount of sodium hydroxide or hydrochloric acid before dissolving the model protein. Excipient compounds used in this example are shown in Table 11 below. Once the excipient solution was prepared, test protein bobbin gamma globulin (BGG) was dissolved at a rate of about 280 mg / mL final protein concentration. BGG solutions in excipient solutions were prepared in 5 mL sterile polypropylene test tubes and stirred at 80-100 rpm overnight on a rotary shaker table. The BGG solution was then placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove the collected air before viscosity measurement.

Viscosity measurements for the formulations prepared above were performed using a DV-IIT LV cone & plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of three collected data points was obtained and recorded as the viscosity of the sample. The viscosity of the excipient-containing solution was normalized to the viscosity of the excipient-free model protein solution, and the results are shown in Table 11 below. The normalized viscosity is the ratio of the viscosity of the excipient-containing model protein solution to the viscosity of the excipient-free model protein solution.

Table 11

exam # Excipient A Excipient B Standardized viscosity designation Concentration (mg / mL) designation Concentration (mg / mL) 20.1 none 0 none 0 1.00 20.2 Aspartame 10 none 0 0.83 saccharin 60 none 0 0.51 20.4 Acesulfame K 80 none 0 0.44 20.5 Theophylline 10 none 0 0.84 20.6 saccharin 30 none 0 0.58 20.7 Acesulfame K 40 none 0 0.61 20.8 Caffeine 15 Taurine 15 0.82 20.9 Caffeine 15 Tyramine 15 0.67

EXAMPLE  21: Protein formulation containing excipients to reduce viscosity and injection pain

Formulations were prepared using excipient compounds, second excipient compounds, and test proteins, and the test proteins were intended to mimic the therapeutic proteins to be used in the therapeutic formulation. The first excipient compound, excipient A, was selected from the group of compounds with local anesthetic properties. The first excipient, excipient A and the second excipient, excipient B are listed in Table 12. These formulations were prepared in 20 mM histidine buffer using excipient A and excipient B in the following manner and their viscosity could be measured. Excipients in the amounts shown in Table 12 were dissolved in 20 mM histidine and the resulting solution was titrated to pH 6 with a small amount of sodium hydroxide or hydrochloric acid prior to model protein dissolution. After the excipient solution was prepared, test protein bobbin gamma globulin (BGG) was dissolved in the excipient solution at a rate at which a final protein concentration of about 280 mg / mL was achieved. BGG solutions in excipient solutions were prepared in 5 mL sterile vials and stirred at 80-100 rpm overnight on a rotary shaker table. The BGG solution was then placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove the collected air before viscosity measurement.

Viscosity measurements for the formulations prepared as above were performed using a DV-IIT LV viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of three collected data points was obtained and recorded as the viscosity of the sample. The viscosity of the excipient-containing solution is normalized to the viscosity of the excipient-free model protein solution, and is shown in Table 12 below. The normalized viscosity is the ratio of the viscosity of the excipient-containing model protein solution to the viscosity of the excipient-free model protein solution.

Table 12

exam # Excipient A Excipient B Standardized viscosity designation Concentration (mg / mL) designation Concentration (mg / mL) 21.1 none 0 none 0 1.00 21.2 Lidocaine 45 none 0 0.73 21.3 Lidocaine 23 none 0 0.74 21.4 Lidocaine 10 Caffeine 15 0.71 21.5 Procaine HCl 40 none 0 0.64 21.6 Procaine HCl 20 Caffeine 15 0.69

EXAMPLE  22: excipient compound and PEG containing formulation

Formulations were prepared using excipient compounds and PEG, PEG was intended to mimic PEGylated therapeutic proteins for use in therapeutic formulations, and excipient compounds were provided in the amounts listed in Table 13. These formulations were prepared by mixing PEG solution in the same volume with excipient solution. These two solutions were prepared in deionized (DI) water.

The PEG solution was prepared by mixing 16.5 g of poly (ethylene oxide) (Aldrich Catalog # 181986) with an average Mw of 100,000 with 83.5 g of deionized water. The mixture was stirred overnight to dissolve completely.

Excipient solutions were prepared by the general method as detailed in Table 13 below: 0.05 g of potassium phosphate was dissolved in deionized water to prepare a solution of about 20 mg / mL potassium phosphate tribasic (Aldrich Catalog # P5629) in deionized water. Prepared. 0.5 mL PEG solution was mixed with 0.5 mL excipient solution to prepare a PEG excipient solution, which was then mixed using vortex for a few seconds. Control samples were prepared by mixing 0.5 mL of PEG solution with 0.5 mL of deionized water. The viscosity was measured and the results are reported in Table 13 below.

Table 13

Exam number Excipient Excipient concentration
(mg / mL) Viscosity (cP) Viscosity
Reduction Rate (%) 22.1 none 0 79.7 0 22.2 Citric Acid Na Salt 10 74.9 6.0 22.3 Potassium phosphate 10 72.3 9.3 22.4 Citric Acid Na Salt / Potassium Phosphate 10/10 69.1 13.3 22.5 Sodium sulfate 10 75.1 5.8 22.6 Citric Acid Na Salt / Sodium Sulfate 10/10 70.4 11.7

EXAMPLE  23: improved processing with excipients in protein solutions

0.25 g of BGG solid was mixed into 4 ml of buffer to make two BGG solutions. Sample A: The buffer is 20 mM histidine buffer (pH = 6.0). Sample B: Buffer is 20 mM histidine buffer containing 15 mg / ml caffeine (pH = 6). The sample was stirred at 100 rpm on a rotary shaker table to dissolve the BGG solids. Buffer solutions containing caffeine excipients were observed to dissolve proteins faster. For the sample with caffeine excipient added (Sample B), BGG completely dissolved within 15 minutes. For the caffeine free sample (Sample A), it took 35 minutes to dissolve.

The next day, the samples were placed in two Amicon Ultra 4 centrifugal filter units with a molecular weight cutoff of 30,000 and the samples were centrifuged at 2,500 rpm at 10 minute intervals. The filtrate volume recovered after centrifugation for 10 minutes each was recorded. The results in Table 14 show that the filtrate is recovered faster in Sample B. In addition, while sample B was concentrated every centrifugation, sample A reached the highest concentration point and no further sample concentration occurred in further centrifugation.

Table 14

Centrifuge time (minutes) Collected Sample A Filtrate  (mL) Collected Sample B Filtrate  (mL) 10 0.28 0.28 20 0.56 0.61 30 0.78 0.88 40 0.99 1.09 50 1.27 1.42 60 1.51 1.71 70 1.64 1.99 80 1.79 2.29 90 1.79 2.39 100 1.79 2.49

EXAMPLE  24: Protein Preparation Containing Multiple Excipients

This example shows whether a combination of caffeine and arginine as an excipient has a beneficial effect on lowering the viscosity of the BGG solution. Four BGG solutions were prepared by mixing 0.18 g of a BGG solid with 0.5 mL of 20 mM histidine buffer at pH 6. Each buffer contained different excipients or combinations of excipients as shown in the table below. The viscosity of the solution was measured as in the previous example. As a result, it was confirmed that the hindered amine excipient and caffeine can be combined with a known excipient such as arginine, and the combination has better viscosity lowering properties than the individual excipient alone.

Table 15

Sample Additive excipients Viscosity (cP) Viscosity Reduction Rate (%) A none 130.6 0 B Caffeine (10mg / ml) 87.9 33 C Caffeine (10mg / ml) / Arginine (25 mg / ml) 66.1 49 D Arginine (25 mg / ml) 76.7 41

Arginine was added to a 280 mg / mL BGG solution in histidine buffer at pH 6. At concentrations higher than 50 mg / mL, further addition of arginine did not further reduce the viscosity as shown in Table 16.

Table 16

Arginine addition amount (mg / mL) Viscosity (cP) Reduced viscosity ( % ) 0 79.0 0% 53 40.9 48% 79 46.1 42% 105 47.8 40% 132 49.0 38% 158 48.0 39% 174 50.3 36% 211 51.4 35%

Caffeine was added to a 280 mg / mL BGG solution in histidine buffer at pH 6. At concentrations higher than 10 mg / mL, the addition of caffeine did not further reduce the viscosity as shown in Table 17.

Table 17

Caffeine added amount (mg / mL) Viscosity (cP) Reduced viscosity ( % ) 0 79 0% 10 60 31% 15 62 23% 22 50 45%

EXAMPLE  25: TFF  Caffeine Effect in Concentration Process

In this example, the bovine gamma globulin (BGG) solution was concentrated using tangential flow filtration (TFF) under caffeine addition and non-addition. Experiments were performed using a Labscale TFF system manufactured by EMD Millipore (Billerica, Mass.). The system was equipped with a Pellicon XL TFF cassette containing an Ultracel membrane of 30 kDa molecular weight cutoff (EMD Millipore, Billerica, Mass.). Nominal membrane surface area was 50 cm ² . The pressure supplied to the cassette was maintained at 30 psi and the retentate pressure was maintained at 10 psi. Filtrate flux was monitored throughout the experiment by gravimetric measurements over time. About 12 g of BGG was dissolved in 500 mL of 15 mg / mL caffeine, 150 mM NaCl and 20 mM histidine containing buffer titrated to pH 6. About 12 g of BGG was dissolved in 500 mL of 150 mM NaCl and 20 mM histidine containing buffer titrated to pH 6 to prepare a control sample. Buffer components were purchased from Sigma-Aldrich. Both solutions were filtered through a 0.2 μm PES filter (VWR, Radnor, PA) before the TFF process. The performance of test and control samples was measured in TFF by mass transfer coefficient. Mass transfer coefficients were obtained for each sample in the following formula (J. Hung, AU Borwankar, BJ Dear, TM Truskett, KP Johnston, High concentration tangential flow ultrafiltration of stable monoclonal antibody solutions with low viscosities. J. Memb. Sci. 508 , 113-126 (2016)):

J = k _c ln (C _w / C _b ) (Equation 3)

Equation 3 is filtered represents the water flow rate (filtrate flux) J, the expression k _c is the mass transfer coefficient and, C _w is the membrane surrounding the protein concentration, C _b is because concentration liquid bulk, mass-transfer coefficient by the equation (3) k _c Can be calculated. The graph for flow rate calculation J according to ln (C _b ) shows a linear curve of slope -kc. Here, the flow rate J is calculated by differentiating the filtrate weight with time, and C _b is calculated using mass balance. The most suitable mass transfer coefficients are listed in Table 18. The addition of 15 mg / mL caffeine increased the mass transfer coefficient by 13% from 22.5 to 25.4 Lm ⁻² hr ⁻¹ (LMH).

Table 18

Sample Mass transfer coefficient k _c  ( LMH ) Control 22.5 ± 0.1 15 mg / mL caffeine 25.4 ± 0.1

EXAMPLE  26: TFF  Caffeine Effect in Concentration Process

In this example, the bovine gamma globulin (BGG) solution was concentrated using tangential flow filtration (TFF) under caffeine addition and non-addition. Experiments were performed using a Labscale TFF system manufactured by EMD Millipore (Billerica, Mass.). The system was equipped with a Pellicon XL TFF cassette containing an Ultracel membrane of 30 kDa molecular weight cutoff (EMD Millipore, Billerica, Mass.). Nominal membrane surface area was 50 cm ² . A control sample was prepared by dissolving about 14.6 g of BGG in 582 mL of 150 mM NaCl and 20 mM histidine containing buffer titrated to pH 6 so that the initial BGG concentration was nominally 25.1 mg / mL. The material was filtered through a 0.2 μm PES filter (VWR, Radnor, PA) and then processed in a TFF apparatus. The pump speed was adjusted so that the feed pressure was initially 30 psi and the retentate valve was adjusted so that the retention pressure was initially 10 psi. The material was concentrated for 4.1 hours without adjusting the pump speed or the retentate valve. Initial concentrations and final concentrations were determined to be 25.4 ± 0.6 and 159 ± 6 mg / mL, respectively, according to Bradford analysis, as shown in Table 19 below. Caffeine-containing samples were prepared by dissolving 14.2 g of BGG in 566 mL of 15 mg / mL caffeine, 150 mM NaCl and 20 mM histidine-containing buffer, titrated to pH 6, so that the initial BGG concentration was nominal 25.1 mg / mL. It was. The material was filtered through a 0.2 μm PES filter (VWR, Radnor, PA) and then processed in a TFF apparatus. The pump speed and the retentate valve were set to the same level as before. The feed pressure and the retentate pressure were found to be 30 psi and 10 psi, respectively, as before. The material was concentrated for 4.1 hours without adjusting the pump speed or the retentate valve. Initial concentrations and final concentrations were determined to be 24.4 ± 0.5 and 225 ± 10 mg / mL, respectively, according to Bradford analysis, as shown in Table 19 below. When caffeine was used in the TFF process, the final protein concentration increased from 159 to 225 mg / mL, an increase of about 42% compared to the control.

Table 19

Sample Initial concentration (mg / mL) Final concentration (mg / mL) Control 25.4 ± 0.6 159 ± 6 15 mg / mL caffeine 24.4 ± 0.5 225 ± 10

EXAMPLE  27: BGG  Caffeine Effect on Sterile Filtration of Solutions

Bovine gamma globulin (BGG), L-histidine and caffeine were purchased from Sigma-Aldrich (St. Louis, MO, trade names G5009, H6034 and C7731). Deionized (DI) water was prepared from tap water using a Direct-Q 3 UV purification system from EMD Millipore (Billerica, Mass.). A 25-mm polyethersulfone (PES) filter with 0.2 μm pore size was purchased from GE Healthcare (Chicago, IL, Catalog No. 6780-2502). 1 mL Luer-Rock syringe was purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, Ref. No. 309628). 20 mM histidine buffer, pH 6.0, was prepared using L-histidine, deionized water and titrated to pH 6.0 by addition of 1 M HCl. Caffeine 15 mg / mL solution was prepared using histidine buffer. Using caffeine-free and caffeine-containing buffer, BGG was reconstituted to a final concentration of about 280 mg / mL. Protein concentration c was calculated by the formula:

(식 4)

(Equation 4)

Where m _p is the protein weight, b is the buffer addition volume, and v is the partial specific volume of BGG, where 0.74 mL / g is considered. The viscosity of each sample was measured using a microVisc rheometer (RheoSense, San Ramon, CA) at a shear rate of 250 s ⁻¹ at 23 ° C. A tensile compression tester (TCT, Instron, Needham, MA, part number 3343) equipped with a 100 N load cell (Instron, Needham, MA, part number 2519-103) is used to measure the energy required to pass the BGG solution through the sterile filter. It was. The syringe plunger was pushed at a rate of 159 mm / min over a 50 mm distance. The energy requirement was calculated by integrating the load-versus-extension curve measured by TCT and the results are summarized in Table 20 below.

Table 20

Sample Protein concentration (mg / mL) Caffeine Concentration (mg / mL) Viscosity (cP) Energy requirement (mJ) One 280 0 106 198 2 280 15.1 68.9 181

EXAMPLE  28: Excipients to Improve Protein A Chromatography Elution

Four experimental grade biosimilar antibodies, ipilimumab, ustekinumab, omalizumab and tocilizumab were purchased from Bioceros (Utrecht, The Netherlands). These antibodies were provided as aliquots frozen at protein concentrations 20, 26, 15 and 23 mg / mL in 40 mM aqueous sodium acetate solution, 50 mM Tris-HCl buffer, pH 5.5, respectively. The protein solution was thawed at room temperature before measurement and then filtered through a 0.2 μm polyethersulfone filter. The filtered protein stock solution was mixed in a 1: 1 ratio of protein stock solution: binding buffer. The binding buffer used to promote antibody binding to Protein A resin consisted of 0.1 M sodium phosphate and 0.15 sodium chloride, pH 7.2 in deionized (DI) water. Deionized water was prepared from tap water using a Direct-Q 3 UV purification system from EMD Millipore (Billerica, Mass.). These solutions were used for Protein A binding and elution experiments using PIERCE ^™ Protein-A spin plates for IgG screening (ThermoFisher Scientific catalog # 45202). The plate was equipped with 96 wells, and 50 μl of Protein A resin was contained in each well. 200 μl of binding buffer was added to each well, and the resin was washed with binding buffer by centrifuging the plate at 1000 × g for 1 minute and then discarding the flow-through. All subsequent centrifugation steps were performed at 1000 xg for 1 minute. This washing procedure was repeated once. After this first wash step, diluted protein samples, i.e. samples containing Ipilimumab, Ustekinumab, Omalizumab and Toxilijumab, were added to the wells of the plate (200 μl per well). The plate was then placed on a Daigger Scientific (Vernon Hills, IL) Labgenius rotary shaker, stirred at 260 rpm for 30 minutes, then the plate was centrifuged and flow-through removed. The wells were then washed by adding 500 μl of binding buffer to each well, centrifuging the plates and removing the flow-through. This washing step was repeated twice. After this wash step, proteins were eluted from the plates using elution buffer with various excipients added. At each elution, 50 μl of neutralization buffer consisting of 1 M sodium phosphate pH 7 was added to each well of the collection plate, followed by 200 μl of elution buffer to each well of the plate. The plate was stirred at 260 rpm for 1 minute and then centrifuged. Flow-through was recovered for analysis. This elution step was repeated once. Excipient-free control buffer included 20 mM citrate and was pH 2.6. Since Protein A elution buffer often contains some salt, 100 mM NaCl elution buffer in citrate buffer was prepared as a second control.

Table 21 lists the excipient solutions used in this example, their concentrations and the final pH of the elution buffer. All of these excipients were purchased from Sigma Aldrich (St. Louis, MO), except aspartame from Herb Store USA (Los Angeles, CA) and trehalose from Cascade Analytical Reagents and Biochemicals (Corvallis, OR). Was purchased from Research Products International (Mt. Prospect, IL, product number S24060). Excipient titrations were mixed in about 10 mL of salt-precitrate buffer control to prepare all excipient-containing elution buffers. Elution buffer was prepared at about 100 mM level of excipient. However, not all excipients are soluble at this concentration; Thus, Table 21 lists the concentrations of all excipients used. The pH of each elution buffer was titrated to pH about 2.6 ± 0.1 using hydrochloric acid or sodium hydroxide as needed.

For each protein sample, ASD high performance size-exclusion chromatography (SEC) using a TSKgel SuperSW3000 column (30 cm x 4.6 mm ID, Tosoh Bioscience, King of Prussia, PA) connected to an HPLC workstation (Agilent HP 1100 system) ) Analysis was performed. Separation was performed at a flow rate of 0.35 mL / min at room temperature. The mobile phase was a buffered aqueous solution consisting of 100 mM sodium phosphate, 300 mM sodium chloride, pH 7. Protein concentration was monitored by measuring absorbance at 280 nm using an Agilent 1100 Series G1315B diode array detector. For each protein, namely Ipilimumab, Ustekinumab, Omalizumab and Toxilijumab, the total amount of protein eluted from the Protein A resin was estimated by integrating chromatograms. The integrated peak areas for each protein, namely Ipilimumab, Ustekinumab, Omalijumab and Toxilijumab, are shown in Table 22-25. In addition, Table 22-25 compares the experimental peak areas with those of the salt-free and salt-containing controls. A value above 100% means that the elution buffer will recover more protein from the Protein A resin than the control, while a value below 100% means less elution buffer is recovered from the Protein A resin compared to the control.

Table 21. EXAMPLE  Excipients Used in 28

Excipient Sigma- of excipients Aldrich  product no Excipient Concentration (mM) pH Caffeine C7731 79 2.6 Acesulfame Potassium 04054 110 2.5 1-methyl-2-pyrrolidone M6762 117 2.6 Aspartame N / A 20 2.6 Taurine T8691 114 2.5 Trehalose N / A 100 2.7 Sucrose N / A 101 2.7 Niacinamide N5535 99 2.7 Sodium chloride control S7653 117 2.6 Control N / A N / A 2.5

Table 22. From Protein A Resin Itilimumap  collection

Excipient Peak area (mAU * min) % Peak area normalized to salt-free control % Peak area normalized to the salt control Site rate 3409 77.9 83.6 Acesulfame Potassium 1567 35.8 38.4 1-methyl-2-pyrrolidone 386 8.8 9.5 Aspartame 4012 91.7 98.3 Taurine 3958 90.4 97.0 Trehalose 3667 83.8 89.9 Sucrose 4585 104.8 112.4 Niacinamide 4295 98.2 105.3 Sodium chloride control 4080 93.2 100.0 Control 4376 100.0 107.2

Table 23. From Protein A resin Ustekinu Map  collection

Excipient Peak Integral Area (mAU * min) % Peak area normalized to salt-free control % Peak area normalized to the salt control Caffeine 2301 86.6 75.2 Acesulfame Potassium 307 16.2 14.0 Aspartame 417 17.4 15.1 1-methyl-2-pyrrolidone 2952 108.8 94.4 Taurine 3257 118.6 103.0 Trehalose 1549 56.6 49.1 Sucrose 1274 51.2 44.4 Niacinamide 3204 116.1 100.8 Sodium chloride control 3176 115.2 100.0

Table 24. From Protein A Resin O'Malley Map  collection

Excipient Peak Integral Area (mAU * min) % Peak area normalized to salt-free control % Peak area normalized to the salt control Caffeine 4040 105.5 117.5 Acesulfame Potassium 3620 94.5 105.3 1-methyl-2-pyrrolidone 3334 87.0 97.0 Aspartame 3605 94.1 104.8 Taurine 4337 113.2 126.1 Trehalose 3571 93.2 103.8 Sucrose 3639 95.0 105.8 Niacinamide 4812 125.6 139.9 Sodium chloride control 3439 89.8 100.0 Control 3831 100.0 111.4

Table 25. From Protein A Resin Tocili County Map  collection

Excipient Peak Integral Area (mAU * min) % Peak area normalized to salt-free control Peak area normalized to the control ( % ) Caffeine 3120 111.2 100.3 Acesulfame Potassium 3083 109.9 99.1 1-methyl-2-pyrrolidone 261 9.3 8.4 Aspartame 556 19.8 17.9 Taurine 3054 108.8 98.2 Trehalose 2781 99.1 89.4 Sucrose 1037 37.0 33.3 Niacinamide 2550 90.9 82.0 Sodium chloride control 3111 110.9 100.0 Control 2806 100.0 90.2

EXAMPLE  29: Excipients to Improve Protein A Chromatography Elution

The test protein used in this example is the same as the protein in Example 28, i. Protein A binding and elution experiments were performed using the same plates as in Example 28. The antibody loading and elution method from the Protein A plate is the same as in Example 28 except for the elution step. In Example 28, eluting wash was performed twice. However, in this example only one wash. As in Example 28, elution buffer was prepared with 20 mM citrate, pH 2.6 control buffer. Elution buffers are shown in Table 26 below. All excipients were purchased from Sigma-Aldrich (St. Louis, Mo.). The recovered protein was analyzed by HPLC in the same manner as in Example 28, and the protein recovery results of each protein, namely, Ipilimumab, Ustekinumab, Omalizumap, and Toxilizumap are shown in Table 27-30 below. .

Table 26. EXAMPLE  Excipients Used in 29

Excipient Sigma- Aldrich  product no Excipient Concentration (mM) pH Control N / A N / A 2.5 Sodium chloride control S7653 117 2.6 Niacinamide N5535 99 2.7 Taurine T8691 114 2.5 Imidazole I5513 100 2.6 4-hydroxybenzenesulfonic acid 171506 107 2.6 Caffeine C7731 79 2.6

Table 27. From Protein A Resin Itilimumap  collection

Excipient Peak area (mAU * min) % Peak area normalized to salt-free control % Peak area normalized to the salt control Control 4841 100.0 88.3 Sodium chloride control 5485 113.3 100.0 Niacinamide 6300 130.1 114.8 Taurine 7557 156.1 137.8 Imidazole 6071 125.4 110.7 4-hydroxybenzenesulfonic acid 5836 120.6 106.4 Caffeine 6051 125.0 110.3

Table 28. From Protein A resin Ustekinu Map  collection

Excipient Peak Integral Area (mAU * min) % Peak area normalized to salt-free control % Peak area normalized to the salt control Control 4572 100.0 107.9 Sodium chloride control 4238 92.7 100.0 Niacinamide 5848 127.9 138.0 Taurine 4744 103.8 112.0 Imidazole 4617 101.0 108.9 4-hydroxybenzenesulfonic acid 4132 90.4 97.5 Caffeine 5084 111.2 120.0

Table 29. From Protein A Resin O'Malley Map  collection

Excipient Peak Integral Area (mAU * min) % Peak area normalized to salt-free control % Peak area normalized to the salt control Control 4194 100.0 91.7 Sodium chloride control 4574 109.1 100.0 Niacinamide 5748 137.0 125.7 Taurine 4676 111.5 102.2 Imidazole 2589 61.7 56.6 4-hydroxybenzenesulfonic acid 3190 76.1 69.7 Caffeine 5807 138.5 127.0

Table 30. From Protein A Resin Tocili County Map  collection

Excipient Peak Integral Area (mAU * min) % Peak area normalized to salt-free control % Peak area normalized to the salt control Control 4667 100.0 97.5 Sodium chloride control 4786 102.6 100.0 Niacinamide 5225 111.9 109.2 Taurine 5396 115.6 112.7 Imidazole 4754 101.9 99.3 4-hydroxybenzenesulfonic acid 4539 97.3 94.8 Caffeine 5656 121.2 118.2

EXAMPLE  30: Protein A Chromatography From the column O'Malley Map  Excipients to Improve Dissolution

Experimental-grade Omalizumap was purchased from Bioceros (Utrecht, The Netherlands) and received frozen at 40 mg sodium acetate solution, 50 mM tris-HCl buffer, 15 mg / mL in pH 5.5. Proteins were thawed at room temperature prior to measurement and then filtered through a 0.2 μm polyethersulfone filter. The filtered material was mixed in a 1: 1 ratio with binding buffer consisting of 20 mM sodium phosphate pH 7 in deionized water. Tap water was purified using a Direct-Q 3 UV purification system from EMD Millipore (Billerica, Mass.) To obtain deionized water. Protein A purification was performed using a HiTrap Protein-A HP 1 mL column from GE Healthcare (Chicago, IL, product # 29048576). In each experiment, the column was first equilibrated with 10 mL of binding buffer. After equilibration, 30 mg of protein was loaded onto a Protein A column. The column was then rinsed with 5 mL of binding buffer. After column washing, bound Omalizumap was eluted from the column using some of the elution buffers listed in Table 31 below. Elution buffer was prepared by dissolving the excipients specified in 20 mM citrate buffer, pH 4.0. All elution buffers were titrated to pH 4.0. Five 1 mL fractions were collected. Finally, the column was washed with 5 mL of 100 mM citrate, pH 3.0 buffer to regenerate Protein A. The flow rate for each stage was 1 mL / min and was maintained using a Fusion 100 infusion pump (Chemyx, Stafford, TX). 10 mL NormJect Luer Lock Syringe (Henke Sass Wolf, Tuttlingen, Germany, reference no. 4100-000V0) was used.

Total protein content was analyzed by high performance size-exclusion chromatography (SEC) analysis of the eluting fractions E1, E2, E3, E4 and E5. SEC analysis was performed using a TSKgel SuperSW3000 column (30 cm × 4.6 mm ID, Tosoh Bioscience, King of Prussia, PA) connected to an HPLC workstation (Agilent HP 1100 system). Separation was performed at a flow rate of 0.35 mL / min at room temperature. The mobile phase is a buffered aqueous solution of 100 mM sodium phosphate, 300 mM sodium chloride, pH 7. Protein concentration was monitored by measuring absorbance at 280 nm using an Agilent 1100 Series G1315B diode array detector. The total amount of protein eluted from the Protein A resin was estimated by integrating the chromatogram.

Since citrate is a common excipient used in Protein A chromatography, it was used herein as a control. Elution fractions of control samples were observed insoluble aggregates when stored overnight at 4 ° C., as confirmed by precipitated phase formation. As such, the peak areas reported in Table 31 below indicate the total amount of soluble protein in the elution fractions. Insoluble aggregates were observed only in the control sample and no such aggregates were observed in the other samples. The higher peak area compared to the control (using citrate excipient) means that the test excipient can be used to more effectively separate proteins from the column.

Table 31. Protein A From the column O'Malley Map  Elution

Eluent excipient Eluent excipient Concentration (mM ) E1 peak area
(mAU * min) E2 peak area
(mAU * min) E3 peak area
(mAU * min) E4 peak area
(mAU * min) E5 peak area
(mAU * min) Total peak area
(mAU
* min) Site rate
(Control) 103 352 9670 4098 4245 2953 21318 Imidazole 100 236 10224 7373 3894 2620 24348 Taurine 125 408 17018 7676 3349 2211 30662 Niacinamide 102 228 14492 5307 2914 2014 24955 Caffeine 81 617 21965 8069 3301 1911 35863

EXAMPLE  31: caffeine excipient variety  Contained in content BGG  Formulation

The formulations were prepared using various molar concentrations of caffeine (concentrations listed in Table 32 below) and test proteins, which were intended to mimic the therapeutic proteins to be used in the therapeutic formulations. The formulations of this example were prepared in 20 mM histidine buffer to measure the viscosity in the following manner. Caffeine 0 and 80 mM stock solutions were prepared in 20 mM histidine and the resulting solution was titrated to pH 6 with a small amount of sodium hydroxide or hydrochloric acid before dissolving the model protein. Additional solutions of varying caffeine concentrations were prepared by mixing the two stock solutions at various volume ratios to provide a series of caffeine-containing solutions of the concentrations listed in Table 32 below. After preparing these excipient solutions, 0.7 mL of each excipient solution was added to 0.25 g of lyophilized BGG powder, so that the test protein bobbin gamma globulin (BGG) was added to each test solution at a final protein concentration of about 280 mg / mL. Was dissolved. BGG-containing solutions were prepared in 5 mL sterile polypropylene test tubes and stirred at 100 rpm overnight on a rotary shaker table. These solutions were then placed in a 2 mL microcentrifuge tube and centrifuged at 2400 rpm for 5 minutes in an IEC MicroMax centrifuge to remove the trapped air prior to viscosity measurements.

Viscosity measurements for the formulations prepared as above were performed using a microVisc viscometer (RheoSense, San Ramon, CA). The viscometer was equipped with an A-10 chip with a channel depth of 100 microns and operated at a shear rate of 250 1 / s at 25 ° C. To determine the viscosity, the test formulation was loaded into a viscometer and care was taken to remove all bubbles from the pipette. Pipettes containing the loaded sample formulation were placed in the apparatus and incubated at the measurement temperature for about 5 minutes. The apparatus was then run until the channel was fully equilibrated with the test fluid, as confirmed by a stable viscosity reading, and the viscosity was recorded in centipoise. The obtained viscosity results are shown in Table 32 below.

Table 32

Caffeine Concentration ( mM ) Viscosity (cP) Standardized viscosity 0 83 1.00 5 67 0.81 10 70 0.84 20 77 0.92 30 63 0.76 40 65 0.78 50 65 0.78 60 57 0.69 70 50 0.60 80 50 0.60

EXAMPLE  32: Deionized water  Preparation of co-solute solution in water

Compounds used as co-solutes to enhance the solubility of caffeine in water were purchased from Sigma-Aldrich (St. Louis, MO), which compounds are niacinamide, proline, procaine HCl, ascorbic acid, 2,5-dihydride Oxybenzoic acid, lidocaine, saccharin, acesulfame K, tyramine and aminobenzoic acid. The dry solids were dissolved in deionized water and in some cases titrated to about 6-8 with 5 M hydrochloric acid or 5 M sodium hydroxide as needed to prepare a solution of each co-solute. The solution was then diluted to a final volume of 25 mL or 50 mL using a Class A scalpel flask and the concentrations were recorded based on the weight of dissolved compound and the final volume of solution. The prepared solution was used as such or diluted with deionized water.

EXAMPLE  33: Caffeine Solubility inspection

The effects of various co-solutes on caffeine solubility at ambient temperature (about 23 ° C.) were evaluated in the following manner. Dry caffeine powder (Sigma-Aldrich, St. Louis, Mo.) was placed in a 20 mL glass scintillation vial and the weight of caffeine was recorded. In some cases 10 mL of the co-solute solution prepared according to Example 32 was added to the caffeine powder; In other cases, a blend of co-solute and deionized water was added to the caffeine powder and the final addition volume was maintained at 10 mL. The extent to which dry caffeine powder contributed to the volume was estimated to be negligible in all these mixtures. A small magnetic rod was placed in the vial and the solution was mixed vigorously by placing on a stirring plate for about 10 minutes. After about 10 minutes, dissolution of the dry caffeine powder in the vial was observed and the results are shown in Table 33 below. These observations indicate that niacinamide, procaine HCl, 2,5-dihydroxybenzoic acid sodium salt, saccharin sodium salt and tyramine chloride salt all reported caffeine dissolution limits (according to Sigma-Aldrich, ˜16 mg / mL), which can dissolve caffeine up to at least about 4 times.

Table 33

Exam number Ball-solute Caffeine Observation designation Concentration (mg / mL) (mg / mL) 33.1 Proline 100 50 DND 33.2 Niacinamide 100 50 CD 33.3 Niacinamide 100 60 CD 33.4 Niacinamide 100 75 CD 33.5 Niacinamide 100 85 CD 33.6 Niacinamide 100 100 CD 33.7 Niacinamide 80 85 CD 33.8 Niacinamide 50 80 CD 33.9 Procaine HCl 100 85 CD 33.10 Procaine HCl 50 80 CD 33.11 Niacinamide 30 80 DND 33.12 Procaine HCl 30 80 DND 33.13 Niacinamide 40 80 MD 33.14 Procaine HCl 40 80 DND 33.15 Ascorbic acid, Na 50 80 DND 33.16 Ascorbic acid, Na 100 80 DND 33.17 2,5 DHBA, Na 40 80 CD 33.18 2,5 DHBA, Na 20 80 MD 33.19 Lidocaine HCl 40 80 DND 33.20 saccharin. Na 90 80 CD 33.21 Acesulfame K 80 80 DND 33.22 Tyramine HCl 60 80 CD 33.23 Na aminobenzoate 46 80 DND 33.24 Saccharin, Na 45 80 DND 33.25 Tyramine HCl 30 80 DND

CD = complete dissolution; MD = mostly dissolved; DND = not soluble

EXAMPLE  34: HUMIRA ^® Profile of

HUMIRA ^® (AbbVie Inc., Chicago, IL) reduces the inflammatory response of autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate to severe chronic psoriasis and juvenile idiopathic arthritis Is a commercially available formulation of the therapeutic monoclonal antibody adalimumab, a TNF-α blocker typically prescribed for. HUMIRA ^® contains 40 mg of adalimumab, 4.93 mg of sodium chloride, 0.69 mg of sodium phosphate monobasic dihydrate, 1.22 mg of sodium phosphate dibasic dihydrate, 0.24 mg of sodium citrate, 1.04 mg of citric acid monohydrate, 9.6 mg of mannitol and polysorbate 80 Commercially available in a single dose of 0.8 mL, containing 0.8 mg. The viscosity versus concentration profile of this formulation was obtained in the following manner. The membrane was washed by charging about 15 mL of deionized water in an Amicon Ultra 15 centrifuge concentrator of 30 kDa molecular weight cutoff (EMD-Millipore, Billerica, Mass.) And centrifuging at 4000 rpm for 10 minutes on Sorvall Legend RT (ThermoFisher Scientific). The residue was then removed and 2.4 mL of HUMIRA ^® liquid formulation was added to the concentrator test tube and centrifuged at 4000 rpm for 60 minutes at 25 ° C. The concentration of the residue was determined by diluting 10 μl of the residue with 1990 μl of deionized water and measuring the absorbance at 280 nm and then calculating the concentration using the dilution factor and extinction coefficient of 1.39 mL / mg-cm. The viscosity of the concentrated sample was measured under a shear rate of 250 sec ⁻¹ and 23 ° C. on a microVisc viscometer (RheoSense, San Ramon, Calif.) Equipped with an A05 chip. After measuring the viscosity, the sample was diluted with a small amount of filtrate and the concentration and viscosity measurements were repeated. Using this process, as shown in Table 34 below, the viscosity value according to the adalimumab concentration variation was obtained.

Table 34

Adalimu map  (mg / mL) Viscosity (cP) 277 125 253 63 223 34 202 20 182 13

EXAMPLE  35: with viscosity-lowering excipient HUMIRA ^®  Reconstitution of the Formulation

The examples below describe the general process of reconstituting in buffers by adding a viscosity-lowering excipient to HUMIRA ^® . About 0.15 g of histidine and 0.75 g of caffeine (Sigma-Aldrich, St. Louis, Mo.) were dissolved in deionized water to prepare a viscosity-lowering excipient solution in 20 mM histidine. The pH of the prepared solution was titrated to about 5 using 5 M hydrochloric acid. Then, deionized water was added to the solution and diluted to a final volume of 50 mL in a flask. HUMIRA ^® was reconstituted at a high mAb concentration using the prepared buffered viscosity-lowering excipient solution. Next, an Amicon Ultra 15 centrifuge tube of 30 kDa molecular weight cutoff was washed, and about 0.8 mL of HUMIRA ^® was added thereto, followed by centrifugation at 4000 rpm at 25 ° C. for 8-10 minutes at Sorvall Legend RT. Thereafter, about 14 mL of a buffered viscosity-lowering excipient solution as described above was prepared and added to the concentrated HUMIRA ^® placed in a centrifuge concentrator. After gentle mixing, the samples were centrifuged at 4000 rpm at 25 ° C. for about 40-60 minutes. The retentate is a concentrated sample of HUMIRA ^® reconstituted in a buffer with the addition of a viscosity-lowering excipient. The viscosity and concentration of the samples were measured and in some cases diluted with a small amount of filtrate to determine the viscosity at lower concentrations. Viscosity measurements were completed using a microVisc viscometer in the same manner as the method using the concentrated HUMIRA ^® formulation in the previous example. Concentrations were determined by Bradford analysis using standard curves prepared with HUMIRA ^® stock solutions diluted in deionized water. The reconstitution of HUMIRA ^® using a viscosity-lowering excipient resulted in a 30% to 60% decrease in viscosity compared to the viscosity value of HUMIRA ^® concentrated in unreconstituted commercial buffer, as shown in Table 35 below.

Table 35

Adalimu map  Concentration (mg / mL) Viscosity (cP) 290 61 273 48 244 20 205 14

EXAMPLE  36: using caffeine as excipient Adalimu map  Improved stability of the solution

After exposing the sample to two different stress environments, the stability of the adalimumab solution with the caffeine excipient and the adalimumab solution without the caffeine excipient was assessed: stirring and freeze-thaw. The adalimumab drug formulation HUMIRA® (AbbVie) with the properties described in more detail in Example 34 was used. HUMIRA® samples were concentrated to 200 mg / mL adalimumab concentration in original buffer as described in Example 39; This concentrated sample was named "Sample 1". A second sample was prepared using adalimumab ˜200 mg / mL and caffeine addition 15 mg / mL as described in Example 40; This concentrated sample with caffeine was named "Sample 2". These two samples were diluted to 1 mg / mL of adalimumab final concentration by adding a diluent as follows: Sample 1 diluent is the original buffer and Sample 2 diluent is 20 mM histidine, 15 mg / mL caffeine, pH = 5 to be. Two HUMIRA® dilutions were filtered through a 0.22 μm syringe filter. For each diluted sample, three batches of 300 μl each were prepared by placing them in a 2 mL Eppendorf tube under a laminar flow hood. The samples were exposed to the following stress environment: For stirring, the samples were placed at 300 rpm for 91 hours on a rotary shaker; For freeze-thaw, the samples were subjected to seven cycles at -17 to 30 ° C for an average of 6 hours per condition.

Table 36

Sample # Additive excipients Stress environment 1-C none none 1-A none Stirring 1-FT none Freeze-thaw 2-C 15 mg / mL caffeine none 2-A 15 mg / mL caffeine Stirring 2-FT 15 mg / mL caffeine Freeze-thaw

EXAMPLE 37: dynamic  Stability Assessment by Light Scattering (DLS)

The hydrodynamic radius of adalimumab molecules in the sample of Example 36 was measured using a Brookhaven Zeta Plus dynamic light scattering device and evidence of aggregate population formation was confirmed. Table 37 shows the DLS results for six samples prepared according to Example 36: Some of these (1-A, 1-FT, 2-A and 2-FT) were exposed to a stressed environment (“stress Exposed samples "); The other samples (1-C and 2-C) were not exposed. The DLS data in Table 37 show that monoclonal antibodies in the caffeine free stress exposed samples form a multimodal particle size distribution. When caffeine was not added as an excipient, stress exposed samples 1-A and 1-FT were observed to have a larger effective diameter than stress non-exposed samples 1-C, which also resulted in a second population of particles with significantly larger diameters. Mean; This larger group of particles is evidence of aggregation into particles that are not visible to the naked eye. Only stress exposed samples containing caffeine (Samples 2-A and 2-FT) formed one particle population with particle diameters similar to stress non-exposed samples 2-C. These results demonstrate that adding caffeine to the sample reduces the formation of aggregates or particles that are not visible to the naked eye.

Table 37

Sample # available diameter  (nm) Group 1 diameter  (nm) Group 1 density On by % Group 2 diameter  (nm) % By concentration of population 2 1-C 10.9 10.8 100 - - 1-A 11.5 10.8 87 28.9 13 1-FT 20.4 11.5 66 112.2 44 2-C 10.5 10.5 100 - - 2-A 10.8 10.8 100 - - 2-FT 11.4 11.4 100 - -

Table 38A and Table 38B show the DLS raw data of the adalimumab sample of Example 36, showing the particle size distribution. In these tables, G (d) is the intensity-weighted differential size distribution. C (d) is the cumulative intensity-weighted differential size distribution.

Table 38A

Table 38B

EXAMPLE  38: Evaluation of stability by size-exclusion chromatography (SEC)

Size exclusion chromatography was used to detect non-visually visible particulates of less than about 0.1 micron in size in the stress exposed adalimumab sample and the stress non-exposed adalimumab sample shown in Example 36. A TSKgel SuperSW3000 column (Tosoh Biosciences, Montgomeryville, Pa.) Equipped with a guide column was used to perform SEC, and the eluate was monitored at 280 nm. A total of 10 μl in each of the stress-exposed and non-exposed samples of Example 36 were eluted by isocratic using pH 6.2 buffer (100 mM phosphate, 325 mM NaCl) at a flow rate of 0.35 mL / min. The residence time of the adalimumab monomer was about 9 minutes. No detectable aggregates were identified in the samples containing the caffeine excipients and the monomer content remained constant in all three samples.

EXAMPLE  39: HERCEPTIN ^®  Formulation  Viscosity decrease

Monoclonal antibodies to the trad Stu jumaep ^{(trastuzumab) (HERCEPTIN ®, Genentech} ) obtained as a lyophilized powder was reconstituted with 21 mg / mL in deionized water. The prepared solution was concentrated as it was by centrifugation at 3500 rpm for 1.5 hours in an Amicon Ultra 4 centrifuge concentrator tube (molecular weight cutoff, 30 kDa). Samples were diluted 200-fold in titration buffer and then absorbance was measured at 280 nm and the concentration was measured by applying an absorption coefficient of 1.48 mL / mg. Viscosity was measured using a RheoSense microVisc viscometer.

Histidine and excipients were dissolved in distilled water, and then the pH was adjusted to an appropriate level to prepare an excipient buffer comprising salicylic acid and caffeine alone or in combination. The conditions of buffer systems 1 and 2 are summarized in Table 39.

Table 39

Buffer  system # Salicylic acid  density Caffeine concentration Osmosis  Concentration (mOsm / kg) pH One 10 mg / mL 10 mg / mL 145 6 2 0 15 mg / mL 86 6

HERCEPTIN ^® solution was diluted in ˜1: 10 ratio in excipient buffer and concentrated in Amicon Ultra 15 (MWCO 30 kDa) concentrator tubes. Concentrations were obtained by Bradford analysis and compared to standard calibration curves prepared from stock HERCEPTIN ^® samples. Viscosity was measured using a RheoSense microVisc viscometer. Concentration and viscosity measurements of the various HERCEPTIN ^® solutions are shown in Table 40 below, where buffer systems 1 and 2 are the buffers shown in Table 39.

Table 40

Excipient Free Control Solution Buffer  system 1: 10 mg / mL caffeine + 10 mg / ml Salicylic acid  adding for liquid Buffer  system 2: 15 mg / mL caffeine addition solution Viscosity (cP) Antibody Concentration (mg / mL) Viscosity (cP) Antibody Concentration (mg / mL) Viscosity (cP) Antibody Concentration (mg / mL) 37.2 215 9.7 244 23.4 236 9.3 161 7.7 167 12.2 200 3.1 108 2.9 122 5.1 134 1.6 54 2.4 77 2.1 101

Buffer System 1, containing both salicylic acid and caffeine, exhibited a 76% peak viscosity reduction at 215 mg / mL compared to the control sample. Caffeine only buffer system 2 showed a viscosity reduction of up to 59% at 200 mg / mL conditions.

EXAMPLE  40: AVASTIN ^®  Lower viscosity of the formulation

AVASTIN ^® received service (monoclonal antibody chopping Park Sihoo jumaep agents, Genentech) in 25 mg / mL solution in Histidine buffer. This sample was concentrated at 3500 rpm in an Amicon Ultra 4 centrifuge concentrator tube (MWCO 30 kDa). The viscosity was measured with a RheoSense microVisc viscometer and the concentration was determined by measuring the absorbance at 280 nm (absorption coefficient, 1.605 mL / mg). Excipient buffer was prepared by adding 10 mg / mL caffeine with 25 mM histidine HCl. AVASTIN ^® stock solutions were diluted with excipient buffer and then concentrated in Amicon Ultra 15 centrifuge concentrator tubes (MWCO 30 kDa). The concentration of excipient samples was determined by Bradford analysis and the viscosity was measured using a RheoSense microVisc viscometer. The results are shown in Table 41 below.

Table 41

Concentration (mg / mL) Viscosity without excipients (cP) 10 mg / mL Caffeine with Caffeine Excipients (cP) Viscosity with Excipients % Reduction 266 297 113 62% 213 80 22 73% 190 21 13 36%

AVASTIN ^® showed a peak viscosity reduction of 73% compared to the control AVASTIN ^® sample when concentrated to 213 mg / mL with the addition of 10 mg / mL caffeine.

EXAMPLE  41: Preparation of formulation containing caffeine, second excipient and test protein

Formulations were prepared using caffeine or a combination of caffeine and a second excipient compound and a test protein as an excipient compound, and the test protein was intended to mimic the therapeutic protein to be used in the therapeutic formulation. These formulations were prepared in 20 mM histidine buffer with the addition of various excipient compounds to determine the viscosity in the following manner. The excipient combinations (excipients A and B, shown in Table 28 below) were dissolved in 20 mM histidine and the resulting solution was titrated to pH 6 by addition of small amounts of sodium hydroxide or hydrochloric acid before dissolving the model protein. Once the excipient solution was prepared, test protein bobbin gamma globulin (BGG) was dissolved at a rate at which a final protein concentration of about 280 mg / mL was achieved. BGG solutions in excipient solutions were prepared in 20 mL glass scintillation vials and then placed on a rotary shaker table and stirred at 80-100 rpm overnight. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for about 10 minutes in an IEC MicroMax microcentrifuge to remove the trapped bubbles prior to viscosity measurements.

Viscosity measurements of the formulations prepared as above were performed using a DV-IIT LV viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of the three collected data points was calculated and recorded in Table 42 below as the viscosity of the sample. The viscosity of the excipient-containing solution was normalized based on the viscosity of the excipient-free model protein solution. The normalized viscosity is the ratio of the viscosity of the excipient-containing model protein solution to the viscosity of the excipient-free model protein solution.

Table 42

Excipient A Excipient B Standardized viscosity designation Concentration (mg / mL) designation Concentration (mg / mL) - 0 - 0 1.00 Caffeine 15 - 0 0.77 Caffeine 15 Sodium acetate 12 0.77 Caffeine 15 Sodium sulfate 14 0.78 Caffeine 15 Aspartic acid 20 0.73 Caffeine 15 CaCl ₂ Dihydrate 15 0.65 Caffeine 15 Dimethyl sulfone 25 0.65 Caffeine 15 Arginine 20 0.63 Caffeine 15 Leucine 20 0.69 Caffeine 15 Phenylalanine 20 0.60 Caffeine 15 Niacinamide 15 0.63 Caffeine 15 ethanol 22 0.65

EXAMPLE  42: Dimethyl Sulfone  Preparation of Formulations Containing Test Proteins

Formulations were prepared using dimethyl sulfone (Jarrow Formulas, Los Angeles, CA) and test proteins as excipient compounds, and the test proteins were intended to mimic the therapeutic proteins to be used in the therapeutic formulations. These formulations were prepared in 20 mM histidine buffer to measure viscosity in the following manner. Dimethyl sulfone was dissolved in 20 mM histidine, the solution obtained was titrated to pH 6 by the addition of a small amount of sodium hydroxide or hydrochloric acid, filtered through a 0.22 μm filter, and the model protein dissolved. Once the excipient solution was prepared, test protein bobbin gamma globulin (BGG) was dissolved at a concentration of about 280 mg / mL. BGG solutions in excipient solutions were prepared in 20 mL glass scintillation vials and then placed on a rotary shaker table and stirred at 80-100 rpm overnight. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for about 10 minutes in an IEC MicroMax microcentrifuge to remove the trapped bubbles prior to viscosity measurements.

Viscosity measurements of the formulations prepared as above were performed using a DV-IIT LV viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 ° C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated for 3 minutes at the desired shear rate and temperature and the measurements collected for 20 seconds. An additional two steps were then performed, consisting of 20 seconds of measurement collection period 1 minute after shear incubation. The average of three collected data points was obtained and recorded as the viscosity of the sample. The viscosity of the excipient-containing solution was normalized based on the viscosity of the excipient-free model protein solution. The normalized viscosity is the ratio of the viscosity of the excipient-containing model protein solution to the viscosity of the excipient-free model protein solution.

Table 43

Dimethyl Sulfone  Concentration (mg / mL) Standardized viscosity 0 1.00 15 0.92 30 0.71 50 0.71 30 0.72

Equivalent

While certain embodiments of the invention have been described herein, the above description is illustrative and not restrictive. While the invention has been described and described in detail by way of the preferred embodiments thereof, those skilled in the art may make various modifications to the form and details without departing from the scope of the invention as set forth in the appended claims. I will understand that. Those skilled in the art will appreciate that many modifications to the present invention will become apparent upon reading the details. Unless otherwise stated, all numbers indicating reaction conditions, amounts of constituents, etc., as used in the specification and claims, are to be understood as being modified in all instances by the term "about." In other words, unless stated to the contrary, the numerical parameters referred to herein are approximations that may vary depending on the desired properties to be obtained by the present invention.

Claims (39)

As a method of improving the parameters of a protein-related process,
Viscosity-lowering excipient additives comprising one or more excipient compounds selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, and diones and sulfones ( providing a viscosity-reducing excipient additive, and
Adding the at least one excipient compound to a carrier solution for a protein-related process in a viscosity-reducing amount
Wherein the parameters are improved,
Wherein the carrier solution comprises a protein of interest. The method of claim 1,
Wherein said parameter is selected from the group consisting of protein production cost, protein yield, protein yield and protein production efficiency. The method of claim 1,
The parameter is a proxy parameter. The method of claim 1,
Wherein the protein-related process is an upstream processing process. The method of claim 4, wherein
The carrier solution for the upstream treatment process is a cell culture medium. The method of claim 5,
Adding the excipient additive to the carrier solution,
A first substep of adding the excipient additive to the supplement medium to produce an excipient-containing supplement medium; And
And a second substep of adding the excipient-containing supplemental medium to the cell culture medium. The method of claim 1,
Wherein the protein-related process is a downstream processing process. The method of claim 7, wherein
Wherein said downstream treatment process is a chromatography process. The method of claim 8,
Wherein said chromatography process is a protein-A chromatography process. The method of claim 8,
The chromatography process recovers the protein of interest,
The subject protein is characterized by an improvement in protein-related parameters selected from the group consisting of improved purity, improved yield, reduced particles, reduced misfolding or reduced aggregation as compared to the control solution. The method of claim 10,
Wherein the improvement of the protein-related parameter is an improvement of the target protein recovery from the chromatography process. The method of claim 1,
The protein-related processes are used for filtration, injection, syringeing, pumping, mixing, centrifugation, membrane separation and lyophilization. The process is selected from the group consisting of. The method of claim 12,
Wherein the process has a lower force consumption compared to a control process. The method of claim 1,
The protein-related process is selected from the group consisting of a cell culture process, a cell culture harvesting process, a chromatography process, a virus inactivation process and a filtration process. The method of claim 14,
The protein-related process is a virus inactivation process,
Wherein the virus inactivation process is performed at a pH of about 2.5 to about 5.0. The method of claim 15,
The protein-related process is a virus inactivation process,
Wherein the virus inactivation process is performed at a higher pH than the control process. The method of claim 14,
Wherein said protein-related process is a filtration process. The method of claim 17,
Wherein said filtration process is a virus removal filtration process or an ultrafiltration / diafiltration process. The method of claim 17,
The filtration process is characterized in that the filtration-related parameters are improved. The method of claim 19,
Wherein the improvement of the filtration-related parameter is a faster filtration rate compared to the filtration rate of the control solution. The method of claim 19,
Wherein the filtration-related parameter improvement is a quantitative decrease in protein aggregates relative to the amount of protein aggregates generated by the control filtration process. The method of claim 19,
Wherein the filtration-related parameter improvement is a higher mass transfer efficiency compared to the mass transfer efficiency of the control filtration process. The method of claim 19,
Wherein the filtration-related parameter improvement is a higher concentration or yield of target protein compared to the concentration or yield of target protein achieved by the control filtration process. The method of claim 1,
And the viscosity-lowering excipient additive comprises two or more excipient compounds. The method of claim 1,
Wherein said at least one excipient compound is a hindered amine. The method of claim 26,
Said at least one excipient compound is selected from the group consisting of caffeine, saccharin, acesulfame potassium, aspartame, theophylline, taurine, 1-methyl-2-pyrrolidone, 2-pyrrolidinone, niacinamide and imidazole , Way. The method of claim 26,
Wherein said at least one excipient compound is selected from the group consisting of caffeine, taurine, niacinamide and imidazole. The method of claim 1,
Wherein said at least one excipient compound is an anionic aromatic excipient. The method of claim 28,
Wherein said anionic aromatic excipient is 4-hydroxybenzenesulfonic acid. The method of claim 1,
Wherein the viscosity-lowering content is from about 1 mg / mL to about 100 mg / mL of one or more excipient compounds. The method of claim 1,
Wherein the viscosity-lowering content is about 1 mM to about 400 mM of one or more excipient compounds. The method of claim 31, wherein
Wherein the viscosity-lowering content is an amount of about 2 mM to about 150 mM. The method of claim 1,
And the carrier solution comprises an additional substance selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers and buffers. The method of claim 1,
Wherein the subject protein is a therapeutic protein. The method of claim 34, wherein
The therapeutic protein is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments, fusion proteins, PEGylated proteins, antibody-drug conjugates, synthetic polypeptides, protein fragments, lipoproteins, enzymes and structural peptides. How. The method of claim 34, wherein
Wherein said therapeutic protein is a recombinant protein. The method of claim 1,
Further comprising adding a second viscosity-lowering excipient to the carrier solution,
Adding the second viscosity-lowering compound further improves the parameter. As a carrier solution,
A liquid medium in which the protein of interest is dissolved and a viscosity-lowering additive,
The carrier solution, wherein the carrier solution has a low viscosity compared to the control solution. The method of claim 38,
The carrier solution further comprises an additional material selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers and buffers.

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