KR102572453B1 - Excipient compounds for protein processing - 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 amount to a carrier solution for a protein-related process comprising the target protein, and a method for improving a parameter of a protein-related process, and the target protein is Disclosed is a carrier solution comprising a dissolved liquid medium and a viscosity-lowering excipient, wherein the carrier solution has a lower viscosity than a control solution that is substantially identical to the carrier solution except that the viscosity-lowering excipient is present.

Description

Excipient compounds for protein processing

related application

This application claims the benefit of U.S. Provisional Application No. 62/459,893, dated February 16, 2017. The entire contents of the above US Provisional Application 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 antibodies 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 household, nutritional, industrial and industrial uses.

Biopolymers used for therapeutic purposes must be formulated to permit incorporation into the body to treat disease. For example, in certain circumstances, it is advantageous to deliver antibody and protein/peptide biopolymer preparations by subcutaneous (SC) or intramuscular (IM) routes rather than administration by intravenous (IV) infusion. To achieve better patient compliance and SC or IM injection convenience, the liquid volume of the syringe is typically limited to 2 to 3 cc, and the viscosity of the formulation is typically should be less than about 20 centipoise (cP). These delivery parameters are not always well suited to 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 high dose levels of antibody using limited liquid volumes, formulations may require high concentrations of antibody in the delivery vehicle, often exceeding the 150 mg/mL level. At this dose level, the viscosity versus concentration curve of the protein solution passes a linear-nonlinear transition, and the viscosity of the formulation rises significantly with increasing concentration. However, the high viscosity is not suitable for standard SC or IM delivery systems. Solutions of biopolymer-based therapeutics are also prone to stability problems such as precipitation, turbidity, opalescence, denaturation and gel formation, and reversible or irreversible aggregation. Stability issues limit the solution's shelf life or require special handling.

One method for preparing protein formulations for injection is to convert a therapeutic protein solution into a reconstituted powder to prepare a suspension suitable for SC or IM delivery. Lyophilization is a 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 further reconstitution. The powder suspension has a low viscosity before re-dissolution (compared to a solution of the same total volume) and therefore may be suitable for SC or IM injection as long as the particles are small enough to pass through a 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 also creates additional problems. There is still a need in the art for techniques to prepare low viscosity protein formulations for therapeutic purposes while avoiding the problems caused by protein powder suspensions.

In addition to the therapeutic uses of the proteins described above, biopolymers such as enzymes, peptides and structural proteins may also have non-therapeutic uses. These non-therapeutic biopolymers can be produced by a number of different routes, for example delivered from plant sources, animal sources, or produced from cell culture.

Non-therapeutic proteins may be prepared, transported, stored and handled as granular or powdered materials or as solutions or typically as dispersions in water. Biopolymers for non-therapeutic use may be globular or fibrous proteins, and certain forms of these materials may have limited water solubility or may be highly viscous upon dissolution. These solution properties can cause problems in the formulation, handling, storage, pumping and performance of non-therapeutic substances, so there is a need for methods to lower the viscosity and improve the solubility and stability of non-therapeutic protein solutions.

Proteins are complex biopolymers, each with a unique folding 3D structure and surface energy map (hydrophobic/hydrophilic regions and charge). In the case of concentrated protein solutions, these macromolecules can interact strongly in complex ways, even inter-lock, depending on their actual conformation and surface energy distribution. Strong and specific interacting "hot-spots" cause protein clustering, increasing the viscosity of the solution. In order to solve this problem, several excipient compounds are used in biotherapeutic formulations to lower the viscosity of the solution by delaying local interaction and clustering. These trials are individually tuned, often empirically, and sometimes through in silico simulation. Combinations of excipient compounds may be provided, but optimization of such combinations again must proceed empirically and on a case-by-case basis.

There is still a need in the art for a truly universal approach to lowering the viscosity of a protein preparation at a given concentration under non-linear conditions. There is also a further need in the art to achieve such a viscosity drop while maintaining protein activity. It would also be desirable to adapt the viscosity-lowering system for use in formulations with a tunable sustained release profile and for use in formulations suitable for depot injection. In addition, improvements in the manufacturing process of proteins and other biopolymers are desired.

The present invention, in an embodiment, 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, wherein the excipient compound has a viscosity-lowering content ( 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 agent is a pharmaceutical composition, the therapeutic agent comprises a therapeutic protein, and the excipient compound is a pharmaceutically acceptable excipient compound. In an embodiment, the agent is a non-therapeutic agent, and the non-therapeutic agent comprises a non-therapeutic protein. In embodiments, the viscosity-lowering amount lowers the viscosity of the formulation to less than the viscosity of the control formulation. In embodiments, 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% less than the viscosity of the control formulation, or at least about 90% less than 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 of protein, or at least about 100 mg/mL of protein, or at least about 200 mg/mL of protein, or at least about 300 mg/mL of protein. Include in mg/mL. In embodiments, the formulation comprises about 5 mg/mL to about 300 mg/mL of the excipient compound, or about 10 mg/mL to about 200 mg/mL of the excipient compound, or about 20 mg of the excipient compound. /mL to about 100 mg/mL, or about 25 mg/mL to about 75 mg/mL of the excipient compound. In an embodiment, the formulation has improved stability compared to a 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 embodiments, the hindered amine is a local injectable anesthetic compound. Hindered amines can have independent pharmacological properties, and hindered amines can be present in a formulation in an amount that has independent pharmacological effects. In embodiments, the hindered amine may be present in the formulation in an amount less than a therapeutically effective amount. The independent pharmacological activity may be local anesthetic activity. In an embodiment, hindered amines with independent pharmacological activity are combined with a second excipient that 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 embodiments, the formulation may include additional substances selected from the group consisting of preservatives, surfactants, sugars, polysaccharides, arginine, proline, hyaluronidase, stabilizers and buffers.

The present invention also includes administering a liquid therapeutic formulation to a mammal; wherein the therapeutic agent comprises a therapeutically effective amount of a 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 method of treating a disease or disorder in a mammal, wherein the therapeutic agent is effective in 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 embodiments, the formulation is administered by subcutaneous or intramuscular or intravenous infusion. In embodiments, the excipient compound is present in the therapeutic formulation in a viscosity-lowering amount, wherein the viscosity-lowering amount reduces the viscosity of the therapeutic formulation to less than the viscosity of the control formulation. In embodiments, the therapeutic agent has improved stability compared to a control agent. In an embodiment, the excipient compound is an essentially pure substance.

The present invention also includes administering the liquid therapeutic formulation by infusion; wherein the therapeutic agent comprises a therapeutically effective amount of a therapeutic protein; wherein the formulation further comprises a pharmaceutically acceptable excipient compound selected from the group consisting of local injectable anesthetic compounds; A pharmaceutically acceptable excipient compound is added to the formulation in a viscosity-lowering amount; reducing the pain experienced by administration of a therapeutic formulation comprising an excipient compound compared to administration of a control therapeutic formulation in a mammal; A method for reducing pain at the site of injection of a therapeutic protein in a mammal in need thereof, wherein the control therapeutic formulation does not contain an excipient compound and is otherwise identical to the therapeutic formulation.

The invention provides, in embodiments, a therapeutic 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; The protein liquid formulation exhibits 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 does not contain an excipient compound and is otherwise identical to the protein liquid formulation. The stability of a liquid formulation may be cold storage conditions stability, room temperature stability or elevated temperature stability.

In addition, the present invention, in an embodiment, 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, wherein the presence of the excipient compound in the formulation is a protein Generates features that improve protein-protein interactions as measured by the diffusion interaction parameter kD or the second viral coefficient B22. In an embodiment, the agent is a therapeutic agent, which comprises a therapeutic protein. In an embodiment, the agent is a non-therapeutic agent, which comprises a non-therapeutic protein.

The present invention also relates, in an embodiment, to a method of improving a protein-related process comprising providing a liquid formulation as described above and using it in a processing treatment 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.

In addition, the present invention is a viscosity containing at least one excipient compound 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. -Protein comprising the step of providing an excipient additive for lowering, and adding one or more excipient compounds in a viscosity-lowering amount to a carrier solution for a protein-related process comprising the protein of interest, whereby the parameter is improved- It relates to methods for improving the parameters of related processes. In an embodiment, the parameter may be selected from the group consisting of protein production cost, protein yield, protein production rate, and protein production efficiency. The parameter may be a proxy parameter. In an embodiment, the protein-related process is an upstream processing 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 a first substep of adding the excipient additive to a supplemental medium to prepare an excipient-containing supplemental medium, and - a second sub-step of adding the containing supplemental medium to the cell culture medium. In other embodiments, the protein-related process is a downstream processing 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 a protein of interest, wherein the protein of interest exhibits an improvement in a protein-related parameter selected from the group consisting of improved purity, improved yield, reduced particle size, reduced misfolding, or reduced aggregation compared to a control solution. to be characterized In an embodiment, the protein-related parameter that is improved is improved recovery of a protein of interest from a chromatography process. In other embodiments, the protein-related process is selected from the group consisting of filtration, injection, syringing, pumping, mixing, centrifugation, membrane separation and lyophilization, wherein the selected process has a physical force ( 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 harvest process, a chromatography process, a virus inactivation process, and a filtration process. In an embodiment, the protein-related process is a virus inactivation process, the virus inactivation process is performed at a pH level of 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 may be a virus removal filtration process or an ultrafiltration/diafiltration process. The filtration process can be characterized by improving filtration-related parameters. An improvement in a filtration-related parameter may be a faster filtration rate than a control solution. An improvement in a filtration-related parameter may result in a lower amount of aggregated protein compared to the amount of aggregated protein generated by the control filtration process. An improvement in a filtration-related parameter may be a mass transfer efficiency higher than that of a control filtration process. An improvement in a filtration-related parameter may be that the concentration or yield of the target protein is higher than the concentration or yield of the target protein achieved by a control filtration process.

Further, the present invention relates to the aforementioned method, 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 one or more excipient compounds is selected from the 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 one or more excipient compound is selected from the group consisting of caffeine, taurine, niacinamide and imidazole. In embodiments, the one or more excipient compounds is an anionic aromatic excipient, and in some embodiments, the anionic aromatic excipient can be 4-hydroxybenzenesulfonic acid. In an embodiment, the viscosity-lowering amount is from about 1 mg/mL to about 100 mg/mL of the one or more excipient compounds, or the viscosity-lowering amount is from about 1 mM to about 400 mM of the one or more excipient compounds, or the viscosity-lowering amount is from about 1 mM to about 400 mM of the one or more excipient compounds. The lowering content is from about 2 mM to about 150 mM. In an embodiment, the carrier solution comprises additional materials selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers and buffers. In an embodiment, the protein of interest is a Therapeutic protein, and the Therapeutic protein can be a recombinant protein, or a monoclonal antibody, polyclonal antibody, antibody fragment, fusion protein, pegylated protein, antibody-drug conjugate, synthetic polypeptide, protein 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 parameter.

Further, the present invention 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 lower viscosity than a control solution. The carrier solution may further contain additional substances selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers and buffers.

1 is a block diagram showing the steps of a fermentation process ("upstream process") for producing a therapeutic protein, eg, a monoclonal antibody.
Figure 2 is a block diagram showing the steps of a purification process ("downstream process") for producing a therapeutic protein, eg, a monoclonal antibody.

The present invention relates to formulations capable of enabling the delivery of protein-concentrated solutions and methods for their preparation. In embodiments, the methods described herein can make a liquid formulation of lower viscosity or higher concentration of a therapeutic protein or non-therapeutic protein, compared to traditional protein solutions. In embodiments, the methods herein can result in liquid formulations with improved stability compared to traditional protein solutions. A stable formulation is one in which the protein contained substantially retains its physical and chemical stability and its therapeutic or non-therapeutic efficacy when stored in a storage environment, whether a cold storage environment, a room temperature environment, or an elevated temperature storage environment. Advantageously, stable formulations may also provide protection against aggregation or precipitation of proteins dissolved in the formulation. For example, a cold storage environment may entail storage in a refrigerator or freezer. In some instances, the cold storage environment may involve storage at temperatures of 10° C. or lower. In an additional 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 involves storage at freezing temperatures, such as about -20°C or lower. In another example, the cold storage environment involves storage at a temperature of about -20°C to about 0°C. A room temperature storage environment may entail storage at ambient temperature, for example from about 10° C. to about 30° C. Elevated storage environments may involve storage at higher than a certain temperature. Stability at elevated temperatures, for example at temperatures of 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.

The tendency of proteins in solution to form entanglements, which can hinder the therapeutic or non-therapeutic efficacy of proteins and limit the translational mobility of entangled chains, is a major challenge in the field of polymer science and engineering. It is well known to those skilled in the art. In embodiments, an excipient compound described herein can inhibit protein clustering due to a specific interaction between an excipient compound and a target protein in solution. The excipient compounds described herein may be natural or synthetic, and are preferably generally recognized as safe (“GRAS”) substances designated by the FDA.

1. Definition

For purposes herein, the term “protein” refers to an amino acid sequence with sufficient chain length to form individual tertiary structures, typically with a molecular weight of 1-3000 kD. In some embodiments, the protein has a molecular weight of about 50-200 kD; In another embodiment, 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 a discrete tertiary structure. A wide variety of biopolymers fall within the scope of the term "protein". For example, the term “protein” may refer to therapeutic or non-therapeutic proteins, including antibodies, aptamers, fusion proteins, pegylated proteins, synthetic polypeptides, protein fragments, lipoproteins, enzymes, structural peptides, and the like. can

By way of non-limiting example, Therapeutic proteins include hormones and prohormones (e.g., insulin and proinsulin, glucagon, calcitonin, thyroid hormone (T3 or T4 or thyroid-stimulating hormone), parathyroid hormone, follicle-stimulating hormone, luteinizing hormone). , growth hormone, growth hormone releasing factor, etc.); Coagulation and anti-coagulation factors (eg tissue factor, von Willebrand's factor), factor VIIIC, factor IX, protein C, plasminogen activator (urokinase, tissue-type plasminogen activator), 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, transforming growth factor, neurotrophic growth factor, insulin-like growth factor, etc.); albumin; collagen and elastin; hematopoietic factors (eg, erythropoietin, thrombopoietin, etc.); osteoinductive factors (eg, bone morphogenetic proteins); receptors (eg, integrins, cadherins, etc.); surface membrane proteins; transport proteins; regulatory proteins; antigenic proteins (eg, viral components that act as antigens); and mammalian proteins, such as antibodies. The term "antibody" herein is used in the broadest sense and includes, but is not limited to, monoclonal antibodies (eg, full length antibodies having an immunoglobulin Fc region), single chain molecules, bispecific and multispecific antibodies, diabodies. , antibody-drug conjugates, antibody compositions with multiple epitope specificities, polyclonal antibodies (eg, polyclonal immunoglobulins used as therapeutic agents for immunocompromised patients), antibody fragments (eg, Fc, Fab, Fv and F(ab') ) is used in the sense of including 2). Antibodies may also be referred to as “immunoglobulins”. An antibody is understood to be directed against a specific protein or non-protein "antigen" that is a biologically important substance; Administration of a therapeutically effective amount of an antibody to a patient can form a complex with the antigen and modify its biological properties to exert a therapeutic effect in the patient.

In an embodiment, the protein is pegylated, ie, means comprising poly(ethylene glycol) ("PEG") and/or poly(propylene glycol) ("PPG") units. PEGylated proteins or PEG-protein conjugates find use in therapeutic applications because of their beneficial properties such as solubility, pharmacokinetics, pharmacodynamics, immunogenicity, renal clearance and stability. Non-limiting examples of pegylated proteins include pegylated interferon (PEG-IFN), pegylated anti-VEGF, PEG protein conjugate drugs, Adagen, Pegaspargase, Pegfil Pegfilgrastim, Pegloticase, Pegvisomant, pegylated epoetin-β and Certolizumab pegol.

PEGylated proteins can be synthesized by various methods, such as reaction of a protein with a PEG reagent having one or more reactive functional groups. Reactive functional groups on PEG reagents can form links with proteins at target protein sites such as lysine, histidine, cysteine and N-terminus. A typical pegylation reagent has a reactive functional group, such as an aldehyde, maleimide or succinimide group, which has a specific reactivity towards the targeted amino acid residue on the protein. The PEGylated reagent may have a PEG chain length of about 1 to about 1000 PEG and/or PPG repeating units. Other pegylation methods include glyco-pegylation, in which the protein is first glycosylated and then the glycosylated residue is pegylated in a second step. Some pegylation processes are aided by enzymes such as sialyltransferase and transglutaminase.

Although pegylated proteins may provide therapeutic advantages over native, non-pegylated proteins, such materials may have physical or chemical properties that make purification, solubilization, filtration, concentration, and administration difficult. PEGylation of proteins can make the solution more viscous compared to native proteins, so PEGylated protein solution preparations should generally be of lower concentrations.

Protein therapeutics are preferably formulated as stable, low-viscosity solutions so that they can be administered to patients with minimal injection volumes. For example, subcutaneous (SC) or intramuscular (IM) injections of drugs generally require small injection volumes, preferably less than 2 mL. The SC and IM injection routes are well suited for self-administration management, which is a less expensive and more accessible form of treatment compared to intravenous (IV) injections performed only under direct medical supervision. For SC or IM injection formulations, the viscosity of the solution should be low, generally less than 30 cP, preferably less than 20 cP, so that the therapeutic solution can pass easily through a small gauge needle. The combination of small injection volume and low viscosity poses problems for using pegylated protein therapeutics for SC or IM injection routes.

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

"Treatment" includes any action intended to cure, cure, alleviate, ameliorate, remedy, or beneficially act on a disorder, including preventing or delaying the onset of symptoms of a disorder and/or alleviating or ameliorating the symptoms of a disorder. Patients in need of treatment include both those already suffering from a particular disorder and those in need of prevention of 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 predisposes a mammal 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, These include autoimmune disorders, gastrointestinal disorders, urinary 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, sport animals, working animals, and the like. "Treatment" can thus include both veterinary and human treatment. For convenience, the mammal undergoing such "treatment" may be referred to as the "patient". In certain embodiments, the patient can be of any age, such as a fetal animal in utero.

In embodiments, treatment comprises providing a therapeutically effective amount of a therapeutic agent to a mammal in need thereof. A "therapeutically effective amount" is at least the minimum concentration of a Therapeutic protein administered to a mammal in need thereof to achieve prevention of a related disorder or treatment of an existing disorder (such treatment or prevention being a "therapeutic intervention"). Therapeutically effective amounts of various therapeutic proteins that can be contained as active ingredients in therapeutic formulations may be familiar in the art; Or, in the case of a Therapeutic protein to be subsequently applied or developed for therapeutic intervention, the therapeutically effective amount can be determined within the scope of routine experimentation by standard techniques performed by those skilled in the art.

Proteins used for non-therapeutic purposes (ie, non-therapeutic purposes), such as household, nutritional, commercial and industrial uses, may be referred to as “non-therapeutic proteins”. Formulations containing non-therapeutic proteins may be referred to as “non-therapeutic formulations”. Non-therapeutic proteins may be derived from plant sources, animal sources, or produced from cell culture; Also, it can be an enzyme or a structural protein. Non-therapeutic proteins can be used for household, nutritional, commercial and industrial uses such as catalysts, human and animal nutrients, processing aids, cleaners and waste disposal.

A major category of non-therapeutic biopolymers are enzymes. Enzymes have many non-therapeutic uses, such as as catalysts, as nutritional components for humans and animals, as processing aids, as cleaners, and as waste treatment agents. Enzymatic 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 human and animal nutrition include nutraceuticals, nutritional sources of protein, chelation or controlled delivery of micronutrients, digestive aids and supplements; It may be derived from amylase, protease, trypsin, lactase and the like. Enzymatic processing aids are used to improve the manufacture 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 amylases, cellulases, pectinases, glucanases, lipases and lactases. Enzymes can also be used in biofuel production. Ethanol for biofuels can be supported, for example, by enzymatic digestion of biomass sources such as cellulosic and lignocellulosic materials. When these raw materials are treated with cellulase and ligninase, biomass is converted into a material that can be fermented as a fuel. In other commercial uses, enzymes are used as detergents, cleaners, and stain lifting aids for laundry, dishwashing, 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 cellulase-based fabric softening, 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 of non-therapeutic biopolymers include fibrous or structural proteins such as keratin, collagen, gelatin, elastin, fibroin, actin, tubulin or hydrolyzed, degraded or derivatized forms thereof. These materials are used in the manufacture and formulation of food ingredients such as gelatin, ice cream, yogurt and confections; It is added to foods as a thickener, rheological modifier, mouthfeel improver and source of nutritional protein. In the cosmetic and personal care industries, collagen, elastin, keratin and hydrolyzed keratin are widely used as ingredients in skin care and hair care products. Another example of a non-therapeutic biopolymer is whey proteins such as beta-lactoglobulin, alpha-lactalbumin and serum albumin. These whey proteins are produced in large quantities as a by-product of dairy operations and have 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 analysis of the present invention, the main analyte peak is generally due to the target protein contained in the preparation, and the main peak of this protein is referred to as the monomer peak. The monomeric peak represents the amount of the target protein, eg, the active component of the protein in the monomeric state, as opposed to the aggregated state (dimeric, trimeric, oligomeric form, etc.) or fragmented state. The monomer peak area can be compared to the total amount of monomeric, aggregated and fragmented peaks associated with the target protein. That is, the stability of a protein-containing formulation can be observed by the relative content of monomers after an elapsed time; Improvement in stability of the protein-containing formulations of the present invention can therefore be measured as a higher % monomer after a certain elapsed time compared to the % monomer in a control formulation without excipients.

In an embodiment, ideal stability is 98 to 100% of the monomer peak as determined by SEC analysis. In an embodiment, the improvement in stability of a protein-containing formulation of the present invention is measured as a higher % monomer after exposure to stress conditions compared to the % monomer of a non-excipient-containing control formulation when the control formulation is exposed to the same stress conditions. It can be. In embodiments, the stress conditions can be cold storage, high temperature storage, exposure to air, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.

In an embodiment, the protein-containing formulations described herein exhibit resistance to protein particle size increase as measured by dynamic light scattering (DLS) analysis. In the DLS analysis herein, the particle size of a protein in a protein-containing preparation can be observed as the median particle diameter. Ideally, the median particle diameter of the target protein represents the active component in its monomeric state, rather than aggregated (dimers, trimers, oligomers, etc.) or fragmented states, and therefore remains relatively unchanged when subjected to DLS analysis. Should not be. An increased median particle diameter may be indicative of aggregated protein. That is, the stability of a 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 exhibit resistance to forming a polydisperse particle size distribution as measured by dynamic light scattering (DLS) analysis. In an embodiment, the protein-containing preparation may comprise a monodisperse particle size distribution of colloidal protein particles. In embodiments, the ideal stability result is less than 10% change in the median particle diameter compared to the initial median particle diameter of the formulation. In embodiments, stability improvement of a protein-containing formulation of the present invention can be measured as a lower percent change in median particle diameter after a given elapsed time compared to the median particle diameter in a non-excipient-containing control formulation. In an embodiment, the improvement in the stability of the protein-containing formulation of the present invention is determined by the % change in median particle diameter after exposure to stress conditions by the percent change in median particle diameter in an excipient-free control formulation exposed to the same stress conditions. Compared to %, it can be measured as less. In embodiments, the stress condition can be cold storage, high temperature storage, exposure to air, exposure to air bubbles, exposure to a shear environment, or exposure to freeze/thaw cycles. In an embodiment, the stability improvement of a protein-containing agent therapeutic formulation of the present invention, as measured by DLS, is a polydisperse particle size distribution in an excipient-free control formulation when exposed to the same stress conditions. Compared to the polydispersity of , it can be measured as low.

In an embodiment, the protein-containing formulations of the invention exhibit resistance to precipitation as measured by turbidity, light scattering and/or particle counting analysis. In turbidity, light scattering or particle counting analyses, a lower value generally means a lower number of particles suspended in the formulation. An increase in turbidity, light scattering or particle count may indicate that the solution of the target protein is not stable. That is, the stability of a protein-containing formulation can be observed by the relative results 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 present invention is such that, after a period of time, the turbidity, light scattering or particle count values are greater compared to the turbidity, light scattering or particle count values of a control formulation containing no excipient. can be measured as low. In an embodiment, the stability improvement of a protein-containing formulation described herein is such that, after exposure to stress conditions, the turbidity, light scattering or particle count is the turbidity of a non-excipient containing control formulation exposed to the same stress conditions, Compared to light scattering or particle counting, it can be measured as low. In embodiments, the stress condition can be cold storage, high temperature storage, exposure to air, exposure to air bubbles, exposure to a shear environment, or exposure to freeze/thaw cycles.

3. Therapeutic formulations

In one aspect, the formulations and methods described herein provide a stable liquid formulation with improved or reduced viscosity comprising a therapeutic protein and an excipient compound in therapeutically effective amounts. In embodiments, the formulation can improve stability while providing an acceptable concentration of the active ingredient and an acceptable viscosity. In embodiments, the formulation provides a stability improvement compared to a control formulation; For purposes herein, a control formulation is a formulation that contains a protein active ingredient that is identical in all respects to the therapeutic formulation on a dry weight basis, except that it lacks an excipient compound. In an embodiment, the stability improvement of the protein containing formulation is in the case of a lower percentage of soluble aggregates, particulates, subvisible particles or gel form compared to a control formulation.

The viscosity of a protein liquid formulation may, by way of non-limiting example, be a property of the protein itself (eg, enzyme, antibody, receptor, fusion protein, etc.); its size, three-dimensional structure, chemical composition and molecular weight; its concentration in the formulation; non-protein components of the formulation; preferred pH range; storage conditions of the formulation; and the method of administration of the agent to the patient, a variety of factors may have an impact. Therapeutic proteins most suitable for use with the excipient compounds described herein are preferably essentially pure, ie free from contaminating proteins. In an embodiment, an “essentially pure” Therapeutic protein comprises at least 90%, or preferably at least 95%, or more preferably at least 99% by weight of the Therapeutic protein, based on the total weight of Therapeutic and contaminating proteins 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 formulations described herein are pharmaceutical-grade formulations, i.e., in a form in which the desired therapeutic effect of the protein active ingredient can be achieved, the formulation to be administered does not contain components toxic to mammals, and is useful for treating mammals. As a preparation for use, it is intended to be used.

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

Excipient compounds of various molecular weights are selected for their particular beneficial properties when used in combination with a protein active ingredient in a formulation. Examples of therapeutic formulations 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 embodiments, an excipient compound described herein is added to a therapeutic formulation in a viscosity-lowering amount. In embodiments, the viscosity-lowering amount is the amount of excipient compound that lowers the viscosity of the formulation by at least 10% as compared to the control formulation; For purposes herein, a control formulation is a formulation containing the same protein active ingredient on a dry weight basis in all manners as the therapeutic formulation except without the excipient compound. In embodiments, the viscosity-lowering amount is the amount of excipient compound that lowers the viscosity of the formulation by at least 30% compared to a control formulation. In embodiments, the viscosity-lowering amount is the amount of excipient compound that lowers the viscosity of the formulation by at least 50% compared to a control formulation. In embodiments, the viscosity-lowering amount is the amount of excipient compound that lowers the viscosity of the formulation by at least 70% as compared to the control formulation. In embodiments, the viscosity-lowering amount is the amount of excipient compound that lowers the viscosity of the formulation by at least 90% as compared to the control formulation.

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

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

These characteristics of the therapeutic formulations described herein are consistent with the clinical situation, i.e., the patient's tolerance for intramuscular injection, the use of small bore needles typical of IM/SC purposes, and the use of acceptable (e.g., 2-3 cc) injection volumes. and in situations where an effective amount of the agent is administered in one injection at one injection site under these conditions, administration of the agent by intramuscular or subcutaneous injection will be permitted. In contrast, SC/IM injection of conventional formulations would not be suitable for clinical situations, as injection of similar doses of therapeutic proteins using conventional formulation techniques would be limited due to the high viscosity of conventional formulations. High concentration solutions of therapeutic proteins formulated with the addition of excipient compounds described herein can be administered to a patient using a syringe or pre-filled syringe.

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

Methods of preparing therapeutic agents may be familiar to those skilled in the art. The therapeutic formulations of the present invention can be prepared, for example, by adding an excipient compound to the formulation before or after adding the therapeutic protein to the solution. A therapeutic agent may be processed, for example, by preparing a combination of a therapeutic protein and an excipient at a first (lower) concentration, followed by filtration or centrifugation to produce a second (higher) concentration of the therapeutic protein. Therapeutic formulations can be prepared using one or more excipient compounds with chaotropes, kosmotropes, hydrotropes and salts. Therapeutic formulations can be formulated with one or more excipient compounds using techniques such as encapsulation, dispersion, liposomes, vesicle formation, and the like. Methods of making therapeutic formulations comprising excipient compounds described herein may include combinations of excipient compounds. In embodiments, combinations of excipients may be useful for lower viscosity, improved stability, or reduced pain at the site of injection. Other additives may be added to the therapeutic formulation during manufacture, such as preservatives, surfactants, sugars, sucrose, trehalose, polysaccharides, arginine, proline, hyaluronidase, stabilizers, buffers, and the like. As used herein, a pharmaceutically acceptable excipient compound is a non-toxic material suitable for administration to animals and/or humans.

4. Non-Therapeutic Agents

In one aspect, the formulations and methods described herein provide a stable liquid formulation with improved or reduced viscosity comprising an effective amount of a non-therapeutic protein and an excipient compound. In embodiments, the formulation improves stability while providing acceptable concentrations of active ingredient and acceptable viscosity. In embodiments, the formulation provides a stability improvement compared to a control formulation; For purposes herein, a control formulation is a formulation containing the same protein active ingredient in all manner on a dry weight basis as the non-therapeutic formulation without the excipient compound.

The viscosity of a protein liquid formulation may, by way of non-limiting example, be determined by the nature of the protein itself (eg, enzyme, structural protein, degree of hydrolysis, etc.); its size, tertiary structure, chemical composition and molecular weight; its concentration in the formulation; constituents of formulations other than proteins; preferred pH range; and storage conditions of the formulation.

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

Excipient compounds of various molecular weights are selected for their particular beneficial properties when combined with the protein active ingredient in a formulation. Examples of non-therapeutic formulations 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 embodiments, an excipient compound described herein is added in a viscosity-lowering amount to a non-therapeutic formulation. In embodiments, the viscosity-lowering amount is the amount of excipient compound that lowers the viscosity of the formulation by at least 10% as compared to the control formulation; For purposes herein, a control formulation is a formulation containing, on a dry weight basis, the same protein active ingredient as the therapeutic formulation except without the excipient compound. In embodiments, the viscosity-lowering amount is the amount of excipient compound that lowers the viscosity of the formulation by at least 30% compared to a control formulation. In embodiments, a lowering amount is an amount of an excipient compound that lowers the viscosity of a formulation by at least 50% as compared to a control formulation. In embodiments, the lowering amount is the amount of excipient compound that lowers the viscosity of the formulation by at least 70% as compared to a control formulation. In embodiments, a lowering amount is an amount of an excipient compound that lowers the viscosity of a formulation by at least 90% as compared to a control formulation.

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

Non-therapeutic agents according to the present invention may have certain beneficial properties. In embodiments, the non-therapeutic agent exhibits resistance to shear degradation, phase separation, clouding out, oxidation, deamidation, aggregation, precipitation and denaturation. In embodiments, a therapeutic formulation can be processed, purified, stored, pumped, filtered, and centrifuged effectively compared to a 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 agent is stored at ambient temperature or in refrigerator conditions for an extended period of time without appreciable loss of efficacy of the non-therapeutic protein. In embodiments, the non-therapeutic formulation is processed to dryness for storage until needed; It can then be reconstituted by adding a suitable solvent, for example water. Advantageously, formulations prepared as described herein are stable for extended periods of time, from several months to several years. If an exceptionally long storage period is desired, the formulation can be preserved (and reactivated in the future) in a freezer without fear of protein denaturation. In embodiments, 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 can be added to the formulation before or after adding the non-therapeutic protein to the solution. A non-therapeutic agent may be prepared at a first (lower) concentration and then brought to a second (higher) concentration by filtration or centrifugation. Non-therapeutic formulations can be prepared using one or more excipient compounds with chaotropes, kosmotropes, hydrotropes and salts. Non-therapeutic formulations can be prepared using one or more excipient compounds using techniques such as encapsulation, dispersion, liposomes, vehicle formation, and the like. Other additives may be added to non-therapeutic formulations during manufacture, including preservatives, surfactants, stabilizers, and the like.

5. Excipient compounds

Several excipient compounds are mentioned herein that are suitable for use with one or more therapeutic or non-therapeutic proteins, and which can be formulated to contain high concentrations of the protein(s). Some of the excipient compound categories discussed below are: (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) diones and sulfones. Without wishing to be bound by theory, it is believed that the excipient compounds referred to herein associate with specific fragments, sequences, structures or sections of a Therapeutic protein that participate in particle-to-particle (i.e., protein-protein) interactions. I think. The association of these excipient compounds with therapeutic or non-therapeutic proteins can mask interactions between proteins so that the proteins can be formulated at high concentrations without excessively increasing the viscosity of the solution. Excipient compounds may advantageously be water soluble and, therefore, are suitable for use with aqueous vehicles. 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 may be derived from materials that are biologically acceptable and non-immunogenic, and are therefore suitable for pharmaceutical use. In therapeutic embodiments, the excipient compound may be metabolized in vivo to produce by-products that are biologically compatible and non-immunogenic.

a. Excipient compound category 1: hindered amines

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 small molecules that contain 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. For the protonated form, the hindered amine may be combined with an anionic counterion such as chloride, hydroxide, bromide, iodide, fluoride, acetate, formate, phosphate, sulfate or carboxylate. Hindered amine compounds usable as excipient compounds include secondary amines, tertiary amines, quaternary ammonium, pyridinium, pyrrolidone, pyrrolidine, peridine, morpholine or guanidinium groups. Hindered amine compounds also contain one or more bulky or sterically hindered groups, such as cyclic aromatic, cycloaliphatic, cyclohexyl or alkyl groups. In an embodiment, the sterically hindered group may be primarily an amine group, such as a 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 means of cationic pi (pi) interactions. In an embodiment, the cationic group of the hindered amine may have an affinity for the electron-rich pi structures of aromatic amino acid residues of the protein, so that it may mask these sites of the protein, reducing the tendency of 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 , betahistine, beta-alanine, 2-methyl-2-imidazoline, 1-butyl-3-methylimidazolium chloride, uric acid, potassium urate, betazole, carnosine, aspartame, It has a chemical structure comprising an imidazole, imidazoline or imidazolidine group or a salt 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 parenteral Anide, poly(hexamethylene biguanide), imidazole, dimethylglycine, agmatine, diazabicyclo[2.2.2]octane, tetramethylethylenediamine, N,N-dimethylethanolamine, Ethanolamine phosphate, glucosamine, choline chloride, phosphocholine, niacinamide, isonicotinamide, 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-taurine adduct, 2-pyridylethylamine, di cyandiamide-benzyl amine adducts, dicyandiamide-alkylamine adducts, dicyandiamide-cycloalkylamine adducts and dicyandiamide-aminomethanephosphonic acid adducts. In an embodiment, a hindered amine compound consistent with the present disclosure is formulated as a protonated ammonium salt. In an embodiment, a hindered amine compound consistent with the present disclosure 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 excipient compounds. In an embodiment, the hindered amine excipient compound is metabolized in the body to form biologically compatible by-products. 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 an additional aspect, the hindered amine excipient compound is present in the formulation at a concentration of about 20 to about 120 mg/ml.

In embodiments, certain hindered amine excipient compounds may have other pharmacological properties. For example, xanthines are a category of hindered amines with 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 xanthines are understood to affect cardiac contractility, heart rate and bronchodilation. 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 are local injectable anesthetic compounds. The local injectable anesthetic compound has 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. It is a hindered amine. Hindered amines in this category are understood to induce local anesthesia by interfering with nerve conduction by inhibiting the influx of sodium ions. The lipophilic aromatic ring of the local anesthetic compound may consist of carbon atoms (eg, a benzene ring) or may include heteroatoms (eg, a thiophene ring). Representative local injectable anesthetic compounds include, but are not limited to, amylocaine, articaine, bupivicaine, butacaine, butanilicaine, chlorprocaine (chlorprocaine), cocaine, cyclomethycaine, dimethocaine, editocaine, hexylcaine, isobucaine, levobupivacaine, lidocaine, metabutethamine, metabutoxycaine, mepivacaine, meprylcaine, propoxycaine, prilocaine, procaine These include procaine, piperocaine, tetracaine, and trimecaine. Local injectable anesthetic compounds can have several advantages over protein therapeutic formulations, such as lowering viscosity, improving stability and reducing 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 embodiments, 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 has a pharmacological effect and/or is present in a therapeutically effective amount. 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, local injectable anesthetic compounds can be used to reduce the viscosity of the formulation and to reduce pain upon injection of the formulation. Reduced injection pain may be caused by anesthetic properties; Alternatively, when the viscosity is lowered by the excipient, it may take less force to inject. In another embodiment, the local injectable anesthetic compound may 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 an anionic aromatic small molecule compound as an excipient compound. 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 contain anionic functional groups such as carboxylates, oxides, phenoxides, sulfonates, sulfates, phosphonates, phosphates or sulfides. Anionic aromatic excipients may be described as acids, sodium salts or otherwise, although it is understood that excipients may be used in a variety of salt forms. While not wishing to be bound by theory, the anionic aromatic excipient compound may associate with and mask the cationic portion of the protein, thereby reducing interactions between protein molecules that cause viscosity of the protein-containing formulation. It is regarded as a bulky, sterically hindered molecule.

In embodiments, examples of 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 adduct, aminophenol, anthranilic acid, cinnamic acid, coumaric acid, adenosine monophosphate, indole acetic acid, potassium urate, furan dicarboxylic acid, furan-2-acrylic acid, and 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 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 a therapeutic protein 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 compatible by-product.

c. Excipient Compound Category 3: Functionalized Amino Acids

High concentration solutions of therapeutic or non-therapeutic proteins can be formulated with one or more functionalized amino acids, wherein one functionalized amino acid or an oligopeptide comprising one or more functionalized amino acids can be used as an excipient compound. there is. In an embodiment, functionalized amino acid compounds include molecules that can be hydrolyzed or metabolized into amino acids ("amino acid precursors"). In embodiments, functionalized amino acids may include aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromatic groups or fused aromatic groups. In embodiments, functionalized amino acid compounds may include 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 as an ionic compound at neutral pH in an 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 the natural substance acetic acid or benzoic acid plus an amino acid. In an embodiment, the functionalized amino acid excipient compound is metabolized in the body to form biologically compatible by-products.

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 to have a structure with charged and bulky portions. In an embodiment, an oligopeptide is composed of 2-10 peptide subunits. An oligopeptide can be bifunctional, for example a cationic amino acid coupled with a non-polar amino acid, or an anionic amino acid coupled with a non-polar amino acid. In an embodiment, an oligopeptide is composed 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, an oligopeptide may have a cross structure such as an ABA repeat pattern. In an embodiment, an oligopeptide may include both anionic and cationic amino acids, such as Arg-Glu. While not wishing to be bound by theory, oligopeptides contain structures capable of associating with proteins in a manner that reduces intermolecular interactions that result in highly viscous solutions; For example, oligopeptide-protein association can be a charge-charge interaction that leaves some non-polar amino acids to break the hydrogen bonds of the hydration layer around the protein, thereby reducing viscosity. In some embodiments, the oligopeptide excipient is present in the composition at less than about 50 mg/ml.

e. Excipient compound category 5: short-chain organic acids

As used herein, the term “short-chain organic acid” refers to C2-C6 organic acid compounds 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 a carboxylic acid, sulfonic acid, phosphonic acid or a salt thereof.

In addition to the four categories of excipients described above, high concentration solutions of therapeutic or non-therapeutic proteins can also be used as excipient compounds in the acid or salt form of short chain organic acids such as sorbic acid, valeric acid, propionic acid, caproic acid and ascorbic acid. Can be formulated with. Examples of excipient compounds belonging to 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 capable of lowering 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 understood to be an organic aliphatic polyacid having a molecular weight < about 1500 and at least two acidic groups, wherein the acidic groups are proton-donating moieties. Non-limiting examples of acidic groups include carboxylate, phosphonate, phosphate, sulfonate, sulfate, nitrate and nitrite groups. The acidic groups of the low molecular weight aliphatic polyacids may be in the form of anionic salts such as carboxylates, phosphonates, phosphates, sulfonates, sulfates, nitrates and nitrites; Its counterion 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. Further examples for low molecular weight aliphatic polyacids in anionic salt form include phosphate (PO ₄ ^{3- )} , hydrogen phosphate (HPO ₄ ^{3- )} , dihydrogen 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 ^- ). The counter ion of an anionic salt can be a Na, Li, K or ammonium ion. Also, these excipients may be used in combination with excipients. Low molecular weight aliphatic polyacids herein may also be alpha hydroxy acids, in which there is also a hydroxy group adjacent to a first acidic group, 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 having more than two acidic groups, such as polyacrylic acid, polyphosphate, polypeptide 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 contain sulfone, sulfonamide or diionic functional groups that are soluble in pure water to greater than 1 g/L at 298K and have a net neutral charge at pH 7. It can be a molecule that Preferably, this molecule has a molecular weight of less than 1000 g/mol, more preferably less than 500 g/mol. Diones and sulfones, which are effective in reducing viscosity, have multiple heterogeneous 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, the double bond feature may allow weak pi-stacking interactions with the protein. In an embodiment, at high protein concentrations, and within proteins that only develop high viscosities at high concentrations, charged excipients are not effective because electrostatic interactions are longer range interactions. Solvated protein surfaces are usually hydrophilic, making them water-soluble. Hydrophobic regions of proteins are normally hidden within a three-dimensional structure, but the structure continues to evolve, unfolding and re-folding (sometimes "breathing"), and contacting adjacent hydrophobic regions of proteins with each other to create hydrophobicity. Aggregation may be caused by interaction. The pi-stacking nature of the dione and sulfone excipients can mask hydrophobic patches that may be exposed during such “breeding”. Another important role of excipients may be to break hydrophobic interactions and hydrogen bonds between closely located proteins, thereby effectively lowering the viscosity of the solution. Dione and sulfone compounds meeting this specification include dimethylsulfone, 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, and butane-2,3-dione; and the like.

6. Protein/Excipient Solutions: Characterization and Process

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 (hereafter, A solution of a therapeutic protein or non-therapeutic protein, formulated with an "excipient additive"), results in improved protein-protein interaction characteristics, as measured by the protein diffusion interaction parameter kD or the second viral coefficient B22. brings As used herein, an “improvement” in one or more protein-protein interaction parameters achieved by a test formulation using a foregoing excipient compound or combination thereof refers to conditions comparable to a corresponding formulation not containing a particular excipient compound or excipient additive. When compared to the test formulation in , it can mean a decrease in attracting protein-protein interactions. Such improvements can be identified by measuring specific parameters applied to the overall process or one aspect thereof, which parameters are any measured parameters pertinent to the process that can quantify the strain and compare it to a previous state or control. The parameter may be related to the process itself, such as efficiency, cost, yield or speed.

Additionally, the parameters may be proxy parameters related to characteristic aspects of larger scale processes. 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 in solution properties. While not wishing to be bound by theory, it is understood that a high negative kD value may indicate that the protein has strong attractive interactions, which may lead to aggregation, instability and rheological problems. When formulated in the presence of certain excipient compounds or combinations thereof described above, the same protein may have an improved proxy parameter that is a lower negative kD value or a kD value close to or greater than zero, such an improved proxy parameter It relates to the improvement of process-related parameters.

In an embodiment, certain excipient compounds or combinations thereof described above, 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 , to improve protein-related processes such as preparation, handling, aseptic filling, purification and analysis of protein-containing solutions using processing methods such as filtration, syringing, transporting, pumping, mixing, heating or cooling. In these and related protein-related processes, the protein of interest is dissolved in a solution that is transported through the processing unit. This solution, referred to herein as a “carrier solution,” includes a cell culture medium (e.g., containing the secreted protein of interest), a cell lysate obtained after cytolysis of host cells (if the protein of interest is present in the cell lysate), It may include an eluate (containing the target protein after chromatographic separation), an electrophoresis solution, a transport solution for transporting the target protein through a tube of a processing device, and the like. A carrier solution containing a protein of interest may also be referred to as a protein-containing solution or protein solution. 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 a parameter of interest in a carrier solution when the parameter is compared to the same parameter measured in a control solution. As used herein, "control solution" means a solution substantially similar to the carrier solution but without viscosity-enhancing excipients. As used herein, a "control process", eg, a control filtration process, a control chromatography process, etc., is a protein-related process that is substantially similar to the protein-related process of interest and is performed with a control solution instead of a carrier solution.

For example, in a process in which a protein-containing solution is pumped through a conduit (e.g., flow chamber, pipe, or tubing), a viscosity-lowering excipient may be added to the protein solution as described above before or during the pumping process to make the solution The forec and power required for pumping can be substantially reduced. It is understood that fluids generally exhibit resistance to flow, i.e., viscosity, and that in order to introduce and amplify flow, a physical force must be applied to the fluid to overcome this viscosity. The power required for pumping, P, is calculated from the head H and capacity Q as shown by the equation:

P ~ HQ (Equation 1)

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

The processing and processing methods of such protein solutions can be improved in efficiency due to lower viscosity, improved solubility or improved stability of the protein in the solution during preparation, processing, purification and analysis steps. Proxy parameter measurements, such as viscosity, solubility or stability of a protein in solution, or process efficiency measurements, 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 processing. For example, protein-containing solutions undergo significant shear stress during manufacture and purification, including, but not limited to, the manipulation of protein solutions through typical process operations such as pumping, mixing, centrifugation and filtration. exposed to a variety of physical stressors. Additionally, during these process steps, air bubbles can be trapped in the fluid to which proteins can adsorb. This interfacial tension, coupled with the typical shear stress encountered during processing, can unfold the adsorbed protein molecules and cause aggregation. Significant protein unfolding can also occur during pump cavitation events and upon exposure to solid surfaces such as ultrafiltration and diafiltration membranes during manufacturing. This phenomenon can deteriorate protein folding and product quality.

For a Newtonian fluid, the stress τ imposed by a given process is determined by the shear rate γ and the fluid's viscosity η, as shown by the equation:

τ = γη (Equation 2)

By formulating the protein solution with one or more of the foregoing 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 processing, as evident, for example, by better or better desirable measurement results of process parameters. Such improved process parameters may include metrics such as reduced levels of protein aggregation, particles or subvisibles (identified macroscopically as turbidity), reduced production losses, or improved overall yield. . As another example of an improved process parameter, lowering the viscosity of the 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 a given specific stress, a decrease in the viscosity of a protein solution by adding an excipient compound or combination thereof described above is accompanied by an increase in the shear rate (γ, Eq. 2), i.e., a reduction in processing time.

While the process is in progress, it is understood that the protein in solution may be the desired protein active ingredient, eg, a therapeutic protein or a non-therapeutic protein. Facilitating the processing of protein active ingredients using the excipients mentioned herein can increase the production rate or yield of protein active ingredients, or improve the efficiency of certain processes, or reduce energy consumption, etc. This optional performance is a process parameter that is improved by using viscosity-lowering excipients. It is also understood that protein contaminants may arise during certain processing techniques, such as during fermentation and purification steps of bioprocessing. If contaminants are removed more quickly, more thoroughly or more effectively, the processing of the desired protein, i.e. the protein active ingredient, can also be improved; This performance is a process parameter that is improved by using excipient compounds or additives for viscosity-lowering. As noted herein, by lowering the viscosity of a solution, improving protein stability, and/or increasing protein solubility, certain excipients described herein may improve the delivery of a desired protein active ingredient and inadvertent protein contamination. can improve the removal of substances; These two effects show that process parameters improved by using viscosity-lowering excipients or additives, which improve the overall process of protein manufacturing.

The engineering of special platform units for therapeutic protein production and purification provides another example of the beneficial use of the viscosity-lowering excipients described herein, and another example of how these excipients or additives improve process parameters. For example, incorporation of one or more of the aforementioned viscosity-enhancing excipients into such production and purification processes can provide substantial molecular stability and recovery improvements, as well as reduced operating costs.

In the art, widely used techniques for producing and purifying therapeutic proteins such as monoclonal antibodies are generally understood to consist of a fermentation process followed by a series of purification process steps. Fermentation or upstream processing (USP) involves culturing the 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, purification or downstream processing (DSP) may include steps such as those shown in FIG. 2 .

As shown in Figure 1, the USP may begin with step 102 of thawing a vial from a master cell bank (MCB). The MCB can be amplified as shown in step 104 to establish a working cell bank (not shown), and/or to create a working stock for further production. Cells are cultured in a series of seed and production bioreactors, as shown in steps 108 and 110, to produce bioreactor product 112, from which the desired product is produced, as shown in step 114. Therapeutic proteins can be harvested. After harvesting step 114, the products may undergo further purification (i.e., DSP, as described in Figure 2 and described in detail below), or these products may typically be frozen and stored at about -80°C. Can be stored in bulk.

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, the desired excipients may be added to the USP in an amount effective to reduce the viscosity of the cell culture medium by at least 20%. In other embodiments, the desired excipients can be added to the USP in an amount effective to reduce the viscosity of the cell culture medium by at least 30%. In an embodiment, a preferred excipient 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 may be added to the cell culture medium in an amount of about 20 mM to about 200 mM. In an embodiment, a preferred excipient may be added to the cell culture medium in an amount of about 25 mM to about 100 mM. The preferred excipient or combination of excipients may be added directly to the cell culture medium, or it may be a component of a more complex supplemental medium, for example, a nutrient-containing solution or "feed" individually formulated and added to the cell culture medium. It may be added as a component of the "solution". In embodiments, a second viscosity-lowering compound may be added to the carrier solution, either directly or via a supplemental medium, and the second viscosity-lowering solution further improves a specific parameter of interest.

As discussed below, there are numerous process-related parameters that can be improved using one or more viscosity-lowering excipients in USP. For example, in embodiments, the use of a viscosity-lowering excipient may improve parameters such as the rate and/or level of cell proliferation in steps such as inoculum amplification (104) and cell culturing (108 and 110) steps, or and/or proxy parameters correlated with improvements in various process parameters. For example, when added to the USP process at a stage such as production bioreactor stage 110, the aforementioned excipients can lower the viscosity of the cell culture medium and thus improve heat transfer efficiency and gas transfer efficiency. Since the cell culture process requires oxygen injection to enable the cells to express proteins, i.e. intracellular oxygen diffusion may be the rate-limiting step, improving the oxygen uptake rate by improving the gas transfer efficiency as the viscosity of the solution decreases. The rate or amount and/or efficiency of protein expression may be improved. In this regard, parameters such as oxygen uptake rate and gas transfer efficiency may be considered proxy parameters, wherein improvements relate to process parameter improvements, ie improved protein expression or improved process efficiencies. Alternatively, the availability of viscosity-lowering excipients may improve proxy parameters such as solubility of protein growth factors required for protein expression, eg, during inoculum amplification step 104 and cell culturing steps 108 and 110. Through this, the process can be improved; Improved solubility of growth factors allows cells to utilize more of these substances, thus stimulating cell proliferation.

In embodiments, process parameters such as protein recovery or protein recovery in the USP can be improved by lowering the viscosity in the USP through several mechanisms. For example, by using the aforementioned excipients, recovery of the therapeutic protein at the end of the cell lysis process in step 114 of harvesting from the completed cell culture may be more efficient or improved. Without wishing to be bound by theory, these viscosity-lowering excipients may increase the efficiency of diffusion of the therapeutic protein away from other cytolytic components by lowering the viscosity of the expressed protein. In addition, separation of membranes and other cellular debris from the protein-containing supernatant can be achieved with a faster separation rate or higher supernatant purity by using viscosity-lowering excipients, i.e. improving the USP efficiency parameters of the process. can do. In addition, the protein separation step using centrifugation or filtration steps can be achieved more quickly using viscosity-lowering excipients, since excipients lower the viscosity of the medium.

In an embodiment, as an added benefit, the use of the aforementioned viscosity-lowering excipients in cell culture reduces protein mis-folding and aggregation, thereby increasing process parameters such as protein yield in USP. As cell culture is optimized to achieve maximum yield of recombinant protein, the protein produced is expressed in a high concentration manner, which can cause mis-folding; It is understood that the addition of viscosity-lowering excipients can increase the amount of intact recombinant protein available at harvest (114) by reducing the detrimental protein-protein interactions that cause mis-folding and aggregation.

The downstream process (DSP) shown in FIG. 2 involves a series of steps to achieve recovery and purification of therapeutic proteins, such as monoclonal antibodies, biopharmaceuticals, vaccines and other biologics, as an exemplary embodiment. Upon completion of the USP, the protein of therapeutic interest is secreted from the host cell and can be dissolved in the cell culture medium. In addition, the therapeutic protein can be dissolved in the fluid medium after cytolysis of the host cells at the end of the USP sequence. DSP involves recovering the protein of interest from a lysed solution (eg, culture medium or host cell lysis medium) and purifying it. During DSP, (i) various contaminants (e.g., insoluble cell debris and particulates) are removed from the medium, (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 freed.

As shown in FIG. 2, the feedstock from cell culture harvest 200 (also shown in FIG. 1) is first subjected to affinity chromatography 204, which typically involves Protein A chromatography or other similar chromatographic 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 proteins (HCP), DNA, charged variants and aggregates. Cation exchange (CEX) chromatography is typically used as the first polishing chromatography step 210, but may be preceded or followed by a second chromatography step 212. A second chromatography step 212 further removes host-cell related impurities (eg, HCP or DNA) or product related impurities such as 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. The final purification step 218 may include ultrafiltration and diafiltration, and preparation for formulation.

Generally as described above, the fermentation process followed by purification processes or DSPs include (1) harvesting the cell culture, (2) chromatography (e.g. Protein A chromatography and polishing chromatography steps, e.g. ion exchange and hydrophobic interactions). chromatography), (3) virus inactivation, and (4) filtration (e.g., virus filtration, sterile filtration, dialysis, and ultrafiltration and diafiltration steps to concentrate and exchange proteins into formulation buffers). can do. Examples are set forth below that illustrate the advantages of using the viscosity-lowering excipients described herein to improve the process parameters associated with these purification processes. Viscosity-lowering excipients or combinations thereof can be introduced at any DSP stage by adding them to the carrier solution or in any other way bringing about contact of the protein of interest with the excipients, whether in soluble or stabilized form. . In an embodiment, a second viscosity-lowering compound may be added to the carrier solution during DSP, the second viscosity-lowering compound additionally improving a particular target parameter.

(1) Cell culture harvesting: Cell culture harvesting generally involves centrifugation and depth filtration operations to physically remove cell debris from a protein-containing solution. The centrifugation step allows more complete separation of soluble proteins from cell debris thanks to the viscosity-lowering excipients. Whether by batch or continuous processing, separation by centrifugation is necessary to maximize recovery of the target protein by making the dense phase as rigid as possible. In embodiments, the addition of the foregoing excipients or combinations thereof may increase the process parameter protein yield, for example, by increasing the yield of protein-containing concentrate flowing out of the dense phase of the separation process by centrifugation. . The depth filtration step is a viscosity-limiting step, i.e. it can be made more efficient by using excipients that lower the solution viscosity. This process can also cause the introduction of air bubbles into the protein solution, which coupled with shear-induced stress can destabilize the therapeutic protein molecule being purified. As noted above, the addition of viscosity-lowering excipients to protein-containing solutions before and/or during cell culture harvest can protect proteins from these stresses, improving the process parameter quantitative product recovery.

(2) Chromatography: After harvesting the cell culture by centrifugation or filtration, chromatography is typically used to separate the therapeutic protein from the fermentation broth. 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 prior to loading into the column to lower its conductivity. In addition, a mimetic resin can also be used as an alternative to Protein A chromatography. These resins provide ligands for binding to immunoglobulins, eg, Ig-binding proteins such as protein G or protein L, synthetic ligands, or protein A-like porous polymers.

Other chromatographic processes can be used for DSP. Ion Exchange Chromatography (IEC) can be used to remove impurities introduced during the preceding process, such as leaked protein A, endotoxins or viruses from the cell line, residual host cell proteins or DNA, or media components. there is. IEC, whether CEX or anion exchange chromatography, can be used directly following Protein A chromatography. Hydrophobic interaction chromatography (HIC) can complement IEC, which is commonly used as a polishing step to remove aggregates. In embodiments, the use of the aforementioned excipients can increase the solubility of host cell proteins and lower their viscosity in the chromatography column loading step. In embodiments, the use of the aforementioned excipients can increase the solubility of the therapeutic protein and lower its viscosity in the chromatography column loading and elution steps.

Chromatographic processes in protein purification include (a) a low pH environment during elution from the Protein A chromatography column, and (b) a local protein concentration elevation in the pore-space of the chromatography resin (often 300-400 mg/mL levels), (c) elevated salt concentrations during ion exchange chromatography, and (d) elevated concentrations of salting-out agents in the eluate from HIC columns. . Addition of the viscosity-lowering excipient to the protein-containing solution as described above before and/or after chromatography allows the protein to pass through the chromatography column to reduce exposure to the potentially harmful environment resulting from the chromatographic process steps. can make it easy. In addition, elevated concentrations of local proteins in the column pore-space can make the material in this space highly viscous, causing significant backpressure in the column. To relieve backpressure, media with relatively large pores are generally used. However, a medium with large pores has lower resolving power than a medium with small pores. The introduction of viscosity-modifying excipients as described above makes it possible to use chromatography media with smaller pores. In an embodiment, the elution step of Protein A chromatography exposes the therapeutic protein to a low pH environment, which may cause a decrease in the solubility of the target protein and an increase in its aggregation: adding an excipient may increase the solubility of the target protein. and thus the recovery yield of the Protein A chromatography step is improved. In other embodiments, the use of an excipient allows elution of the target protein from the Protein A resin at a higher pH, which reduces chemical stress on the target protein, thereby reducing the amount of protein degradation that occurs during the processing process. The parameter protein yield can be improved.

(3) Virus inactivation : The virus inactivation process typically involves maintaining a protein solution at a low pH, eg below pH 4, for a long period of time. However, this environment can make the therapeutic protein unstable. Formulating proteins in the presence of viscosity-lowering excipients, for example by adding viscosity-lowering excipients before and/or during the virus inactivation process, results in process parameters such as protein stability or solubility, or overall yield thereof. can improve

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

(a) Viral filtration purifies the protein solution by removing viral particles, which can be twice the size of recombinant human monoclonal antibodies. Therefore, the filtration membrane for virus filtration may have nano-sized pores. Because of the small pore size that proteins must pass through, the filtration step can cause stress on proteins, accompanied by a significant level of membrane fouling from protein aggregating particles. The addition of viscosity-lowering excipients, for example, before and/or during filtration, as described above, can lower a measurable parameter such as backpressure in a filtration system by increasing the collective diffusivity. It can reduce the tendency of membrane fouling 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) The ultrafiltration and diafiltration (UF/DF) process concentrates the protein solution and replaces the buffer system by passing the protein-containing solution through a filter membrane with a specific molecular weight cutoff that is less than the protein of interest. At this stage, the protein solution is exposed to high shear stress within the filter unit, elevated protein concentration, and adsorption of proteins to the hydrophobic membranes typically used in UF/DF processes, all of which can increase protein aggregation. As mentioned above, the addition of viscosity-lowering excipients, eg, before and/or during the UF/DF process, will increase the overall diffusion coefficient (eg, as measured by kD increase), which will reduce back pressure in the filtration system. can This not only reduces membrane-wide shear stress but also promotes reduced back-diffusion from the filter membrane, lowering the effective protein concentration at the membrane interface and increasing the permeate flux. As a result, viscosity-lowering excipients can be used to improve parameters associated with higher throughput, reduced production losses and increased overall yield in these filtration processes. Also, passing viscous fluids through ultra- and diafilters can create a significant pressure drop across the filter device, making the separation inefficient. As described above, when the protein solution is formulated in the presence of a viscosity-lowering excipient, the pressure drop across the entire filter device can be substantially reduced, thereby reducing both process parameters, operation cost and process time. This can be improved.

After addition of an excipient to complete upstream protein processing or downstream purification, the excipient may remain as part of the drug substance mixture or may be separated from the protein active ingredient. For the separation of excipients from protein active components, typical small molecule separation methods such as buffer exchange, ion exchange, ultrafiltration and dialysis can be used. In addition to the beneficial effect on the protein purification process as described above, the use of the above-mentioned excipients can protect and maintain the equipment used for protein production, processing and purification. For example, device-related processes such as cleaning, sanitizing and maintenance of protein processing devices may be facilitated by the use of the aforementioned excipients due to reduced fouling, reduced denaturation, improved low viscosity and solubility of proteins. , and the parameters associated with these processing improvements are likewise improved.

Although the use of excipient compounds to improve upstream and/or downstream processes has been extensively described herein, it is understood that combinations of excipients may be added together to achieve a desired effect, such as improvement of a parameter of interest. The term “excipient additive” can refer to one excipient compound that induces a desired effect or parameter improvement, or a combination of excipient compounds that achieves a desired effect or parameter improvement.

Example

ingredient:

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

Histidine, Sigma Aldrich

Other materials mentioned in the examples below were purchased from Sigma Aldrich unless otherwise noted.

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

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

Example 2: Viscosity measurement

Viscosity measurements for formulations prepared as described in Example 1 were performed using a DV-IIT LV cone & plate viscometer (Brookfield Engineering, Middleboro, Mass.). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. An additional two steps were then performed consisting of 1 minute of shear incubation followed by a 20 second measurement collection period. The average of the three data points collected was taken 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 protein concentration in the test 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 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, to which excipient compounds were added to some samples. In the assay, dimethylcyclohexylamine (DMCHA), dicyclohexylmethylamine (DCHMA), dimethylaminopropylamine (DMAPA), triethanolamine (TEA), dimethylethanolamine (DMEA) and hydrochloride salts of niacinamide was investigated as an example of a hindered amine excipient compound. In addition, taurine-dicyandiamide adducts and hydroxybenzoic acid salts 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, which shows the viscosity lowering effect of the added excipient compound.

Table 1

test number added excipients Excipient concentration (mg/mL) Viscosity (cP) viscosity reduction rate 4.1 doesn't exist 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 aromatic excipient compounds added

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

Table 2

test number added excipients Excipient concentration (mg/mL) Viscosity (cP) viscosity reduction rate 5.1 doesn't exist 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) with free amine at the N terminus and free acid at the C terminus were prepared by NeoBioLab Inc. (Woburn, MA) with >95% purity. Dipeptides (n=2) were synthesized with 95% purity from LifeTein LLC (Somerset, NJ). 280 mg/mL BGG formulations were prepared as in Example 1, with excipient compounds added to some samples. The viscosity of each solution was measured as in Example 2, and the results are presented in Table 3 below, which shows the viscosity lowering effect of the added excipient compound.

Table 3

test number added excipients Excipient concentration (mg/mL) Viscosity (cP) viscosity reduction rate 6.1 doesn't exist 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: Guanyl taurine excipient synthesis

Guanyl taurine was prepared according to the method described in US Patent 2,230,965. Mix 3.53 parts of taurine (Sigma-Aldrich, St. Louis, MO) with 1.42 parts of dicyandiamide (Sigma-Aldrich, St. Louis, MO), then grind with a mortar and pestle until a homogeneous mixture is obtained. did 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 compounds

Formulations were prepared using an excipient compound and a test protein, the test protein intended to mimic either a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. These formulations were prepared in an aqueous solution of 50 mM histidine hydrochloride buffer to which various excipient compounds were added to measure the viscosity in the following manner. First, histidine hydrochloride buffer solution by dissolving 1.94 g of histidine in distilled water, titrating to pH about 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 volumetric flask. was prepared first. Then, excipient compounds were dissolved in 50 mM histidine HCl buffer. A list of excipients is given in Example 4. In some cases, excipient compounds were dissolved in 50 mM histidine HCl buffer, and the resulting solution was titrated to pH 6 with a small amount of sodium hydroxide or hydrochloric acid before dissolving the model protein. In some cases, excipient compounds were titrated to pH 6 prior to dissolution 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 about 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 isolate the solid excipient. Once the excipient solution in 50 mM histidine HCl was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a rate that achieved a final protein concentration of approximately 280 mg/mL. A solution of BGG in 50 mM histidine HCl with added excipients was prepared in a 20 mL vial and stirred overnight at 100 rpm on a rotary shaker table. Then, the BGG solution was placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove trapped air before measuring the viscosity.

Viscosity measurements of the formulations prepared as described above were performed using a DV-IIT LV viscometer (cone & plate viscometer) (Brookfield Engineering, Middleboro, MA). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. Then, an additional two steps consisting of 1 minute of shear incubation followed by a measurement collection period of 20 seconds were performed. The average of the three data points collected was taken and recorded as the viscosity of the sample. The viscosity of the excipient-containing solution was normalized to that of the non-excipient-containing model protein solution. The normalized viscosity is the ratio of the viscosity of the model protein solution with an excipient to the viscosity of the model protein solution without the excipient.

Table 4

test number added 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 agmatine 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 the test protein

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

Viscosity measurements of the formulations prepared as described above were performed using a DV-IIT LV viscometer (cone & plate viscometer) (Brookfield Engineering, Middleboro, MA). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. Then, an additional two steps consisting of 1 minute of shear incubation followed by a measurement collection period of 20 seconds were performed. The average of the three data points collected was taken and recorded as the viscosity of the sample. The viscosities of the excipient-containing solutions were normalized to those of the non-excipient-containing model protein solutions, summarized in Table 5 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with an excipient to the viscosity of the model protein solution without the excipient. This example shows that a combination of a first excipient and a second excipient can provide superior results than a single excipient.

table 5

first excipient
Second excipient
Normalized Viscosity test number designation Concentration (mg/mL) designation Concentration (mg/mL) 10.1 salicylic acid 30 doesn't exist 0 0.79 10.2 salicylic acid 25 imidazole 4 0.59 10.3 4-hydroxybenzoic acid 30 doesn't exist 0 0.61 10.4 4-hydroxybenzoic acid 25 imidazole 5 0.57 10.5 4-hydroxybenzene sulfonic acid 31 doesn't exist 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 doesn't exist 0 Caffeine 10 0.73 10.9 doesn't exist 0 imidazole 5 0.75

Example 11: excipient combination and Preparation of formulations containing the test protein

Formulations were prepared using a first excipient compound, a second excipient compound and a test protein, the test protein intended to mimic either a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a 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 with a non-ionic or cationic charge and an imidazoline or benzene ring at pH 6 as listed in Table 6 below. Formulations with these excipients were prepared in distilled water for viscosity measurement in the following manner. The combination of the first excipient and the second excipient was dissolved in distilled water, and the obtained 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, the test protein bovine gamma globulin (BGG) was dissolved at a rate that achieved a final protein concentration of approximately 280 mg/mL. A solution of BGG in distilled water with added excipients was prepared in a 20 mL vial and stirred overnight at 100 rpm on a rotary shaker table. Then, the BGG solution was placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove trapped air before measuring the viscosity.

Viscosity measurements of the formulations prepared as described above were performed using a DV-IIT LV viscometer (cone & plate viscometer) (Brookfield Engineering, Middleboro, MA). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. Then, an additional two steps consisting of 1 minute of shear incubation followed by a measurement collection period of 20 seconds were performed. The average of the three data points collected was taken and recorded as the viscosity of the sample. The viscosities of the excipient-containing solutions were normalized to those of the non-excipient-containing model protein solutions, summarized in Table 6 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with an excipient to the viscosity of the model protein solution without the excipient. This example shows that a combination of a first excipient and a second excipient can provide superior results than a single excipient.

table 6

first excipient Second excipient standardized
viscosity test number designation Concentration (mg/mL) designation Concentration (mg/mL) 11.1 salicylic acid 20 doesn't exist 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 formulations containing excipient compounds and PEG

Materials: All materials were purchased from Sigma-Aldrich (St. Louis, MO). Formulations are prepared using excipient compounds and PEG, which is intended to mimic pegylated therapeutic proteins used in therapeutic formulations. These formulations were prepared by mixing equal volumes of PEG solutions with excipient solutions. These two solutions were prepared in Tris buffer (pH 7.3) consisting of 10 mM Tris, 135 mM NaCl, 1 mM trans-cinnamic acid.

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

An example of the 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 with a minimum amount of 10 M NaOH, to a pH of about 80 mg/ml of citric acid in Tris buffer. A mL solution was prepared.

A PEG excipient solution was prepared by mixing 0.5 mL of the PEG solution and 0.5 mL of the excipient solution, and then mixed using a 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: Viscosity measurement of formulations containing excipient compounds and PEG

Viscosity measurements of the prepared formulations were performed using a DV-IIT LV viscometer (cone & plate viscometer) (Brookfield Engineering, Middleboro, MA). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. Then, an additional two steps consisting of 1 minute of shear incubation followed by a measurement collection period of 20 seconds were performed. The average of the three data points collected was taken and recorded as the viscosity of the sample.

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

table 7

test number excipient Excipient concentration (mg/mL) Viscosity (cP) viscosity reduction rate 13.1 doesn't exist 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 etidronic acid/K salt 24 71.91 31% 13.21 etidronic acid/K salt 12 80.5 23% 13.22 etidronic acid/Na salt 24 71.6 32% 13.23 etidronic acid/Na salt 12 79.4 24%

Example 14: BSA PEG per molecule with 1 chain pegylated BSA Manufacturing

To a 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 bar. Then 400 mg of methoxy polyethylene glycol maleimide, MW=5,000, (Aldrich Cat. # 63187) was 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 products were characterized by SDS-PAGE and SEC, pegylated BSA was clearly observed. The reaction mixture was placed in an Amicon centrifuge tube with a molecular weight cutoff (MWCO) of 30,000 and concentrated to several ml. The sample was then diluted 20-fold in 50 mM histidine buffer, approximately pH 6, and then concentrated until a highly viscous 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 of 0.6678. As a result, it was confirmed that the final concentration of BSA in the solution was 342 mg/mL.

Example 15: BSA PEG per molecule with multiple chains pegylated BSA Manufacturing

A solution of 5 mg/mL BSA (Aldrich A7906) in 25 mM phosphate buffer, pH 7.2, was prepared by mixing 0.5 g of BSA with 100 mL of buffer. 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 next 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 with MWCO 30,000 to reach a concentration of about 150 mg/mL.

Example 16: Lysozyme PEG per molecule with multiple chains pegylated Lysozyme manufacturing

A solution of 5 mg/mL lysozyme (Aldrich L6876) in 25 mM phosphate buffer, pH 7.2, was prepared by mixing 0.5 g of lysozyme with 100 mL of buffer. 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 with 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 of 2.63. As a result, the final concentration of lysozyme in the solution was confirmed to be 140 mg/mL.

Example 17: BSA PEG per molecule with 1 chain pegylated BSA's Influence of Excipients on Viscosity

An excipient added PEGylated BSA formulation (of Example 14) was prepared by adding 6 or 12 mg of the excipient salt to 0.3 mL of a PEGylated BAS solution. The solutions were mixed by gentle agitation and the viscosity was measured by a 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 compounds.

Table 8

test number excipient Excipient concentration (mg/mL) Viscosity (cP) viscosity reduction rate 17.1 doesn't exist 0 228.6 0% 17.2 alpha-cyclodextrin sulfated sodium salt 20 151.5 34% 17.3 K Acetate 40 89.5 60%

Example 18: BSA PEG per molecule with multiple chains pegylated BSA's Influence of Excipients on Viscosity

8 mg of the excipient salt was added to 0.2 mL of the pegylated BAS solution to prepare a pegylated BSA formulation (of Example 15) with Na citrate added as an excipient. The solutions were mixed by gentle agitation and the viscosity was measured by a 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 compounds.

Table 9

test number added excipients Excipient concentration (mg/mL) Viscosity (cP) viscosity reduction rate 18.1 doesn't exist 0 56.8 0% 18.2 Citric acid Na salt 40 43.5 23%

Example 19: Lysozyme PEG per molecule with multiple chains pegylated lysozyme Influence of Excipients on Viscosity

6 mg of the excipient salt was added to 0.3 mL of the pegylated lysozyme solution to prepare a pegylated lysozyme formulation (of Example 16) with potassium acetate added as an excipient. The solutions were mixed by gentle agitation and the viscosity was measured by a 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 compounds.

Table 10

test number excipient Excipient concentration (mg/mL) Viscosity (cP) viscosity reduction rate 19.1 doesn't exist 0 24.6 0% 19.2 K Acetate 20 22.6 8%

Example 20: Excipient combination Protein formulations containing

Formulations were prepared using an excipient compound or two excipient compounds and a test protein, the test protein 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 measure 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. The excipient compounds used in this example are shown in Table 11 below. Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a rate resulting in a final protein concentration of approximately 280 mg/mL. A solution of BGG in the excipient solution was prepared in a 5 mL sterile polypropylene test tube and stirred overnight at 80-100 rpm on a rotary shaker table. Then, the BGG solution was placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove trapped air before measuring the viscosity.

Viscosity measurements of the formulations prepared above were performed using a DV-IIT LV viscometer (cone & plate viscometer) (Brookfield Engineering, Middleboro, MA). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. An additional two steps were then performed consisting of 1 minute of shear incubation followed by a 20 second measurement collection period. The average of the three data points collected was taken and recorded as the viscosity of the sample. The viscosity of the excipient-containing solution was normalized to that of the non-excipient-containing model protein solution, and the results are shown in Table 11 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with an excipient to the viscosity of the model protein solution without the excipient.

Table 11

test # Excipient A Excipient B Normalized Viscosity designation Concentration (mg/mL) designation Concentration (mg/mL) 20.1 doesn't exist 0 doesn't exist 0 1.00 20.2 aspartame 10 doesn't exist 0 0.83 saccharin 60 doesn't exist 0 0.51 20.4 Acesulfame K 80 doesn't exist 0 0.44 20.5 theophylline 10 doesn't exist 0 0.84 20.6 saccharin 30 doesn't exist 0 0.58 20.7 Acesulfame K 40 doesn't exist 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

A formulation was prepared using an excipient compound, a second excipient compound and a test protein, the test protein intended to mimic the therapeutic protein 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 of the contents shown in Table 12 were dissolved in 20 mM histidine, and the obtained 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, the test protein bovine gamma globulin (BGG) was dissolved in the excipient solution at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in excipient solution was prepared in a 5 mL sterile vial and stirred overnight at 80-100 rpm on a rotary shaker table. Then, the BGG solution was placed in a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax centrifuge to remove trapped air before measuring the viscosity.

Viscosity measurements of the formulations prepared as described above were performed using a DV-IIT LV viscometer (cone & plate viscometer) (Brookfield Engineering, Middleboro, MA). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. Then, an additional two steps consisting of 1 minute of shear incubation followed by a measurement collection period of 20 seconds were performed. The average of the three data points collected was taken and recorded as the viscosity of the sample. The viscosity of the excipient-containing solution was normalized to that of the non-excipient-containing model protein solution and is shown in Table 12 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with an excipient to the viscosity of the model protein solution without the excipient.

Table 12

test # Excipient A Excipient B Normalized Viscosity designation Concentration (mg/mL) designation Concentration (mg/mL) 21.1 doesn't exist 0 doesn't exist 0 1.00 21.2 lidocaine 45 doesn't exist 0 0.73 21.3 lidocaine 23 doesn't exist 0 0.74 21.4 lidocaine 10 Caffeine 15 0.71 21.5 Procaine HCl 40 doesn't exist 0 0.64 21.6 Procaine HCl 20 Caffeine 15 0.69

Example 22: Formulations containing excipient compounds and PEG

Formulations were prepared using an excipient compound and PEG, the PEG intended to mimic the pegylated therapeutic protein to be used in the therapeutic formulation, and the excipient compounds provided in the amounts listed in Table 13. These formulations were prepared by mixing equal volumes of PEG solutions with excipient solutions. These two solutions were prepared in deionized (DI) water.

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

The excipient solution was prepared by the general method as detailed in Table 13 below: 0.05 g of potassium phosphate was dissolved in deionized water to obtain a solution of about 20 mg/mL potassium phosphate tribasic (Aldrich Catalog # P5629) in deionized water. manufactured. A PEG excipient solution was prepared by mixing 0.5 mL of the PEG solution with 0.5 mL of the excipient solution, which was then mixed using a vortex for several seconds. A control sample was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of deionized water. Viscosity was measured, and the results are recorded in Table 13 below.

Table 13

test number excipient excipient concentration
(mg/mL) Viscosity (cP) viscosity
Decrease rate (%) 22.1 doesn't exist 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 Sulphate 10/10 70.4 11.7

Example 23: Improving the processing of protein solutions using excipients

0.25 g of BGG solid was mixed in 4 ml of buffer to make 2 BGG solutions. Sample A: Buffer is 20 mM histidine buffer (pH=6.0). Sample B: Buffer is 20 mM histidine buffer with 15 mg/ml caffeine (pH=6). The sample was stirred at 100 rpm on a rotary shaker table to dissolve the BGG solids. It was observed that the buffer solution containing the caffeine excipient dissolved the protein faster. For the sample with added caffeine excipient (Sample B), BGG was completely dissolved within 15 minutes. For the sample without caffeine (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 for 10 minute intervals. The volume of filtrate recovered after each 10-minute centrifugation was recorded. The results in Table 14 show that sample B recovers the filtrate faster. In addition, sample B was concentrated with each centrifugation, but sample A reached the highest concentration point, and no additional sample concentration occurred with additional centrifugation.

Table 14

Centrifugation time (minutes) Sample A's collected filtrate (mL) Sample B's collected 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 formulation with multiple excipients

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

Table 15

Sample added excipients Viscosity (cP) Viscosity reduction rate (%) A doesn't exist 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 solution of 280 mg/mL BGG in histidine buffer, pH 6. At concentrations higher than 50 mg/mL, the addition of more arginine did not further reduce the viscosity as shown in Table 16.

Table 16

Addition of arginine (mg/mL) Viscosity (cP) Decreased 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 solution of 280 mg/mL BGG in histidine buffer at pH 6. At concentrations higher than 10 mg/mL, the viscosity did not decrease any further, as shown in Table 17, even with the addition of more caffeine.

Table 17

Caffeine Added Amount (mg/mL) Viscosity (cP) Decreased viscosity ( % ) 0 79 0% 10 60 31% 15 62 23% 22 50 45%

Example 25: TFF Effects of Caffeine in the Concentration Process

In this example, bovine gamma globulin (BGG) solutions were concentrated with and without caffeine using tangential flow filtration (TFF). Experiments were performed using a Labscale TFF system produced by EMD Millipore (Billerica, MA). The system was equipped with a Pellicon XL TFF cassette containing an Ultracel membrane with a 30 kDa molecular weight cutoff (EMD Millipore, Billerica, Mass.). The 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. The filtrate flux was monitored throughout the experiment gravimetrically over time. Approximately 12 g of BGG was dissolved in 500 mL of a buffer containing 15 mg/mL caffeine, 150 mM NaCl and 20 mM histidine, titrated to pH 6. A control sample was prepared by dissolving approximately 12 g of BGG in 500 mL of buffer containing 150 mM NaCl and 20 mM histidine, titrated to pH 6. 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 the test and control samples was measured during TFF by mass transfer coefficient. The mass transfer coefficient was obtained for each sample by 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 represents the filtrate flux J, where k _c is the mass transfer coefficient, C _w is the protein concentration around the membrane, and C _b is the concentration in the bulk of the liquid, so by Equation 3 the mass transfer coefficient k _c can be calculated. The graph of the calculated flow rate J as a function of ln(C _b ) shows a linear curve with a slope -kc. Here, the flow rate J is calculated by differentiating the weight of the filtrate with time, and C _b is calculated using the mass-balance. The most suitable mass transfer coefficients are listed in Table 18. 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 group 22.5 ± 0.1 15 mg/mL caffeine 25.4 ± 0.1

Example 26: TFF Effects of Caffeine in the Concentration Process

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

Table 19

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

Example 27: BGG Effects of Caffeine on Sterile Filtration of Solutions

Bovine gamma globulin (BGG), L-histidine and caffeine were purchased from Sigma-Aldrich (St. Louis, MO, product names G5009, H6034 and C7731, respectively). Deionized (DI) water was prepared from tap water using a Direct-Q 3 UV purification system from EMD Millipore (Billerica, MA). A 25-mm polyethersulfone (PES) filter with 0.2 μm pore size was purchased from GE Healthcare (Chicago, IL, catalog number 6780-2502). A 1 mL luer-lock syringe was purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, reference number 309628). A 20 mM histidine buffer, pH 6.0, was prepared using L-histidine, deionized water and titrated to pH 6.0 with the addition of 1 M HCl. A 15 mg/mL solution of caffeine was prepared using histidine buffer. BGG was reconstituted to a final concentration of approximately 280 mg/mL using caffeine-free and caffeine-containing buffers. Protein concentration c was calculated with the formula:

(Equation 4)

In the equation, m _p is the protein weight, b is the buffer addition volume, and ν is the partial specific volume of BGG, which is considered to be 0.74 mL/g here. 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 a sterile filter. did The syringe plunger was pushed at a rate of 159 mm/min for a distance of 50 mm. The energy demand 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 chromatographic elution

Four experimental grade biosimilar antibodies, ipilimumab, ustekinumab, omalizumab and tocilizumab were purchased from Bioceros (Utrecht, The Netherlands). These antibodies were provided as frozen aliquots at protein concentrations of 20, 26, 15 and 23 mg/mL, respectively, in 40 mM sodium acetate aqueous solution, 50 mM Tris-HCl buffer, pH 5.5. 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, MA). 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 has 96 wells, and 50 µl of protein A resin is accommodated in each well. Resin was washed with binding buffer by adding 200 μl of binding buffer to each well, centrifuging the plate at 1000 xg for 1 minute and discarding the flow-through. All subsequent centrifugation steps were performed at 1000 xg for 1 min. This washing procedure was repeated once. After this first washing step, diluted protein samples, i.e., samples containing ipilimumab, ustekinumab, omalizumab and toxylizumab, 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 and agitated at 260 rpm for 30 minutes, then the plate was centrifuged and the flow-through was removed. The wells were then washed by adding 500 μL of Binding Buffer to each well, centrifuging the plate and removing the flow-through. This washing step was repeated twice. After this washing step, proteins were eluted from the plate using an 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 agitated at 260 rpm for 1 minute and then centrifuged. The flow-through was recovered for analysis. This elution step was repeated once. The excipient-free control buffer contained 20 mM citrate and was pH 2.6. Since Protein A elution buffers often contain 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), trehalose from Cascade Analytical Reagents and Biochemicals (Corvallis, OR), and sucrose was purchased from Research Products International (Mt. Prospect, IL, product number S24060). All excipient-containing elution buffers were prepared by mixing an appropriate amount of excipient into about 10 mL of salt-free citrate buffer control. Elution buffer was prepared at a level of about 100 mM of the excipient. However, not all excipients are soluble at this concentration; Table 21 therefore lists the concentrations used for all excipients. The pH of each elution buffer was titrated to about 2.6 ± 0.1 using hydrochloric acid or sodium hydroxide as needed.

For each protein sample, ASD High Performance Size-Exclusion Chromatography (SEC ) analysis was performed. Separation was performed at room temperature at a flow rate of 0.35 mL/min. 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 toxilizumab, the total amount of protein eluted from the protein A resin was estimated by integrating the chromatogram. The integrated peak areas for each protein, i.e., ipilimumab, ustekinumab, omalizumab, and toxilizumab, are shown in Tables 22-25. Tables 22-25 also compare the experimental peak areas to those of salt-free and salt-containing controls. A value higher than 100% means that the elution buffer recovers more protein from the Protein A resin than the control, and a value less than 100% means that less elution buffer recovers 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 group N/A N/A 2.5

Table 22. from protein A resin ipilimumab collect

excipient Peak area (mAU*min) Peak area (%) normalized to salt-free control Peak area (%) normalized to salt control citrate 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 group 4376 100.0 107.2

Table 23. from protein A resin Ustekinumab collect

excipient Peak integral area (mAU*min) Peak area (%) normalized to salt-free control Peak area (%) normalized to 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's Map collect

excipient Peak integral area (mAU*min) Peak area (%) normalized to salt-free control Peak area (%) normalized to 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 group 3831 100.0 111.4

Table 25. from protein A resin tosilizumab collect

excipient Peak integral area (mAU*min) Peak area (%) normalized to salt-free control Peak area normalized to 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 group 2806 100.0 90.2

Example 29: excipients to improve protein A chromatographic elution

The test proteins used in this example are the same as those in Example 28, namely ipilimumab, ustekinumab, omalizumab and toxilizumab. 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, elution washes were performed twice. However, in this example, only one wash was performed. As in Example 28, the elution buffer was prepared as a 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 proteins were analyzed by HPLC in the same manner as in Example 28, and the protein recovery results of each protein, i.e., ipilimumab, ustekinumab, omalizumab and toxylizumab are shown in Tables 27-30 below. .

Table 26. Example Excipients used in 29

excipient Sigma- Aldrich product no Excipient concentration (mM) pH control group 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 ipilimumab collect

excipient Peak area (mAU*min) Peak area (%) normalized to salt-free control Peak area (%) normalized to salt control control group 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 Ustekinumab collect

excipient Peak integral area (mAU*min) Peak area (%) normalized to salt-free control Peak area (%) normalized to salt control control group 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's Map collect

excipient Peak integral area (mAU*min) Peak area (%) normalized to salt-free control Peak area (%) normalized to salt control control group 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 tosilizumab collect

excipient Peak integral area (mAU*min) Peak area (%) normalized to salt-free control Peak area (%) normalized to salt control control group 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 column O'Malley's Map Excipients to improve dissolution

Experimental-grade omalizumab was purchased from Bioceros (Utrecht, The Netherlands) and supplied frozen at 15 mg/mL in 40 mM aqueous sodium acetate, 50 mM tris-HCl buffer, pH 5.5. Proteins were thawed at room temperature before 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, MA) to obtain deionized water. Protein A purification was performed using a HiTrap Protein-A HP 1 mL column from GE Healthcare (Chicago, IL, catalog number 29048576). In each experiment, the column was first equilibrated with 10 mL of binding buffer. After equilibration, 30 mg of protein was loaded onto the Protein A column. The column was then rinsed with 5 mL of binding buffer. After washing the column, the bound omalizumab was eluted from the column partially using one of the elution buffers listed in Table 31 below. Elution buffer was prepared by dissolving the indicated excipients in 20 mM citrate buffer, pH 4.0. Elution buffers were all 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 step was 1 mL/min and was maintained using a Fusion 100 infusion pump (Chemyx, Stafford, TX). A 10 mL NormJect Luer Lock syringe (Henke Sass Wolf, Tuttlingen, Germany, reference number 4100-000V0) was used.

Elution fractions E1, E2, E3, E4 and E5 were analyzed for total protein content by high-performance size-exclusion chromatography (SEC) analysis. SEC analysis was performed 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). Separation was performed at room temperature at a flow rate of 0.35 mL/min. 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.

Citrate is a common excipient used in Protein A chromatography, so it was used as a control here. Elution fractions of the control sample showed insoluble aggregates when stored overnight at 4°C, as confirmed by precipitate formation. Accordingly, the peak area recorded in Table 31 below represents 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 the citrate excipient) indicates that the protein can be more effectively separated from the column using the test excipient.

Table 31. Protein A from column O'Malley's Map Elution

dissolution excipient dissolution 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) citrate
(control group) 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 Several included in the content BGG formulation

Formulations were prepared using different 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 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 obtained 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 in varying volume ratios to provide a series of caffeine-containing solutions of 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, and the test protein bovine gamma globulin (BGG) was added to each test solution at a rate at which a final protein concentration of about 280 mg/mL was achieved. was dissolved with BGG-containing solutions were prepared in 5 mL sterile polypropylene test tubes and stirred overnight at 100 rpm on a rotary shaker table. Then, these solutions were placed in a 2 mL microcentrifuge tube and centrifuged at 2400 rpm for 5 minutes in an IEC MicroMax centrifuge to remove trapped air before measuring the viscosity.

Viscosity measurements of the formulations prepared as described 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 25° C. with a shear rate of 250 1/s. To measure the viscosity, the test formulation was loaded into the viscometer, taking care to remove all air bubbles from the pipette. The pipette containing the sample preparation to be loaded was placed in the device and incubated at the measurement temperature for about 5 minutes. The device was then run until the channels were fully equilibrated with the test fluid, as confirmed by stable viscosity readings, then the viscosity was recorded in centipoise. The obtained viscosity results are shown in Table 32 below.

Table 32

Caffeine concentration ( mM ) Viscosity (cP) Normalized 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 solutions in

Compounds used as co-solutes to increase caffeine solubility in water were purchased from Sigma-Aldrich (St. Louis, MO), and these compounds were niacinamide, proline, procaine HCl, ascorbic acid, 2,5-dihydride hydroxybenzoic acid, lidocaine, saccharin, acesulfame K, tyramine and aminobenzoic acid. Solutions of each co-solute were prepared by dissolving the dry solid in deionized water and titrating the pH to about 6-8 with 5 M hydrochloric acid or 5 M sodium hydroxide as needed in some cases. The solution was then diluted to a final volume of 25 mL or 50 mL using a Class A volumetric flask and the concentration based on the weight of the dissolved compound and the final volume of the solution was recorded. The prepared solution was used as is or diluted with deionized water.

Example 33: caffeine soluble test

The effect of different co-solutes on caffeine solubility at ambient temperature (about 23° C.) was 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 a 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, keeping the final addition volume at 10 mL. The contribution of dry caffeine powder to volume was assumed to be negligible for all of these mixtures. A small magnetic rod was placed in the vial and the solution was placed on a stir plate for about 10 minutes to mix vigorously. 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 suggest that niacinamide, procaine HCl, 2,5-dihydroxybenzoic acid sodium salt, saccharin sodium salt, and tyramine chloride salt all exceed the reported caffeine solubility limits (at room temperature, ~16 mg/day according to Sigma-Aldrich). mL) to solubilize caffeine by at least about 4-fold.

Table 33

test number co-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 soluble; DND = not dissolved

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 preparation of the therapeutic monoclonal antibody adalimumab, a TNF-α blocker typically prescribed for HUMIRA ^® contains adalimumab 40 mg, sodium chloride 4.93 mg, sodium phosphate monobasic dihydrate 0.69 mg, sodium phosphate dibasic dihydrate 1.22 mg, sodium citrate 0.24 mg, citric acid monohydrate 1.04 mg, mannitol 9.6 mg and polysorbate 80 It is marketed as 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. An Amicon Ultra 15 centrifugal concentrator with a 30 kDa molecular weight cutoff (EMD-Millipore, Billerica, MA) was charged with approximately 15 mL of deionized water and the membrane washed by centrifugation at 4000 rpm for 10 minutes in a 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, measuring the absorbance of the diluted sample at 280 nm and calculating the concentration using the dilution factor and extinction coefficient of 1.39 mL/mg-cm. The viscosity of the concentrated sample was measured at a shear rate of 250 sec ⁻¹ and 23° C. in a microVisc viscometer (RheoSense, San Ramon, CA) equipped with an A05 chip. After measuring the viscosity, the sample was diluted with a small amount of filtrate, and concentration and viscosity measurements were repeated. Using this process, as shown in Table 34 below, the viscosity value according to the concentration change of adalimumab was obtained.

Table 34

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

Example 35: viscosity-lowering excipient added HUMIRA ^® Reconstitution of formulation

The examples below describe the general process for reconstitution in buffer with the addition of viscosity-lowering excipients to HUMIRA ^® . A viscosity-lowering excipient solution was prepared in 20 mM histidine by dissolving about 0.15 g of histidine and 0.75 g of caffeine (Sigma-Aldrich, St. Louis, Mo.) in deionized water. The pH of the prepared solution was titrated to about 5 using 5 M hydrochloric acid. The solution was then diluted to a final volume of 50 mL in a volumetric flask by the addition of deionized water. HUMIRA ^® was reconstituted at a high concentration of mAb using the prepared buffered viscosity-lowering excipient solution. Next, an Amicon Ultra 15 centrifuge tube with a 30 kDa molecular weight cutoff was washed, to which approximately 0.8 mL of HUMIRA ^® was added and centrifuged at 4000 rpm at 25° C. for 8-10 minutes in a Sorvall Legend RT. Then, about 14 mL of the buffered viscosity-lowering excipient solution as above was prepared and added to the concentrated HUMIRA ^® placed in a centrifugal 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 to which viscosity-lowering excipients have been added. Samples were measured for viscosity and concentration, in some cases diluted with a small amount of filtrate to measure viscosity at lower concentrations. Viscosity measurements were completed using a microVisc viscometer in the same manner as with the concentrated HUMIRA ^® formulation in the previous examples. Concentrations were determined by Bradford assay using a standard curve prepared with HUMIRA ^® stock solutions diluted in deionized water. As a result of reconstitution of HUMIRA ^® using a viscosity-lowering excipient, as shown in Table 35 below, the viscosity decreased by 30% to 60% compared to the viscosity values of HUMIRA ^® concentrated in unreconstituted commercial buffer.

Table 35

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

Example 36: using caffeine as an excipient Adalimumab Improved solution stability

After exposing the samples to two different stress environments, the stability of adalimumab solutions with and without caffeine excipients was evaluated: agitated and freeze-thawed. Adalimumab drug formulation HUMIRA® (AbbVie), with properties described in more detail in Example 34, was used. HUMIRA® samples were concentrated to a concentration of 200 mg/mL adalimumab in original buffer as described in Example 39; This concentrated sample was designated "Sample 1". A second sample was prepared as described in Example 40 using ~200 mg/mL of adalimumab and 15 mg/mL of caffeine added; This concentrated sample with added caffeine was designated "Sample 2". These two samples were diluted to a final concentration of 1 mg/mL of adalimumab by adding diluents as follows: sample 1 diluent was original buffer, sample 2 diluent was 20 mM histidine, 15 mg/mL caffeine, pH=5. am. Two HUMIRA® dilutions were filtered through a 0.22 μm syringe filter. For each diluted sample, 3 batches of 300 μl each were prepared by placing them in 2 mL Eppendorf tubes under a laminar flow hood. The samples were exposed to stress environments as follows: for agitation, the samples were placed on a rotary shaker at 300 rpm for 91 hours; For freeze-thaw, samples were subjected to 7 cycles between -17 and 30°C for an average of 6 hours per condition.

Table 36

Sample # added excipients stress environment 1-C doesn't exist doesn't exist 1-A doesn't exist stirring 1-FT doesn't exist freeze-thaw 2-C 15 mg/mL caffeine doesn't exist 2-A 15 mg/mL caffeine stirring 2-FT 15 mg/mL caffeine freeze-thaw

Example 37: dynamic Stability evaluation by light scattering (DLS)

A Brookhaven Zeta Plus dynamic light scattering device was used to measure the hydrodynamic radius of the adalimumab molecules in the samples of Example 36 and to confirm evidence of aggregate population formation. Table 37 shows the DLS results for six samples prepared according to Example 36: some of them (1-A, 1-FT, 2-A and 2-FT) were exposed to a stress environment ("stress exposed samples"); The other samples (1-C and 2-C) were not exposed. The DLS data in Table 37 shows that the monoclonal antibodies in the non-caffeinated stressed samples form a multimodal particle size distribution. In the absence of caffeine as an excipient, stress-exposed samples 1-A and 1-FT were observed to have larger effective diameters than non-stress-exposed samples 1-C, which also resulted in a second particle population with a significantly larger diameter. means; These groups of larger diameter particles are evidence of aggregation into particles invisible to the naked eye. Only stress-exposed samples containing caffeine (Samples 2-A and 2-FT) formed one particle population with a particle diameter similar to that of non-stress-exposed sample 2-C. These results demonstrate that the addition of caffeine to the sample reduces the formation of aggregates or sub-visible particles.

Table 37

Sample # available diameter (nm) Group 1's diameter (nm) Group 1's density On by % group 2 diameter (nm) % by concentration in group 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 - -

Tables 38A and 38B show DLS raw data of samples of adalimumab from Example 36, showing 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)

Using size exclusion chromatography, invisible particulates less than about 0.1 micron in size were detected in the stress-exposed and non-stress-exposed adalimumab samples 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 from each of the stress exposed and non-exposed samples of Example 36 was eluted isocratically using pH 6.2 buffer (100 mM phosphate, 325 mM NaCl) at a flow rate of 0.35 mL/min. The retention time of the adalimumab monomer was about 9 minutes. No detectable aggregates were identified in the samples containing the caffeine excipient, and the monomer content remained constant in all three samples.

Example 39: HERCEPTIN ^® formulation decrease in viscosity

The monoclonal antibody trastuzumab ( ^HERCEPTIN® , Genentech) was obtained as a lyophilized powder and reconstituted at 21 mg/mL in deionized water. The prepared solution was concentrated as is by centrifugation at 3500 rpm for 1.5 hours in an Amicon Ultra 4 centrifugal concentrator tube (molecular weight cutoff, 30 kDa). The sample was diluted 200-fold in the titration buffer, and then the absorbance was measured at 280 nm and an extinction coefficient of 1.48 mL/mg was applied to determine the concentration. Viscosity was measured using a RheoSense microVisc viscometer.

An excipient buffer containing salicylic acid and caffeine alone or in combination was prepared by dissolving histidine and an excipient in distilled water and then adjusting the pH to an appropriate level. The conditions for buffer systems 1 and 2 are summarized in Table 39.

Table 39

buffer system # salicylic acid density caffeine concentration osmolarity Concentration (mOsm/kg) pH One 10 mg/mL 10 mg/mL 145 6 2 0 15 mg/mL 86 6

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

Table 40

Control solution without added excipients buffer system 1: 10mg /mL caffeine + 10mg /ml salicylic acid adding dragon liquid buffer system 2: 15mg /mL caffeinated 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 showed the highest viscosity reduction rate of 76% at 215 mg/mL compared to the control sample. Buffer system 2 containing only caffeine showed a maximum decrease in viscosity of 59% at 200 mg/mL.

Example 40: AVASTIN ^® Lowering the viscosity of the formulation

AVASTIN ^® (monoclonal antibody bevacizumab formulation, Genentech) was given as a 25 mg/mL solution in histidine buffer. This sample was concentrated at 3500 rpm in an Amicon Ultra 4 centrifugal concentrator tube (MWCO 30 kDa). The concentration was determined by measuring the viscosity with a RheoSense microVisc viscometer and measuring the absorbance at 280 nm (extinction coefficient, 1.605 mL/mg). An excipient buffer was prepared by adding 10 mg/mL caffeine along with 25 mM histidine HCl. AVASTIN ^® stock solutions were diluted with excipient buffer and then concentrated in Amicon Ultra 15 centrifugal concentrator tubes (MWCO 30 kDa). Concentrations of excipient samples were determined by Bradford assay, and viscosities were measured using a RheoSense microVisc viscometer. Results are shown in Table 41 below.

Table 41

Concentration (mg/mL) Viscosity without additives (cP) 10mg Viscosity (cP) upon addition of /mL caffeine excipient Viscosity by excipient Decrease % 266 297 113 62% 213 80 22 73% 190 21 13 36%

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

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

Formulations were prepared using caffeine or a combination of caffeine and a second excipient compound as an excipient compound and a test protein, the test protein 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 measure 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 pH-titrated to pH 6 by adding a small amount of sodium hydroxide or hydrochloric acid before dissolving the model protein. Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a rate to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in excipient solution was prepared in a 20 mL glass scintillation vial and then placed on a rotary shaker table and stirred overnight at 80-100 rpm. Then, the BGG solution was transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for about 10 minutes in an IEC MicroMax microcentrifuge to remove trapped air bubbles before measuring the viscosity.

Viscosity of the formulation prepared as described above was measured using a DV-IIT LV viscometer (cone & plate viscometer) (Brookfield Engineering, Middleboro, MA). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. Then, an additional two steps consisting of 1 minute of shear incubation followed by a measurement collection period of 20 seconds were performed. The three collected data points were averaged and reported in Table 42 below as the viscosity of the sample. The viscosity of the excipient-containing solutions was normalized based on the viscosity of the non-excipient-containing model protein solution. The normalized viscosity is the ratio of the viscosity of the model protein solution with an excipient to the viscosity of the model protein solution without the excipient.

Table 42

Excipient A Excipient B Normalized 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 and Preparation of formulations containing the test protein

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

Viscosity of the formulation prepared as described above was measured using a DV-IIT LV viscometer (cone & plate viscometer) (Brookfield Engineering, Middleboro, MA). A CP-40 cone was mounted on the viscometer and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL, incubated at a given shear rate and temperature for 3 minutes and then measurements were taken for 20 seconds. Then, an additional two steps consisting of 1 minute of shear incubation followed by a measurement collection period of 20 seconds were performed. The average of the three data points collected was taken and recorded as the viscosity of the sample. The viscosity of the excipient-containing solutions was normalized based on the viscosity of the non-excipient-containing model protein solution. The normalized viscosity is the ratio of the viscosity of the model protein solution with an excipient to the viscosity of the model protein solution without the excipient.

Table 43

dimethyl sulfone Concentration (mg/mL) Normalized Viscosity 0 1.00 15 0.92 30 0.71 50 0.71 30 0.72

equivalent

While specific embodiments of the present invention have been described herein, the above description is illustrative and not limiting. Although the present invention has been specifically described and described with preferred embodiments, various changes in form and detail may be made to those skilled in the art without departing from the scope of the invention as encompassed by the appended claims. you will understand that Numerous variations on the present invention will become apparent to those skilled in the art upon reading the details. Unless otherwise stated, all numbers expressing reaction conditions, amounts of constituents, etc., 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 recited herein are approximations that may vary depending on the desired properties sought to be obtained by the present invention.

Claims (39)

As a method for improving the parameters of a protein production process,
The method comprises the steps of preparing an excipient-protein composition by adding one or more excipients selected from imidazole, taurine, niacinamide or caffeine in a viscosity-reducing amount to a protein composition, and the excipient-protein Applying the composition to a protein production process,
The parameter includes filtration rate, aggregated protein production, transfer efficiency or protein yield, wherein the parameter is improved compared to performing the protein production process on a control solution, wherein the control solution has the viscosity- and does not contain said one or more excipients in descending amounts. The method of claim 1, wherein the parameter further comprises a protein production unit cost or a protein production rate. The method of claim 1 , wherein the protein production process comprises a chromatography process. 4. The method of claim 3, wherein said chromatography process comprises a Protein-A chromatography process. The method of claim 1 , wherein the parameter comprises protein yield. The method of claim 1 , wherein the protein production process comprises a filtration process. The method according to claim 6, wherein the filtration step is a virus removal filtration step or an ultrafiltration/diafiltration step. 7. The method of claim 6, wherein the filtration process is characterized by improved filtration-related parameters. 9. The method of claim 8, wherein the improved filtration-related parameter is a filtration rate greater than that of the control solution. 9. The method of claim 8, wherein the improved filtration-related parameter is the production of an amount of aggregated protein that is less than the amount of aggregated protein produced by the filtration process relative to the control solution. 9. The method of claim 8, wherein the improved filtration-related parameter is a higher mass transfer efficiency than the mass transfer efficiency of the filtration process relative to the control solution. 9. The method of claim 8, wherein the improved filtration-related parameter is a higher concentration or higher yield than the concentration or yield of the target protein produced by the filtration process relative to the control solution. The method of claim 1 , wherein the viscosity-lowering amount is between 1 mg/mL and 100 mg/mL of the one or more excipients. The method of claim 1 , wherein the viscosity-lowering amount is between 1 mM and 400 mM of the one or more excipients. 15. The method according to claim 14, wherein the viscosity-lowering content is in the range of 2 mM to 150 mM. The method of claim 1, wherein the excipient-protein composition comprises additional substances selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers and buffers. 2. The method of claim 1, wherein said protein composition comprises a therapeutic protein. 18. The method of claim 17, wherein the therapeutic protein is a monoclonal antibody, polyclonal antibody, antibody fragment, fusion protein, PEGylated protein, antibody-drug conjugate, synthetic polypeptide, protein fragment, lipoprotein, enzyme and structure. A method selected from the group consisting of peptides. 18. The method of claim 17, wherein the therapeutic protein is a recombinant protein. 2. The method of claim 1 , further comprising adding a second viscosity-lowering excipient to the excipient-protein composition, wherein adding the second viscosity-lowering compound imparts an additional improvement to the parameter. how it would be. 21. The method of claim 20, wherein the second viscosity-lowering excipient is theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin, acesulfame potassium, niacinamide or isonicotinamide. The method of claim 1 , wherein said one or more excipients comprises imidazole. 2. The method of claim 1, wherein said one or more excipients comprises taurine. The method of claim 1 , wherein said one or more excipients comprises niacinamide. 2. The method of claim 1, wherein said one or more excipients comprises caffeine. The method of claim 1 , wherein the parameter includes filtration rate. The method of claim 1 , wherein the parameter comprises aggregated protein production. 2. The method of claim 1, wherein the parameter includes transfer efficiency. The method of claim 1 , wherein the parameter further comprises the energy required for the protein to pass through the sterile filter. The method of claim 1 , wherein the parameter further comprises protein elution. 26. The method of claim 25, wherein the viscosity-lowering amount of caffeine is less than or equal to 22 mg/ml. 26. The method of claim 25, wherein the viscosity-lowering amount of caffeine is less than or equal to 15 mg/ml. delete delete delete delete delete delete delete