JP7256816B2 - Excipient compounds for protein formulations - Google Patents

Description

RELATED APPLICATIONS This application is the subject of U.S. Provisional Application No. 62/639,950, filed March 7, 2018, and U.S. Provisional Application No. 62/679,647, filed June 1, 2018. claim the interests of The entire contents of each of the above applications are incorporated herein by reference.

This application relates generally to biopolymer formulations, such as protein formulations, with stabilizing excipients.

Biopolymers can be used for therapeutic or non-therapeutic purposes. Biopolymer-based therapeutic agents, such as formulations containing proteins, antibodies, or enzymes, are widely used to treat disease. Non-therapeutic biopolymers such as formulations containing enzymes, peptides, or structural proteins are useful in non-therapeutic applications such as household, nutritional, commercial, and industrial applications.

Of particular interest are therapeutic and non-therapeutic uses of protein biopolymers. Proteins are complex biopolymers, each with its own unique folded 3D structure and surface energy map (hydrophobic/hydrophilic regions and charge). In concentrated protein solutions, these macromolecules can interact strongly and entangle in more complex ways, depending on their precise shape and surface energy distribution. "Hot spots" for strong specific interactions cause protein clustering and increase solution viscosity. To address these issues, many excipient compounds are used in biopharmaceutical formulations, aiming to reduce solution viscosity by preventing local interactions and clustering. These efforts are individually tailored, often guided empirically and sometimes by in silico simulations. Combinations of excipient compounds may be provided, but optimizing such combinations must proceed empirically and on a case-by-case basis.

Biopolymers such as proteins used in therapeutic applications need to be formulated for introduction into the body for the treatment of disease. For example, under certain circumstances it is advantageous to deliver antibody and protein/peptide biopolymer formulations by subcutaneous (SC) or intramuscular (IM) routes instead of administering these formulations by intravenous (IV) injection. is. However, for better patient compliance and comfort with SC or IM injections, the volume of liquid in the syringe is typically limited to 2-3 mL and the viscosity of the formulation is typically less than about 20 centipoise (cP). , so conventional medical devices and small-bore needles can be used to deliver the formulation. These delivery parameters do not always match well with the dosage requirements of the delivered formulation.

For example, antibodies may need to be delivered at high dosage levels to exert their intended therapeutic effect. Delivery of high dose levels of antibody formulations using limited fluid volumes may require high concentrations of antibody in the delivery vehicle and may exceed levels of 150 mg/mL. At this dosage level, the viscosity versus concentration plots of the protein solutions are beyond their linear-nonlinear transition, and the viscosity of the formulation increases dramatically with increasing concentration. However, the increased viscosity is incompatible with standard SC or IM delivery systems. Solutions of biopolymer-based or protein-based therapeutics experience stability problems such as precipitation, fragmentation, oxidation, deamidation, turbidity, opacification, denaturation, and gel formation, reversible or irreversible aggregation. easier. Stability issues limit the shelf life of solutions or require special handling.

One approach to producing injectable protein formulations is to convert therapeutic protein solutions into powders that can be reconstituted to form suspensions suitable for SC or IM delivery. Freeze-drying is a standard technique for producing protein powders. Lyophilization, spray drying, further precipitation, followed by supercritical fluid extraction have been used to produce protein powders for subsequent reconstitution. Powder suspensions may be suitable for SC or IM injections if the particles are small enough to pass through a needle due to their low viscosity (compared to solutions of the same total dose) prior to reconstitution. However, protein crystals present in powders have an inherent risk of triggering an immune response. Uncertain protein stability/activity after reconstitution raises additional concerns. There remains a need in the art for techniques to produce low viscosity protein formulations for therapeutic purposes while avoiding the limitations introduced by protein powder suspensions.

More complex antibody formulations such as antibody drug conjugates (ADCs) are particularly vulnerable to viscosity and stability issues. ADCs attach small molecule drugs to monoclonal antibodies (mAbs) via chemical linkers, which target specific antigens on aberrant "target cells," and the small molecule drugs Selected to have a specific antigenic effect. When the mAb contacts the target cell antigen, it and its bound drug are taken up by the cell and translocated inside the cell. Inside the cell, the mAb and/or linker are cleaved, releasing the drug and exerting its biological effect on the cell. Drugs are usually chemotherapeutic agents and are highly toxic when released systemically. ADCs bring chemotherapeutic agents into direct contact with their target cancer cells. This conjugation of small molecules to mAbs can exacerbate the viscosity and stability problems that affect therapeutic protein formulations. Payload compounds are typically small, hydrophobic molecules, and increasing drug-to-antibody ratios can significantly affect the stability, solubility, and solution-interaction properties of larger ADCs. High salt concentrations in the formulation increase the hydrophobic interactions between the ADC complexes, making ADC solubility more sensitive to salt than unconjugated antibody. Processing or storage of ADC solutions can cause aggregation or precipitation of ADC species, especially if the drug-to-antibody ratio (DAR) is high. Drug binding can also affect the conformational stability of mAbs, particularly their Fc domains. In addition, drug conjugation also reduces the net surface charge of the mAb, which can affect ADC solubility.

In addition to the therapeutic uses of proteins described above, biopolymers such as enzymes, peptides, and structural proteins can be used for non-therapeutic uses. These non-therapeutic biopolymers can be produced from many different routes, eg, derived from plant sources, animal sources, or produced from cell culture.

Non-therapeutic proteins can be produced, transported, stored and processed as granular or powdered materials, or as solutions or dispersions, usually in water. Biopolymers for non-therapeutic applications may be spherical or fibrous proteins, and certain forms of these materials may have limited or aqueous solubility or exhibit high viscosity upon dissolution. The properties of these solutions can present challenges to the formulation, handling, storage, pumping, and performance of non-therapeutic materials, so reducing the viscosity and improving solubility and stability of non-therapeutic protein solutions need a way to

There remains a need in the art for a truly universal approach to reducing the viscosity and/or improving the stability of protein formulations, especially at high protein concentrations. There is an additional need in the art to achieve this viscosity reduction while maintaining protein activity. Additionally, it is desirable to adapt the viscosity reducing system for use with formulations that have a tunable sustained release profile and for use with formulations that are compatible with depot injection. Additionally, it is desirable to improve processes for producing proteins and other biopolymers.

In embodiments, disclosed herein are liquid formulations composed of proteins and hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, and low molecular weight aliphatic polyacids. and an excipient compound selected from the group, wherein the excipient compound is added in a viscosity-lowering amount. In embodiments, the protein is a PEGylated protein and the excipient is a low molecular weight aliphatic polyacid. In embodiments, the formulation is a pharmaceutical composition, the therapeutic formulation comprises a therapeutic protein, and the excipient compound is a pharmaceutically acceptable excipient compound. In embodiments, the formulation is a non-therapeutic formulation, and the non-therapeutic formulation comprises a non-therapeutic protein. In embodiments, the viscosity reducing amount reduces the viscosity of the formulation to a viscosity lower than that of the control formulation. In embodiments, the viscosity of the formulation is at least about 10% lower than the viscosity of the control formulation, or at least about 30% lower than the viscosity of the control formulation, or at least about 50% lower than the viscosity of the control formulation, or or at least about 90% lower than the viscosity of the control formulation. In embodiments, 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 embodiments, the excipient compound has a molecular weight of less than 5000 Da, or less than 1500 Da, or less than 500 Da. In embodiments, the formulation comprises at least about 25 mg/mL protein, or at least about 100 mg/mL protein, or at least about 200 mg/mL protein, or at least about 300 mg/mL protein. In embodiments, the formulation comprises from about 5 mg/mL to about 300 mg/mL of excipient compound, or from about 10 mg/mL to about 200 mg/mL of excipient compound, or from about 20 mg/mL to about 100 mg/mL of excipient compound. mL, or from about 25 mg/mL to about 75 mg/mL of excipient compound. In embodiments, the formulation has improved stability when compared to a control formulation. In embodiments, the excipient compound is a hindered amine. In embodiments, the hindered amine is selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin, and acesulfame potassium. In embodiments, the hindered amine is caffeine. In embodiments, the hindered amine is a locally injectable anesthetic compound. Hindered amines can have independent pharmacological properties, and hindered amines can be present in the formulation in amounts that have independent pharmacological effects. In embodiments, the hindered amine may be present in the formulation in an amount that is less than the therapeutically effective amount. An independent pharmacological activity can be local anesthesia. In embodiments, the hindered amine with independent pharmacological activity is combined with a second excipient compound that further reduces the viscosity of the formulation. The second excipient compound can be selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin, and acesulfame potassium. In embodiments, the formulation can include additional agents selected from the group consisting of preservatives, surfactants, sugars, polysaccharides, arginine, proline, hyaluronidase, stabilizers, and buffers.

Further disclosed herein is a method of treating a disease or disorder in a mammal comprising administering to the mammal a liquid therapeutic formulation, wherein the therapeutic formulation comprises a therapeutically effective amount of and the formulation comprises a pharmaceutically acceptable excipient selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, and low molecular weight aliphatic polyacids. A therapeutic formulation, further comprising a compound compound, is effective in treating a disease or disorder. In embodiments, the therapeutic protein is a PEGylated protein and the excipient compound is a low molecular weight aliphatic polyacid. In embodiments, the excipient is a hindered amine. In embodiments, the hindered amine is a local anesthetic compound. In embodiments, the formulation is administered by subcutaneous, intramuscular, or intravenous injection. 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 a lower viscosity than that of the control formulation. In embodiments, the therapeutic formulation has improved stability when compared to the control formulation. In embodiments, the excipient compound is essentially pure.

Further disclosed herein is a method of reducing pain at the site of injection of a therapeutic protein in a mammal in need thereof, comprising administering a liquid therapeutic formulation by injection, The formulation comprises a therapeutically effective amount of a therapeutic protein, the formulation further comprises a pharmaceutically acceptable excipient compound selected from the group consisting of local injectable anesthetic compounds, and is pharmaceutically acceptable The excipient compound is added to the formulation in a viscosity-lowering amount, and the mammal experiences less pain upon administration of the therapeutic formulation containing the excipient compound than upon administration of the control therapeutic formulation, and the control therapeutic formulation. The therapeutic formulation does not contain excipient compounds and is otherwise identical to the therapeutic formulation.

In embodiments, disclosed herein are methods of improving the stability of liquid protein formulations, wherein therapeutic proteins and hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and an excipient compound selected from the group consisting of low molecular weight aliphatic polyacids, wherein the liquid protein formulation has improved stability compared to the control liquid protein formulation. and the control liquid protein formulation contains no excipient compound and is otherwise identical to the liquid protein formulation. The stability of liquid formulations can be cold storage conditions stability, room temperature stability or elevated temperature stability.

Also, in embodiments, disclosed herein are selected from the group consisting of proteins and hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, and low molecular weight aliphatic polyacids. and an excipient compound, wherein the presence of the excipient compound in the formulation results in improved protein-protein interaction properties as measured by the protein diffusion interaction parameter kD, or the second virial coefficient B22. bring. In embodiments, the formulation is a therapeutic formulation and comprises a therapeutic protein. In embodiments, the formulation is a non-therapeutic formulation and comprises a non-therapeutic protein.

In embodiments, further disclosed herein are methods of improving protein-related processes comprising providing a liquid formulation as described above and using it in a method of treatment. In embodiments, processing methods include filtration, pumping, mixing, centrifugation, diafiltration, lyophilization, or chromatography. In embodiments, the processing method is selected from the group consisting of cell culture harvest, chromatography, virus inactivation, and filtration. In embodiments, the processing method is a chromatographic process or a filtration process. In embodiments, the filtration process is a virus filtration process or an ultrafiltration/diafiltration process.

Also disclosed herein are methods for improving parameters of protein-related processes, including hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, and at least one excipient compound selected from the group consisting of diones and sulfones; Including adding a compound, the carrier solution containing the protein of interest, thereby improving the parameter. In embodiments, the parameter may be selected from the group consisting of cost of protein production, amount of protein production, rate of protein production, and efficiency of protein production. A parameter may be a proxy parameter. In embodiments, the protein-related process is an upstream processing process. A carrier solution for upstream processing can be cell culture media. In an embodiment, when the carrier solution is a cell culture medium, adding an excipient additive to the carrier solution comprises first adding the excipient additive to the supplemental medium to form an excipient-containing supplemental medium. and a second substep of adding an excipient-containing supplement medium to the cell culture medium. In other embodiments, the protein-related process is a downstream process. The downstream process can be a chromatographic process and the chromatographic process can be a Protein-A chromatographic process. In embodiments, the chromatographic process is to recover the protein of interest, wherein the protein of interest exhibits improved purity, improved yield, fewer particles, less misfolding, or characterized by an improved protein-related parameter selected from the group consisting of less aggregation. In embodiments, the improved protein-related parameter is improved yield of the protein of interest from the chromatography process. In other embodiments, the protein-related process is a process selected from the group consisting of filtration, infusion, injection, pumping, mixing, centrifugation, diafiltration, and lyophilization, wherein the selected process is greater than the control process. less force is required. In embodiments, 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 embodiments, 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 at a pH higher than a control process. done. In other embodiments, the protein-related process is a filtration process. The filtration process can be a virus removal filtration process or an ultrafiltration/diafiltration process. Filtration processes can be characterized by improved filtration-related parameters. An improved filtration-related parameter can be a filtration rate that is greater than that of the control solution. An improved filtration-related parameter can be the production of an amount of aggregated protein that is less than the amount of aggregated protein produced by a control filtration process. An improved filtration-related parameter can be a mass transfer efficiency that is higher than that of a control filtration process. An improved filtration-related parameter can be a higher concentration or yield of target protein than that produced by a control filtration process.

Further disclosed herein is the above method, wherein the viscosity-lowering excipient additive comprises two or more excipient compounds. In embodiments, at least one excipient compound is a hindered amine. In embodiments, the at least one excipient compound is selected from the group consisting of caffeine, saccharin, acesulfame potassium, aspartame, theophylline, taurine, 1-methyl-2-pyrrolidone, 2-pyrrolidinone, niacinamide, and imidazole. be. In embodiments, the at least one excipient compound is selected from the group consisting of caffeine, taurine, niacinamide, and imidazole. In embodiments, at least one excipient compound is an anionic aromatic excipient, and in some embodiments the anionic aromatic excipient can be 4-hydroxybenzenesulfonic acid. In embodiments, the viscosity-lowering amount is from about 1 mg/mL to about 100 mg/mL of the at least one excipient compound, or the viscosity-lowering amount is from about 1 mM to about 400 mM of the at least one excipient compound. , or the viscosity-lowering amount is an amount from about 2 mM to about 150 mM. In embodiments, the carrier solution comprises additional agents selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers, and buffers. In embodiments, the protein of interest is a therapeutic protein and the therapeutic protein can be a recombinant protein or monoclonal antibody, polyclonal antibody, antibody fragment, fusion protein, pegylated protein, antibody drug conjugate, synthetic It may be selected from the group consisting of polypeptides, protein fragments, lipoproteins, enzymes, and structural peptides. In embodiments, the method further comprises adding a second viscosity-lowering excipient to the carrier solution, wherein adding the second viscosity-lowering compound adds further improvement to the parameter.

Further, a carrier solution is disclosed herein, comprising a liquid medium in which the protein of interest is dissolved, and a viscosity-lowering additive, wherein the carrier solution has a viscosity lower than that of the control solution. The carrier solution can further comprise additional agents selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers, and buffers.

Further, the present disclosure relates to stability-enhanced formulations comprising a therapeutic protein and a stability-improving amount of a stabilizing excipient, wherein the stability-enhanced formulation lacks a stabilizing excipient except that the stability is improved. It is characterized by improved stability parameters compared to a control formulation that is identical to the fortified formulation. In embodiments, the therapeutic protein is an antibody, and the antibody can be an antibody drug conjugate. Stabilizing excipients include hindered amine compounds, anionic aromatic compounds, functionalized amino acid compounds, oligopeptides, short chain organic acids, low molecular weight polyacids, dione or sulfone compounds, zwitterionic compounds, or hydrogen bonding elements. It may be a crowding agent comprising: In embodiments, the stabilizing excipient is in an amount from about 1 mM to about 500 mM, or from about 5 mM to about 250 mM, or from about 10 mM to about 100 mM, or from about 5 mg/mL to about 50 mg/mL. can be added to the formulation at An improved stability parameter can be, for example, thermal storage stability, which is improved at temperatures of about 10°C to 30°C. In embodiments, the improved stability parameter is improved freeze/thaw stability or improved shear stability. In embodiments, the stability-enhanced formulation has a lower particle count as compared to the control. In embodiments, the stability-enhanced formulation has improved biological activity compared to the control.

Also disclosed herein is a method of improving the stability of a therapeutic formulation, comprising adding a stability-improving amount of a stabilizing excipient to the therapeutic formulation, thereby improving the stability of the therapeutic formulation. Including improved stability, the stability of a therapeutic formulation is measured relative to the stability of a control formulation that is identical to the therapeutic formulation but lacking the stabilizing excipient. Stabilizing excipients can be hindered amines, anionic aromatic compounds, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight polyacids, diones, sulfones, zwitterionic compounds, or crowding agents with hydrogen bonding. could be. In embodiments, measuring the stability of the therapeutic formulation comprises measuring a stability-related parameter, e.g., a parameter selected from the group consisting of heat storage stability, freeze/thaw stability, and shear stability. can include In embodiments, the therapeutic formulation comprises a therapeutic protein, which can be an antibody, and the antibody can be an antibody drug conjugate. Further disclosed herein is a method of improving a parameter of a protein-related process comprising adding a stability-improving amount of a stabilizing excipient to a protein-related process carrier solution, wherein the carrier solution comprises , comprising a protein of interest thereby improving a parameter, the protein of interest may be a therapeutic protein. In embodiments, the parameter may be selected from the group consisting of cost of protein production, amount of protein production, rate of protein production, and efficiency of protein production.

Figure 2 shows a graph of particle size distribution for solutions of monoclonal antibodies under stressed and unstressed conditions as assessed by dynamic light scattering. The data curves in Figure 1 have a baseline offset to allow comparison. The curve for Sample 1-A is offset by 100 intensity units and the curve for Sample 1-FT is offset by 200 intensity units on the Y-axis. shows a graph measuring the sample diameter versus multimodal size distribution of several molecular populations as assessed by dynamic light scattering. The data curves in Figure 2 have a baseline offset to allow comparison. The curve for Sample 2-A is offset by 100 intensity units and the curve for Sample 2-FT is offset by 200 intensity units on the Y-axis. Shows a size exclusion chromatogram of a monoclonal antibody solution with a main peak of monomer at a retention time of 8-10 minutes. The data curves in Figure 3 have a baseline offset to allow comparison. The curves for samples 2-C, 2-A and 2-FT are offset along the Y-axis. presents a block diagram showing the steps of the fermentation process (“upstream processing”) for producing therapeutic proteins such as monoclonal antibodies. presents a block diagram showing the steps of a purification process (“downstream processing”) for producing therapeutic proteins such as monoclonal antibodies.

Disclosed herein are formulations and methods for their production that enable the delivery of concentrated protein solutions. In embodiments, the approaches disclosed herein can result in higher concentrations of therapeutic or non-therapeutic proteins in lower viscosity liquid formulations or in liquid formulations compared to conventional protein solutions. .

In embodiments, the approaches disclosed herein can result in liquid formulations with improved stability compared to conventional protein solutions. In one aspect, a stable formulation substantially retains its physical and chemical stability or integrity, and its therapeutic or non-therapeutic efficacy when the protein contained therein is subjected to stress conditions. It is a thing. In another aspect, a stable formulation is one in which the protein contained therein substantially retains its soluble, monomeric, or non-aggregated state. As used herein, stress conditions are physical or chemical conditions that adversely affect a protein in a formulation. Advantageously, a stable formulation can also provide protection against aggregation or precipitation of proteins dissolved therein.

Examples of physical stress conditions include mechanical shear, contact with air/liquid interfaces, freeze-thaw cycles, long-term storage under storage conditions (either cold storage conditions, room temperature conditions, or high temperature storage conditions) or other conditions. physical perturbations such as exposure to denaturing conditions of For example, cold storage conditions may require storage in a refrigerator or freezer. In some instances, cryopreservation conditions may require storage at temperatures of 10°C or less. In additional examples, cryopreservation conditions require storage at temperatures from about 2°C to about 10°C. In another example, cryopreservation conditions require storage at a temperature of about 4°C. In additional examples, cryopreservation conditions require storage at freezing temperatures, such as about -20°C or lower. In another example, cryopreservation conditions require storage at temperatures from about -80°C to about 0°C. Room temperature storage conditions may require storage at ambient temperatures of, for example, about 10°C to about 30°C. High temperature storage conditions may require storage at temperatures above about 30°C. For example, high temperature stability at temperatures from about 30° C. to about 50° C. can be used as part of accelerated aging testing to predict long-term storage under normal ambient (10-30° C.) conditions. Stress conditions can also include chemical perturbations, such as changes in pH, which can affect the stability or integrity of a protein in formulation, for example by affecting its tertiary structure.

As is well known to those skilled in the fields of polymer science and engineering, proteins in solution tend to form entanglements, limiting translational movement of the entangled chains and reducing the therapeutic or non-therapeutic effects of proteins. may be hindered. In embodiments, the excipient compounds disclosed herein can inhibit protein clustering through specific interactions between the excipient compound and the target protein in solution. The excipient compounds disclosed herein can be natural or synthetic and desirably are substances generally recognized as safe by the FDA (“GRAS”).

1. DEFINITIONS For the purposes of this disclosure, the term "protein" refers to a sequence of amino acids usually having a molecular weight of 1-3000 kDa and having a chain length long enough to form a discrete tertiary structure. In some embodiments, the protein has a molecular weight of about 50-200 kDa. In other embodiments, the protein has a molecular weight of about 20-1000 kDa, or about 20-2000 kDa. In contrast to the term "protein", the term "peptide" refers to a sequence of amino acids with no individual tertiary structure. A wide variety of biopolymers are included within the scope of the term "protein". For example, the term "protein" can 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.

a. Therapeutic Biopolymers and Related Definitions These biopolymers that contain proteins with therapeutic effects are sometimes referred to as "therapeutic biopolymers." Those proteins that have therapeutic effects are sometimes referred to as "therapeutic proteins."

As non-limiting examples, therapeutic proteins include hormones and prohormones such as insulin and proinsulin, glucagon, calcitonin, thyroid hormone (T3 or T4 or thyroid stimulating hormone), parathyroid hormone, follicle stimulating hormone, luteinizing hormones, growth hormone, growth hormone releasing factor, etc.); coagulation and anticoagulation factors (e.g. tissue factor, von Willebrand factor, factor VIIIC, factor IX, protein C, plasminogen activator (urokinase, tissue type plasminogen activator), thrombin); cytokines, chemokines, and inflammatory mediators; interferons; colony-stimulating factors; interleukins (eg, IL-1 to IL-10); , fibroblast growth factor, platelet-derived growth factor, transforming growth factor, transforming growth factor, neurotrophic growth factor, insulin-like growth factor, etc.); albumin; collagen and elastin; hematopoietic factors (e.g., erythropoietin, thrombopoietin, etc.) osteoinductive factors (e.g., bone morphogenetic proteins); receptors (e.g., integrins, cadherins, etc.); membrane surface proteins; transport proteins; regulatory proteins; of mammalian proteins.

In certain embodiments, a therapeutic protein can be an antibody. The term "antibody" is used herein in its broadest sense and non-limiting examples include monoclonal antibodies (including, for example, full length antibodies having an immunoglobulin Fc region), single chain molecules, double Specific and multispecific antibodies, diabodies, antibody-drug conjugates, antibody compositions with polyepitopic specificity, polyclonal antibodies (such as polyclonal immunoglobulins used as therapeutics for immunocompromised patients), and fragments of antibodies (including, for example, Fc, Fab, Fv, nanobodies, and F(ab')2). Antibodies are also called "immunoglobulins". It is understood that antibodies are directed against specific protein or non-protein "antigens" that are biologically important substances. Administration of a therapeutically effective amount of an antibody to a patient forms a complex with the antigen, thereby altering its biological properties and allowing the patient to experience a therapeutic effect.

In embodiments, the antibody may be an antibody drug conjugate (ADC). Antibody-drug conjugates are a category of therapeutic proteins that combine the highly specific targeting ability of antibodies with therapeutically active compounds such as cytotoxic compounds. ADCs consist of an antibody linked to a therapeutically active agent via a biodegradable chemical linker. More specifically, ADCs may include human or humanized mAbs that are specific for antigens that have minimal or no expression in normal cells but are expressed in aberrant "target" cells. . ADCs also include potent agents such as cytotoxic agents that can destroy target cells. Such agents are generally systemically toxic and therefore not suitable for general systemic administration. The targeting ability of the mAb component of the ADC allows the drug to be directed specifically to, be taken up by, and exert its effect within target cells, rather than being distributed all over the body. . To form ADCs, mAbs are linked to drugs with labile bonds that are stable in the extracellular environment (e.g., intravenous and interstitial circulation), but degrade when the ADC is taken up intracellularly. be. When the bond between the ADC and the drug is broken, the drug is released inside the cell and affects the cell. ADCs are suitable for use with cytotoxic agents, especially when these compounds are too toxic to be used alone. In cancer chemotherapy, for example, some of the agents selected for use in ADCs are orders of magnitude more toxic than conventional anticancer agents. Examples include anti-microtubule agents, alkylating agents, and DNA minor groove binders, which may be too toxic to be successfully administered, but mAbs that bind antigens expressed only on cancer cells can be used to target cancer cells. Once the ADC localizes to the tumor and binds to surface target cell antigens, the complex can be internalized into the cell into vesicles. The internalized vesicles fuse together and enter the endosomal-lysosomal pathway where they are contacted by proteases and the mAb and/or linker molecules are digested, thereby releasing the drug payload. The payload (eg, cytotoxic agent) then traverses the lysosomal membrane and enters the cytoplasm and/or nucleus where it exerts its pharmaceutical (eg, cytotoxic) effect. This focused delivery of highly potent pharmaceutical compounds maximizes the intended therapeutic effect while minimizing normal tissue exposure to these agents. Formulations containing ADC are suitable for intravenous or topical administration so that the ADC reaches the target cells to be treated.

In certain embodiments, the therapeutic proteins are pegylated, meaning that they contain poly(ethylene glycol) (“PEG”) and/or poly(propylene glycol) (“PPG”) units. PEGylated proteins, or PEG-protein conjugates, find utility in therapeutic applications due to beneficial properties such as solubility, pharmacokinetics, pharmacodynamics, immunogenicity, renal clearance, and stability. Non-limiting examples of pegylated proteins are pegylated interferon (PEG-IFN), pegylated anti-VEGF, pegylated protein binders, adagen, pegasparagase, pegfilgrastim, pegroticase, pegvisomant, pegylated epoetin-beta , and certolizumab pegol.

PEGylated proteins can be synthesized by a variety of methods, including reaction of the protein with a PEG reagent having one or more reactive functional groups. Reactive functional groups of PEG reagents can form linkages with proteins at target protein sites such as lysine, histidine, cysteine, the N-terminus, and the like. Typical PEGylation reagents have reactive functional groups such as aldehyde, maleimide, succinimide, etc., and have specific reactivity with target amino acid residues on proteins. The PEGylation reagent can have a PEG chain length and/or PPG repeat unit from about 1 to about 1000. Other methods of PEGylation include glyco-PEGylation, where the protein is first glycosylated and then the glycosylated residue is PEGylated in a second step. Certain PEGylation processes are assisted by enzymes such as sialyltransferases and transglutaminases.

PEGylated proteins can offer therapeutic advantages over native non-PEGylated proteins, but these materials may have physical or chemical properties that make them difficult to purify, dissolve, filter, concentrate, and administer. There is PEGylation of proteins can result in higher solution viscosities compared to native proteins, which generally requires formulation of PEGylated protein solutions at lower concentrations.

It is desirable to formulate protein therapeutics in stable, low-viscosity solutions so that minimal injection volumes can be administered to patients. 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 preferred for self-administered care. It is less costly and easier to handle than intravenous (IV) injections, which are performed only under direct medical supervision. Formulations for SC or IM injection require low solution viscosities, generally less than 30 cP, preferably less than 20 cP, so that the therapeutic solution can be easily flowed through fine gauge needles. This combination of small injection volume and low viscosity requirements presents challenges to the use of PEGylated protein therapeutics by SC or IM injection routes.

A formulation that includes a therapeutically effective amount of a therapeutic protein may be referred to as a "therapeutic formulation." A therapeutic protein included in a therapeutic formulation is sometimes referred to as its "protein active ingredient." Generally, therapeutic formulations contain a therapeutically effective amount of an active protein ingredient and excipients, with or without other optional ingredients. As used herein, the term "therapeutic" includes both treatment of existing disorders and prevention of disorders. Therapeutic proteins include, for example, proteins such as bevacizumab, trastuzumab, adalimumab, infliximab, etanercept, darbepoetin alfa, epoetin alfa, cetuximab, filgrastim, and rituximab. Other therapeutic proteins will be familiar to those of skill in the art.

"Treatment" includes preventing or delaying the onset of symptoms and/or alleviating or ameliorating symptoms of a disorder, curing, healing, alleviating, ameliorating, correcting, or otherwise beneficially affecting the disorder. including any means intended to These patients in need of treatment include both those who already have the particular disorder and those for whom prevention of the disorder is desired. A disorder is any condition that alters the homeostatic health of a mammal, including an acute or chronic disease, or a condition that predisposes the mammal to an acute or chronic disease. Non-limiting examples of disorders include cancer, metabolic disorders (e.g. diabetes), allergic disorders (e.g. asthma), skin disorders, cardiovascular disorders, respiratory disorders, blood disorders, musculoskeletal disorders, inflammatory or rheumatic disorders, autoimmune disorders, gastrointestinal disorders, urological disorders, sexual and reproductive disorders, neurological disorders, and the like. The term "mammal" for therapeutic purposes can refer to any animal classified as a mammal, including humans, domestic animals, companion animals, farm animals, sporting animals, working animals, and the like. Thus, "treatment" can include both veterinary and human treatments. For convenience, a mammal undergoing such "treatment" may be referred to as a "patient." In certain embodiments, the patient can be of any age, including fetal animals in utero.

In embodiments, treatment comprises providing a therapeutically effective amount of a therapeutic formulation to a mammal in need thereof. A "therapeutically effective amount" is at least the minimal concentration of a therapeutic protein administered to a mammal in need thereof to treat an existing disorder or prevent an anticipated disorder (such treatment or treatment). It is effective in prevention, which is “therapeutic intervention”. The therapeutically effective amounts of various therapeutic proteins that can be included as active ingredients in therapeutic formulations may be well known in the art. Alternatively, for therapeutic proteins to be discovered in the future or applied for therapeutic intervention, the therapeutically effective amount can be determined by standard techniques practiced by those skilled in the art using only routine experimentation. .

b. Non-Therapeutic Biopolymers and Related Definitions Those proteins that are used for non-therapeutic purposes (i.e., non-therapeutic purposes), e.g., household, nutritional, commercial, and industrial uses, are referred to as "non-therapeutic proteins." Sometimes. Formulations containing non-therapeutic proteins are sometimes referred to as "non-therapeutic formulations." Non-therapeutic proteins may be derived from plant sources, animal sources, or produced from cell culture. They can also be enzymes or structural proteins. Non-therapeutic proteins can be used in household, nutritional, commercial, and industrial applications such as catalysts, human and animal nutrition, processing aids, cleaning agents, and waste disposal.

An important category of non-therapeutic biopolymers is that of enzymes. Enzymes have several non-therapeutic uses such as, for example, catalysis, human and animal nutritional components, processing aids, cleaning agents, and waste treatment agents. Enzyme catalysts are used to facilitate a variety of chemical reactions. Examples of enzymatic catalysts for non-therapeutic use include catalase, oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase. Uses of enzymes for human and animal nutrition include dietary supplements, protein sources, chelation or controlled delivery of micronutrients, digestive aids, and supplements. These can be derived from amylases, proteases, trypsin, lactase, and the like. Enzymatic processing aids are used to improve the production of food and beverage products in operations such as baking, brewing, fermentation, juice processing, and winemaking. Examples of these food and beverage processing aids include amylases, cellulases, pectinases, glucanases, lipases, and lactases. Enzymes can also be used to produce biofuels. For example, ethanol for biofuels is aided by enzymatic degradation of biomass feedstocks such as cellulosic and lignocellulosic materials. Treatment of such feedstock materials with cellulases and ligninases ferments the biomass into fuel substrates. In other commercial applications, enzymes are used as detergents, cleaners, and soil lifting aids in 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 softening of textiles, leather processing, waste treatment, contaminated sediment treatment, water treatment, pulp bleaching, pulp softening and exfoliation. be done. Typical enzymes for these purposes 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 preparation and formulation of food ingredients such as gelatin, ice cream, yogurt and confectionery. They are also added to foods as thickeners, rheology modifiers, mouthfeel improvers and as a source of nutritional protein. Collagen, elastin, keratin, and hydrolyzed keratin are widely used in skin and hair care formulations in the cosmetic and personal care industry. Yet other examples of non-therapeutic biopolymers are whey proteins such as beta-lactoglobulin, alpha-lactalbumin, and serum albumin. These whey proteins are produced in large quantities as a by-product from dairy operations and have been used in a variety of non-therapeutic applications.

2. Measurements In embodiments, the protein-containing formulations described herein are tolerant to monomer loss, as measured by size exclusion chromatography (SEC) analysis. In the SEC analysis used herein, the main peak of the analyte is generally associated with the target protein contained in the formulation, and this main peak of protein is called the monomer peak. The monomer peak represents the amount of target protein, eg, protein active ingredient, in its monomeric state, as opposed to its aggregated (dimer, trimer, oligomer, etc.) or fragmented state. The monomer peak area can be compared to the total area of monomer, aggregate and fragment peaks associated with the target protein. Thus, the stability of protein-containing formulations can be monitored by the relative amount of monomer over time. The improved stability of the protein-containing formulations of the present invention can therefore be measured as a higher percent monomer after a period of time compared to the percent monomer in a control formulation containing no excipients. can.

In embodiments, the ideal stability result is to have a 98-100% monomer peak as determined by SEC analysis. In embodiments, the improved stability of the protein-containing formulations of the invention is such that such control formulations have the same It can be measured as a higher percent monomer when exposed to stress conditions. In embodiments, the stress condition can be cold storage, high temperature storage, exposure to air, exposure to light, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.

In embodiments, the protein-containing formulations described herein are resistant to an increase in protein particle size, as measured by dynamic light scattering (DLS) analysis. In the DLS analysis used herein, the particle size of protein in protein-containing formulations can be observed as the median particle size. Ideally, the median particle size of the target protein should remain relatively unchanged when subjected to DLS analysis, because the particle size varies depending on the aggregated state (dimers, trimers, oligomers, etc.) or fragments. This is to represent the active ingredient in its monomeric state, as opposed to its enzymatic state. An increase in median particle size may represent aggregated protein. Therefore, the stability of protein-containing formulations can be monitored by the relative change in median particle size over time.

In embodiments, the protein-containing formulations described herein are resistant to forming polydisperse particle size distributions as determined by DLS analysis. In embodiments, the protein-containing formulation can comprise a monodisperse particle size distribution of colloidal protein particles. In embodiments, the ideal stability result is less than 10% change in median particle size compared to the initial median particle size of the formulation. In embodiments, the improved stability of a protein-containing formulation of the invention is measured as a lower percent change in median particle size after a period of time compared to the median particle size of a control formulation containing no excipients. can be In embodiments, the improved stability of the protein-containing formulations of the present invention, compared to the percent change in median particle size for a control formulation containing no excipients after exposure to stress conditions, is such a control formulation. can be measured as the lower percent change in the median particle size when subjected to the same stress conditions. In embodiments, the stress condition can be cold storage, high temperature storage, exposure to air, exposure to light, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles. In embodiments, the improved stability of a protein-containing pharmaceutical therapeutic formulation of the invention is measured by the percent change in polydisperse particle size distribution in a control formulation containing no excipients, compared to It can be measured as a less polydisperse particle size distribution as measured by DLS when subjected to the same stress conditions.

In embodiments, the protein-containing formulations disclosed herein are resistant to particle formation, denaturation, or precipitation as measured by turbidity, light scattering, and/or particle counting analysis. For turbidity, light scattering, or particle count analysis, lower values generally represent fewer suspended particles in the formulation. Increased turbidity, light scattering, or particle counts may indicate that the target protein solution is not stable. Thus, the stability of protein-containing formulations can be monitored by the relative amount of turbidity, light scattering, or particle counting over time. In embodiments, the ideal stability result is to have low and relatively constant turbidity, light scattering, or particle counts. In embodiments, the improved stability of the protein-containing formulations described herein, compared to turbidity, light scattering, or particle counts for control formulations containing no excipients, after a period of time: It can be measured as lower turbidity, lower light scattering, or lower particle counts. In embodiments, the improved stability of the protein-containing formulations described herein is such a control formulation compared to the turbidity, light scattering, or particle count of a control formulation containing no excipients, It can be measured as lower turbidity, lower light scattering, or lower particle count when exposed to the same stress conditions. In embodiments, the stress condition can be cold storage, high temperature storage, exposure to air, exposure to light, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles. In embodiments, protein-containing formulations disclosed herein retain a higher percentage of biological activity compared to control formulations. Biological activity can be monitored via binding assays or via therapeutic effects in mammals.

3. Therapeutic Formulations In one aspect, the formulations and methods disclosed herein provide stable liquid formulations of improved or reduced viscosity comprising therapeutically effective amounts of therapeutic proteins and excipient compounds. do. In embodiments, the formulations may have improved stability while providing acceptable active ingredient concentrations and acceptable viscosities. In embodiments, the formulation provides improved stability when compared to a control formulation. For the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is identical in all respects, on a dry weight basis, to the therapeutic formulation except that it lacks an excipient compound. In embodiments, the formulations are stable under stress conditions of long-term storage, elevated temperatures such as 25-45° C., freeze/thaw conditions, shear or mixing, injection, dilution, bubble exposure, oxygen exposure, light exposure, and freeze-drying. provide sexual improvement. In embodiments, the improved stability of protein-containing formulations is in the form of a lower percentage of soluble aggregates, particles, subvisible particles, or gel formation compared to control formulations.

It is understood that the viscosity of liquid protein formulations can be influenced by a variety of factors, including but not limited to the nature of the protein itself (e.g., enzymes, antibodies, receptors, fusion proteins, etc.), its size, tertiary Included are the original structure, chemical composition, and molecular weight, its concentration in the formulation, the components of the formulation other than protein, the desired pH range, the storage conditions of the formulation, and the method of administering the formulation to a patient. Therapeutic proteins most suitable for use with the excipient compounds described herein are preferably essentially pure, ie free of contaminating proteins. In embodiments, an "essentially pure" therapeutic protein is at least 90%, or preferably at least 95% by weight of the therapeutic protein, all based on the total weight of therapeutic protein and contaminating proteins in the composition. , or more preferably at least 99% by weight. For clarity, proteins added as excipients are not intended to be included in this definition. The therapeutic formulations described herein are pharmaceutical grade formulations, i.e., formulations intended for use in treating mammals, wherein the desired therapeutic effect of the protein active ingredient is achievable and where the formulation is administered. It is a form that does not contain ingredients that are toxic to mammals.

In embodiments, the therapeutic formulation comprises at least 25 mg/mL of protein active ingredient. In other embodiments, the therapeutic formulation comprises at least 100 mg/mL of protein active ingredient. In other embodiments, the therapeutic formulation comprises at least 10 mg/mL of protein active ingredient. In other embodiments, the therapeutic formulation comprises at least 50 mg/mL of protein active ingredient. In other embodiments, the therapeutic formulation comprises at least 200 mg/mL of protein active ingredient. In still other embodiments, the therapeutic formulation solution comprises at least 300 mg/mL of protein active ingredient. Generally, excipient compounds disclosed herein are added to therapeutic formulations in amounts of about 5 to about 300 mg/mL. In embodiments, excipient compounds may be added in amounts of about 10 to about 200 mg/mL. In embodiments, excipient compounds may be added in amounts of about 20 to about 100 mg/mL. In embodiments, excipients may be added in amounts of about 25 to about 75 mg/mL.

Excipient compounds of various molecular weights are selected for their particular advantageous properties when combined with the protein active ingredient in the formulation. Examples of therapeutic formulations containing excipient compounds are provided below. In embodiments, the excipient compound has a molecular weight of less than 5000 Da. In embodiments, the excipient compound has a molecular weight of less than 1000 Da. In embodiments, the excipient compound has a molecular weight of less than 500 Da.

In embodiments, an excipient compound disclosed herein is added to a therapeutic formulation in a viscosity-lowering amount. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 10% when compared to a control formulation. For the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is identical in all respects on a dry weight basis to the therapeutic formulation except that it lacks an excipient compound. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 30% when compared to a control formulation. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 50% when compared to a control formulation. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 70% when compared to a control formulation. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 90% when compared to a control formulation.

In embodiments, the viscosity-lowering amount results in a therapeutic formulation having a viscosity of less than 100 cP. In other embodiments, the therapeutic formulation has a viscosity of less than 50 cP. In other embodiments, the therapeutic formulation has a viscosity of less than 20 cP. In still other embodiments, the therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term "viscosity" refers to kinematic viscosity values as measured by the method disclosed herein.

A therapeutic formulation according to the present disclosure has certain advantageous properties that improve the stability of the formulation. In embodiments, the therapeutic formulation is resistant to shear degradation, phase separation, clouding, oxidation, deamidation, aggregation, precipitation, and denaturation. In embodiments, therapeutic formulations are processed, purified, stored, injected, dosed, filtered, and centrifuged more effectively than control formulations.

In embodiments, therapeutic formulations are administered to patients at high concentrations of therapeutic proteins. In embodiments, the therapeutic formulations are administered to the patient with less infusion and/or less discomfort than experienced with similar formulations lacking the therapeutic excipient. In embodiments, the therapeutic formulation is administered to the patient using a finer gauge needle or less syringe force than would be required for a similar formulation lacking the therapeutic excipient. In embodiments, the therapeutic formulation is administered as a depot injection. In embodiments, the therapeutic formulation extends the half-life of the therapeutic protein in the body.

These features of the therapeutic formulations disclosed herein are useful in the clinical setting, namely the use of small-bore needles for typical IM/SC purposes, and the use of acceptable infusion volumes (eg, 2-3 mL). and, under these conditions, an effective amount of the formulation can be administered in a single injection at a single injection site, in situations where consent for intramuscular injection is obtained from the patient. allows administration of such formulations. In contrast, SC/IM injection of conventional formulations is preferred in clinical settings because injection of equivalent doses of therapeutic proteins using conventional formulation techniques is limited by the higher viscosity of conventional formulations. isn't it.

A therapeutic formulation according to this disclosure may possess certain advantageous properties consistent with improved stability. In embodiments, the therapeutic formulation is resistant to shear degradation, phase separation, clouding, precipitation, oxidation, deamidation, aggregation, and/or denaturation. In embodiments, therapeutic formulations are processed, purified, stored, injected, dosed, filtered, and/or centrifuged more effectively than control formulations.

In embodiments, the therapeutic formulations disclosed herein are resistant to monomer loss, as measured by size exclusion chromatography (SEC) analysis. In SEC analysis, the main analyte peak is generally associated with the active ingredient of the formulation, such as a therapeutic protein, and this main peak of the active ingredient is called the monomer peak. The monomer peak represents the amount of active ingredient in the monomeric state as opposed to aggregates (dimers, trimers, oligomers, etc.). Highly concentrated solutions of therapeutic proteins formulated with excipient compounds described herein can be administered to patients using syringes or pre-filled syringes. Therefore, the stability of therapeutic formulations can be monitored by the relative amounts of monomers over time. In embodiments, the improved stability of a therapeutic formulation disclosed herein is a higher percent monomer after a period of time compared to the percent monomer in a control formulation containing no excipients. It can be measured as a mer. In embodiments, the improved stability of a therapeutic formulation disclosed herein is compared to percent monomer in a control formulation without excipients (after exposure to stress conditions) after exposure to stress. can be measured as a higher percent monomer. In embodiments, the stress condition 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 embodiments, the therapeutic formulations of the invention are resistant to protein particle size increases, as measured by dynamic light scattering (DLS) analysis. In DLS analysis, the therapeutic protein particle size can be observed as the median particle size. Ideally, the median particle size of therapeutic proteins should remain relatively unchanged. Therefore, an increase in median particle size may represent aggregated protein. Therefore, the stability of therapeutic formulations can be monitored by the relative change in median particle size over time. In embodiments, the therapeutic formulations disclosed herein are resistant to forming polydisperse particle size distributions as determined by dynamic light scattering (DLS) analysis. In embodiments, the improved stability of a therapeutic formulation of the present invention is measured as a lower percent change in median particle size after a period of time compared to the median particle size of a control formulation containing no excipients. can be In embodiments, the improved stability of the therapeutic formulations disclosed herein is a median It can be measured as a lower percent change in particle size. In other words, in embodiments, improved stability prevents an increase in particle size and is measured by light scattering. In embodiments, the stress condition 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 embodiments, the improved stability of the therapeutic formulations disclosed herein is a less polydisperse particle size distribution as measured by DLS compared to a polydisperse particle size distribution in the excipient-free control formulation. It can be measured as a dispersed particle size distribution.

In embodiments, the therapeutic formulations disclosed herein are resistant to sedimentation as measured by turbidity, light scattering, or particle counting analysis. In embodiments, the improved stability of the therapeutic formulations disclosed herein is compared to turbidity, light scatter, or particle counts for control formulations containing no excipients after a period of time. , lower turbidity, lower light scattering, or lower particle counts. In embodiments, the improved stability of the therapeutic formulations disclosed herein is compared to turbidity, light scattering, or particle counts of control formulations containing no excipients after exposure to stress conditions. , lower light scattering, or lower particle counts. In embodiments, the stress condition 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 embodiments, the therapeutic excipient has antioxidant properties that stabilize the therapeutic protein against oxidative damage, thereby improving its stability. In embodiments, the therapeutic formulations are stored at ambient temperature or under refrigerated conditions for extended periods of time without appreciable loss of potency of the therapeutic protein. In embodiments, the therapeutic formulation is dried for storage until it is needed and then reconstituted with a suitable solvent such as water. Advantageously, formulations prepared as described herein can be stable for extended periods of time, from months to years. If an exceptionally long period of storage is desired, the formulation can be stored in the freezer (later reactivated) without concern for protein denaturation. In embodiments, formulations may be prepared for long-term storage that does not require refrigeration.

In embodiments, the excipient compounds disclosed herein are added to therapeutic formulations in stability-improving amounts. In embodiments, a stability-improving amount is an amount of excipient compound that reduces the viscosity of the formulation by at least 10% when compared to a control formulation. For the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar on a weight basis to the therapeutic formulation, but lacking an excipient compound. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 30% when compared to a control formulation. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 50% when compared to a control formulation. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 70% when compared to a control formulation. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 90% when compared to a control formulation.

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

4. Non-Therapeutic Formulations In one aspect, the formulations and methods disclosed herein provide stable liquid formulations of improved or reduced viscosity comprising effective amounts of non-therapeutic proteins and excipient compounds. In embodiments, the formulations have improved stability while providing acceptable concentrations of active ingredients and acceptable viscosities. In embodiments, the formulation provides improved stability when compared to a control formulation. For the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is identical in all respects on a dry weight basis to the non-therapeutic formulation except that it lacks an excipient compound.

It is understood that the viscosity of a liquid protein formulation can be influenced by a variety of factors, including but not limited to the properties of the protein itself (e.g., enzymes, structural proteins, degree of hydrolysis, etc.), its size, tertiary Included are the original structure, chemical composition, and molecular weight, its concentration in the formulation, the components of the formulation other than protein, the desired pH range, and the storage conditions of the formulation.

In embodiments, the non-therapeutic formulation comprises at least 25 mg/mL of protein active ingredient. In other embodiments, the non-therapeutic formulation comprises at least 100 mg/mL of protein active ingredient. In other embodiments, the non-therapeutic formulation comprises at least 200 mg/mL of protein active ingredient. In still other embodiments, the non-therapeutic formulation solution comprises at least 300 mg/mL protein active ingredient. Generally, excipient compounds disclosed herein are added to non-therapeutic formulations in amounts of about 5 to about 300 mg/mL. In embodiments, excipients are added in amounts of about 10 to about 200 mg/mL. In embodiments, excipients are added in amounts of about 20 to about 100 mg/mL. In embodiments, excipients are added in amounts of about 25 to about 75 mg/mL.

Excipient compounds of various molecular weights are selected for their particular advantageous properties when combined with the protein active ingredient in the formulation. Examples of non-therapeutic formulations containing excipient compounds are provided below. In embodiments, the excipient compound has a molecular weight of less than 5000 Da. In embodiments, the excipient compound has a molecular weight of less than 1000 Da. In embodiments, the excipient compound has a molecular weight of less than 500 Da.

In embodiments, an excipient compound disclosed herein is added to a non-therapeutic formulation in a viscosity-lowering amount. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 10% when compared to a control formulation. For the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is identical in all respects on a dry weight basis to the therapeutic formulation except that it lacks an excipient compound. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 30% when compared to a control formulation. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 50% when compared to a control formulation. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 70% when compared to a control formulation. In embodiments, a viscosity-lowering amount is an amount of an excipient compound that reduces the viscosity of a formulation by at least 90% when compared to a control formulation.

In embodiments, the viscosity-lowering amount results in a non-therapeutic formulation having a viscosity of less than 100 cP. In other embodiments, non-therapeutic formulations have a viscosity of less than 50 cP. In other embodiments, non-therapeutic formulations have a viscosity of less than 20 cP. In still other embodiments, the non-therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term "viscosity" refers to kinematic viscosity values.

Non-therapeutic formulations according to the present disclosure can have certain advantageous properties that improve the stability of the formulation. In embodiments, non-therapeutic formulations are resistant to shear degradation, phase separation, cloudiness, oxidation, deamidation, aggregation, precipitation, and denaturation. In embodiments, therapeutic formulations can be processed, purified, stored, pumped, filtered, and centrifuged more effectively than control formulations.

In embodiments, the non-therapeutic excipient has antioxidant properties that stabilize the non-therapeutic protein against oxidative damage, thereby improving its stability. In embodiments, the non-therapeutic formulations are stored at ambient temperature or under refrigerated conditions for extended periods of time without appreciable loss of potency of the non-therapeutic protein. In embodiments, the non-therapeutic formulation can be dried for storage until needed and then reconstituted with a suitable solvent such as water. Advantageously, formulations prepared as described herein are stable for extended periods of time, from months to years. If an exceptionally long period of storage is desired, the formulation can be stored in the freezer (later reactivated) without concern for protein denaturation. In embodiments, formulations are prepared for long-term storage that does not require refrigeration.

In embodiments, the excipient compounds disclosed herein are added to non-therapeutic formulations in stability-improving amounts. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 10% when compared to a control formulation. For the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar on a weight basis to the therapeutic formulation, but lacking an excipient compound. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 30% when compared to a control formulation. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 50% when compared to a control formulation. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 70% when compared to a control formulation. In embodiments, a stability-improving amount is an amount of an excipient compound that reduces degradation of a formulation by at least 90% when compared to a control formulation.

Methods for preparing non-therapeutic formulations containing the excipient compounds disclosed herein will be familiar to those skilled in the art. For example, an excipient compound can be added to the formulation before or after the non-therapeutic protein is added to the solution. A non-therapeutic formulation can be produced at a first (lower) concentration and then filtered or centrifuged to produce a second (higher) concentration. Non-therapeutic formulations can be made with one or more excipient compounds, including chaotropes, kosmotropes, hydrotropes, and salts. Non-therapeutic formulations can be made with one or more excipient compounds using techniques such as encapsulation, dispersion, liposomes, vesicle formation, and the like. Other additives, including preservatives, surfactants, stabilizers, etc., can be introduced into the non-therapeutic formulations during their manufacture.

5. Excipient Compounds A number of excipient compounds are described herein, each suitable for use with one or more therapeutic or non-therapeutic proteins, each containing high concentrations of protein The formulation can be configured as follows. Some of the categories of excipient compounds described 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, (7) diones and sulfones, (8) zwitterionic excipients, and (9) crowding agents containing hydrogen bonding elements. Without being bound by theory, the excipient compounds described herein are specific fragments, sequences, Considered to be associated with a structure or area. The association of these excipient compounds with therapeutic or non-therapeutic proteins masks interactions between proteins, allowing the proteins to be formulated at high concentrations without causing excessive solution viscosity. In embodiments, excipient compounds can provide more stable protein-protein interactions. Protein-protein interactions can be measured by the protein diffusion parameter kD, the second virial coefficient of osmotic pressure B22, or other techniques well known to those of skill in the art.

Excipient compounds may advantageously be water soluble and thus suitable for use with aqueous vehicles. In embodiments, the excipient compound has a water solubility of greater than 1 mg/mL. In embodiments, the excipient compound has a water solubility greater than 10 mg/mL. In embodiments, the excipient compound has a water solubility greater than 100 mg/mL. In embodiments, the excipient compound has a water solubility greater than 500 mg/mL. In embodiments, a cosolute or hydrotrope can be added in combination with the excipient compound to increase the solubility of the excipient compound. For example, certain excipients may have limited solubility in aqueous solutions containing therapeutic proteins, and cryopreservation conditions may further reduce this solubility. Co-solutes or hydrotropes can be added to increase the solubility of excipients in solution under cryogenic or ambient room or elevated temperature conditions. Examples of co-solutes and hydrotropes include benzoate, benzyl alcohol, phenylalanine, nicotinamide, proline, procaine, 2,5-dihydroxybenzoate, tyramine, and saccharin. Advantageously, for therapeutic proteins, excipient compounds can be obtained from materials that are both biologically acceptable and non-immunogenic, and are therefore suitable for pharmaceutical use. In therapeutic embodiments, an excipient compound can be metabolized in the body to yield biologically compatible, non-immunogenic byproducts.

a. Excipient Compound Category 1: Hindered Amines Solutions of therapeutic or non-therapeutic proteins can be formulated with hindered amine small molecules as excipient compounds. As used herein, the term "hindered amine" refers to small molecules that contain at least one bulky or sterically hindering group consistent with the following examples. Hindered amines can be used in the free base form, in the protonated form, or a combination of the two. In protonated form, hindered amines have anions such as chloride, hydroxide, bromide, iodide, fluoride, acetate, formate, phosphate, sulfate, or carboxylate. can associate with a sex counterion. Hindered amine compounds useful as excipient compounds can contain secondary amine, tertiary amine, quaternary ammonium, pyridinium, pyrrolidone, pyrrolidine, piperidine, morpholine, or guanidinium groups, thus excipient compounds. has a cationic charge in aqueous solution at neutral pH. Hindered amine compounds also contain at least one bulky or sterically hindering group, such as a cyclic aromatic, cycloaliphatic, cyclohexyl, or alkyl group. In embodiments, the sterically hindering group can itself be an amine group such as a dialkylamine, trialkylamine, guanidinium, pyridinium, or quaternary ammonium group. 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 through cationic pi interactions. In embodiments, since the cationic groups of hindered amines may have an affinity for the electron-rich pi-structures of aromatic amino acid residues in proteins, they may be able to reduce such reduces the propensity of the shielded proteins to associate and aggregate.

In embodiments, the hindered amine excipient compound is an imidazole, imidazoline, or imidazolidine group, or salts thereof, such as imidazole, 1-methylimidazole, 4-methylimidazole, 1-hexyl-3-methylimidazolium chloride, 1 , 3-dimethyl-2-imidazolidinone, histamine, 4-methylhistamine, alpha-methylhistamine, betahistine, beta-alanine, 2-methyl-2-imidazoline, 1-butyl-3-methylimidazolium chloride, uric acid, It has a chemical structure that includes potassium urate, betasol, carnosine, aspartame, saccharin, acesulfame potassium, xanthine, theophylline, theobromine, caffeine, and anserine. In embodiments, the hindered amine excipient compound is dimethylethanolamine, dimethylaminopropylamine, triethanolamine, dimethylbenzylamine, dimethylcyclohexylamine, diethylcyclohexylamine, dicyclohexylmethylamine, hexamethylenebiguanide, poly(hexamethylenebiguanide) , imidazole, lysine, methylglycine, sarcosine, dimethylglycine, agmatine, diazabicyclo[2.2.2]octane, folinic acid sodium salt, folinic acid calcium salt, tetramethylethylenediamine, N,N-dimethylethanolamine, ethanolamine phosphate , glucosamine, choline chloride, phosphocholine, niacinamide, isonicotinamide, N,N-diethylnicotinamide, nicotinic acid sodium salt, isonicotinate, tyramine, 3-aminopyridine, 2,4,6-trimethylpyridine, 3 - pyridinemethanol, nicotinamide adenine dinucleotide, biotin, morpholine, N-methylpyrrolidone, 2-pyrrolidinone, dipyridamole, procaine, lidocaine, dicyandiamide-taurine adduct, 2-pyridylethylamine, 6-hydroxypyridine-2-carboxylic acid, selected from the group consisting of dicyandiamide-benzylamine adducts, dicyandiamide-alkylamine adducts, dicyandiamide-cycloalkylamine adducts, and dicyandiamide-aminomethanephosphonic acid adducts. In embodiments, the hindered amine excipient compound is 1-(1-adamantyl)ethylamine, 1-aminobenzotriazole, 2-dimethylaminoethanol, 2-methyl-2-imidazoline, 2-methylimidazole, 3-aminobenzamide, 3-indoleacetic acid, 4-aminopyridine, 6-amino-1,3-dimethyluracil, acetylcholine, agmatine sulfate, benzalkonium chloride, ethyl 3-aminobenzoate, sulfacetamide, butyl anthranilate, aminohippuric acid, benzamide oxime , benzethonium chloride, benzylamine, berberine chloride, castanospermine, clemizole, cycloserine, phenylserine, DL-3-phenylserine, cysteamine, cytidine, diethanolamine, diphenhydramine, DL-norepinephrine, dopamine, emtricitabine, ethanolamine, guanfacine, isonicotinamide, lithium chloride, meglumine, methylcytidine, myristyl gamma picolinium chloride, niacinamide, phenylethylamine, polyethyleneimine, pyridoxine, rasagiline mesylate, serotonin, synephrine, neamine, spermine, spermidine, 1,3-diaminopropane, Adenosine, chloroquine phosphate, cystamine, pyridylethylamine, tetramethylethylenediamine, tryptamine, tyramine, 1-methylimidazole, spectinomycin, cyclohexanemethylamine, N,N-dimethylphenethylamine, phenethylamine, tetraethylammonium, tetramethylammonium acetate, dicyclomine , hordenine, methylaminoethylpyridine, nicotinamide riboside, 1-butylimidazole, 1-hexylimidazole, 1-methylimidazole, 2-ethylimidazole, 2-N-butylimidazole, 2-methylimidazole, 1-dodecylimidazole, other imidazoles alkylated with a C ₁ -C ₁₂ hydrocarbon chain at the 1 or 2 position, pridinol methanesulfonic acid, hemin, N,N-dimethylphenethylamine, voglibose, N-ethyl-L-glutamine, nicotine, selected from the group consisting of piperazine, demeclocycline, and salts thereof; In embodiments, the hindered amine excipient compound is phenethylamine, diphenhydramine, N-methylphenethylamine, N,N-dimethylphenethylamine, β,3-dihydroxyphenethylamine, β,3-dihydroxy-N-methylphenethylamine, 3-hydroxyphenethylamine, It can have phenethylamine functional groups such as 4-hydroxyphenethylamine, tyrosinol, tyramine, N-methyltyramine, and hordenine. Preferably, the phenethylamine-containing structure is a non-psychotropic compound.

Suitable salts of hindered amine structures can be chlorides, bromides, acetates, citrates, sulfates and phosphates. In embodiments, hindered amine compounds consistent with the present disclosure are formulated as protonated ammonium salts. In embodiments, hindered amine compounds consistent with the present disclosure are formulated as salts with inorganic or organic anions as counterions.

In embodiments, high concentration solutions of therapeutic or non-therapeutic proteins are formulated with caffeine in combination with benzoic acid, hydroxybenzoic acid, or benzenesulfonic acid as excipient compounds. In embodiments, the hindered amine excipient compound is metabolized in the body to produce biologically compatible byproducts. 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 from about 10 mg/mL to about 200 mg/mL. In yet 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, this hindered amine category of viscosity-lowering excipients may include methylxanthines such as caffeine and theophylline, but their use is typically limited due to their low water solubility. In some applications, it may be advantageous to have a more concentrated solution of these viscosity-lowering excipients despite their low water solubility. For example, in processing, it may be advantageous to have a concentrated excipient solution that can be added to a concentrated protein solution, thereby adding excipients without diluting the protein below the desired final concentration. can be added. In other cases, additional viscosity-lowering excipients may be required to achieve the desired viscosity reduction, stability, tonicity, etc. of the final protein formulation despite its low water solubility. be. In embodiments, the high concentration excipient solution is used as a viscosity-lowering excipient (i) at a concentration 1.5-50 times higher than the effective viscosity-lowering amount, or (ii) as reported in the literature At concentrations 1.5 to 50 times higher than the solubility in pure water at 298 K (reported, for example, in The Merck Index; Royal Society of Chemistry; Fifteenth Edition, (April 30, 2013)), viscosity-reducing excipients It can be formulated as an agent.

Certain co-solutes can significantly increase the solubility limits of these low-solubility, viscosity-reducing excipients, allowing excipient solutions at concentrations several times higher than solubility values reported in the literature. have been discovered. These co-solutes can be grouped into the general category of hydrotropes. The co-solutes found to provide the greatest improvement in solubility for this application are generally very soluble in water (>0.25 M) at ambient temperature and physiological pH and carry either a pyridine or benzene ring. contained. Examples of compounds useful as cosolutes include aniline HCl, isoniacinamide, niacinamide, n-methyltyramine HCl, phenol, procaine HCl, resorcinol, saccharin calcium salt, saccharin sodium salt, sodium aminobenzoate, sodium benzoate, Sodium parahydroxybenzoate, sodium metahydroxybenzoate, sodium 2,5-dihydroxybenzoate, sodium salicylate, sodium sulfanilate, sodium parahydroxybenzenesulfonate, synephrine, and tyramine HCl.

In embodiments, certain hindered amine excipient compounds may have other pharmacological properties. As an example, xanthines are a category of hindered amines that generally have independent pharmacological properties, including stimulant and bronchodilator properties when absorbed systemically. 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. Locally injectable anesthetic compounds are hindered amines with a ternary molecular structure of (a) a lipophilic aromatic ring, (b) an intermediate ester or amide linkage, and (c) a secondary or tertiary amine. This category of hindered amines is understood to block nerve conduction by inhibiting the influx of sodium ions, thereby inducing local anesthesia. The lipophilic aromatic ring of the local anesthetic compound may be formed from carbon atoms (eg, benzene ring) or may contain heteroatoms (eg, thiophene ring). Representative local injectable anesthetics include amilocaine, articaine, bupivacaine, butacaine, butanilicaine, chloroprocaine, cocaine, cyclomethicaine, dimethocaine, editocaine, hexylcaine, isobucaine, levobupivacaine, lidocaine, metabutetamine, Metabutoxycaine, mepivacaine, meprilcaine, propoxycaine, prilocaine, procaine, pipelocaine, tetracaine, trimecaine and the like. Locally injectable anesthetic compounds offer multiple advantages in protein therapeutic formulations, including reduced viscosity, increased stability, and reduced pain on 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 that has independent pharmacological properties is present in an amount that has no pharmacological effect and/or is not therapeutically effective. In other embodiments, excipient compounds that have independent pharmacological properties are present in amounts that are pharmacologically effective and/or therapeutically effective. In certain embodiments, the hindered amine with independent pharmacological properties is used in combination with another excipient compound selected to reduce formulation viscosity, wherein the hindered amine with independent pharmacological properties is It is used to benefit from its pharmacological activity. For example, local injectable anesthetic compounds may be used to reduce the viscosity of the formulation and reduce pain upon injection of the formulation. Relief of injection pain may be caused by anesthetic properties. Also, if the excipients reduce the viscosity, the force of injection should be reduced. Alternatively, a locally injectable anesthetic compound can be used to provide the desirable pharmacological benefit of reducing local sensation during injection of the formulation while in combination with another excipient compound that reduces the viscosity of the formulation.

b. Excipient Compound Category 2: Anionic Aromatic Solutions of therapeutic or non-therapeutic proteins can be formulated with anionic aromatic small molecule compounds as excipient compounds. Anionic aromatic excipient compounds can contain aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, phenol, hydroxyaryl, heteroaromatic groups, or fused aromatic groups. Anionic aromatic excipient compounds can also contain anionic functional groups such as carboxylate, oxide, phenoxide, sulfonate, sulfate, phosphonate, phosphate, or sulfide. Anionic aromatic excipients may be described as acids, sodium salts, or otherwise, but it is understood that the excipients may be used in various salt forms. Without wishing to be bound by theory, anionic aromatic excipient compounds are believed to be bulky, sterically hindered molecules that are capable of associating with the cationic moieties of proteins, thus reducing the Moieties can be shielded, thereby reducing interactions between protein molecules that make protein-containing formulations viscous or cause stability problems.

In embodiments, examples of anionic aromatic excipient compounds include salicylic acid, aminosalicylic acid, hydroxybenzoic acid, aminobenzoic acid, para-aminobenzoic acid, benzenesulfonic acid, hydroxybenzenesulfonic acid, 4-phenylbutyric acid, naphthalenesulfone. acid, 1,5-naphthalenedisulfonic acid, 2,6-naphthalenedisulfonic acid, 2,7-naphthalenedisulfonic acid, hydroquinonesulfonic acid, sulfanilic acid, vanillic acid, homovanillic acid, vanillin, vanillin-taurine adduct, aminophenol, Anthranilic acid, cinnamic acid, menadione sodium bisulfite, 4-hydroxy-3-methoxycinnamic acid, caffeic acid, chlorogenic acid, gentisic acid, coumaric acid, adenosine monophosphate, indoleacetic acid, potassium urate, furandicarboxylic acid , furan-2-acrylic acid, 2-furanpropionic acid, sodium phenylpyruvate, sodium hydroxyphenylpyruvate, trimethoxybenzoic acid, dihydroxybenzoic acid, ferrocenecarboxylic acid, trihydroxybenzoic acid, pyrogallol, benzoic acid, and the aforementioned acid salts and the like. In embodiments, the anionic aromatic excipient compound is formulated in the form of an ionized salt. In embodiments, the anionic aromatic compound is formulated as a salt of a hindered amine such as dimethylcyclohexylammonium hydroxybenzoate. In embodiments, the anionic aromatic excipient compounds are formulated with various counterions such as organic cations. In embodiments, high concentration solutions of therapeutic or non-therapeutic proteins are formulated with an anionic aromatic excipient compound and caffeine. In embodiments, the anionic aromatic excipient compound is metabolized in the body to produce biologically compatible byproducts.

In embodiments, examples of aromatic excipient compounds include phenols and polyphenols. As used herein, the term "phenol" refers to an organic molecule consisting of at least one aromatic or fused aromatic group bonded to at least one hydroxyl group, and the term "polyphenol" refers to two Refers to an organic molecule consisting of one or more phenolic groups. Such excipients may be advantageous under certain circumstances, for example, when used in formulations containing highly concentrated solutions of therapeutic or non-therapeutic PEGylated proteins to reduce solution viscosity. Non-limiting examples of phenols include benzenediol resorcinol (1,3-benzenediol), catechol (1,2-benzenediol) and hydroquinone (1,4-benzenediol), benzenetriol hydroxyquinol (1,2 ,4-benzenetriol), pyrogallol, (1,2,3-benzenetriol), and phloroglucinol (1,3,5-benzenetriol), benzenetetrol 1,2,3,4-benzenetetrol and 1,2,3,5-Benzenetetrol, and benzenepentaol and benzenehexaol. Non-limiting examples of polyphenols include tannic acid, ellagic acid, epigallocatechin gallate, catechins, tannins, ellagitannins, and gallotannins. More generally, phenolic and polyphenolic compounds include, but are not limited to, flavonoids, lignans, phenolic acids, and stilbenes. Flavonoid compounds include, but are not limited to, anthocyanins, chalcone, dihydrochalcones, dihydroflavanols, flavanols, flavanones, flavones, flavonols, and isoflavonoids. Phenolic acids include, but are not limited to, hydroxybenzoic acid, hydroxycinnamic acid, hydroxyphenylacetic acid, hydroxyphenylpropanoic acid, and hydroxyphenylpentanoic acid. Other polyphenolic compounds include alkylmethoxyphenols, alkylphenols, curcuminoids, hydroxybenzaldehydes, hydroxybenzoketones, hydroxycinnamaldehydes, hydroxycoumarins, hydroxyphenylpropenes, methoxyphenols, naphthoquinones, hydroquinones, phenolterpenes, resveratrol, and tyrosol. including but not limited to: In embodiments, the polyphenol is tannic acid. In embodiments, the phenol is gallic acid. In embodiments, the phenol is pyrogallol. In embodiments, the phenol is resorcinol. Without wishing to be bound by theory, the hydroxyl groups of phenolic compounds such as gallic acid, pyrogallol, and resorcinol form hydrogen bonds with the ether oxygen atoms in the backbone of the PEG chain, thus underlying the solution structure of PEG. solution viscosity is reduced by forming a phenol/PEG complex that converts to Polyphenolic compounds, such as tannic acid, derive their viscosity-lowering properties from their respective phenolic building blocks, such as gallic acid, pyrogallol, and resorcinol. The specific structure of the phenolic groups within the polyphenolic compounds can lead to complex behavior, and the viscosity reduction achieved by the addition of phenols is enhanced by the addition of smaller amounts of the respective polyphenols.

c. Excipient Compound Category 3: Functionalized Amino Acids Solutions of therapeutic or non-therapeutic proteins can be formulated with one or more functionalized amino acids, either a single functionalized amino acid or one or Oligopeptides containing multiple functionalized amino acids can be used as excipient compounds. In embodiments, functionalized amino acid compounds include molecules that can be hydrolyzed or metabolized to yield amino acids (“amino acid precursors”). In embodiments, functionalized amino acids can include aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromatic groups, or fused aromatic groups. In embodiments, functionalized amino acid compounds are methylated, ethylated, propylated, butylated, benzylated, cycloalkylated, glycerylated, hydroxyethylated, hydroxypropylated, PEGylated, and esterified such as PPG esters. It can contain amino acids. In embodiments, 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 embodiments, the functionalized amino acid compound is a charged ionic compound in aqueous solution at neutral pH. For example, single amino acids can be derivatized by forming esters such as acetates and benzoates, and the hydrolysis products are acetic acid or benzoic acid, both natural sources, as well as amino acids. is. In embodiments, the functionalized amino acid excipient compound is metabolized in the body to produce biologically compatible by-products.

d. Excipient Compound Category 4: Oligopeptides Solutions of therapeutic or non-therapeutic proteins can be formulated using oligopeptides as excipient compounds. In embodiments, the oligopeptide is designed such that its structure has a charged portion and a bulky portion. In embodiments, the oligopeptide consists of 2-10 peptide subunits. Oligopeptides can be bifunctional, eg, cationic amino acids linked to non-polar ones, or anionic amino acids linked to non-polar ones. In embodiments, the oligopeptide consists of 2-5 peptide subunits. In embodiments, the oligopeptides are homopeptides such as polyglutamic acid, polyaspartic acid, polylysine, polyarginine, and polyhistidine. In embodiments, the oligopeptide has a net cationic charge. In other embodiments, the oligopeptide is a heteropeptide such as Trp2Lys3. In embodiments, an oligopeptide can have an alternating structure, such as an ABA repeat pattern. In embodiments, the oligopeptide contains both anionic and cationic amino acids, such as Arg-Glu, Lys-Glu, His-Glu, Arg-Asp, Lys-Asp, His-Asp, Glu-Arg, Glu-Lys , Glu-His, Asp-Arg, Asp-Lys, Asp-His. Without wishing to be bound by theory, oligopeptides have structures that allow them to bind proteins in such a way as to reduce intermolecular interactions that lead to high viscosity solutions and stability problems. For example, oligopeptide-protein associations are interactions between charges, leaving amino acids somewhat non-polar, disrupting the hydrogen bonding of the hydrated layer around the protein and thus reducing viscosity or stabilizing. improve sexuality. In some embodiments, the oligopeptide excipient is present in the composition at a concentration of about 50 mg/mL or less.

e. Excipient Compound Category 5: Short Chain Organic Acids Solutions of therapeutic or non-therapeutic proteins can be formulated using short chain organic acids as excipient compounds. As used herein, the term "short chain organic acid" refers to _C2 - _C6 organic acid compounds and their salts, esters, amides, or lactones. This category includes saturated and unsaturated carboxylic acids, hydroxy-functionalized carboxylic acids, amides, and linear, branched or cyclic carboxylic acids. In embodiments, the acidic group of the short chain organic acid is carboxylic acid, sulfonic acid, phosphonic acid, or salts thereof.

Solutions of therapeutic or non-therapeutic proteins are formulated using, for example, short-chain organic acids in the acid or salt form of sorbic acid, valeric acid, propionic acid, caproic acid, and ascorbic acid as excipient compounds. can do. Examples of excipient compounds in this category include potassium sorbate, calcium gluconate, glucuronic acid, calcium lactate, 2-hydroxylactic acid, sodium glycolate, potassium glycolate, ammonium glycolate, sodium valproate, taurine, aceto Included are hydroxamic acid, acetone sodium bisulfite adduct, acetylhydroxyproline, calcium propionate, magnesium propionate, sodium propionate, sodium ascorbate, and salts thereof.

f. Excipient Compound Category 6: Low Molecular Weight Polyacids Solutions of therapeutic or non-therapeutic proteins or PEGylated proteins are formulated with specific excipient compounds that allow for reduced solution viscosity or improved stability. and such excipient compounds are low molecular weight polyacids. Low molecular weight polyacids may include organic polyacids or inorganic polyacids. These low molecular weight polyacid excipients can also be used in combination with other excipients.

In embodiments, organic polyacids may be structured as low molecular weight aliphatic polyacids. As used herein, the term "low molecular weight aliphatic polyacid" refers to an organic aliphatic polyacid having a molecular weight of less than about 1500 Da and having at least two acidic groups, the acidic groups understood to be part. Non-limiting examples of acidic groups include carboxylate, phosphonate, phosphate, sulfonate, sulfate, nitrate, nitrite. The acidic groups of low molecular weight aliphatic polyacids can be in the form of anionic salts such as carboxylates, phosphonates, phosphates, sulfonates, sulfates, nitrates, and nitrites, and their counterions are sodium, potassium, lithium, and It can be an ammonium ion. Specific examples of low molecular weight aliphatic polyacids useful for interaction with the PEGylated proteins described herein include oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, succinic acid, maleic acid. acid, tartaric acid, glutaric acid, malonic acid, itaconic acid, methylmalonic acid, azelaic acid, citric acid, 3,6,9-trioxaundecanedioic acid, ethylenediaminetetraacetic acid (EDTA), aspartic acid, pyrrolidonecarboxylic acid, pyrro Glutamic acid, glutamic acid, alendronic acid, medronic acid, etidronic acid and salts thereof.

In other embodiments, the low molecular weight polyacid is an inorganic polyacid. Further examples of low molecular weight polyacids in the form of their anionic salts are phosphate (PO ₄ ^3- ), hydrogen phosphate (HPO ₄ ^2- ), dihydrogen phosphate (H ₂ PO ₄ ^- ), sulfate, bisulfate (HSO ₄ ^- ), pyrophosphate (P ₂ O ₇ ^4- ), hexametaphosphate, borate, carbonate (CO ₃ ^2- ), bicarbonate (HCO ₃ ^- ). Counterions for anionic salts can be Na, Li, K, or ammonium ions.

In embodiments, the low molecular weight aliphatic polyacid can also be an alpha hydroxy acid with hydroxyl groups adjacent to the first acidic group, such as glycolic, lactic, and gluconic acids and salts thereof. In embodiments, the low molecular weight aliphatic polyacids are oligomeric forms with three or more acidic groups, such as polyacrylic acids, polyphosphates, polypeptides and salts thereof. In some embodiments, the low molecular weight aliphatic polyacid excipient is present in the composition at a concentration of about 50 mg/mL or less.

g. Excipient Compound Category 7: Diones and Sulfones Effective viscosity-lowering or stabilizing excipients are soluble in pure water to at least 1 g/L at 298 K, have a net neutral charge at pH 7, and are sulfones. , sulfonamide, or dione functional groups. Preferably the molecule has a molecular weight of less than 1000 g/mol, more preferably less than 500 g/mol. Diones and sulfones that are effective in reducing viscosity and/or improving stability have multiple double bonds, are water soluble, have no net or anionic charge at pH 7, and strong hydrogen Not a binding donor. Without wishing to be bound by theory, the properties of double bonds allow weak pi-stacking interactions with proteins. In embodiments, with proteins that develop high viscosities at high protein concentrations, and only at high concentrations, charged excipients are not effective because electrostatic interactions are long-range interactions. The surfaces of solvated proteins are predominantly hydrophilic, making them water-soluble. Hydrophobic regions of proteins are generally shielded within the three-dimensional structure, but structures are constantly evolving, unfolding and refolding (sometimes called "breathing"), and the hydrophobic regions of neighboring proteins may come into contact with each other, leading to aggregation due to hydrophobic interactions. The pi-stacking function of dione and sulfone excipients can mask hydrophobic patches that may be exposed during such "breathing". Another important role of excipients is to disrupt hydrophobic interactions and hydrogen bonds between adjacent proteins, effectively reducing solution viscosity. Dione and sulfone compounds that fit this description include dimethylsulfone, ethylmethylsulfone, ethylmethylsulfonylacetate, ethylisopropylsulfone, sodium cyclamate, bis(methylsulfonyl)methane, methanesulfonamide, methioninesulfone, 1,2-cyclo Included are pentanedione, 1,3-cyclopentanedione, 1,4-cyclopentanedione, and butane-2,3-dione.

h. Excipient Compound Category 8: Zwitterionic Excipients Solutions of therapeutic or non-therapeutic proteins contain certain zwitterionic compounds as excipients to improve stability or reduce viscosity. can be formulated using As used herein, the term "zwitterion" refers to a compound having a cationic and anionic charge portion. In embodiments, the zwitterionic excipient compound is an amine oxide. In embodiments, the opposite charges are separated from each other by 2-8 chemical bonds. In embodiments, the zwitterionic excipient compound can be a small molecule, such as those having a molecular weight of about 50 to about 500 g/mole, or a small molecule, such as those having a molecular weight of about 500 to about 2000 g/mole. It can be an intermediate molecular weight molecule or it can be a high molecular weight molecule such as a polymer having a molecular weight of about 2000 to about 100,000 g/mole.

Examples of zwitterionic excipient compounds include (3-carboxypropyl)trimethylammonium chloride, 1-aminocyclohexanecarboxylic acid, homocycloleucine, 1-methyl-4-imidazoleacetic acid, 3-(1-pyridinio) -1-propanesulfonate, 4-aminobenzoic acid, alendronic acid, aminoethylsulfonic acid, aminohippuric acid, aspartame, aminotris(methylenephosphonic acid) (ATMP), calcobutrol, carteridol, cocamidopropyl betaine, Cocamidopropyl hydroxysultaine, creatine, cytidine monophosphate, diaminopimelic acid, diethylenetriaminepentaacetic acid, dimethylphenylalanine, methylglycine, sarcosine, dimethylglycine, zwitterionic dipeptides (eg: Arg-Glu, Lys-Glu, His- Glu, Arg-Asp, Lys-Asp, His-Asp, Glu-Arg, Glu-Lys, Glu-His, Asp-Arg, Asp-Lys, Asp-His), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), dipalmitoylphosphatidylcholine, ectoine, ethylenediaminetetra(methylenephosphonic acid) (EDTMP), folic acid benzoic acid mixture, folic acid niacinamide mixture, gelatin, hydroxyproline, iminodiacetic acid, isogubacin, lecithin, myristamine oxide, nicotinamide adenine dinucleotide ( NAD), N-methylaspartic acid, N-methylproline, N-trimethyllysine, ornithine, oxolinic acid, risedronic acid, allylcysteine, S-allyl-L-cysteine, somapacitan, taurine, theanine, trigonelline, vigabatrin, ectoine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, o-octylphosphorylcholine, nicotinamide mononucleotide, triglycine, tetraglycine, β-guanidinopropionic acid, 5-aminolevulinic acid, picolinic acid, lidofenin, phosphocholine , 1-(5-carboxypentyl)-4-methylpyridin-1-ium bromide, L-anserine nitrate, reduced L-glutathione, N-ethyl-L-glutamine, N-methylproline, (Z)-1-[ N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate), (Z)-1-[N-(3-amino Propyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate (DPTA-NONate), and zoledronic acid.

Without wishing to be bound by theory, zwitterionic excipient compounds interact with proteins, e.g., through charge interactions, hydrophobic interactions, and steric interactions to reduce viscosity or stabilize Protein formulations such as electrolyte contribution, reduction of surface tension, change in amount of available unbound water, or change in dielectric constant that exert an effect and make the protein more resistant to aggregation Affects the bulk properties of water in

i. Excipient Compound Category 9: Crowding Agents Containing Hydrogen Bonding Elements Solutions of therapeutic or non-therapeutic proteins may contain hydrogen bonding elements as excipients to improve stability or reduce viscosity. It can be formulated with a crowding agent comprising: As used herein, the term "crowding agent" refers to formulation additives that decrease the amount of water available to dissolve proteins in solution and increase the effective protein concentration. In embodiments, the crowding agent can reduce the particle size of the protein or reduce the amount of unfolding of the protein in solution. In embodiments, the crowding agent can act as a solvent modifier that causes water structuring through hydrogen bonding and hydration effects. In embodiments, the crowding agent can reduce the amount of intermolecular interactions between proteins in solution. In embodiments, the crowding agent has a structure that includes at least one hydrogen bond donor element, such as hydrogen bonded to oxygen, sulfur, or nitrogen atoms. In embodiments, the crowding agent has a structure comprising at least one weakly acidic hydrogen bond donor element having a pKa of about 6 to about 11. In embodiments, the crowding agent has a structure comprising from about 2 to about 50 hydrogen bond donor elements. In embodiments, the crowding agent has a structure that includes at least one hydrogen bond acceptor element, such as a Lewis base. In embodiments, the crowding agent has a structure comprising from about 2 to about 50 hydrogen bond acceptor elements. In embodiments, the crowding agent has a molecular weight of about 50-500 g/mole. In embodiments, the crowding agent has a molecular weight of about 100-350 g/mole. In other embodiments, the crowding agent may have a molecular weight greater than 500 g/mole, such as raffinose, inulin, pullulan, or sinistrin.

Examples of crowding agent excipients containing hydrogen bonding elements include 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidione, 15-crown-5, 18-crown-6 , 2-butanol, 2-butanone, 2-phenoxyethanol, acetaminophen, allantoin, arabinose, arabitol, benzyl acetoacetate, benzyl alcohol, chlorobutanol, cholestanol tetraacetyl-b-glucoside, cinnamaldehyde, cyclohexanone, deoxyribose, Diethyl carbonate, dimethyl carbonate, dimethyl isosorbide, dimethylacetamide, dimethylformamide, dimethylolethylene urea, dimethyluracil, epilactose, erythritol, erythrose, ethyl lactate, ethyl maltol, ethylene carbonate, formamide, fucose, galactose, genistein, gentisic acid ethanolamide, gluconolactone, glyceraldehyde, glycerol, glycerol carbonate, glycerol formal, glycerol urethane, glycyrrhizic acid, gossypin, harpagoside, hederacoside C, icodextrin, iditol, imidazolidone, inositol, inulin, isomaltitol, kojic acid, Lactitol, lactobionic acid, lactose, lactulose, lyxose, madecassoside, maltotriose, mangiferin, mannose, melezitose, methyl lactate, methylpyrrolidone, mogroside V, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, N -methylacetamide, N-methylformamide, N-methylpropionamide, pentaerythritol, pinoresinol diglucoside, piracetam, propyl gallate, propylene carbonate, psicose, pullulan, pyrogallol, quinic acid, raffinose, rebaudioside A, rhamnose, ribitol , ribose, ribulose, saccharin, sedoheptulose, sinistrin, solketal, stachyose, sucralose, tagatose, t-butanol, tetraglycol, triacetin, N-acetyl-d-mannosamine, nystose, kestose, turanose, acarbose, D-saccharic acid 1, 4-lactone, thiodigalactoside, fucoidan, hydroxysafrole yellow A, shikimic acid, diosmin, pravastatin sodium salt, D-altrose, L-gulon γ-lactone, neomycin, rubusoside dihydroartemisinin, phloroglucinol, naringin, baicalein , hesperidin, apigenin, pyrogallol, morin, salsalate, kaempferol, myricetin, 3′,4′,7-trihydroxyisoflavone, (±)-taxifolin, silybin, perseitol diformal, 4-hydroxy phenylpyruvate, sulfacetamide, isopropyl β-D-1-thiogalactopyranoside, ethyl 2,5-dihydroxybenzoate, spectinomycin, resveratrol, quercetin, kanamycin sulfate, 1-(2-pyrimidyl)piperazine, 2-(2-pyridyl)ethylamine, 2-imidazolidone, DL-1,2-isopropylideneglycerol, metformin, m-xylylenediamine, demeclocycline, tripropylene glycol, tubeimoside I, verbenaroside, xylitol, and xylose. be done.

6. Protein/Excipient Solutions: Properties and Processes In certain embodiments, hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic Excipients, therapeutic or non-therapeutic formulated with the above-identified excipient compounds or combinations thereof, such as excipients and crowding agents containing hydrogen-bonding elements (hereinafter "excipient excipients") A solution of proteins for protein yields improved protein-protein interaction properties as measured by the protein diffusion interaction parameter k _D or the second virial coefficient B ₂₂ . As used herein, an "improvement" in one or more protein-protein interaction parameters achieved by a test formulation using the above-identified excipient compounds or combinations thereof is the excipient It can refer to a decrease in attractive protein-protein interactions when a test formulation is compared under comparable conditions with a comparable formulation without compound or excipient additives. Such improvements can be manifested by measuring specific parameters that apply to the process as a whole or aspects thereof, where a parameter is any metric associated with a process that quantifies change and Can be compared to conditions or controls. Parameters can relate to the process itself, such as efficiency, cost, yield, speed. Improving the stability of protein-containing formulations during processing has the advantages of improved yield, increased biological activity, and reduced presence of particles in the formulation.

A parameter can also be a proxy parameter that relates to a feature or aspect of a larger process. By way of example, parameters such as the k _D or B ₂₂ parameters can be referred to as proxy parameters. Measurement of k _D and B ₂₂ can be performed using industry standard techniques and can be indicative of process-related parameters such as improved solution properties and stability of proteins in solution. Without wishing to be bound by theory, it is understood that highly negative _kD values indicate strong attractive interactions with proteins, which can lead to aggregation, instability, and rheological problems. . The same proteins, when formulated in the presence of the above-identified excipient compounds or combinations thereof, have improved protein properties with less negative _kD values, or _kD values near zero or greater. The improved proxy parameters are associated with improved process-related parameters.

In embodiments, clouds containing hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic excipients, and hydrogen bonding elements Some of the excipient compounds or combinations thereof described above, such as sling agents, can be used for filtration, syringe, transfer, pumping, mixing, heating or cooling by heat transfer, gas transfer, centrifugation, chromatography, membrane separation, Protein-related processes such as manufacturing, processing, aseptic filling, purification, and analysis of protein-containing solutions using processing methods such as centrifugal concentration, tangential flow filtration, radial flow filtration, axial flow filtration, freeze-drying, and gel electrophoresis used to improve In these and related protein-related processes, the protein of interest is dissolved in a solution that carries it through the processor. Such solutions, referred to herein as "carrier solutions", include cell culture media (e.g., containing a secreted protein of interest), lysate after host cell lysis (wherein the protein of interest is present in the lysate), , an elution solution (containing the protein of interest after chromatographic separation), an electrophoresis solution, a transport solution for carrying the protein of interest through conduits within the processor, 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 of the above-identified excipient compounds or combinations thereof can be added to protein-containing solutions to improve various aspects of processing. As used herein, the terms "improve," "improve," etc., refer to an improvement in a parameter of interest in a carrier solution when that parameter is compared to the same parameter measured in a control solution. Point to change. As used herein, "control solution" means a solution lacking viscosity-lowering excipients but otherwise substantially similar to the carrier solution. As used herein, a "control process", e.g., a control filtration process, a control chromatography process, etc., is substantially similar to the protein-related process of interest and is performed using a control solution instead of a carrier solution. related processes.

For example, in processes in which a protein-containing solution is pumped through a conduit (e.g., flow chamber, tubing or tubing), it may be advantageous to add the above-identified excipient compounds or combinations thereof to reduce viscosity. is. As described above, adding a viscosity-lowering excipient to the protein solution prior to or during the pumping process can substantially reduce the force and power required to pump the solution. . It is understood that fluids generally exhibit a resistance to flow, or viscosity, and a force must be applied to the fluid to overcome this viscosity in order to induce and propagate flow. The power P required for the pumping scale by the lift H and the discharge amount Q is shown in the following equation.
P to HQ (Formula 1)

Viscous fluids tend to increase pump power requirements, reduce pump efficiency, reduce pump lift and capacity, and increase frictional resistance in piping. Addition of the above-described viscosity-lowering excipients to the protein solution before or during pumping will reduce either or both the lift (H, Eq.1) or the volume (Q, Eq.1), resulting in Costs can be greatly reduced. The advantage of reduced viscosity is manifested by, for example, improved throughput, improved yield, or reduced processing time. Additionally, frictional losses due to the transmission of fluids through conduits can account for a significant portion of the costs associated with transporting such fluids. Adding the viscosity-lowering excipients described above to the protein solution before or during pumping can significantly reduce processing costs by reducing the friction associated with the pumping process. The processing cost measure represents a processing parameter that can be improved by using viscosity-lowering excipients.

These processes and methods of treating protein solutions can improve efficiency by reducing viscosity, improving solubility, or improving stability of proteins in solution during manufacturing, processing, purification, and analytical steps. Measurement of processing efficiency or measurement of surrogate parameters such as viscosity, solubility, or stability of proteins in solution represent processing parameters that can be improved by using viscosity-lowering excipients. It is understood that several different factors adversely affect protein viscosity, solubility, and stability during processing. For example, protein-containing solutions are subject to various physical stressors during manufacturing and purification by manipulating the protein solution through routine processing operations including, but not limited to, pumping, mixing, centrifugation, and filtration. Significant shear stress induced is included. Furthermore, during these processing steps, air bubbles can become entrained in the fluid to which proteins can be adsorbed. Such interfacial tension, coupled with normal shear stresses encountered during processing, can unfold and aggregate adsorbed protein molecules. In addition, significant protein unfolding can occur during pump cavitation events and exposure to solid surfaces such as ultrafiltration and diafiltration membranes during manufacture. Such events can compromise protein folding and product quality.

For a Newtonian fluid, the stress τ imposed by a given process scale, where γ is the shear rate of the fluid and η is the viscosity of the fluid, is given by:
τ = γη (equation 2)

The viscosity of the solution can be reduced by formulating the protein solution with one or more of the above excipient compounds or combinations thereof, thus reducing the shear stress encountered by the protein solution. can. A reduction in shear stress can improve the stability of formulations being processed, manifested by, for example, better or more desirable measurements of processing parameters. Such improved processing parameters include metrics such as reduced levels of protein aggregates, particles, or sub-visible particles, reduced product loss, or improved overall yield. As another example of improved processing parameters, reducing the viscosity of the protein-containing solution can reduce the processing time of the solution. Processing time for a given unit operation is generally inversely proportional to shear rate. Therefore, for a given characteristic stress, a reduction in the viscosity of a protein solution due to the addition of an excipient compound or combination thereof as described above is associated with an increase in shear rate (γ, see Equation 2) and thus the treatment Relates to time savings. Additionally, the addition of some of the excipient compounds identified above or combinations thereof can improve the stability of protein solutions during different stages of processing.

It is understood that the protein in solution during processing may be the desired protein active ingredient, eg, therapeutic or non-therapeutic protein. Ease of processing of such protein active ingredients using the excipients described herein may increase the yield or production rate of the protein active ingredient, or improve the efficiency of a particular process. , or reduced energy usage, etc., all of which represent improved processing parameters through the use of viscosity-lowering excipients. It is also understood that protein contaminants may be formed during certain processing techniques, such as fermentation and purification steps of bioprocesses. Faster, more complete, or more efficient removal of contaminants can also improve processing of the desired protein, ie, the protein active ingredient. These results represent improved processing parameters through the use of viscosity-lowering excipient compounds or additives. As described herein, certain excipients described herein may reduce solution viscosity, improve protein stability, and/or increase protein solubility to achieve desired It can improve the transport of protein active ingredients and improve the removal of unwanted protein contaminants. Both effects represent improved processing parameters through the use of viscosity-lowering excipients or additives, indicating that these excipients or additives improve the overall process of protein production. Reduction of misfolded proteins, microparticles, denatured proteins, or other artifacts of destabilized proteins in solution can be achieved through the use of stabilizing excipients during processing steps.

Operation of specific platform units for the production and purification of therapeutic proteins provides further examples of the advantageous use of excipient compounds or combinations thereof identified above, and the use of these excipients to improve processing parameters. Further examples of excipients or excipients are provided. For example, as described below, the introduction of one or more of the above-identified excipient compounds or combinations thereof into these production and purification processes can significantly enhance the stability and recovery of the molecule. Improvements can be provided, as well as reduced operating costs.

It is understood in the art that widely practiced techniques for producing and purifying therapeutic proteins such as monoclonal antibodies generally consist of a series of steps for fermentation processes followed by purification treatments. be done. Fermentation, or upstream processing (USP), involves growing therapeutic proteins in bioreactors, usually using bacterial or mammalian cell lines. In embodiments, the USP may include steps as shown in FIG. In embodiments, purification or downstream processing (DSP) may include steps as shown in FIG.

As shown in FIG. 4, USP may begin with step 102 of thawing a vial from a master cell bank (MCB). As shown in step 104, the MCB can be expanded to form working cell banks (not shown) and/or to generate working stock for further generation. As shown in steps 108 and 110, cell culture is performed in a series of seed and production bioreactors to produce bioreactor product 112, from which the desired therapeutic protein is harvested, as shown in step 114. obtain. Following harvest 114, the products may be subjected to further purification (i.e., DSP as described in more detail below and shown in FIG. 5), or these products are typically frozen and It can be stored in bulk by storing at a temperature of about -80°C.

In embodiments, protein production by cell culture techniques can be improved through the use of the excipients identified above, manifested by improvements in process-related parameters. In embodiments, desired excipients can be added to the USP in amounts effective to reduce the viscosity of the cell culture medium by at least 20%. In other embodiments, desired excipients can be added in USP in amounts effective to reduce the viscosity of the cell culture medium by at least 30%. In embodiments, desired excipients may be added to the cell culture medium in amounts of about 1 mM to about 400 mM. In embodiments, desired excipients may be added to the cell culture medium in amounts of about 20 mM to about 200 mM. In embodiments, desired excipients may be added to the cell culture medium in amounts of about 25 mM to about 100 mM. The desired excipient or combination of excipients is added directly to the cell culture medium, or components of more complex supplemental media, such as solutions or " It can be added as a "feeding" solution. In embodiments, a second excipient, e.g., a viscosity-lowering compound, can be added to the carrier solution, either directly or via a supplement medium, wherein the second viscosity-lowering compound is the desired Add further improvements to specific parameters.

As described below, there are a number of process-related parameters in the USP that can be improved using one or more of the above identified excipient compounds or combinations thereof. For example, in embodiments, the use of viscosity-reducing excipients can improve parameters such as the rate and/or extent of cell growth during steps such as seed growth 104 and cell culture 108 and 110 and/or or correlated with improvements in various process parameters. For example, adding some of the above-identified excipients to the USP process, in steps such as bioreactor production step 110, can reduce the viscosity of the cell culture medium and subsequently improve heat transfer efficiency and gas transfer. Efficiency can be improved. Cell culture processes require oxygen injection into the cells to allow protein expression, and thus diffusion of oxygen into the cells may be the rate-limiting step, thus reducing the viscosity of the solution. Improving oxygen uptake rate by improving transfer efficiency can improve the rate or amount of protein expression and/or its efficiency. In this context, parameters such as the rate of oxygen uptake rate and gas transfer efficiency can be considered proxy parameters, and their improvement correlates with improved protein expression or process parameter improvements for improved processing efficiency. . As another example, the availability of viscosity-lowering excipients, e.g., during seed growth step 104 and cell culture steps Treatment can be improved by improving proxy parameters such as (required for protein expression with improved growth factor solubility thereby promoting cell proliferation).

In embodiments, process parameters such as amount of protein recovery or percent protein recovery during USP can be improved by reducing viscosity during USP by several mechanisms. For example, the harvesting of therapeutic proteins at the end of the lysis step during harvesting 114 from completed cell cultures may be more efficient or improved in ways other than the use of excipients identified above. obtain. Without wishing to be bound by theory, by reducing the viscosity of the expressed protein, these viscosity-lowering excipients can increase the efficiency of diffusion of the therapeutic protein away from other dissolved components. Furthermore, separation of membranes and other cellular debris from protein-containing supernatants can be achieved with faster separation rates or higher supernatant purities using viscosity-lowering excipients, thereby USP efficiency process parameters are improved. In addition, protein separation steps using centrifugation or filtration steps can be accomplished faster using viscosity-lowering excipients because the excipients reduce the viscosity of the medium. Excipients can also improve the stability of therapeutic protein solutions, so upstream and downstream processing of proteins can benefit from the use of these excipients. In embodiments, excipients can improve the stress tolerance of proteins during processing, which can reduce the amount of protein aggregation or denaturation during processing steps.

In embodiments, as an added benefit, the use of excipient compounds or combinations thereof as described above, e.g., viscosity-reducing excipients, in cell culture reduces protein misfolding and aggregation, thus reducing USP Process parameters such as the yield of protein in the medium can be increased. It is understood that when cell culture is optimized for maximum yield of recombinant protein, the resulting protein may be expressed in a highly concentrated manner, causing misfolding. . Addition of the above-identified excipient compounds or combinations thereof, such as viscosity-lowering excipients, reduces attractive protein-protein interactions that lead to misfolding and aggregation, thereby making available for harvesting 114 The amount of intact recombinant protein is increased.

In an exemplary embodiment, the downstream processing (DSP) shown in Figure 5 includes a series of steps that result in the recovery and purification of therapeutic proteins such as monoclonal antibodies, biopharmaceuticals, vaccines, and other biologics. be At the end of USP, the therapeutic protein of interest can be secreted from the host cell and dissolved in the cell culture medium. The therapeutic protein can also be dissolved in liquid medium following host cell lysis at the end of the USP sequence. DSP is performed to recover and purify the protein of interest from the solution in which it is dissolved (eg, culture medium or host cell lysate). During DSP, (i) various contaminants (such as insoluble cell debris and particles) are removed from the medium and (ii) protein products are isolated through techniques such as extraction, precipitation, adsorption or ultrafiltration. (iii) purifying the protein product through techniques such as affinity chromatography, precipitation or crystallization, and (iv) further polishing the product to remove viruses.

As shown in FIG. 5, the feedstock from cell culture harvest 200 (also depicted in FIG. 4) is first subjected to affinity chromatography 204, typically involving protein A chromatography or other similar chromatography steps. be. The virus inactivation step 208 typically requires the feedstock to be held at low pH. One or more polishing chromatography steps 210 and 212 are performed to remove impurities such as host cell proteins (HCPs), DNA, charge variants, and aggregates. Cation exchange (CEX) chromatography is commonly used as an initial polishing chromatography step 210, but may be preceded or followed by a second chromatography step 212. The 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 second chromatography step 212 . Virus filtering 214 is performed to perform virus removal. A final purification step 218 may include ultrafiltration and diafiltration, as well as formulation preparation.

As generally described above, purification processes or DSPs following fermentation processes include (1) cell culture harvesting, (2) chromatography (e.g., Protein A chromatography, and ion exchange and hydrophobic interaction chromatography). (3) viral inactivation, and (4) filtration (e.g., viral filtration, sterile filtration, dialysis, and ultrafiltration or ultrafiltration to concentrate the protein and exchange the protein into the formulation buffer). diafiltration step). The examples are to illustrate the benefits of using the excipient compounds identified above or combinations thereof, such as viscosity-lowering excipients, to improve the process parameters associated with these purification processes. , provided below. The above-identified excipient compounds or combinations thereof, e.g., viscosity-lowering excipients, may be used to solubilize or stabilize the protein of interest and excipients by adding it to the carrier solution or otherwise. It is understood that by modifying the contacts in a non-enhanced form, they can be introduced at any stage of the DSP. In embodiments, a second excipient, such as a viscosity-lowering compound, may be added to the carrier solution during DSP, the second compound adding additional improvement to a particular parameter of interest.

(1) Cell culture harvesting: Cell culture harvesting generally involves centrifugation and depth filtration operations to physically remove cell debris from the protein-containing solution. The centrifugation step can provide a more complete separation of soluble proteins from cell debris due to the advantage of viscosity-lowering excipients. Centrifugation, whether done batchwise or continuously, should compact the dense phase as much as possible to maximize recovery of the target protein. In embodiments, the addition of the above-identified excipients or combinations thereof improves the process parameter of protein yield, e.g., by increasing the yield of protein-containing separate liquid exiting the high-density phase of the centrifugation process. can be increased. Since the depth filtration step is a viscosity limited step, it can be made more efficient by using excipients that reduce the viscosity of the solution. These processes can also introduce air bubbles into the protein solution, which combined with shear-induced stress can destabilize the purified therapeutic protein molecule. As described above, the addition of viscosity-lowering excipients to protein-containing solutions before and/or during harvesting of cell cultures protects proteins from these stresses, thereby reducing potential protein aggregation. Reduced and quantified product recovery process parameters can be improved.

(2) Chromatography: After harvesting the cell culture by centrifugation or filtration, chromatography is typically used to separate the therapeutic protein from the fermentation broth. Protein A chromatography is used when the therapeutic protein is an antibody. Protein A is selective for IgG antibodies and binds dynamically at high flow rates and volumes. Cation exchange (CEX) chromatography can be used as a cost-effective alternative to Protein A chromatography. When using CEX, it is necessary to adjust the pH of the feed solution to lower its conductivity before loading it onto the column in order to optimize the dynamic binding capacity. Mimetic resins can also be used as an alternative to Protein A chromatography. These resins provide ligands for binding immunoglobulins, Ig binding proteins such as protein G and protein L, synthetic ligands, or porous polymers such as protein A.

Other chromatographic processes can be used during DSP. Ion exchange chromatography (IEC) was used to remove impurities introduced during previous processes, such as leached protein A from cell lines, endotoxins or viruses, residual host cell proteins or DNA, or media components. can be removed. IEC can be applied directly after Protein A chromatography, whether CEX or anion exchange chromatography. Hydrophobic interaction chromatography (HIC) can complement IEC, which is commonly used as a polishing step to remove aggregates. In embodiments, the excipients identified above can be used to increase the solubility and decrease the viscosity of host cell proteins during the loading step of the chromatography column. In embodiments, the excipients identified above can be used to increase the solubility and decrease the viscosity of the therapeutic protein during the loading and elution steps of the chromatography column.

In the chromatographic process during protein purification, (a) low pH conditions during elution from the Protein-A chromatography column, (b) increased local protein concentration within the pores of the chromatography resin (often around 300-400 mg/mL Among them, (c) increased salt concentration during ion-exchange chromatography, (d) increased concentration of salting-out agent during elution from the HIC column, and other stringent requirements are imposed on protein formulations. As noted above, the addition of viscosity-lowering excipients to protein-containing solutions prior to and/or during chromatography facilitates migration of the protein through the chromatography column and thus is imposed by the chromatography step. Less exposure to potentially damaging conditions. In addition, high local protein concentrations within the column pores create viscous material within this space, creating large back pressures on the column. To relieve this backpressure, media with relatively large pores are typically used. However, the resolution of large pore media is lower than that of small pore media. Incorporation of the viscosity modifying excipients described above allows the use of smaller pores in the chromatographic media. In embodiments, the elution step from Protein A chromatography exposes therapeutic proteins to low pH conditions that can reduce solubility and increase aggregation. Addition of excipients can increase the solubility of the target protein and thus improve recovery from the Protein A chromatography step. In other embodiments, the use of excipients allows the target protein to be eluted from the Protein A resin at a higher pH, thereby reducing chemical stress on the target protein, resulting in increased processing efficiency. The amount of proteolysis in the medium is reduced, improving the process parameter of protein yield.

(3) Virus inactivation: The virus inactivation process usually involves holding the protein solution at a low pH, eg below 4, for an extended period of time. However, this environment can destabilize therapeutic proteins. For example, by adding a viscosity-lowering excipient prior to and/or during the virus inactivation process, formulating the protein in the presence of the viscosity-lowering excipient may improve the stability or solubility of the protein, or Process parameters such as its net yield can be improved.

(4) Filtration: Filtration processes include viral filtration processes (nanofiltration) to remove viral particles, and ultrafiltration/diafiltration processes to concentrate protein solutions and to exchange buffer systems. is included.

(a) Virus filtration purifies the protein solution by removing virus particles, which can be on the order of twice the size of recombinant human monoclonal antibodies. Therefore, filtration membranes for virus filtration may require nano-sized pores. Proteins have to pass through small pore sizes, and as a result, this filtration step can introduce stress to proteins, with significant levels of membrane fouling by aggregated particles of protein. As described above, the addition of viscosity-lowering excipients, e.g., before and/or during filtration, reduces measurable parameters such as filtration system back pressure by increasing the cooperative diffusion rate. and the propensity for membrane fouling can be reduced as the protein-protein interactions that cause it are relaxed. The net result is an improvement in these parameters that indicates improved performance of the virus filtration unit during protein purification.

(b) the ultrafiltration and diafiltration (UF/DF) process concentrates a protein solution by passing the protein-containing solution through a filter membrane having a characteristic molecular weight cut-off lower than the protein of interest; Change the buffer system. In this step, the protein solution is subjected to high shear stress within the filter unit, increasing protein concentration, protein adsorption to hydrophobic membranes commonly used during the UF/DF process. As described above, the addition of viscosity-lowering excipients, e.g., before and/or during the UF/DF process, results in an increase in cooperative diffusion rate (e.g., as measured by an increase in _k ), the back pressure of the filtration system can be reduced. This not only reduces shear stress across the membrane, but also promotes back diffusion through the filter membrane, thus reducing the effective protein concentration at the membrane interface and increasing permeation flux. As a result, the use of viscosity-lowering excipients improves parameters associated with increased throughput during these filtration processes, resulting in reduced product losses and increased net yields. Additionally, passing viscous fluids through ultra- and dialysis filters can create large pressure drops across the filter apparatus, resulting in inefficient separations. As noted above, formulating protein solutions in the presence of viscosity-lowering excipients can significantly reduce the pressure drop across the filter device, thereby reducing both operating costs and processing time. The reduction improves process parameters.

After upstream protein processing or downstream purification is completed with added excipients, the excipients can either remain part of the drug substance mixture or be separated from the protein active ingredient. Typical small molecule separation methods such as buffer exchange, ion exchange, ultrafiltration, and dialysis can be used to separate the excipients from the protein active ingredient. In addition to the beneficial effects on the protein purification process outlined above, the use of the excipients identified above can protect and preserve the equipment used in protein manufacturing, processing, and purification. For example, equipment-related processes such as cleanup, sterilization, and maintenance of protein processing equipment can benefit from reduced fouling, reduced denaturation, reduced viscosity, and improved protein solubility through the use of the excipients identified above. As a result, the parameters associated with these process improvements are improved as well.

Although the use of excipient compounds to improve upstream and/or downstream processing is described extensively herein, excipient compounds may be used to achieve a desired effect, such as improving a desired parameter. It is understood that the agents can be added together in combination. The term "excipient additive" refers to a single excipient compound or a combination of excipient compounds that produces a desired effect or improved parameter, wherein the combination cause the parameter to be changed.

material:
• Bovine Gamma Globulin (BGG), >99% Purity, Catalog No. G5009, Sigma Aldrich
● Histidine, Sigma Aldrich
• Materials other than those described in the examples below were obtained from Sigma Aldrich unless otherwise specified.

Example 1 Preparation of Formulations Comprising Excipient Compounds and Test Proteins Formulations are prepared using excipient compounds and test proteins, where the test proteins are therapeutic proteins used in therapeutic formulations or non-therapeutic proteins. It is intended to mimic any of the non-therapeutic proteins used in therapeutic formulations. Such formulations were prepared in 50 mM histidine hydrochloride with different excipient compounds for viscosity measurements in the following manner. First, 1.94 g of histidine was dissolved in distilled water, the pH was adjusted to about 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.), and then a final volume of 250 mL was added with distilled water in a volumetric flask. Histidine hydrochloride was prepared by diluting to volume. Excipient compounds were then dissolved in 50 mM histidine HCl. A list of excipients is provided in Examples 4, 5, 6, and 7 below. In some cases, excipient compounds were adjusted to pH 6 before dissolving in 50 mM histidine HCl. In this case, the excipient compound was first dissolved in deionized water at about 5 wt% and the pH was adjusted to about 6.0 using hydrochloric acid or sodium hydroxide. The prepared salt solution was then placed in a convection laboratory oven 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 ratio of approximately 0.336 g BGG per mL of excipient solution. This resulted in a final protein concentration of approximately 280 mg/mL. A solution of BGG in 50 mM histidine HCl containing excipients was formulated in 20 mL vials and shaken overnight at 100 rpm on an orbital shaker table. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurements.

Example 2 Viscosity Measurements Viscosity measurements of formulations prepared as described in Example 1 were performed using a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer at a volume of 0.5 mL and incubated at the given shear rate and temperature for 3 minutes followed by a measurement collection time of 20 seconds. This was followed by two additional steps consisting of a 1 minute shear incubation followed by a 20 second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample.

Example 3 Measurement of Protein Concentration The concentration of protein in the experimental solution was determined by measuring the absorbance of the protein solution at a wavelength of 280 nm with a UV/VIS spectrometer (Perkin Elmer Lambda 35). The instrument was first calibrated to zero absorbance with 50 mM histidine buffer at pH 6. The protein solution was then diluted 300-fold with the same histidine buffer and the absorbance at 280 nm was recorded. The final concentration of protein in solution was calculated using an extinction coefficient value of 1.264 mL/(mg x cm).

Example 4 Formulations Containing Hindered Amine Excipient Compounds Formulations containing 280 mg/mL BGG were prepared as described in Example 1 using several samples with added excipient compound. bottom. In these tests, the hydrochloride salts of dimethylcyclohexylamine (DMCHA), dicyclohexylmethylamine (DCHMA), dimethylaminopropylamine (DMAPA), triethanolamine (TEA), dimethylethanolamine (DMEA), and niacinamide were tested as hindered amines. Tested as an example of an excipient compound. Hydroxybenzoate of DMCHA and taurine-dicyandiamide adducts were also tested as examples of hindered amine excipient compounds. The viscosity of each protein solution was measured as described in Example 2 and the results are presented in Table 1 below, demonstrating the viscosity-lowering benefits of the added excipient compounds.

Example 5 Formulations Containing Anionic Aromatic Excipient Compounds Formulations of 280 mg/mL BGG were prepared as described in Example 1 using several samples with added excipient compounds. prepared. The viscosity of each solution was measured as described in Example 2 and the results are presented in Table 2 below, demonstrating the viscosity-lowering benefits of the added excipient compounds.

Example 6: Formulations Containing Oligopeptide Excipient Compounds Oligopeptides (n=5) were obtained from NeoBioLab Inc. (Woburn, Mass.) with greater than 95% purity at the N-terminus as the free amine and the C-terminus as the free acid. Dipeptides (n=2) were synthesized with 95% purity by LifeTein LLC (Somerset, NJ). Formulations of 280 mg/mL BGG were prepared as described in Example 1 using several samples containing synthetic oligopeptides as added excipient compounds. The viscosity of each solution was measured as described in Example 2 and the results are presented in Table 3 below, demonstrating the viscosity-lowering benefits of the added excipient compounds.

Example 7: Synthesis of excipients of guanyl taurine Guany taurine was prepared according to the method described in US Patent No. 2,230,965. 3.53 parts taurine (Sigma-Aldrich, St. Louis, MO) was mixed with 1.42 parts dicyandiamide (Sigma-Aldrich, St. Louis, MO) and ground with a mortar and pestle until a uniform mixture was obtained. . The mixture was then placed in a flask and heated at 200° C. for 4 hours. The product was used without further purification.

Example 8: Protein Formulations Comprising Excipient Compounds Formulations are prepared using excipient compounds and test proteins, where the test proteins are used in therapeutic formulations or in non-therapeutic formulations. It is intended to mimic any of the non-therapeutic proteins currently being used. Such formulations were prepared in 50 mM histidine hydrochloride buffer containing different excipient compounds for viscosity measurements in the following manner. First, 1.94 g of histidine was dissolved in distilled water, the pH was adjusted to about 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.), and then a final volume of 250 mL was added with distilled water in a volumetric flask. Histidine-HCl buffer was first prepared by diluting to volume. Excipient compounds were then dissolved in 50 mM histidine HCl buffer. Table 4 provides a list of excipient compounds. In some cases, excipient compounds were dissolved in 50 mM histidine HCl buffer and the pH of the resulting solution was adjusted to pH 6 with small amounts of sodium hydroxide or hydrochloric acid prior to dissolving the model protein. In some cases, excipient compounds were adjusted to pH 6 before dissolving in 50 mM histidine HCl. In this case, the excipient compound was first dissolved in deionized water at about 5 wt% and the pH was adjusted to about 6.0 using hydrochloric acid or sodium hydroxide. The prepared salt solution was then placed in a convection laboratory oven 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 in a ratio to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in 50 mM histidine HCl containing excipients was formulated in 20 mL vials and shaken overnight at 100 rpm on an orbital shaker table. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurements.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer at a volume of 0.5 mL and incubated at the given shear rate and temperature for 3 minutes followed by a measurement collection time of 20 seconds. This was followed by two additional steps consisting of a 1 minute shear incubation followed by a 20 second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of solutions containing excipients were normalized to the viscosities of model protein solutions without excipients. Normalized viscosity is the ratio of the viscosity of a model protein solution containing excipients to the viscosity of a model protein solution without excipients.

Example 9 Preparation of Formulations Containing Excipient Combinations and Test Proteins Formulations are prepared using a primary excipient compound, a secondary excipient compound and a test protein, where the test protein is a therapeutic formulation. It is intended to mimic either therapeutic proteins used or non-therapeutic proteins used in non-therapeutic formulations. Primary excipient compounds were selected from compounds with both anionic and aromatic functionality, as listed in Table 5 below. Secondary excipient compounds are selected from compounds with either a nonionic or cationic charge at pH 6 and either an imidazoline ring or a benzene ring, as listed in Table 5 below. was done. Formulations of these excipients were prepared in 50 mM histidine-HCl buffer for viscosity measurements in the following manner. First, 1.94 g of histidine was dissolved in distilled water, the pH was adjusted to about 6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.), and then a final volume of 250 mL was added with distilled water in a volumetric flask. Histidine hydrochloride was prepared by diluting to volume. Individual primary or secondary excipient compounds were then dissolved in 50 mM histidine HCl. The combination of primary and secondary excipients was dissolved in 50 mM histidine HCl and the pH of the resulting solution was adjusted with small amounts of sodium hydroxide or hydrochloric acid to pH 6 before dissolving the model protein. Once the excipient solutions were prepared as described above, the test protein bovine gamma globulin (BGG) was dissolved in each test solution in a ratio to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in 50 mM histidine HCl containing excipients was formulated in 20 mL vials and shaken overnight at 100 rpm on an orbital shaker table. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurements.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer at a volume of 0.5 mL and incubated at the given shear rate and temperature for 3 minutes followed by a measurement collection time of 20 seconds. This was followed by two additional steps consisting of a 1 minute shear incubation followed by a 20 second measurement collection time. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of solutions with excipients were normalized to the viscosities of model protein solutions without excipients and are summarized in Table 5 below. Normalized viscosity is the ratio of the viscosity of a model protein solution containing excipients to the viscosity of a model protein solution without excipients. This example shows that a combination of primary and secondary excipients can give better results than a single excipient.

Example 10 Preparation of Formulations Containing Excipient Combinations and Test Proteins Formulations are prepared using a primary excipient compound, a secondary excipient compound and a test protein, where the test protein is a therapeutic formulation. It is intended to mimic therapeutic proteins used or non-therapeutic proteins used in non-therapeutic formulations. Primary excipient compounds were selected from compounds with both anionic and aromatic functionality, as listed in Table 6 below. Secondary excipient compounds are selected from compounds with either a nonionic or cationic charge at pH 6 and either an imidazoline ring or a benzene ring, as listed in Table 6 below. was done. Formulations of these excipients were prepared in distilled water for viscosity measurements in the following manner. The combination of primary and secondary excipients was dissolved in distilled water and the pH of the resulting solution was adjusted with a small amount of sodium hydroxide or hydrochloric acid to pH 6 before dissolving the model protein. Once the excipient solution in distilled water was prepared, the test protein bovine gamma globulin (BGG) was dissolved in a ratio to achieve a final protein concentration of approximately 280 mg/mL. Solutions of BGG in distilled water containing excipients were formulated in 20 mL vials and shaken overnight at 100 rpm on an orbital shaker table. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurements.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer at a volume of 0.5 mL and incubated at the given shear rate and temperature for 3 minutes followed by a measurement collection time of 20 seconds. This was followed by two additional steps consisting of a 1 minute shear incubation followed by a 20 second measurement collection time. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of solutions with excipients were normalized to the viscosities of model protein solutions without excipients and are summarized in Table 6 below. Normalized viscosity is the ratio of the viscosity of a model protein solution containing excipients to the viscosity of a model protein solution without excipients. This example shows that a combination of primary and secondary excipients can give better results than a single excipient.

Example 11 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 therapeutic PEGylated proteins used in therapeutic formulations. Such formulations were prepared by mixing equal volumes of PEG solution with excipient solutions. Both solutions were prepared in Tris buffer consisting of 10 mM Tris pH 7.3, 135 mM NaCl, 1 mM trans-cinnamic acid. A PEG solution was prepared by mixing 3 g of poly(ethylene oxide) average molecular weight of about 1,000,000 (Aldrich Catalog # 372781) with 97 g of Tris buffer. The mixture was stirred overnight for complete dissolution.

An example of preparation of an excipient solution is as follows. An approximately 80 mg/mL solution of citric acid in Tris buffer was prepared by dissolving 0.4 g of citric acid (Aldrich cat. #251275) in 5 mL of Tris buffer and adjusting the pH to 7.0 with a minimal volume of 10 M NaOH solution. adjusted to 3. A PEG excipient solution was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of excipient solution and mixed by using a vortex for a few seconds. A control sample was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of Tris buffer.

Example 12 Viscosity Measurements of Formulations Containing Excipient Compounds and PEG Viscosity measurements of prepared formulations were performed using a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer at a volume of 0.5 mL and incubated at the given shear rate and temperature for 3 minutes followed by a measurement collection time of 20 seconds. This was followed by two additional steps consisting of a 1 minute shear incubation followed by a 20 second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample.

The results presented in Table 7 demonstrate the viscosity-reducing effect of the added excipient compounds.

Example 13: Preparation of PEGylated BSA with one PEG chain per BSA molecule To a beaker was added 200 mL of phosphate buffered saline (Aldrich Cat. #P4417) and 4 g of BSA (Aldrich Cat. #A7906). , was mixed using a magnetic bar. Then 400 mg of methoxypolyethylene 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 quenched by adding 20 drops of 0.1M HCl. The reaction product was characterized by SDS-PAGE and SEC, clearly showing PEGylated BSA. The reaction mixture was placed in an Amicon centrifuge tube with a 30 kDa molecular weight cutoff (MWCO) and concentrated to a few milliliters. The sample was then diluted 20-fold with a 50 mM histidine buffer with a pH of about 6, followed by concentration until a highly viscous liquid was obtained. Final concentrations of protein solutions were obtained by measuring absorbance at 280 nm and using an extinction coefficient of 0.6678 for BSA. Results indicated that the final concentration of BSA in solution was 342 mg/mL.

Example 14: Preparation of PEGylated BSA with multiple PEG chains per BSA molecule Prepared by mixing 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 reaction was allowed to proceed overnight at room temperature. 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 Amicon centrifuge tubes (MWCO 30 kDa) until a concentration of approximately 150 mg/mL was reached. bottom.

Example 15: Preparation of PEGylated Lysozyme with Multiple PEG Chains per Lysozyme Molecule Prepared by mixing with 100 mL of buffer. Then 1 g of methoxy PEG propionaldehyde Mw=5,000 (JenKem Technology, Plano, Tex. 75024) was added, followed by 0.12 g of sodium cyanoborohydride (Aldrich 156159). The reaction was allowed to proceed overnight at room temperature. The next day, the reaction mixture was diluted 49-fold with phosphate buffer (25 mM, pH 7.2) and concentrated using Amicon centrifuge tubes (MWCO 30 kDa). The final concentration of the protein solution was obtained by measuring the absorbance at 280 nm and using the extinction coefficient of 2.63 for lysozyme. The final concentration of lysozyme in solution was 140 mg/mL.

Example 16: Effect of excipients on viscosity of PEGylated BSA with one PEG chain per BSA molecule Formulation salts were prepared by adding 0.3 mL of PEGylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc with an A10 channel (100 microns deep) at a shear rate of 500 s ⁻¹ . Viscometer measurements were completed at ambient temperature. The results presented in Table 8 demonstrate the viscosity-reducing effect of the added excipient compounds.

Example 17: Effect of Excipients on Viscosity of PEGylated BSA with Multiple PEG Chains per BSA Molecule Prepared by adding milligrams of excipient salt to 0.2 mL of PEGylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc with an A10 channel (100 microns deep) at a shear rate of 500 s ⁻¹ . Viscometer measurements were completed at ambient temperature. The results presented in Table 9 demonstrate the effect of added excipient compounds on reducing viscosity.

Example 18: Effect of excipients on viscosity of PEGylated lysozyme with multiple PEG chains per lysozyme molecule Excipient salts were prepared by adding to 0.3 mL of pegylated lysozyme solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc with an A10 channel (100 microns deep) at a shear rate of 500 s ⁻¹ . Viscometer measurements were completed at ambient temperature. The results presented in Table 10 demonstrate the viscosity-lowering benefit of the added excipient compound.

Example 19: Protein Formulations Comprising Combinations of Excipients Formulations are prepared using a combination of an excipient compound or two excipient compounds and a test protein, where the test protein is used in a therapeutic formulation. It is intended to mimic therapeutic proteins. These formulations were prepared in 20 mM histidine buffer containing different excipient compounds for viscosity measurements in the following manner. The excipient combination was dissolved in 20 mM histidine and the pH of the resulting solution was adjusted with small amounts of sodium hydroxide or hydrochloric acid to pH 6 before dissolving the model protein. The excipient compounds of this example are listed in Table 11 below. Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved in a ratio to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in excipient solution was formulated into a 5 mL sterile polypropylene tube and shaken overnight at 80-100 rpm on an orbital shaker table. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for approximately 10 minutes to remove entrained air prior to viscosity measurements.

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

Example 20: Protein Formulations Containing Excipients to Reduce Viscosity and Injection Pain A formulation is prepared using an excipient compound, a second excipient compound, and a test protein, wherein , is intended to mimic therapeutic proteins used in therapeutic formulations. The first excipient compound, Excipient A, was selected from a 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 using excipient A and excipient B in a 20 mM histidine buffer as follows so that their viscosities could be measured. The amounts of excipients disclosed in Table 12 were dissolved in 20 mM histidine and the pH of the resulting solution was adjusted to pH 6 with a small amount of sodium hydroxide or hydrochloric acid prior to dissolving the model protein. . Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved in the excipient solution in a ratio to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in excipient solution was formulated into a 5 mL sterile polypropylene tube and shaken overnight at 80-100 rpm on an orbital shaker table. The BGG-excipient solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for approximately 10 minutes to remove entrained air prior to viscosity measurements.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer at a volume of 0.5 mL and incubated at the given shear rate and temperature for 3 minutes followed by a measurement collection time of 20 seconds. This was followed by two additional steps consisting of a 1 minute shear incubation followed by a 20 second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of solutions containing excipients were normalized to the viscosities of model protein solutions without excipients and the results are shown in Table 12 below. Normalized viscosity is the ratio of the viscosity of a model protein solution containing excipients to the viscosity of a model protein solution without excipients.

Example 21: Formulations Comprising Excipient Compounds and PEG Formulations were prepared using excipient compounds and PEG, where PEG was intended to mimic therapeutic PEGylated proteins used in therapeutic formulations. and excipient compounds were provided in the amounts shown in Table 13. These formulations were prepared by mixing equal volumes of PEG solution with excipient solutions. Both solutions were prepared in deionized (DI) water. A PEG solution was prepared by mixing 16.5 g of poly(ethylene oxide) average molecular weight of about 100,000 (Aldrich Catalog #181986) with 83.5 g of DI water. The mixture was stirred overnight for complete dissolution.

Excipient solutions are prepared according to this general method, as detailed in Table 13 below. An approximately 20 mg/mL solution of aqueous tripotassium phosphate (Aldrich catalog #P5629) in DI water was prepared by dissolving 0.05 g of potassium phosphate in 5 mL of DI water. A PEG excipient solution was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of excipient solution and mixed by using a vortex for a few seconds. A control sample was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of DI water. The viscosity is measured and the results recorded in Table 13 below.

Example 22 Treatment of Improved Protein Solutions with Excipients Two BGG solutions were prepared by mixing 0.25 g of solid BGG with 4 mL of buffer. For sample A: the buffer was 20 mM histidine buffer (pH=6.0). For Sample B: Buffer was 20 mM histidine buffer containing 15 mg/mL caffeine (pH=6). Dissolution of solid BGG was performed by placing the sample on an orbital shaker set at 100 rpm. Buffer samples containing caffeine excipients were observed to dissolve proteins faster. For the sample containing the caffeine excipient (Sample B), complete dissolution of BGG was achieved in 15 minutes. For the caffeine-free sample (Sample A), dissolution took 35 minutes. The samples were then placed in two separate Amicon Ultra 4 Centrifugal Filter Units with a 30 kDa molecular weight cutoff and the samples were centrifuged at 2,500 rpm for 10 minute intervals. The volume of filtrate collected was recorded each time centrifugation was performed for 10 minutes. The results in Table 14 show that sample B filtrate recovery is fast. Furthermore, while sample B continued to enrich with each run, sample A reached its maximum enrichment point and did not result in further enrichment of the sample.

Example 23: Protein formulation with multiple excipients This example shows how the combination of caffeine and arginine as excipients has a beneficial effect on reducing the viscosity of BGG solutions. there is Four BGG solutions were prepared by mixing 0.18 g of solid BGG with 0.5 mL of 20 mM histidine buffer at pH 6. Each buffer contained a different excipient or combination of excipients as described in the table below (Table 15). Solution viscosities were measured as described in previous examples. The results show that the hindered amine excipient caffeine can be combined with known excipients such as arginine, and that the combination has better viscosity-lowering properties than the individual excipients themselves. ing.

Arginine was added to a 280 mg/mL solution of BGG in pH 6 histidine buffer. As shown in Table 16, at levels above 50 mg/mL, additional addition of arginine did not further reduce viscosity.

Caffeine was added to a 280 mg/mL solution of BGG in pH 6 histidine buffer. As shown in Table 17, at levels above 10 mg/mL, further addition of caffeine did not further reduce viscosity.

Example 24: Effect of caffeine during the TFF enrichment process was concentrated. Experiments were performed using a lab-scale TFF system manufactured by EMD Millipore (Billerica, Mass.). The system was fitted with a Pellicon XL TFF cassette containing a 30 kDa molecular weight cutoff Ultracel membrane (EMD Millipore, Billerica, MA). The nominal membrane surface area was 50 cm ² . Feed pumping to the cassette was maintained at 30 psi and retentate pressure was maintained at 10 psi. The filtrate flux was monitored during the course of the experiment by measuring its mass as a function of time. Approximately 12 grams of BGG was dissolved in 500 mL of buffer containing 15 mg/mL caffeine, 150 mM NaCl, and 20 mM histidine and adjusted to pH 6. A control sample was prepared by dissolving 12 grams of BGG in 500 mL of buffer containing 150 mM NaCl and 20 mM histidine and adjusting the pH to 6. Buffer components were purchased from Sigma-Aldrich. Both solutions were filtered through a 0.2 μm polyethersulfone (PES) filter (VWR, Radnor, PA) before TFF treatment. The performance of test and control samples in TFF was measured by mass transfer coefficients. Mass transfer coefficients were determined for each sample using the following equation (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= _kcln ( _Cw / _Cb ) (Equation 3)

Equation 3 represents the flux J near the filtrate, where k _c is the mass transfer coefficient, C _w is the protein concentration near the membrane, and C _b is the concentration in the liquid bulk, whereby in Eq. Calculation of the transfer coefficient k _c becomes possible. A graph of the calculated flux J versus ln(C _b ) gives a linear plot with a slope of −kc. where the flux J is calculated by taking the derivative of the filtrate mass with respect to time and C _b is calculated using the mass balance. The best approximation mass transfer coefficients are shown in Table 18. Introduction of 15 mg/mL caffeine increased mass transfer coefficient values by approximately 13% from 22.5 to 25.4 Lm ⁻² hr ⁻¹ (LMH).

Example 25: Effect of caffeine during the TFF enrichment process was concentrated. Experiments were performed using a lab-scale TFF system manufactured by EMD Millipore (Billerica, Mass.). The system was fitted with a Pellicon XL TFF cassette containing a 30 kDa molecular weight cutoff Ultracel membrane (EMD Millipore, Billerica, MA). The nominal membrane surface area was 50 cm ² . The control sample was adjusted to a nominal initial BGG concentration of 25.1 mg/mL by dissolving 14.6 grams of BGG in 582 mL of buffer containing 150 mM NaCl and 20 mM histidine adjusted to pH 6. was prepared to Materials were filtered through a 0.2 μm PES filter (VWR, Radnor, Pa.) and then processed with a TFF apparatus. The pump speed was adjusted so that the feed pressure was initially 30 psi and the retentate valve was adjusted so that the retentate pressure was initially 10 psi. The material was concentrated without adjusting either the pump speed or the retentate valve for 4.1 hours. The initial and final concentrations were determined by Bradford assay to be 25.4±0.6 and 159±6 mg/mL, respectively, as shown in Table 19 below. A caffeine-containing sample was 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 adjusted to pH 6 so that the initial BGG concentration was Prepared to nominally 25.1 mg/mL. Materials were filtered through a 0.2 μm PES filter (VWR, Radnor, Pa.) and then processed with a TFF apparatus. The pump speed and retentate valve were set at the same levels as before. As before, the feed and retentate pressures were determined to be 30 psi and 10 psi, respectively. The material was concentrated without adjusting either the pump speed or the retentate valve for 4.1 hours. The initial and final concentrations were determined by Bradford assay to be 24.4±0.5 and 225±10 mg/mL, respectively, as shown in Table 19 below. The use of caffeine during TFF treatment increased the final protein concentration by approximately 42% from 159 to 225 mg/mL when compared to controls.

式中、ｍ_pは、タンパク質の質量、ｂは、添加された緩衝液の容量、およびνは、ＢＧＧの部分比容で、ここでは０．７４ｍＬ／ｇとした。各サンプルの粘度は、２３℃の温度で、２５０ｓ^-1ノ剪断速度で、ｍｉｃｒｏＶｉｓｃレオメーター（ＲｈｅｏＳｅｎｓｅ、Ｓａｎ Ｒａｍｏｎ、ＣＡ）を使用して測定した。ＢＧＧ溶液を滅菌フィルターに通すのに必要なエネルギーは、１００Ｎのロードセル（Ｉｎｓｔｒｏｎ、Ｎｅｅｄｈａｍ、ＭＡ、部品番号２５１９－１０３）を装着した引張圧縮試験機（Ｔｅｎｓｉｌｅ Ｃｏｍｐｒｅｓｓｉｏｎ Ｔｅｓｔｅｒ（ＴＣＴ）、Ｉｎｓｔｒｏｎ、Ｎｅｅｄｈａｍ、ＭＡ、部品番号３３４３）を使用して測定した。シリンジのプランジャーは、５０ｍｍの距離を１５９ｍｍ／ｍｉｎの速度で押し下げた。エネルギー所要量は、ＴＣＴで測定された負荷対伸展曲線を積分することによって計算され、結果を、以下の表２０にまとめる。

Example 26: Effect of caffeine during sterile filtration of BGG solutions Bovine gamma globulin (BGG), L-histidine, and caffeine were obtained from Sigma-Aldrich (St. ) was purchased from Deionized (DI) water was produced from tap water using a Direct-Q 3 UV purification system from EMD Millipore (Billerica, Mass.). 25 mm polyethersulfone (PES) filters with 0.2 μm pores were purchased from GE Healthcare (Chicago, Ill., catalog number 6780-2502). 1-mL Luer-Lok syringes were 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, DI water and titrated to pH 6.0 with 1 M HCl. A 15 mg/mL solution of caffeine was prepared using histidine buffer. Caffeine-free and caffeine-containing buffers were used to reconstitute BGG to a final concentration of approximately 280 mg/mL. Protein concentration c was calculated using the following formula.

where _mp is the mass of protein, b is the volume of buffer added, and ν is the partial specific volume of BGG, here taken to be 0.74 mL/g. The viscosity of each sample was measured using a microVisc rheometer (RheoSense, San Ramon, Calif.) at a temperature of 23° C. and a shear rate of 250 s ⁻¹ . The energy required to force the BGG solution through the sterile filter was measured using a Tensile Compression Tester (TCT), Instron, Needham, MA, equipped with a 100 N load cell (Instron, Needham, MA, part number 2519-103). , part number 3343). The syringe plunger was pushed down a distance of 50 mm at a speed of 159 mm/min. Energy requirements were calculated by integrating the TCT-measured load vs. extension curves and the results are summarized in Table 20 below.

Example 27 Excipients to Improve Protein A Chromatography Elution Four purified research-grade biosimilar antibodies, ipilimumab, ustekinumab, omalizumab, and tocilizumab, were purchased from Bioceros (Utrecht, The Netherlands). These were provided as frozen aliquots of 20, 26, 15, and 23 mg/mL protein concentrations in aliquots of 40 mM aqueous sodium acetate, 50 mM Tris-HCl buffer (pH 5.5), respectively. Prior to measurement, protein solutions were thawed at room temperature and then filtered through 0.2 μm polyethersulfone filters. The filtered protein stock solution was mixed so that the ratio of protein stock solution to binding buffer was 1:1. The binding buffer used to facilitate antibody binding to protein A resin was composed of 0.1 M sodium phosphate and 0.15 sodium chloride at pH 7.2 in deionized (DI) water. . DI water was produced from tap water using a Direct-Q 3 UV purification system from EMD Millipore (Billerica, Mass.). These solutions were used to perform Protein A binding and elution studies using the PIERCE™ Protein-A Spin Plate for IgG Screening (ThermoFisher Scientific Catalog #45202). The plate had 96 wells, each containing 50 μL Protein A resin. The resin was washed with binding buffer by adding 200 μL of binding buffer to each well, the plate was centrifuged at 1000×g for 1 minute, and the flow-through was discarded. All subsequent centrifugation steps were performed at 1000 xg for 1 minute. This washing procedure was repeated once more. Following these initial washing steps, diluted protein samples, ie samples containing ipilimumab, ustekinumab, omalizumab, and tocilizumab, were added to the wells of the plate (200 μL per well). The plate was then placed on a Daigger Scientific (Vernon Hills, Ill.) Labgenius orbital shaker and agitated at 260 rpm for 30 minutes, followed by centrifugation of the plate and discarding the flow-through. The wells were then washed by adding 500 μL of binding buffer to each well, centrifuging the plate, and discarding the flow-through. This washing step was repeated twice. After these washing steps, proteins were eluted from the plates using elution buffers supplemented with different excipients. For each elution, 50 μL of neutralization buffer consisting of 1M sodium phosphate pH 7 was added to each well of the collection plate, and then 200 μL of elution buffer was added to each well of the plate. The plate was agitated at 260 rpm for 1 minute and then centrifuged. Flow-through was collected for analysis. This elution step was repeated one more time. Control buffers without excipients contained 20 mM citric acid and had a pH of 2.6. Since protein A elution buffers often contain some amount of salt, an elution buffer of 100 mM NaCl in citrate buffer was prepared as a secondary control.

Table 21 shows the excipient solutions used in this example, their concentrations, and the final pH of the elution buffer. excluding aspartame purchased from Herb Store USA (Los Angeles, Calif.), trehalose purchased from Cascade Analytical Reagents and Biochemicals (Corvallis, OR), and crosose purchased from Research Products International (Mt. Prospect, IL, product number S2406). All excipients were purchased from Sigma Aldrich (St. Louis, Mo.). All elution buffers containing excipients were prepared by mixing appropriate amounts of excipients with approximately 10 mL of no-salt citrate buffer control. Elution buffer was prepared at approximately 100 mM excipient. However, not all excipients are soluble at this level. Therefore, Table 21 shows all excipient concentrations used. The pH of each elution buffer was adjusted to approximately 2.6±0.1 using hydrochloric acid or sodium hydroxide as needed.

For each protein sample, ASD high performance size exclusion chromatography (ASD) 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). SEC) analysis was performed. Separations were performed at room temperature with a flow rate of 0.35 mL/min. The mobile phase was 100 mM sodium phosphate, 300 mM sodium chloride, pH 7 aqueous buffer. Protein concentration was monitored by absorbance at 280 nm using an Agilent 1100 series G1315B diode array detector. The total amount of protein eluted from the Protein A resin for each protein, ipilimumab, ustekinumab, omalizumab, and tocilizumab, was estimated by integrating the chromatograms. The integrated peak areas for each protein ipilimumab, ustekinumab, omalizumab, and tocilizumab are shown in Tables 22-25. Tables 22-25 also compare the experimental peak areas to the peak areas of the no-salt and salt-containing controls. Values greater than 100% indicate that more protein was recovered from the Protein A resin in the elution buffer than the control, while values less than 100% indicated less protein in the elution buffer than the control. It indicates that it was recovered from the A resin.

Example 28 Excipients to Improve Protein A Chromatography Elution The test proteins used in this example are the same as in Example 27, namely ipilimumab, ustekinumab, omalizumab, and tocilizumab . Protein A binding and elution studies were performed using plates identical to those in Example 27. The method of loading and eluting antibody from protein A plates was identical to that of Example 27, except for the elution step. In Example 27, two elution washes were performed. However, in this example only one wash was performed. As in Example 27, elution buffer was prepared from 20 mM citrate (pH 2.6), control buffer. Excipients are described in Table 26 below. All excipients were purchased from Sigma-Aldrich (St. Louis, Mo.). Proteins recovered in the same manner as in Example 27 were analyzed by HPLC and the results of protein recovery for each protein ipilimumab, ustekinumab, omalizumab, and tocilizumab are shown in Tables 26-30 below.

Example 29: Excipients that improve omalizumab elution from protein A chromatography columns ) was provided frozen at 15 mg/mL. Prior to experiments, proteins were thawed at room temperature and filtered through 0.2 μm polyethersulfone filters. The filtered material was mixed at a 1:1 ratio with a binding buffer consisting of 20 mM sodium phosphate (pH 7) in DI water. Tap water was purified with an EMD Millipore (Billerica, MA) Direct-Q 3 UV purification system to produce DI water. Protein A purification was performed using GE Healthcare HiTrap Protein-A HP 1 mL columns (Chicago, Ill., product number 29048576). For 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 washed with 5 mL binding buffer. After washing the column, bound omalizumab was eluted from the column using one fraction of elution buffer containing the excipients shown in Table 31 below. Elution buffers were prepared by dissolving the indicated excipients in 20 mM citrate buffer (pH 4.0). All elution buffers were adjusted to pH 4.0. Five 1 mL fractions were collected. Finally, Protein A was renatured by washing the column with 5 mL of 100 mM citrate (pH 3.0) buffer. The flow rate for each step was 1 mL/min and was maintained with a Fusion 100 infusion pump (Chemyx, Stafford, Tex.). A 10 mL NormJect Luer Lok syringe was used (Henke Sass Wolf, Tuttlingen, Germany, reference number 4100-000V0).

Eluted fractions, E1, E2, E3, E4, and E5, were determined 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). Separations were performed at room temperature with a flow rate of 0.35 mL/min. The mobile phase was 100 mM sodium phosphate, 300 mM sodium chloride, pH 7 aqueous buffer. Protein concentration was monitored by 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 chromatograms.

Citric acid was used here as a control as it is a common excipient used in Protein A chromatography. Eluted fractions of the control sample showed insoluble aggregates upon overnight storage at 4° C. as evidenced by the formation of a precipitated phase. Therefore, the peak areas reported in Table 31 below represent the amount of total soluble protein in the elution fractions. We note that insoluble aggregates were only observed in the control sample, none of the other samples showed such aggregates. A peak area greater than that of the control (using the citrate vehicle) indicates that the protein can be more efficiently separated from the column using the test vehicle.

Example 30: Formulations of BGG with Different Amounts of Caffeine Excipient is intended to mimic therapeutic proteins used in therapeutic formulations. The formulations of this example were prepared in 20 mM histidine buffer for viscosity measurements in the following manner. Stock solutions of 0 and 80 mM caffeine were prepared in 20 mM histidine and the pH of the resulting solutions was adjusted with small amounts of sodium hydroxide or hydrochloric acid to pH 6 before dissolving the model proteins. Additional solutions of varying caffeine concentrations were prepared by mixing the two stock solutions in varying volume ratios to provide a range of caffeine-containing solutions at the concentrations shown in Table 32 below. Once these excipient solutions were prepared, the test protein, bovine gamma globulin (BGG), was added to approximately Each test solution was dissolved in a ratio to achieve a final protein concentration of 280 mg/mL. Solutions containing BGG were prepared in 5 mL sterile polypropylene tubes and shaken overnight at 100 rpm on an orbital shaker table. These solutions were then transferred to 2 mL microcentrifuge tubes and centrifuged in an IEC MicroMax microcentrifuge at 2400 rpm for approximately 5 minutes to remove entrained air prior to viscosity measurements.

Viscosity measurements of formulations prepared as described above were performed with a microVisc viscometer (RheoSense, San Ramon, Calif.). The viscometer was equipped with an A-10 tip with a 100 micron deep channel and was operated at 25° C. with a shear rate of 250 s ⁻¹ . To measure the viscosity, the test formulation was loaded into the viscometer taking care to remove all air bubbles from the pipette. A pipette containing the loaded sample formulation was placed in the instrument and incubated at the measured temperature for approximately 5 minutes. The instrument was then run until the channel was fully equilibrated with the test fluid (indicated by a stable viscosity reading), after which the viscosity was recorded in centipoise. The viscosity results obtained are presented in Table 32 below.

Example 31 Preparation of Solutions of Co-Solutes in Deionized Water The compounds used as co-solutes to enhance the solubility of caffeine in water were obtained from Sigma-Aldrich (St. Included were proline, procaine HCl, ascorbic acid, 2,5-dihydroxybenzoic acid, lidocaine, saccharin, acesulfame K, tyramine, and aminobenzoic acid. A solution of each co-solute is prepared by dissolving the dry solids in deionized water, optionally adjusting the pH with 5 M hydrochloric acid or 5 M sodium hydroxide from a pH of about 6 to about pH values between 8 were adjusted. The solution was then diluted to a final volume of either 25 mL or 50 mL using a Class A volumetric flask and the concentration was recorded based on the mass of compound dissolved and the final volume of solution. The prepared solutions were used either neat or diluted with deionized water.

Example 32: Testing Caffeine Solubility The effect of different co-solutes on the solubility of caffeine at ambient temperature (approximately 23°C) was evaluated in the following manner. Dry caffeine powder (Sigma-Aldrich, St. Louis, Mo.) was added to 20 mL glass scintillation vials and the mass of caffeine recorded. In some cases, 10 mL of cosolute solution prepared according to Example 31 was added to the caffeine powder. In other cases, a blend of cosolute solution and deionized water was added to the caffeine powder to keep the final addition volume at 10 mL. The volume contribution of dry caffeine powder was considered negligible in any of these mixtures. A small magnetic stir bar was placed into the vial and the solution was vigorously mixed on a stir plate for approximately 10 minutes. Dissolution of the dry caffeine powder was observed in the vial after about 10 minutes, the results are shown in Table 33 below. These observations indicate that niacinamide, procaine HCl, 2,5-dihydroxybenzoic acid sodium salt, saccharin sodium salt, and tyramine chloride salt all fall below the reported caffeine solubility limits (Sigma-Aldrich, room temperature It allowed caffeine to dissolve at least about 4 times as much as about 16 mg/mL at

Example 33 Profile of HUMIRA® HUMIRA® (AbbVie Inc., Chicago, Ill.) is a commercial formulation of the therapeutic monoclonal antibody adalimumab, a TNF-alpha blocker commonly used in rheumatoid arthritis, It is prescribed to reduce the inflammatory response of autoimmune diseases such as psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate to severe chronic psoriasis and juvenile idiopathic arthritis. HUMIRA® is marketed in a single dose of 0.8 mL and contains 40 mg adalimumab, 4.93 mg sodium chloride, 0.69 mg monosodium phosphate dihydrate, 1.22 mg 0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 9.6 mg mannitol, and 0.8 mg polysorbate 80. A viscosity versus concentration profile for this formulation was generated in the following manner. An Amicon Ultra 15 centrifugal concentrator (EMD-Millipore, Billerica, Mass.) with a 30 kDa molecular weight cutoff was filled with approximately 15 mL of deionized water and centrifuged at 4000 rpm for 10 minutes in a Sorvall Legend RT (ThermoFisher Scientific) to remove membranes. Rinse. Residual water was then removed and 2.4 mL of HUMIRA® liquid formulation was added to the concentrate tube and centrifuged at 4000 rpm for 60 minutes at 25°C. The concentration of the retentate was determined by diluting 10 μL of the retentate with 1990 μL of deionized water, measuring the absorbance of the diluted sample at 280 nm, and using the dilution factor and an extinction coefficient of 1.39 mL/mg-cm to determine the concentration. Calculated. The viscosity of the concentrated samples was measured using a microVisc viscometer with an A05 tip (RheoSense, San Ramon, Calif.) at 23° C. and a shear rate of 250 s ⁻¹ . After viscosity measurement, the sample was diluted with a small amount of filtrate and the concentration and viscosity measurement were repeated. Using this process, viscosity values were generated at different adalimumab concentrations, as shown in Table 34 below.

Example 34 Reformulation of HUMIRA® with Viscosity-Lowering Excipients Describe the process. A solution of viscosity-lowering excipient in 20 mM histidine was prepared by dissolving approximately 0.15 g of histidine and 0.75 g of caffeine (Sigma-Aldrich, St. Louis, Mo.) in deionized water. The pH of the resulting solution was adjusted to about 5 with 5M hydrochloric acid. This solution was then diluted with deionized water to a final volume of 50 mL in a volumetric flask. The resulting buffered viscosity-lowering excipient solution was then used to reformulate HUMIRA® at high mAb concentrations. Approximately 0.8 mL of HUMIRA® is then added to rinsed Amicon Ultra 15 centrifugal concentrator tubes (30 kDa molecular weight cutoff) and centrifuged for 8-10 minutes at 4000 rpm at 25° C. in a Sorvall Legend RT. separated. Approximately 14 mL of the buffered viscosity-lowering excipient solution prepared as described above was then added to the concentrated HUMIRA® in the centrifugal concentrator. After gentle mixing, the samples were centrifuged at 4000 rpm at 25° C. for approximately 40-60 minutes. The retentate was a concentrated sample of HUMIRA® reformulated in buffer with viscosity-lowering excipients. Samples were measured for viscosity and concentration, and in some cases were diluted with a small amount of filtrate to measure viscosity at a lower concentration. Viscosity measurements were completed using a microVisc viscometer as was the case for the concentrated HUMIRA® formulations in the previous example. Concentrations were determined by the Bradford assay using a standard curve generated from HUMIRA® stock solutions diluted in deionized water. Reformulation of HUMIRA® with viscosity-lowering excipients resulted in increased viscosity values for HUMIRA® concentrated in non-reformulated commercial buffers compared to the viscosity values shown in Table 35 below. resulted in a viscosity reduction of 30% to 60% as indicated.

Example 35: Improved Stability of Adalimumab Solutions with Caffeine as an Excipient was evaluated after exposure to . The adalimumab drug formulation HUMIRA® (AbbVie) was used and has the properties described in more detail in Example 33. HUMIRA® samples were concentrated in the original buffer to an adalimumab concentration of 200 mg/mL as described in Example 38. This concentrated sample is referred to as "Sample 1". A second sample was prepared as described in Example 40 with approximately 200 mg/mL adalimumab and 15 mg/mL added caffeine. This concentrated sample with added caffeine is called "Sample 2". Both samples were diluted with diluent to a final concentration of 1 mg/mL of adalimumab as follows. Sample 1 diluent is the original buffer solution and Sample 2 diluent is 20 mM histidine, 15 mg/mL caffeine, pH=5. Both HUMIRA® dilutions were filtered through a 0.22 μm syringe filter. Three batches of 300 μL each in 2 mL Eppendorf tubes were prepared for each diluted sample in a laminar flow hood. The samples were subjected to the following stress conditions. For agitation, samples were placed on an orbital shaker at 300 rpm for 91 hours. For freeze-thaw, samples were repeated 7 times for an average of 6 hours at −17 to 30° C. for each condition. Table 36 shows the samples prepared.

Example 36 Evaluation of Stability by Dynamic Light Scattering (DLS) A Brookhaven Zeta Plus dynamic light scattering instrument was used to measure the hydrodynamic radius of the adalimumab molecules in the samples of Example 35 and Looked for evidence of formation. Table 37 shows DLS results for six samples prepared according to Example 35. Some of them (1-A, 1-FT, 2-A and 2-FT) were subjected to stress conditions ("stress samples"). The others (1-C and 2-C) were not stressed. The DLS data in Table 37 and accompanying Figures 1, 2, and 2 show the multimodal particle size distribution of the monoclonal antibody in the caffeine-free stress samples. In the absence of caffeine as an excipient, the stressed samples 1-A and 1-FT had a larger effective diameter than the unstressed sample 1-C, and also had a significantly larger diameter. 2 populations of particles are shown. This new group of particles with larger diameters is evidence of agglomeration into invisible particles. The caffeine-containing stressed samples (Samples 2-A and 2-FT) are similar in size to the unstressed sample 2-C, exhibiting only one particle population. These results indicate that the addition of caffeine to these samples reduced the formation of aggregates or sub-visible particles.

Tables 38A and 38B display the DLS raw data for the adalimumab samples from Example 36, showing the particle size distribution. In these tables, G(d) is the intensity-weighted difference size distribution. C(d) is the cumulative intensity-weighted difference size distribution.

Example 37: Evaluation of Stability by Size Exclusion Chromatography (SEC) Using size exclusion chromatography, stressed and unstressed adalimumab samples described in Example 36 showed a size of about 0.1. Sub-micron sub-micron sub-visible particles were detected. To perform SEC, a TSKgel SuperSW3000 column (Tosoh Biosciences, Montgomeryville, Pa.) equipped with a guard column was used and elution was monitored at 280 nm. A total of 10 μL of each stressed and unstressed sample from Example 36 was aliquoted with pH 6.2 buffer (100 mM phosphate, 325 mM NaCl) at a flow rate of 0.35 mL/min. , eluted isocratically. The retention time for adalimumab monomer was approximately 9 minutes. No detectable aggregates were observed in the samples containing the caffeine excipient and the amount of monomer in all three samples remained constant.

Example 38 Viscosity Reduction of HERCEPTIN® Formulations Monoclonal antibody trastuzumab (HERCEPTIN®, Genentech) was received as a lyophilized powder and reconstituted to 21 mg/mL with DI water. The resulting solution was concentrated in situ by centrifugation at 3500 rpm for 1.5 hours in Amicon Ultra 4 centrifugal concentration tubes (molecular weight cutoff, 30 kDa). Concentration was determined by diluting the sample 200-fold with the appropriate buffer and measuring absorbance at 280 nm using an extinction coefficient of 1.48 mL/mg. Viscosity was measured using a RheoSense microVisc viscometer.

Excipient buffers were prepared by dissolving salicylic acid and caffeine alone or in combination, dissolving histidine and excipients in distilled water and adjusting the pH to the appropriate level. The conditions for buffer systems 1 and 2 are summarized in Table 39.

The HERCEPTIN® solution was diluted with excipient buffer at a ratio of approximately 1:10 and concentrated with Amicon Ultra 15 (MWCO 30 kDa) concentration tubes. Concentrations were determined using the Bradford assay and compared to a standard calibration curve prepared from stock HERCEPTIN® samples. Viscosity was measured using a RheoSense microVisc viscometer. Table 40 below shows concentration and viscosity measurements for various HERCEPTIN® solutions, with buffer systems 1 and 2 referencing those listed in Table 39.

Buffer system 1 containing both salicylic acid and caffeine reduced viscosity by up to 76% at 215 mg/mL compared to the control sample. Buffer system 2, containing only caffeine, reduced viscosity by up to 59% at 200 mg/mL.

Example 39 Viscosity Reduction of AVASTIN® Formulations AVASTIN® (monoclonal antibody bevacizumab, formulation marketed by Genentech) was received as a 25 mg/mL solution in histidine buffer. Samples were concentrated in Amicon Ultra 4 centrifugal concentration tubes (MWCO 30 kDa) at 3500 rpm. Viscosity was measured with a RheoSense microVisc and concentration was determined by absorbance at 280 nm (extinction coefficient, 1.605 mL/mg). Excipient buffer was prepared by adding 10 mg/mL caffeine with 25 mM histidine HCl. AVASTIN® stock solutions were diluted in excipient buffer and then concentrated in Amicon Ultra 15 centrifugal concentrator tubes (MWCO 30 kDa). Excipient sample concentrations were determined by the Bradford assay and viscosities were measured using a RheoSense microVisc. The results are shown in Table 41 below.

When AVASTIN® was concentrated to 213 mg/ml with 10 mg/mL caffeine, it showed a viscosity reduction of up to 73% compared to control AVASTIN® samples.

Example 40 Preparation of Formulations Containing Caffeine, a Secondary Excipient and a Test Protein The test protein prepared using, is intended to mimic the therapeutic protein used in the therapeutic formulation. Such formulations were prepared in 20 mM histidine buffer containing different excipient compounds for viscosity measurements in the following manner. Excipient combinations (Excipients A and B listed in Table 42 below) were dissolved in 20 mM histidine and the pH of the resulting solution was adjusted to a small amount of hydroxylation prior to dissolving the model protein. Adjusted to pH 6 with sodium or hydrochloric acid. Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved in a ratio to achieve a final protein concentration of approximately 280 mg/mL. Solutions of BGG in excipient solution were formulated into 20 mL glass scintillation vials and shaken overnight at 80-100 rpm on an orbital shaker table. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for approximately 10 minutes to remove entrained air prior to viscosity measurements.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer at a volume of 0.5 mL and incubated at the given shear rate and temperature for 3 minutes followed by a measurement collection time of 20 seconds. This was followed by two additional steps consisting of a 1 minute shear incubation followed by a 20 second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample in Table 42 below. The viscosities of solutions containing excipients were normalized to the viscosities of model protein solutions without excipients. Normalized viscosity is the ratio of the viscosity of a model protein solution containing excipients to the viscosity of a model protein solution without excipients.

Example 41 Preparation of Formulations Containing Dimethylsulfone and Test Proteins Formulations were prepared using dimethylsulfone (Jarrow Formulas, Los Angeles, Calif.) as the excipient compound and the test protein, which was used in the therapeutic formulation. It is intended to mimic therapeutic proteins used in Such formulations were prepared in 20 mM histidine buffer for viscosity measurements in the following manner. Dimethyl sulfone is dissolved in 20 mM histidine and the pH of the resulting solution is adjusted with a small amount of sodium hydroxide or hydrochloric acid to pH 6 and then filtered through a 0.22 micron filter before dissolving the model protein. bottom. Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a concentration of approximately 280 mg/mL. Solutions of BGG in excipient solution were formulated into 20 mL glass scintillation vials and shaken overnight at 80-100 rpm on an orbital shaker table. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for approximately 10 minutes to remove entrained air prior to viscosity measurements.

Viscosity measurements of formulations prepared as described above were performed using a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer at a volume of 0.5 mL and incubated at the given shear rate and temperature for 3 minutes followed by a measurement collection time of 20 seconds. This was followed by two additional steps consisting of a 1 minute shear incubation followed by a 20 second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of solutions containing excipients were normalized to the viscosities of model protein solutions without excipients. The normalized viscosity recorded in Table 43 is the ratio of the viscosity of the model protein solution with excipient to the viscosity of the model protein solution without excipient.

Example 42: Preparation of Buffers A buffer of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), 50 mM glycine, 35 mM caffeine was added to 0.392 g MES monohydrate, 0.374 g of glycine, and 0.682 g of caffeine were prepared by dissolving in 90 mL of Milli-Q ultrapure water. After all contents were dissolved, the pH of the solution was adjusted to 5.5 and the final volume was brought to 100 mL by adding Milli-Q ultrapure water to the volumetric flask. The buffer was then vacuum filtered through a 0.2 μm PES filter using a bottle top filter apparatus. A similar buffer containing 20 mM histidine, 50 mM glycine, and 35 mM caffeine was similarly prepared.

A control buffer of 20 mM tris(hydroxymethyl)aminomethane (TRIS), 100 mM sodium chloride, 55 mM mannitol and 0.1 mM diethylenetriaminepentaacetic acid (DTPA) was 1.211 g TRIS, 2.938 g sodium chloride. , was prepared by dissolving 2.098 g of mannitol and 0.019 g of DTPA in 450 mL of Milli-Q ultrapure water. After all contents were dissolved, the pH of the solution was adjusted to 7.0 and the volume was adjusted to 500 mL by adding Milli-Q ultrapure water to the volumetric flask. The buffer was vacuum filtered through a 0.2 μm PES filter using a bottle top filter apparatus.

Example 43: Ipilimumab Formulations A sample of the monoclonal antibody ipilimumab was obtained from Bioceros (The Netherlands) and buffered using Amicon Ultra 15 centrifugal concentrator tubes with a molecular weight cutoff of 30 kDa (EMD Millipore, Billerica, Mass.). Replaced with the three prepared buffers of Example 42. The target final protein concentration was 20 mg/mL and the final concentration was measured by absorbance at 280 nm (A280) using a Synergy HT plate reader (BioTek, Winooski, Vt.). The absorbance of the protein solution was subtracted from the absorbance of the blank buffer. The absorbance of the blank-subtracted protein solution is divided by the reported extinction coefficient and then multiplied by the protein dilution factor (20-fold) to determine the final protein concentration. Since caffeine interferes with absorbance measurements at 280 nm, protein concentrations of solutions containing caffeine were determined by mass balance against protein solutions measured at A280. This gave an approximate concentration of the A280 protein solution determined on a mass basis.

The prepared protein solutions were then added to 384-microwell plates (Aurora Microplates, Whitefish, MT). Each solution was loaded into triplicate wells at 35 μL per well. Microwell plates were then centrifuged at 400×g in a Sorvall Legend RT centrifuge to remove any trapped air pockets. Pre-cut pressure-sensitive sealing tape (Thermo Scientific) was applied to the top of the microwell plate to prevent evaporation before being placed in the DLS instrument (DynaPro II DLS plate reader, Wyatt Technology Corp., Goleta, Calif.). bottom. The sample compartment of the DLS instrument was kept at 65° C. and the particle size of the protein solution was recorded for 9 hours. Table 44 shows the radial size of ipilimumab in three different formulations at 1 hour measurements.

Example 44 Testing the Stability of Protein Formulations by Urea Denaturation Urea is known to denature and unfold proteins in solution. The screening methodology in this example involved adding a specific concentration of urea to a therapeutic protein solution, such as ustekinumab. This example is based on the hypothesis that protective excipients prevent or reduce unfolding of therapeutic proteins in the presence of urea, and by measuring the amount of unfolded protein, This makes it possible to identify excipients that are effective in stabilizing proteins. One method of tracking protein unfolding involves the use of exogenous fluorescent dyes such as Sypro orange. Sypro orange binds to hydrophobic regions of unfolded protein structures, leading to an increase in the observed fluorescence signal. Therefore, measuring the difference in fluorescence intensity of unfolded protein-Sypro orange complexes in the presence of various excipients can identify the stabilizing effect.

All excipients used in this example, listed in Table 45 below, were of the highest purity and were obtained from Sigma Aldrich (St. Louis, Mo.) or Cayman Chemical (Ann Arbor, Mich.) . Stock solutions of excipients were prepared by dissolving each excipient at a concentration of 100 mg/mL in 20 mM histidine buffer (pH 6.0). Histidine buffer was prepared by dissolving 1.55 g of histidine in 0.500 L of Milli-Q water and adjusting the pH to 6.0 using 1 M HCl. Protein formulations containing excipients were then prepared by mixing each excipient preparation (5 mg/mL final concentration) with ustekinumab (1 mg/mL final concentration). For use in this example, a 9 M urea stock solution was prepared by dissolving 27 g of urea in the same histidine buffer and adjusting the pH to 6.0 using 1 M HCl. This urea stock solution was then added to a final concentration of 6M to form the test solution (excipients + protein + urea). Sypro orange dye from the stock solution (5000X) was then added respectively to a final concentration of 20X. The pH of the mixture was checked again and found to be pH 6.0. The test solutions were incubated for 30 minutes at room temperature. 200 μL of samples were transferred to Greiner CellStar blackwell clear flat-bottom 96-well plates and the fluorescence of each sample was measured using a BioTek Synergy HT plate reader with 485 nm excitation and 590/20 nm emission filters. The fluorescence intensity of different test formulations is compared to that of a control formulation (protein and urea, without any excipients) and those test formulations that show decreased fluorescence contain stabilizing excipients. I thought. As shown in Table 45 below, several excipients reflected increased stability compared to the control when their fluorescence was compared to that of the control. These conclusions were drawn from the ability of excipients to stabilize proteins to correlate with their ability to reduce the fluorescence measured during the experiment. Stabilizing excipients prevent or reduce protein unfolding, leading to a decrease in protein-Sypro orange interactions, which is subsequently manifested as a decrease in fluorescence intensity. The results of these tests are summarized in Table 45 below.

Example 45: Stabilization of Protein Formulations at Low pH Therapeutic proteins, particularly antibodies, are exposed to low pH solution conditions during various stages of processing, particularly during purification and virus removal. This exposure to acidic pH conditions can cause conformational changes, which in turn lead to protein unfolding and aggregation. Screening methodologies to identify stabilizing excipients include incubating therapeutic proteins such as omalizumab at acidic pH. Protective excipients prevent or reduce denaturation of therapeutic proteins at low pH and are therefore effective in stabilizing proteins in the presence of low pH when measured by the amount of unfolded protein. excipients can be identified. The unfolding of therapeutic proteins at acidic pH can be followed using an exogenous fluorescent dye such as Sypro orange. Addition of Sypro orange (Thermo-Fisher, Waltham Mass.), as done in this example, binds the dye to the hydrophobic regions of the unfolded protein, leading to an increase in fluorescence signal. Therefore, measuring the difference in fluorescence intensity of the unfolded protein-Sypro orange in the presence of different excipients allows the identification of stabilizers.

All excipients used in this example, listed in Table 46 below, were of the highest purity and were obtained from Sigma Aldrich (St. Louis, Mo.) or Cayman Chemical (Ann Arbor, Mich.) . Stock solutions of excipients were prepared by dissolving each excipient at a concentration of 100 mg/mL in 0.15 M glycine buffer (pH 2.6). The acidification buffer was prepared by dissolving 1.65 g of histidine in 0.09 L of Milli-Q water, adjusting the pH to 2.6 using 1 M HCl and bringing the volume to 0.100 L. Protein formulations containing each excipient were then prepared by mixing the excipient preparation (5 mg/mL final concentration) with ustekinumab (1 mg/mL final concentration). Glycine acidified buffer was then added to the excipient-protein mixture, followed by Sypro orange dye from the stock solution (5000X) to a final concentration of 20X. 200 μL of samples were transferred to Greiner CellStar blackwell clear flat-bottom 96-well plates and the fluorescence of each sample was measured using a BioTek Synergy HT plate reader with an excitation wavelength of 485 nm and an emission filter wavelength of 590/20 nm. The fluorescence intensity of the different test formulations was compared to that of the control formulation (protein and glycine buffer, without any excipients) and those showing decreased fluorescence were those containing the stabilizing excipients. thought. As shown in Table 46, several excipients reflected increased stability compared to the control when their fluorescence was compared to that of the control. These conclusions were drawn from the ability of excipients to stabilize proteins to correlate with their ability to reduce the fluorescence measured during the experiment. Stabilizing excipients prevent or reduce protein unfolding, leading to a decrease in protein-Sypro orange interactions, which is subsequently manifested as a decrease in fluorescence intensity. The results of these tests are summarized in Table 46 below.

Example 46 Testing Excipients as Thermal Stabilizers Therapeutic proteins are frequently exposed to temperature fluctuations, which can lead to changes in tertiary and secondary structural elements. This can lead to protein aggregation and reduce the amount of active native protein. Excipients that protect against heat stress were identified in this example by heat degradation studies in the presence or absence of the excipients listed in Table 47 below. All excipients used in this example, listed in Table 47 below, were of the highest purity and were obtained from Sigma Aldrich (St. Louis, Mo.) or Cayman Chemical (Ann Arbor, Mich.) . Stock solutions of excipients were prepared by dissolving each excipient at a concentration of 100 mg/mL in 20 mM histidine buffer (pH 6.0). Histidine buffer was prepared by dissolving 1.55 g of histidine in 0.500 L of Milli-Q water and adjusting the pH to 6.0 using 1 M HCl. Protein formulations containing excipients were then prepared by mixing each excipient preparation (5 mg/mL final concentration) with ustekinumab (1 mg/mL final concentration). The formulation was aliquoted into 0.2 mL microcentrifuge tubes and incubated in a heating block at 65° C. for 120 minutes. Aliquots were taken at 0, 15, 30, 60, 90, and 120 minutes. Samples were chilled on ice for 5 minutes and spun down at 5000 rpm for 10 minutes. Supernatants were then loaded onto an Agilent 1100 HPLC system equipped with a TSKgel SW3000 column (30 cm x 4.8 mm ID) and an Agilent G1351B diode array detector set at 280 nm and samples were analyzed by size exclusion HPLC. A mobile phase of 50 mM phosphate buffer, 100 mM NaCl, pH 6.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the monomer peak area and the change in integrated peak area was plotted as a function of time. Thermal stability correlated with the percentage of monomer remaining at the end of the 2 hour incubation.

Example 47 Testing Excipients as Stabilizers Against Mechanical Shear Stress Therapeutic proteins are often subjected to mechanical stress by agitation, agitation, etc., and the applied shear stress causes protein aggregation. there is a possibility. For excipients that provide protection against shear stress, stir the therapeutic protein solution in the presence of the test excipient (listed in Table 48 below) and observe the change in the number of aggregated particles. identified by All excipients used in this example, listed in Table 48 below, were of the highest purity and were obtained from Sigma Aldrich (St. Louis, Mo.) or Cayman Chemical (Ann Arbor, Mich.) . Stock solutions of excipients were prepared by dissolving each excipient at a concentration of 100 mg/mL in Milli-Q water. Excipient-containing protein formulations were then prepared by combining each excipient preparation at a final concentration of 0.5 mg/mL with omalizumab at a final concentration of 2 mg/mL. Samples were transferred to 0.5 mL cryogenic vials (ThermoFisher, Waltham, Mass.) and fixed on an orbital shaker. They were then incubated for 72 hours at 22° C. with agitation set at 300 rpm. At the end of the incubation period, 100 μL of each sample was transferred to a 96-well plate and absorbance was measured at 350 nm. Increased aggregation is observed by observing changes in light scattering at 350 nm and comparing those changes to the light scattering of a control formulation (prepared identically to the test sample, without added excipients) at 350 nm. measured by Excipients that are protective in nature were identified by their ability to slow the rate of aggregation and reduce A350 absorbance values after the agitation step.

Example 48: Freeze-Thaw Stability of Protein Solutions Containing Excipients All excipients used in this example, listed in Table 49 below, were purchased from Sigma Aldrich (St. It was obtained from Cayman Chemical (Ann Arbor, Mich.). Stock solutions of test excipients (listed in Table 49) were prepared by dissolving each excipient at a concentration of 100 mg/mL in 20 mM histidine buffer (pH 6.0). A buffer was prepared by dissolving 1.55 g of histidine in 0.500 L of Milli-Q water and adjusting the pH to 6.0 using 1 M HCl. Excipient-containing protein formulations were then prepared by combining each excipient preparation at a final concentration of 5 mg/mL with omalizumab at a final concentration of 5 mg/mL. 0.4 mL of each formulation was transferred to wells in a 96-well polypropylene plate (Advangene, Ill.). Samples were frozen to −80° C. in a freezer and thawed at room temperature for at least 5 cycles before transferring 100 μL of sample to a Greiner CellStar Blackwell clear flat-bottom 96-well plate. The stabilizing effect of excipients was measured using light scattering analysis at 350 nm to measure the formation of protein aggregates and their changes compared to control formulations at 350 nm (no excipients added, tested (prepared similarly to the sample) were analyzed by comparing the light scattering. Excipients that are protective in nature were identified by their ability to slow the rate of aggregation and reduce A350 absorbance values after the agitation step.

Example 49: Examination of excipients by DLS diffusion interaction parameter _k Histidine (Sigma-Aldrich, St. Louis, Mo.) was prepared by dissolving it in Type 1 ultrapure water. The resulting solution was titrated to pH 6 by dropwise addition of 1M hydrochloric acid. After pH adjustment, the buffer was diluted with type 1 ultrapure water to a final volume of 1 L in a volumetric flask. All excipients used in this example, listed in Table 50 below, were of the highest purity and were obtained from Sigma Aldrich (St. Louis, Mo.) or Cayman Chemical (Ann Arbor, Mich.) .

Using the proteins listed in Table 50, a series of six test protein solutions with protein concentrations ranging from about 4 mg/mL to about 20 mg/mL were prepared, all in 20 mM His HCl buffer at pH 6. . In a 384-well microplate (Aurora Microplates, Whitefish, MT), 15 μL of protein solution was prepared in 20 mM HisHCl buffer at pH 6 using the excipients listed in Table 50 to obtain each Excipients were tested at 6 different protein concentrations. Microplates containing protein-excipient combinations were centrifuged at 400×g in a Sorvall Legend RT centrifuge and shaken on a plate shaker to mix the samples well. A second centrifugation step was completed to remove air bubbles. The diffusion interaction parameter (k _D ) of these protein-excipient formulations has been evaluated using dynamic light scattering (DLS) in dilute solutions as a method to investigate the effect of excipients on protein-protein interactions (PPI). Measured by To perform DLS studies, the microplates prepared above were loaded into a DynaPro II DLS plate reader (Wyatt Technologies Corp., Goleta, CA) and the diffusion coefficient of each sample was measured at 25°C. For test solutions containing each excipient, the measured diffusion coefficient was plotted as a function of protein concentration and the slope of the linear fit of the data was recorded as _kD . A more negative k _D indicated a stronger net attractive PPI and a more positive k _D indicated a stronger net repulsive PPI. Table 50 shows the _kD values for the test solutions containing each excipient, and these test values for each excipient were compared to those of the control solution (protein in histidine buffer, but no excipient). _kD values can be compared.

Example 50 Testing Excipients for Viscosity Reduction The biosimilar monoclonal antibodies Omalizumab and Ustekinumab obtained from Bioceros (Netherlands) were buffer exchanged into 20 mM His HCl buffer at pH 6 and treated with Amicon Ultra with a molecular weight cutoff of 30 kDa. 15 centrifugal concentration tubes (EMD Millipore, Billerica, Mass.) were used to concentrate. The resulting concentrate was prepared by serially diluting the concentrate with 20 mM His HCl and loading 100 μL of each dilution into a UV-clear 96 half-well microplate (Greiner Bio-One, Austria) and run on a Synergy HT plate reader ( Protein concentration was analyzed by absorbance at 280 nm by measuring absorbance at 280 nm on a BioTek, Winooski, Vt.). The blank pathlength-corrected absorbance measurements were then divided by the respective extinction coefficient and multiplied by the dilution factor to determine the protein concentration. Excipient solutions were prepared at 10X the desired final concentration in 20 mM HisHCl (pH 6) or at the solubility limit of the compound and adjusted to pH 6 with concentrated hydrochloric acid or sodium hydroxide as necessary. The concentrated protein formulations were then added to 384-well microplates (Aurora Microplates, Whitefish, MT) in a 10X excipient solution of the excipients listed in Table 51 below (9 parts protein, 1 part of excipient solution or buffer). All excipients used in this example, listed in Table 51 below, were of the highest purity and were obtained from Sigma Aldrich (St. Louis, Mo.) or Cayman Chemical (Ann Arbor, Mich.) . Each sample was diluted in the same volume, so the protein concentration in each sample was the same. Microplates were then centrifuged at 400 xg in a Sorvall Legend RT centrifuge and shaken on a plate shaker. After shaking, a 5-fold dilution of polyethylene glycol surface-modified gold nanoparticles (nanoComposix, San Diego, Calif.) in 2 μL of 20 mM His HCl was added to each sample well. The microplate was shaken again to mix the gold nanoparticles with the sample and then placed in a DynaPro II DLS plate reader (Wyatt Technology Corp., Goleta, Calif.) to determine the apparent particle size of the gold nanoparticles at 25°C. Measured in The ratio of the apparent particle size of the gold nanoparticles in the protein formulation to the known particle size of the gold nanoparticles in water was used to determine the viscosity of the protein formulation according to the Stokes-Einstein equation. In this example, the ratio of the apparent radius of the gold nanoparticles to the actual radius was multiplied by the viscosity of water at 25° C. to calculate the viscosity of the protein formulation in centipoise (cP). The results of these tests are summarized in Table 51 below.

Example 51: Thermal Degradation Assay Using Infliximab REMICADE® Infliximab was obtained from the Clinigen Group and reconstituted according to the Janssen package insert instructions, supplemented with 50 mg/ml sucrose and 0.05 mg/mL polysorbate 80. A 10 mg/mL solution of infliximab in 5 mM phosphate buffer (approximately pH 7) containing The reconstituted drug product was then mixed 1:1 by volume with 50 mM sodium acetate buffer (pH 5). The resulting solution was then injected onto a small scale preparative scale cation exchange column (GE Healthcare, Chicago, Ill.). After loading the infliximab onto the column, the column was washed with 10 column volumes of 50 mM sodium acetate buffer (pH 5). Infliximab was then eluted from the column with 5 column volumes of 250 mM sodium chloride, 50 mM sodium acetate buffer (pH 5). The eluted infliximab was then buffer exchanged into 20 mM phosphate buffer, pH 7 using Amicon Ultra 15 (EMD Millipore, Billerica, Mass.) centrifugal concentrators with a molecular weight cutoff of 30 kDa (EMD Millipore, Billerica, MA). Stock solutions of 4 or 8 mg/mL infliximab in 20 mM phosphate buffer at pH 7 were then used in subsequent studies.

All excipients used in this example, listed in Table 52 below, were of the highest purity and were obtained from Sigma Aldrich (St. Louis, Mo.) or Cayman Chemical (Ann Arbor, Mich.) . The stock infliximab solution was mixed with a stock excipient solution containing the excipients listed in Table 52 formulated in 20 mM phosphate buffer, pH 7 to give a final concentration of infliximab of approximately 2 mg/ml in each sample. Make up to mL. Samples containing infliximab solution and stock excipient solutions were then dispensed into PCR tubes in five 100 μL aliquots. Heat stress was applied by incubating aliquots in dry bath (Benchmark Scientific, Sayreville, NJ) at 55° C. for different times ranging from 15 minutes to about 3 hours. Samples were then removed from the dry bath and placed on ice to quench thermal aggregation. These stressed samples were then subjected to high performance size exclusion chromatography (HP-SEC) for monomer content using an Agilent 1100 series HPLC equipped with a diode array detector monitoring absorbance at 280 nm. ). HPLC was performed through a TSKgel SuperSW3000 4.6 mm x 30 cm column (Tosoh Bioscience, Tokyo, Japan) at a flow rate of 0.35 mL/min using a column temperature of 25° C. and a mobile phase of 100 mM phosphoric acid, 300 mM NaCl, pH 7. operated. For each sample, the monomer peak area was divided by the monomer peak area obtained from the same unstressed sample to determine the percent monomer remaining after exposure to heat stress. . The percent monomer remaining in the unstressed samples was then plotted as a function of incubation time and the absolute value of the slope of the linear fit to the data was recorded as the percent loss of monomer. The determined percent monomer loss was then normalized by dividing the percent monomer loss by the percent monomer loss of the buffer control without excipients and the results are shown in Table 52 below.

Example 52: DLS Viscosity Measurement of Concentrated Omalizumab Using Nicotinamide Mononucleotide and Itaconic Acid Nicotinamide mononucleotide (NMN) was collected from nutritional supplement capsules purchased from Genex Formulas (Orlando, FL) and itaconic acid Sigma-Aldrich (St. Louis, Mo.). These substances were used as excipients in the following experiments.

The biosimilar monoclonal antibody Omalizumab obtained from Bioceros (Netherlands) was buffer exchanged into 20 mM His HCl buffer at pH 6 and transferred to Amicon Ultra 15 centrifugal concentrator tubes (EMD Millipore, Billerica, Mass.) with a 30 kDa molecular weight cutoff. was used to concentrate. A buffer was prepared by dissolving 1.55 g of histidine in 0.5 L of Milli-Q water and adjusting the pH to 6.0 using 1 M HCl. The resulting concentrate was prepared by serially diluting the concentrate with 20 mM His HCl and loading 100 μL of each dilution into a UV-clear 96 half-well microplate (Greiner Bio-One, Austria) and run on a Synergy HT plate reader ( Protein concentration was analyzed by A280 by measuring absorbance at 280 nm on a BioTek, Winooski, Vt.). The blank pathlength-corrected A280 measurement of each sample was then divided by the respective extinction coefficient and multiplied by the dilution factor to determine the protein concentration. Stock excipient solutions were prepared at 20 mM HisHCl (pH 6) using the excipients described above at 1 M or the solubility limit of the compound, and diluted with concentrated hydrochloric acid or sodium hydroxide as needed. The pH was adjusted to 6. The concentrated protein formulation is then mixed with the stock excipient solution or control at a ratio of 9 parts protein formulation: 1 part excipient solution or buffer (as control) and aliquots are placed in 384-well microplates. (Aurora Microplates, Whitefish, MT) wells. Microplates were then centrifuged at 400 xg in a Sorvall Legend RT and shaken on a plate shaker. After shaking the microplate, a 5-fold dilution of polyethylene glycol surface-modified gold nanoparticles with a diameter of 100 nm (nanoComposix, San Diego, Calif.) in 2 μL of 20 mM His HCl was added to each sample well. The microplate was shaken again to mix the gold nanoparticles with the sample, which was then placed in a DynaPro II DLS plate reader (Wyatt Technology Corp., Goleta, Calif.) to determine the apparent particle size of the gold nanoparticles. Measured at 25°C. The ratio of the apparent particle size of the gold nanoparticles in the protein formulation to the apparent particle size of the gold nanoparticles in the buffer (no protein) was used to determine the viscosity of the protein formulation according to the Stokes-Einstein equation. . In this example, the ratio of the apparent radius of the gold nanoparticles to the actual radius was multiplied by the viscosity of water at 25° C. to calculate the viscosity of the protein formulation in centipoise (cP). Results using two different excipients are shown in Table 53 below.

Example 53: DLS Viscosity Measurements of Concentrated Omalizumab 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), dicyclomine hydrochloride, pridinol methanesulfonic acid, 1-butylimidazole, 1-hexylimidazole are available from Sigma It was purchased from Aldrich (St. Louis, Mo.) and used in this example to prepare excipient solutions. O-(octylphosphoryl)choline was purchased from Sigma Aldrich (St. Louis, Mo.) as a 1 M solution and used as the stock excipient solution in this example.

A biosimilar concentrate preparation of the monoclonal antibody omalizumab obtained from Bioceros (The Netherlands) was prepared as described in Example 52 and analyzed for protein concentration as described in Example 52. Stock excipient solutions using the excipients described above were prepared in 20 mM HisHCl (pH 6) buffer (prepared as described in Example 52) at 1 M or the solubility limit of the compound and and adjusted to pH 6 with concentrated hydrochloric acid or sodium hydroxide. O-(octylphosphoryl)choline was used as a stock excipient without additional preparation. The concentrated protein formulation is then mixed with the stock excipient solution or control at a ratio of 9 parts protein formulation: 1 part excipient solution or buffer (as control) and aliquots are placed in 384-well microplates. (Aurora Microplates, Whitefish, MT) wells. Microplates were then centrifuged at 400 xg in a Sorvall Legend RT and shaken on a plate shaker. After shaking the microplate, a 5-fold dilution of polyethylene glycol surface-modified gold nanoparticles with a diameter of 100 nm (nanoComposix, San Diego, Calif.) in 2 μL of 20 mM His HCl was added to each sample well. The microplate was shaken again to mix the gold nanoparticles with the sample, which was then placed in a DynaPro II DLS plate reader (Wyatt Technology Corp., Goleta, Calif.) to determine the apparent particle size of the gold nanoparticles. Measured at 25°C. The ratio of the apparent particle size of the gold nanoparticles in the protein formulation to the apparent particle size of the gold nanoparticles in the buffer (no protein) was used to determine the viscosity of the protein formulation according to the Stokes-Einstein equation. . In this example, the ratio of the apparent radius of the gold nanoparticles to the actual radius was multiplied by the viscosity of water at 25° C. to calculate the viscosity of the protein formulation in centipoise (cP). Results using five different excipients are shown in Table 54 below.

Example 54: DLS Viscosity Measurement of Concentrated Omalizumab In this example, the following agents were used to prepare stock solutions of excipients: tetraethylammonium chloride, tetramethylammonium acetate, 1-methylimidazole, 1-butylimidazole, 1 -hexylimidazole, 2-ethylimidazole, 2-methylimidazole, and spectinomycin were used and purchased from Sigma Aldrich (St. Louis, Mo.). Triglycine, tetraglycine, and 2-butylimidazole were purchased from Chem-Impex (Wood Dale, Ill.). Hordenine HCl was purchased from Bulk Supplements (Henderson, NV).

A biosimilar concentrate preparation of the monoclonal antibody omalizumab obtained from Bioceros (The Netherlands) was prepared as described in Example 52 and analyzed for protein concentration as described in Example 52. Stock excipient solutions using the excipients described above were prepared in 20 mM HisHCl (pH 6) buffer (prepared as described in Example 52) at 1 M or the solubility limit of the compound and and adjusted to pH 6 with concentrated hydrochloric acid or sodium hydroxide. The concentrated protein formulation was then mixed with the stock excipient solution or control at a ratio of 9 parts protein: 1 part excipient solution or buffer (as control) and aliquots were placed in 384-well microplates ( Aurora Microplates, Whitefish, MT) wells. Microplates were then centrifuged at 400 xg in a Sorvall Legend RT and shaken on a plate shaker. After shaking the microplate, a 5-fold dilution of polyethylene glycol surface-modified gold nanoparticles with a diameter of 100 nm (nanoComposix, San Diego, Calif.) in 2 μL of 20 mM His HCl was added to each sample well. The microplate was shaken again to mix the gold nanoparticles with the sample, which was then placed in a DynaPro II DLS plate reader (Wyatt Technology Corp., Goleta, Calif.) to determine the apparent particle size of the gold nanoparticles. Measured at 25°C. The ratio of the apparent particle size of the gold nanoparticles in the protein formulation to the apparent particle size of the gold nanoparticles in the buffer (no protein) was used to determine the viscosity of the protein formulation according to the Stokes-Einstein equation. . In this example, the ratio of the apparent radius of the gold nanoparticles to the actual radius was multiplied by the viscosity of water at 25° C. to calculate the viscosity of the protein formulation in centipoise (cP). Results using 12 different excipients are shown in Table 55 below.

Example 55: Excipients that enhance thermostability Therapeutic proteins are often exposed to temperature fluctuations, which can lead to changes in their tertiary and secondary structural elements. This leads to protein aggregation and loss of active native species. Excipients that protect against heat stress were tested in thermal degradation studies in the presence or absence of the excipient. Excipient stocks were prepared by dissolving the excipients listed in Table 56 below at a concentration of 100 mg/mL in 20 mM histidine buffer, pH 6.0 (described in Example 52). prepared as follows). Each test sample was prepared by adding an excipient stock to a final concentration of 5 mg/mL of excipient in buffer and ustekinumab stock in histidine buffer to a final concentration of 20 mg/mL. (prepared as described in Example 51) by diluting the protein to 1 mg/mL of The formulation was aliquoted into 0.2 mL microcentrifuge tubes and incubated in a heat block at 65° C. for 120 minutes. Aliquots were collected at 0, 30, 60, 90 and 120 minutes. Samples were then chilled on ice for 5 minutes and spun down at 9000 rpm for 10 minutes. Following this, a sample of the supernatant was analyzed by SE-HPLC as follows: equipped with a TSKgel SW3000 size exclusion chromatography column (30 cm x 4.8 mm ID) and an Agilent G1351B diode array detector monitoring at 280 nm. The supernatant was loaded onto an Agilent 1100 HPLC system equipped with. A mobile phase of 0.5% phosphoric acid, 150 mM NaCl, pH 3.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the peak area under the monomer peak and plotting the change in integrated peak area as a function of time. Thermal stability was correlated with the fraction of monomer remaining at the end of the 2 hour incubation, reported as percent increase in monomer compared to control (no additive). The results for the seven excipients tested are summarized in Table 56 below.

Example 56: Excipients that Improve Thermal Stability of ADC Antibody Drug Conjugates (ADCs) Conjugation of Small Molecules to Monoclonal Antibodies Via Chemical Linkers Allowing Site-Specific Delivery of Small Molecule Drugs is a therapeutic protein produced by The binding linker and small molecule combination alters the chemical and physical properties (charge, hydrophobicity, etc.) of the ADC compared to its protein precursor, leading to additional stability concerns. The ustekinumab-FITC compound of Example 59 was used as the model ADC compound for these studies. Excipients that protect model ADCs from thermal stress were tested in thermal degradation studies in the presence or absence of excipients. Excipient stocks were prepared by dissolving the excipients listed in Table 57 below at a concentration of 100 mg/mL in 20 mM histidine buffer, pH 6.0 (described in Example 52). prepared as follows). Each test sample was prepared by adding an excipient stock to a final concentration of 5 mg/mL in buffer and ustekinumab-FITC (described in Example 59 below) in histidine buffer at a final concentration of ustekinumab-FITC was prepared by adding to 1 mg/mL. The formulations were aliquoted into 0.2 mL microcentrifuge tubes and incubated on a heat block at 65° C. for 120 minutes. Aliquots were collected at 0, 30, 60, 90 and 120 minutes. Samples were then chilled on ice for 5 minutes and spun down at 9000 rpm for 10 minutes. Samples were analyzed by SE-HPLC and supernatants were loaded on an Agilent 1100 HPLC system monitored at 280 nm with a TSK gel SW3000 size exclusion chromatography column (30 cm x 4.8 mm ID) and an Agilent G1351B diode array detector. A mobile phase of 0.5% phosphoric acid, 150 mM NaCl, pH 3.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the peak area under the monomer peak and the change in the integrated peak area plotted as a function of time and expressed as percent monomer compared to the control (no excipient). recorded an increase. Thermal stability correlated with the percentage of monomer remaining at the end of the 2 hour incubation.

Example 57: Excipients that Protect Against Freeze/Thaw Stress Therapeutic proteins should be treated with a Often kept cold. In some cases this may be done by freezing the formulation until use. However, low temperatures, concentration gradients, and ice formation during repeated freezing and thawing can stress proteins. Excipients that protect against heat stress were tested and identified by thermal degradation studies in the presence or absence of excipients. Excipient stocks were prepared as described in Example 52, prepared by dissolving the excipients listed in Table 58 below at a concentration of 1 M in 20 mM histidine buffer, pH 6.0. bottom. Each test sample was spiked with excipient stock to a final concentration of 100 mM excipient and ustekinumab stock in histidine buffer (prepared as described in Example 50). Prepared by diluting the protein from 20 mg/mL to a final concentration of 2 mg/mL. A control was prepared in the same manner, but without the additive stock. The formulation was then aliquoted into 0.5 mL cryovials and frozen at -80°C for 120 minutes. The samples were then thawed using a water bath kept at room temperature. After repeating this freeze-thaw cycle six times, each sample was aliquoted into 0.2 mL microcentrifuge tubes and spun down at 9000 rpm for 10 minutes. Supernatants were loaded onto an Agilent 1100 HPLC system equipped with a TSK gel SW3000 size exclusion chromatography column (30 cm x 4.8 mm ID) and an Agilent G1351B diode array detector monitoring at 280 nm and samples were analyzed by SE-HPLC. analyzed. A mobile phase of 0.5% phosphoric acid, 150 mM NaCl, pH 3.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the peak area under the monomer peak and plotting the change in integrated peak area as a function of time.

Example 58: Accelerated Degradation Study Excipients that protect against heat stress were tested in a thermal degradation study in the presence or absence of the excipient. Excipient stocks were prepared as described in Example 52, prepared by dissolving the excipients listed in Table 59 below at a concentration of 1 M in 20 mM histidine buffer, pH 6.0. bottom). Each test sample was prepared by adding excipients to a final concentration of 100 mM and diluting the protein from a 20 mg/mL ustekinumab stock in histidine buffer to a final concentration of 2 mg/mL. prepared. A control was prepared in the same manner, but without the additive stock. 1 mL of each sample was aliquoted into 2 mL glass vials (West Pharmaceutical Services, PA) and incubated at 40° C. for 4 weeks. Aliquots were collected after 0, 1, 2, 3, and 4 weeks. The supernatant was loaded onto an Agilent 1100 HPLC system equipped with a TSKgel SW3000 size exclusion chromatography column (30 cm x 4.8 mm ID) and an Agilent G1351B diode array detector monitoring at 280 nm and samples were analyzed by SE-HPLC. . A mobile phase of 0.5% phosphoric acid, 150 mM NaCl, pH 3.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the peak area under the monomer peak and plotting the change in integrated peak area as a function of time. Thermal stability correlated with the percentage of monomer remaining at the end of 4 weeks of incubation.

Example 59: Synthesis of ustekinumab FITC A model compound representing an antibody drug conjugate (ADC) was synthesized as follows. Ustekinumab was purchased from Bioceros (Utrecht, The Netherlands) as frozen aliquots at a mAb concentration of 26 mg/mL in 40 mM aqueous sodium acetate, 50 mM Tris-HCl buffer (pH 5.5). Samples were buffer exchanged into pH 9.2 carbonate buffer and then incubated with 5 equivalents of fluorescein isothiocyanate (FITC) dissolved in anhydrous dimethylsulfoxide to give 1.6 equivalents of FITC per equivalent of ustekinumab. took in. The average molar ratio of FITC to ustekinumab was determined by measuring the absorbance at 280 nm (representing protein + FITC) and at 495 nm (representing FITC). Calculations give the mAb extinction coefficient at 280 nm of 1.61 L/g. cm, 148,600 as the MW of the mAb, and 68,000 L/g.m. as the extinction coefficient of FITC at 495 nm. cm was used. Excess unreacted FITC was removed by dialysis against pH 6, 20 mM histidine buffer. The samples were then concentrated to 15 mg/mL using Amicon centrifuge tubes with a MWCO of 30 kDa.

Example 60: Stability of ustekinumab-FITC The ADC model compound of Example 59 was diluted in 20 mM histidine buffer at pH 6 (prepared as described in Example 52) to a mAb concentration of 1 mg/mL. Samples were prepared using the additive excipients listed in Table 60 below to test their ability to protect model ADC compounds from mechanical shear stress. The samples were mechanically stressed by placing them on a shaker table at 300 rpm for 72 hours at 23°C. After stressing the solution, the particle size of the ADC complex was determined by dynamic light scattering. The particle radius of the control sample after shearing was 143 nm, indicating significant aggregation compared to the unstressed sample (5.5 nm radius). Samples containing excipients showed no significant increase in particle radius compared to the unstressed control sample (radius 5.5 nm), indicating a protective effect against mechanical shear stress. The results are shown in Table 60 below.

Example 61: Effect of co-solutes on caffeine solubility in aqueous buffers during refrigerated storage Prepared by titrating and diluting to a final volume of 100 mL with Milli-Q water. Buffers were then used to prepare a 50 mM co-solute solution and the following excipients: sodium benzoate, 1-methyl-2-pyrrolidone, proline, phenylalanine, arginine monohydrochloride, benzyl alcohol, and nicotinamide. ,It was used. Approximately 0.1 g of caffeine was dissolved in a 5 mL aliquot of each resulting solution to give a caffeine concentration of 20 mg/mL. 20 mg/mL caffeine with different volume ratios of co-solutes and corresponding solutions with excipient solutions but no caffeine were prepared in triplicate in 96-well microplates and in all cases total wells The volume was kept at 300 μL. The resulting microplates were then sealed with microplate tape and stored in a refrigerator keeping the temperature in the range of 2-5°C. During storage, the microplates were visually inspected for evidence of precipitation within the wells. For each condition, the three earliest observed wells of sedimentation are recorded and the results are summarized in Table 61 below.

Example 62: Testing Excipients to Reduce Viscosity of Antibody Solutions A stock buffer solution of 20 mM Histidine-HCl pH 6.0 (His HCl) was added to 1.55 g histidine (Sigma-Aldrich, St. Louis, Mo.). State) was prepared by dissolving in Type 1 ultrapure water. The contents were completely dissolved and the pH was adjusted to 6.0 using hydrochloric acid solution. After pH adjustment, the volumetric flask was brought to a final volume of 0.5 L. All excipients were dissolved in His HCl and prepared at 10x concentration (1 M) or compound solubility limit. The pH of the excipient solutions was measured and adjusted to pH 6.0 if necessary.

In this example, the viscosity of protein solutions with excipient concentrations of 0.1 M or less was measured. The biosimilar monoclonal antibody Omalizumab, purchased from Bioceros (Netherlands), was buffer exchanged into His HCl and centrifuged using a pre-rinsed Amicon-15 centrifuge with a 30 kDa molecular weight cut-off (EMD Millipore, Billerica, Mass.). Concentrated to 200 mg/mL. A dispersion of polyethylene glycol surface-modified gold nanoparticles (nanoComposix, San Diego, Calif.) was mixed thoroughly and diluted 5-fold in His HCl. In a separate PCR tube, 2.1 μL of gold nanoparticles, 5.3 μL of 10× excipient solution, and 47.6 μL of concentrated Omalizumab were combined and mixed thoroughly. Duplicate 25 μL volumes of each solution were transferred to a 384-well plate (Aurora Microplates, Whitefish, Mont.) and centrifuged at 400×g for 1 minute (Sorvall Legend RT). A tape seal was used to prevent sample evaporation. Plates were then transferred to a DynaPro II DLS plate reader (Wyatt Technology Corp., Goleta, CA) to measure the apparent particle size of the gold nanoparticles at 25°C. The ratio of the measured apparent radius to the particle size of known radius was calculated to determine the viscosity of the protein formulation according to the Stokes-Einstein equation.

In this example, the diffusion interaction parameter (k _D ) of dilute protein solutions was determined by DLS in the presence of excipients up to 0.1 M. 0.2 M excipient solutions were prepared separately from previously prepared excipient stock solutions. k _D was measured using 5 different protein concentrations ranging from 10 mg/mL to 0.6 mg/mL in the presence of 0.1 M excipient. 15 μL of protein solution and 15 μL of 0.2 M excipient solution (1:1 mix) were mixed in a 384-well plate (Aurora Microplates, Whitefish, MT). After loading the samples, the well plate was shaken on a plate shaker to mix the contents for 5 minutes. Once mixed, the well plate was centrifuged at 400 xg for 1 minute in a Sorvall Legend RT to expel air pockets. The well plate was then loaded into a DynaPro II DLS plate reader (Wyatt Technologies Corp., Goleta, CA) and the diffusion coefficient of each sample was measured at 25°C. For each excipient, the measured diffusion coefficient was plotted as a function of protein concentration and the slope of the linear fit of the data was recorded as _kD . Results are summarized in Tables 62A and 62B below for two different series of tests.

Equivalents Although specific embodiments of the present invention have been disclosed herein, the above specification is illustrative and not restrictive. Although the invention has been particularly shown and described with reference to preferred embodiments thereof, various changes may be made therein in form and detail without departing from the scope of the invention falling within the scope of the appended claims. It is understood by those skilled in the art to obtain Many variations of the invention will become apparent to those skilled in the art upon review of this specification. Unless otherwise stated, all numbers expressing reaction conditions, amounts of ingredients, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about." should. Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained from the present invention.
Embodiments of the present invention include the following.
Item 1
A stability-enhanced formulation comprising a therapeutic protein and a stability-improving amount of a stabilizing excipient, compared to a control formulation that is identical to said stability-enhancing formulation but lacking said stabilizing excipient. and stability-enhanced formulations characterized by improved stability parameters.
Item 2
3. The stability-enhanced formulation of paragraph 1, wherein said therapeutic protein is an antibody.
Item 3
3. The stability-enhanced formulation of paragraph 2, wherein the antibody is an antibody-drug conjugate.
Item 4
2. The stability-enhanced formulation of paragraph 1, wherein said stabilizing excipient is a hindered amine compound.
Item 5
2. The stability-enhanced formulation of paragraph 1, wherein said stabilizing excipient is an anionic aromatic compound.
Item 6
2. The stability-enhanced formulation of paragraph 1, wherein said stabilizing excipient is a functionalized amino acid compound.
Item 7
2. The stability-enhanced formulation of paragraph 1, wherein said stabilizing excipient is an oligopeptide.
Item 8
2. The stability-enhanced formulation of paragraph 1, wherein said stabilizing excipient is a short chain organic acid.
Item 9
2. The stability-enhanced formulation of paragraph 1, wherein said stabilizing excipient is a low molecular weight polyacid.
Item 10
2. The stability-enhanced formulation of paragraph 1, wherein the stabilizing excipient is a dione compound or a sulfone compound.
Item 11
2. The stability-enhanced formulation of paragraph 1, wherein said stabilizing excipient is a zwitterionic compound.
Item 12
2. The stability-enhanced formulation of paragraph 1, wherein the stabilizing excipient is a crowding agent having a hydrogen-bonding element.
Item 13
The stability-enhanced formulation of paragraph 1, wherein said stabilizing excipient is added in an amount of about 1 mM to about 500 mM.
Item 14
14. The stability-enhanced formulation of paragraph 13, wherein said stabilizing excipient is added in an amount of about 5 mM to about 250 mM.
Item 15
15. The stability-enhanced formulation of paragraph 14, wherein said stabilizing excipient is added in an amount of about 10 mM to about 100 mM.
Item 16
16. The stability-enhanced formulation of paragraph 15, wherein said stabilizing excipient is added in an amount of about 5 mg/mL to about 50 mg/mL.
Item 17
2. The stability-enhanced formulation of paragraph 1, wherein said improved stability parameter is heat storage stability.
Item 18
18. The stability-enhanced formulation of paragraph 17, wherein said heat storage stability is improved at a temperature of about 10°C to 30°C.
Item 19
2. The stability-enhanced formulation of paragraph 1, wherein said improved stability parameter is improved freeze/thaw stability.
Item 20
2. The stability-enhanced formulation of paragraph 1, wherein said improved stability parameter is improved shear stability.
Item 21
2. The stability-enhanced formulation of paragraph 1, wherein said formulation has a reduced particle count compared to said control formulation.
Item 22
2. The stability-enhanced formulation of paragraph 1, wherein said formulation has improved biological activity compared to said control formulation.
Item 23
1. A method of improving the stability of a therapeutic formulation comprising adding a stability-improving amount of a stabilizing excipient to said therapeutic formulation, thereby improving said stability of said therapeutic formulation; A method, wherein said stability of said therapeutic formulation is measured relative to the stability of a control formulation, which is identical to said therapeutic formulation but lacking said stabilizing excipient.
Item 24
cloudy where the stabilizing excipient has hindered amines, anionic aromatic compounds, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight polyacids, diones, sulfones, zwitterionic compounds, and hydrogen bonding elements; 24. The method of paragraph 23, wherein the method is selected from the group consisting of:
Item 25
24. The method of Paragraph 23, wherein said step of measuring said stability of said therapeutic formulation relative to said stability of said control formulation comprises measuring a stability-related parameter.
Item 26
26. The method of paragraph 25, wherein said stability-related parameter is selected from the group consisting of heat storage stability, freeze/thaw stability, and shear stability.
Item 27
24. The method of Paragraph 23, wherein said therapeutic formulation comprises a therapeutic protein.
Item 28
28. The method of Paragraph 27, wherein said therapeutic protein is an antibody.
Item 29
29. The method of Paragraph 28, wherein said antibody is an antibody drug conjugate.
Item 30
A method of improving parameters of a protein-related process comprising adding a stability-improving amount of a stabilizing excipient to a carrier solution of said protein-related process, said carrier solution comprising a protein of interest, thereby A method of improving said parameter.
Item 31
31. The method of Paragraph 30, wherein said parameter is selected from the group consisting of cost of protein production, amount of protein production, rate of protein production, and efficiency of protein production.
Item 32
32. The method of Paragraph 31, wherein said protein of interest is a therapeutic protein.

Claims (11)

A liquid formulation comprising a therapeutic protein and a viscosity-lowering amount of hordenine, wherein the therapeutic protein is an antibody, and the viscosity of said formulation is at least 30 % lower than that of a control formulation and less than 100 cP. and wherein said control formulation does not contain said hordenine but is otherwise identical to said formulation on a dry weight basis. 2. The formulation of claim 1 , wherein said antibody is an antibody drug conjugate. A formulation according to claim 1 , wherein said hordenine is added in an amount of 1 mM to 500 mM. 4. The formulation of claim 3 , wherein said hordenine is added in an amount of 5 mM to 250 mM. A formulation according to claim 4 , wherein said hordenine is added in an amount of 10 mM to 100 mM. 6. The formulation of claim 5 , wherein said hordenine is added in an amount of 5 mg/mL to 50 mg/mL. A method of reducing the viscosity of a liquid therapeutic formulation comprising a therapeutic protein, wherein the therapeutic protein is an antibody, the method comprising adding a viscosity-lowering amount of hordenine to the formulation , thereby reducing the viscosity of the formulation. is reduced by at least 30 % relative to the viscosity of a control formulation, wherein the viscosity of said formulation is less than 100 cP, said control formulation does not comprise said hordenine, but otherwise said formulation . and the method on a dry weight basis. 8. The method of claim 7 , wherein said antibody is an antibody drug conjugate. 2. The formulation of claim 1, wherein the viscosity of said formulation is at least 30% lower than the viscosity of a control formulation. 2. The formulation of claim 1, wherein the formulation has a viscosity of less than 50 cP. 2. The formulation of claim 1, wherein the formulation comprises at least 200 mg/mL antibody .

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