KR20230018549A - Excipient compounds for protein formulations - Google Patents

Abstract

Disclosed herein are stability-enhancing formulations comprising a therapeutic protein and a stability-enhancing amount of a stabilizing excipient, wherein the stability-enhancing formulation is otherwise identical to the stability-enhancing formulation, but has improved stability parameters compared to a control formulation lacking the stabilizing excipient. characterized by Further disclosed herein are methods of improving the stability of a therapeutic formulation or improving parameters of a protein-related process.

Description

Excipient compounds for protein formulation {EXCIPIENT COMPOUNDS FOR PROTEIN FORMULATIONS}

related application

This application claims the benefit of U.S. Provisional Application Serial No. 62/639,950, filed on March 7, 2018, and U.S. Provisional Application Serial No. 62/679,647, filed on June 1, 2018. The entire contents of each of the above applications are incorporated herein by reference.

field of application

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

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

Of particular interest are protein biopolymers for therapeutic and non-therapeutic uses. Proteins are complex biopolymers, each with a uniquely folded 3-D structure and surface energy map (hydrophobic/hydrophilic regions and charge). In concentrated protein solutions, these macromolecules can strongly interact and even interconnect in 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 problems, a number of excipient compounds are used in biotherapeutic formulations that aim to reduce solution viscosity by disrupting localized interactions and clustering. These efforts are individually tailored, often made empirically, and sometimes guided by in silico simulations. Combinations of excipient compounds may be provided, but again optimizing the combination must proceed empirically and on a case-by-case basis.

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

For example, antibodies may need to be delivered at high dose levels to exert their intended therapeutic effect. The use of limited liquid volumes to deliver high dose levels of antibody formulations may require high concentrations of antibody in the delivery vehicle, sometimes exceeding levels of 150 mg/mL. At this dosage level, the protein solution's viscosity versus concentration plot crosses a linear-nonlinear transition and the viscosity of the formulation increases rapidly with increasing concentration. However, the increased viscosity is not compatible with standard SC or IM delivery systems. Solutions of biopolymer-based or protein-based therapeutics are also susceptible to stability problems such as precipitation, fragmentation, oxidation, deamidation, hazing, opalescence, denaturation and gel formation, reversible or irreversible aggregation. Stability issues limit the shelf life of the solution or require special handling.

One approach to producing injectable protein formulations is to convert a therapeutic protein solution into a powder that can be reconstituted to form a suspension suitable for SC or IM delivery. Lyophilization is a standard technique for producing protein powders. Freeze-drying, spray drying and even precipitation followed by supercritical fluid extraction have been used to create protein powders for subsequent reconstitution. The powdered suspension has a low viscosity (compared to the same total volume of solution) prior to redissolution and therefore may be suitable for SC or IM injection if the particles are small enough to pass through a needle. However, protein crystals present in the powder have an inherent risk of triggering an immune response. Uncertain protein stability/activity after re-dissolution raises additional concerns. There remains a need in the art for techniques to create 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 susceptible to viscosity and stability problems. ADCs link small molecule drugs to monoclonal antibodies (mAbs) via chemical linkers; mAbs target specific antigens on abnormal “target cells,” and small molecule drugs are selected to have specific effects on those target cells. When the mAb comes into contact with a target cell antigen, the mAb and its attached drug are taken up by the cell and enter the interior of the cell. Inside the cell, the mAb and/or linker is cleaved and the drug is released to exert its biological effect on the cell. Typically, the drug is a chemotherapeutic agent that is too toxic for systemic release. ADCs bring chemotherapeutic agents into direct contact with their target cancer cells. This attachment of small molecules to the mAb can exacerbate viscosity and stability issues affecting therapeutic protein formulations. Payload compounds are typically hydrophobic small molecules, which can exert significant effects on the stability, solubility and solution interaction properties of larger ADCs as the drug-antibody ratio increases. The high salt concentration of the formulation can increase the hydrophobic interactions between the ADC complexes, making the ADC's solubility more sensitive to salt effects than unconjugated antibodies. Handling or storage of ADC solutions can stimulate aggregation or precipitation of ADC species, especially at high drug-to-antibody ratios (DAR). Drug conjugation can also affect the conformational stability of a mAb, particularly its Fc domain. In addition, drug conjugation may also affect the solubility of the ADC by reducing the net surface charge of the mAb.

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

Non-therapeutic proteins may be produced, transported, stored, and handled as granular or powdered materials or generally as solutions or dispersions in water. Biopolymers for non-therapeutic applications may be globular or fibrous proteins, and certain forms of these materials may have limited solubility in water or exhibit high viscosity upon dissolution. Since these solution properties can cause problems in formulation, handling, storage, pumping and performance of non-therapeutic substances, methods are needed to reduce the viscosity, improve solubility and stability of non-therapeutic protein solutions.

There is 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 a further need in the art to achieve this viscosity reduction while preserving the activity of the protein. It would be further desirable to adapt the viscosity reducing system for use with formulations having tunable and sustained release profiles and for use with formulations adapted for depot injection. It is also desirable to improve processes for producing proteins and other biopolymers.

In an embodiment, a liquid formulation comprising a protein and an excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and low molecular weight aliphatic polyacids is As disclosed herein, the excipient compound is added in a viscosity reducing amount. In an embodiment, the protein is a PEGylated protein and the excipient is a low molecular weight aliphatic polyacid. In an embodiment, the formulation is a pharmaceutical composition and the therapeutic formulation comprises a therapeutic protein, wherein the excipient compound is a pharmaceutically acceptable excipient compound. In an embodiment, the formulation is a non-therapeutic formulation and the non-therapeutic formulation comprises a non-therapeutic protein. In embodiments, the viscosity reducing amount reduces the viscosity of the formulation to a viscosity less than the viscosity of the control formulation. In embodiments, the viscosity of the formulation is at least about 10% less than the viscosity of the control formulation, at least about 30% less than the viscosity of the control formulation, at least about 50% less than the viscosity of the control formulation, or at least about 70% less than the viscosity, or at least about 90% less than the viscosity of the control formulation. In an embodiment, the viscosity is less than about 100 cP, less than about 50 cP, less than about 20 cP, or less than about 10 cP. In an embodiment, the excipient compound has a molecular weight of <5000 Da, or <1500 Da, or <500 Da. In an embodiment, the formulation contains at least about 25 mg/mL of protein, or at least about 100 mg/mL of protein, or at least about 200 mg/mL of protein, or at least about 300 mg/mL of protein. In embodiments, the formulation comprises from about 5 mg/mL to about 300 mg/mL of the excipient compound, or from about 10 mg/mL to about 200 mg/mL of the excipient compound, or from about 20 mg/mL to about 100 mg/mL of the excipient compound. mg/mL, or from about 25 mg/mL to about 75 mg/mL of the excipient compound. In embodiments, the formulation has improved stability when compared to a control formulation. In an embodiment, the excipient compound is a hindered amine. In an embodiment, the hindered amine is selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin and acesulfame potassium. In an embodiment, the hindered amine is caffeine. In an embodiment, the hindered amine is a locally injectable anesthetic compound. Hindered amines can have independent pharmacological properties, and hindered amines can be present in formulations in amounts that have independent pharmacological effects. In embodiments, the hindered amine may be present in the formulation in an amount less than a therapeutically effective amount. The independent pharmacological activity may be local anesthetic activity. In an embodiment, a hindered amine having independent pharmacological activity is combined with a second excipient compound that further reduces the viscosity of the formulation. The second excipient compound may be selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin and acesulfame potassium. In embodiments, the formulation may include additional 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, the therapeutic formulation comprising a therapeutically effective amount of a therapeutic protein, the formulation comprising a hindered amine further comprising a pharmaceutically acceptable excipient compound selected from the group consisting of anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids and low molecular weight aliphatic polyacids, wherein the therapeutic formulation is effective for the treatment of a disease or disorder am. In an embodiment, the therapeutic protein is a PEGylated protein and the excipient compound is a low molecular weight aliphatic polyacid. In an embodiment, the excipient is a hindered amine. In an embodiment, the hindered amine is a local anesthetic compound. In embodiments, the formulation is administered by subcutaneous injection or intramuscular or intravenous injection. In embodiments, the excipient compound is present in the therapeutic formulation in a viscosity reducing amount, and the viscosity reducing amount reduces the viscosity of the therapeutic formulation to a viscosity less than that of the control formulation. In embodiments, the therapeutic formulation has improved stability when compared to a control formulation. In an embodiment, 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 an animal in need thereof comprising administering a liquid therapeutic formulation by injection, said therapeutic formulation comprises a therapeutically effective amount of a therapeutic protein, said formulation further comprising a pharmaceutically acceptable excipient compound selected from the group consisting of locally injectable anesthetic compounds, said pharmaceutically acceptable excipient compound in a viscosity reducing amount. is added to a formulation, wherein the mammal experiences less distress from administration of a therapeutic formulation comprising an excipient compound than administration of a control therapeutic formulation, wherein the control therapeutic formulation does not contain the excipient compound and is otherwise identical to the therapeutic formulation. .

In an embodiment, comprising preparing a liquid protein formulation comprising a therapeutic protein and an excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides and short chain organic acids and low molecular weight aliphatic polyacids. Disclosed herein is a method of improving the stability of a liquid protein formulation, wherein the liquid protein formulation exhibits improved stability compared to a control liquid protein formulation, wherein the control liquid protein formulation does not contain an excipient compound and otherwise liquid protein formulation is the same as The stability of a liquid formulation may be cold storage condition stability, room temperature stability or elevated temperature stability.

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

Further disclosed herein is a method of improving a protein-related process comprising, in an embodiment, providing the liquid formulation described above and using it in a method of treatment. In an embodiment, the processing method comprises filtration, pumping, mixing, centrifugation, membrane separation, lyophilization or chromatography. In an embodiment, the treatment method is selected from the group consisting of cell culture harvesting, chromatography, virus inactivation and filtration. In an embodiment, the treatment method is a chromatography process or a filtration process. In an embodiment, the filtration process is a viral filtration process or an ultrafiltration/diafiltration process.

Provided is a viscosity reducing excipient additive comprising at least one excipient compound selected from the group consisting of hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids and diones and sulfones, Also disclosed herein is a method of improving a parameter of a protein-related process comprising adding a decreasing amount of at least one excipient compound to a carrier solution for the protein-related process, thereby improving the parameter, the carrier solution of interest Contains protein. In an embodiment, the parameter may be selected from the group consisting of protein production cost, protein yield, protein production rate and protein production efficiency. Parameters may be proxy parameters. In an embodiment, the protein-related process is an upstream processing process. The carrier solution for the upstream treatment process may be a cell culture medium. In an embodiment, when the carrier solution is a cell culture medium, adding an excipient additive to the carrier solution comprises a first substep of adding the excipient additive to the supplemented medium to form a supplemented medium containing the excipient and the supplemented medium containing the excipient to the cell culture medium. A second sub-step of adding to the medium is included. In another embodiment, the protein-related process is a downstream processing process. The downstream process may be a chromatography process, and the chromatography process may be a Protein-A chromatography process. In an embodiment, the chromatographic process recovers a protein of interest, wherein the protein of interest is an improvement selected from the group consisting of improved purity, improved yield, fewer particles, less misfolding, or less aggregation as compared to a control solution. characterized by protein-related parameters. In an embodiment, an improved protein-related parameter is an improved yield of a protein of interest from a chromatography process. In another embodiment, the protein-related process is a process selected from the group consisting of filtration, infusion, injection, pumping, mixing, centrifugation, membrane separation and lyophilization, wherein the selected process will require less force than the control process. can In an embodiment, the protein-related process is selected from the group consisting of a cell culture process, a cell culture harvest process, a chromatography process, a virus inactivation process, and a filtration process. In an embodiment, the protein-related process is a virus inactivation process, the virus inactivation process is performed at a pH level of about 2.5 to about 5.0, or the virus inactivation process is performed at a higher pH than the control process. In another embodiment, the protein-related process is a filtration process. The filtration process may be a virus removal filtration process or an ultrafiltration/diafiltration process. The filtration process may be characterized by improved filtration-related parameters. An improved filtration-related parameter may be a filtration rate 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 may be a higher mass transfer efficiency than that of a control filtration process. The improved filtration related parameter may be a higher concentration or higher yield of the target protein than the concentration or yield of the target protein produced by the control filtration process.

Further disclosed herein is a method as described above wherein the viscosity reducing excipient additive comprises two or more excipient compounds. In an embodiment, at least one excipient compound is a hindered amine. In an embodiment, 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. is chosen In an embodiment, the at least one excipient compound is selected from the group consisting of caffeine, taurine, niacinamide and imidazole. In embodiments, the at least one excipient compound is an anionic aromatic excipient, and in some embodiments, the anionic aromatic excipient can be 4-hydroxybenzenesulfonic acid. In an embodiment, the viscosity reducing amount is from about 1 mg/mL to about 100 mg/mL of at least one excipient compound, or the viscosity reducing amount is from about 1 mM to about 400 mM of at least one excipient compound, or the viscosity reducing amount is from about 2 mM to about 150 mM. In an embodiment, the carrier solution comprises an additional agent selected from the group consisting of preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers and buffers. In an embodiment, the protein of interest is a Therapeutic protein, and the Therapeutic protein can be a recombinant protein, or a monoclonal antibody, polyclonal antibody, antibody fragment, fusion protein, PEGylated protein, antibody-drug conjugate, synthetic polypeptide, protein fragments, lipoproteins, enzymes and structural peptides. In an embodiment, the method further comprises adding a second viscosity reducing excipient to the carrier solution, wherein adding the second viscosity reducing compound adds a further improvement to the parameter.

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

The present disclosure also relates to a stability-enhancing formulation comprising a therapeutic protein and a stability-enhancing amount of a stabilizing excipient, wherein the stability-enhancing formulation is compared to a control formulation that is otherwise identical to the stability-enhancing formulation but lacks the stabilizing excipient. characterized by improved stability parameters. In an embodiment, the therapeutic protein is an antibody, and the antibody may be an antibody-drug conjugate. Stabilizing excipients are hindered amine compounds, anionic aromatic compounds, functionalized amino acid compounds, oligopeptides, short chain organic acids, low molecular weight polyacids, diionic or sulfonic compounds, zwitterionic compounds, or crowding agents with hydrogen bonding elements. agent). In an embodiment, the stabilizing excipient is in an amount of about 1 mM to about 500 mM, or in an amount of about 5 mM to about 250 mM, or in an amount of about 10 mM to about 100 mM, or in an amount of about 5 mg/mL to about 50 mg/mL. It can be added to the formulation in any amount. The improved stability parameter can be heat storage stability, for example, heat storage stability is improved at temperatures of about 10°C to 30°C. In an embodiment, the improved stability parameter is improved freeze/thaw stability or improved shear stability. In an embodiment, the stability enhancing formulation has a reduced number of particles compared to the control. In embodiments, the stability enhancing formulation has improved biological activity compared to a control.

Also disclosed herein is a method of improving the stability of a therapeutic formulation comprising adding a stability-enhancing amount of a stabilizing excipient to the therapeutic formulation, thereby improving the stability of the therapeutic formulation, wherein the stability of the therapeutic formulation determines the stability of the therapeutic formulation. and the stability of a control formulation otherwise identical to but lacking stabilizing excipients. 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 elements. In embodiments, measuring the stability of the therapeutic formulation may include measuring a stability related parameter, eg, a parameter selected from the group consisting of heat storage stability, freeze/thaw stability and shear stability. In an embodiment, the therapeutic formulation comprises a therapeutic protein, which may be an antibody, and the antibody may be an antibody-drug conjugate. Further disclosed herein is a method of improving a parameter of a protein-related process comprising adding a stability-enhancing amount of a stabilizing excipient to a carrier solution for the protein-related process, wherein the carrier solution contains a protein of interest, whereby the parameter and the protein of interest may be a therapeutic protein. In an embodiment, the parameter may be selected from the group consisting of protein production cost, protein yield, protein production rate and protein production efficiency.

1 presents a graph of particle size distribution for solutions of monoclonal antibodies under stressed and non-stressed conditions evaluated by dynamic light scattering. The data curves in Figure 1 have baseline offsets that allow for comparison: on the Y-axis, 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. do.
2 presents a graph measuring sample diameter versus multimodal size distribution for several populations of molecules evaluated by dynamic light scattering. The data curves in FIG. 2 have baseline offsets that allow for comparison: on the Y-axis 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. do.
Figure 3 presents a size exclusion chromatogram of a monoclonal antibody solution with a major monomer peak at 8-10 min retention time. The data curves in FIG. 3 have baseline offsets that allow for comparison: the curves for samples 2-C, 2-A and 2-FT are offset in the Y-axis direction.
4 provides a block diagram showing steps in a fermentation process (“upstream processing”) to produce a therapeutic protein, eg, a monoclonal antibody.
5 provides a block diagram showing steps in a purification process (“downstream processing”) to produce a therapeutic protein, eg, a monoclonal antibody.

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

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

Examples of physical stress conditions are mechanical shear, contact with the air/water interface, freeze-thaw cycles, long-term storage under storage conditions (whether cold storage conditions, room temperature conditions, or elevated temperature storage conditions), or other denaturing conditions. This includes physical disturbances such as exposure to For example, cold storage conditions may entail storage in a refrigerator or freezer. In some instances, the cold storage condition may involve storage at a temperature of 10° C. or lower. In a further example, the cold storage condition entails storage at a temperature of about 2°C to about 10°C. In another example, the cold storage condition entails storage at a temperature of about 4°C. In a further example, the cold storage condition entails storage at a freezing temperature, such as about -20°C or lower. In another example, the cold storage condition entails storage at a temperature of about -80°C to about 0°C. Room temperature storage conditions may entail storage at ambient temperatures, for example, between about 10° C. and about 30° C. Elevated storage conditions may involve storage at temperatures above about 30°C. For example, elevated temperature stability at temperatures of about 30° C. to about 50° C. can be used as part of an accelerated aging study to predict long-term storage at typical ambient (10-30° C.) conditions. Stress conditions can also include chemical perturbations, such as pH changes, which can affect the stability or integrity of proteins in a formulation, for example by affecting the tertiary structure of the protein.

It is well known to those skilled in the art of polymer science and engineering that proteins in solution tend to form entanglements, which can limit the translational mobility of entangled chains and interfere with the therapeutic or non-therapeutic efficacy of proteins. In an embodiment, an excipient compound as disclosed herein is capable of inhibiting protein clustering due to specific interactions between the excipient compound and the target protein in solution. An excipient compound as disclosed herein may be natural or synthetic, and is preferably a substance generally recognized as safe by the FDA ("GRAS").

1. Definition

For purposes of this disclosure, the term "protein" refers to a sequence of amino acids having a chain length long enough to give rise to a discrete tertiary structure, typically with a molecular weight of 1-3000 kDa. In some embodiments, the molecular weight of the protein is between about 50-200 kDa; In another embodiment, the molecular weight of the protein is 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 that does not have a distinct 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 comprising proteins with therapeutic effects may be referred to as "therapeutic biopolymers". These proteins that have therapeutic effects may be referred to as “therapeutic proteins”.

By way of non-limiting example, Therapeutic proteins include mammalian proteins such as hormones and prohormones (eg, insulin and proinsulin, glucagon, calcitonin, thyroid hormones (T3 or T4 or thyroid-stimulating hormone), parathyroid glands). hormones, follicle-stimulating hormone, luteinizing hormone, growth hormone, growth hormone releasing factor, etc.); coagulation and anti-coagulation 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; interferon; colony stimulating factor; interleukins (eg, IL-1 to IL-10); growth factors (eg, vascular endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, neurotrophic growth factor, insulin-like growth factor, etc.); albumin; collagen and elastin; hematopoietic factors (eg, erythropoietin, thrombopoietin, etc.); osteoinductive factors (eg, bone morphogenetic proteins); receptors (eg, integrins, cadherins, etc.); surface membrane proteins; transport proteins; regulatory proteins; antigenic proteins (eg, viral components that act as antigens); and antibodies.

In certain embodiments, a therapeutic protein may be an antibody. The term "antibody" includes, but is not limited to, monoclonal antibodies (including, for example, full length antibodies having an immunoglobulin Fc region), single chain molecules, bispecific and multispecific antibodies, diabodies. , antibody-drug conjugates, antibody compositions having polyepitope specificity, polyclonal antibodies (eg, polyclonal immunoglobulins used as therapeutics for immune-compromised patients), and fragments of antibodies (eg, is used herein in its broadest sense to include Fc, Fab, Fv, nanobodies and F(ab')2). Antibodies may also be referred to as “immunoglobulins”. Antibodies are understood to be 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 can form a complex with the antigen and alter its biological properties such that the patient experiences a therapeutic effect.

In an embodiment, an antibody may be an antibody-drug conjugate (ADC). Antibody-drug conjugates are a category of therapeutic proteins that combine the highly specialized targeting ability of antibodies with therapeutically active compounds, such as cytotoxic compounds; ADCs consist of antibodies linked to therapeutically active agents via biodegradable chemical linkers. More specifically, an ADC may include a human or humanized mAb specific for an antigen that is expressed on abnormal "target" cells, but with minimal or no expression on normal cells. ADCs further include potent pharmacological agents such as cytotoxic agents capable of destroying target cells; Since these agents are typically systemically toxic, they are not suitable for generalized systemic administration. The targeting ability of the mAb component of an ADC allows the pharmaceutical agent to be specifically directed to target cells, taken up by target cells, and exert its effect within these cells without systemic distribution. To form the ADC, the mAb is linked to a pharmaceutical agent with a labile bond that is stable in the extracellular environment (eg, intravenous and interstitial circulation), but is degraded when the ADC is internalized into the cell. As the bond between the ADC and the pharmaceutical agent breaks down, the agent is released inside the cell to exert its effect on the cell. ADCs are particularly suitable for use with cytotoxic agents, especially when these compounds are too toxic for use as a stand-alone therapy. For example, in cancer chemotherapy, some of the agents selected for use in ADCs are several orders of magnitude more toxic than traditional anti-cancer agents. Examples include anti-microtubule agents, alkylating agents and DNA minor groove binding agents, which may be too toxic to administer successfully, but can be targeted to cancer cells using the specificity of mAbs to bind antigens expressed only by cancer cells. there is. Once the ADC localizes to the tumor and binds to a target cell antigen on its surface, the complex can be internalized into the cells of the vesicle. The internalized vesicles fuse with each other and enter the endosome-lysosomal pathway, where they encounter proteases and/or linker molecules that degrade the mAb and release the pharmaceutical payload. The payload (eg, cytotoxic agent) then crosses the lysosomal membrane and enters the cytoplasm and/or nucleus, where it exerts its pharmacological (eg, cytotoxic) effect. This focused delivery of highly potent pharmaceutical compounds maximizes their intended therapeutic effect while minimizing normal tissue exposure of these agents. Formulations comprising ADC are suitable for intravenous or topical administration to allow the ADC to reach the target cells to be treated.

In certain embodiments, 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 are useful in therapeutic applications due to their beneficial properties such as solubility, pharmacokinetics, pharmacodynamics, immunogenicity, renal clearance and stability. Non-limiting examples of PEGylated proteins include PEGylated interferon (PEG-IFN), PEGylated anti-VEGF, PEG protein conjugate drugs, Adagen, Pegaspargase, Pegfilgrastim ), Pegloticase, Pegvisomant, PEGylated epoetin-β, and Certolizumab pegol.

PEGylated proteins can be synthesized by a variety of methods, such as reaction of a protein with a PEG reagent having one or more reactive functional groups. Reactive functional groups on PEG reagents can form links with proteins at targeted protein sites such as lysine, histidine, cysteine and N-terminus. Typical PEGylation reagents have reactive functional groups such as aldehyde, maleimide or succinimide groups that have specific reactivity with the targeted amino acid residue on the protein. The PEGylation reagent can have a PEG chain length of about 1 to about 1000 PEG and/or PPG repeat units. Another method of PEGylation includes glyco-PEGylation, wherein the protein is first glycosylated and then the glycosylated residues are PEGylated in a second step. Certain PEGylation processes are assisted by enzymes such as sialyltransferase and transglutaminase.

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

It is desirable to formulate protein therapeutics as stable, low-viscosity solutions so that they can be administered to patients with minimal injection volumes. For example, subcutaneous (SC) or intramuscular (IM) injections of drugs generally require small injection volumes, preferably less than 2 mL. The SC and IM injection routes are well suited for self-administration management, which is a less expensive and more accessible form of treatment compared to intravenous (IV) injections performed only under direct medical supervision. Formulations for SC or IM injection require low solution viscosities, generally less than 30 cP, preferably less than 20 cP, to permit easy flow of the therapeutic solution through the narrow gauge needle. The combination of these small injection volume and low viscosity requirements present challenges to the use of PEGylated protein therapeutics in the SC or IM injection route.

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

“Treatment” means treating, curing, alleviating, ameliorating, rescuing, or otherwise benefiting a disorder, including preventing or delaying the onset of symptoms and/or alleviating or ameliorating the symptoms of the disorder. Include any action to influence. Patients in need of treatment include both those who already have the particular disorder and those for whom prevention of the disorder is desirable. A disorder is any condition that alters the homeostatic well-being of a mammal, including an acute or chronic disease or pathological condition predisposing the mammal to an acute or chronic disease. Non-limiting examples of disorders include cancer, metabolic disorders (eg diabetes), allergic disorders (eg asthma), dermatological disorders, cardiovascular disorders, respiratory disorders, blood disorders, musculoskeletal disorders, inflammatory or rheumatoid disorders, autologous Includes immune disorders, gastrointestinal disorders, urological disorders, sexual and reproductive disorders, neurological disorders, and the like. The term "mammal" for therapeutic purposes may refer to any animal classified as a mammal, including humans, livestock, pets, farm animals, sport animals, working animals, and the like. Thus, "treatment" can include both veterinary and human treatment. For convenience, the mammal undergoing the "treatment" may be referred to as the "patient". In certain embodiments, the patient may be of any age, including fetal animals in utero.

In embodiments, treating comprises providing a therapeutically effective amount of a therapeutic formulation to a mammal in need thereof. A “therapeutically effective amount” is at least a minimal concentration of a Therapeutic protein administered to a mammal in need thereof to effect the treatment of a pre-existing disorder or the prophylaxis of a probable disorder (wherein said treatment or said prophylaxis is defined as a “therapeutic intervention”). "am). Therapeutically effective amounts of various therapeutic proteins that can be included as active ingredients in a therapeutic formulation may be familiar in the art; For therapeutic proteins subsequently discovered or subjected to therapeutic intervention, the therapeutically effective amount can be determined by standard techniques performed by those skilled in the art using experimentation that does not exceed routine.

b. Non-Therapeutic Biopolymers and Related Definitions

Proteins used for non-therapeutic purposes (i.e., purposes that do not involve therapy), such as household, nutritional, commercial and industrial applications, may be referred to as “non-therapeutic proteins”. Formulations containing non-therapeutic proteins may be referred to as “non-therapeutic formulations”. Non-therapeutic proteins can be derived from plant sources, animal sources, or can be produced from cell culture; They may 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, detergents and waste treatment.

An important category of non-therapeutic biopolymers is the enzyme category. Enzymes have many non-therapeutic applications, such as, for example, catalysts, human and animal nutritional ingredients, processing aids, cleaning agents, and waste treatment agents. Enzymatic catalysts are used to accelerate various chemical reactions. Examples of enzyme catalysts for non-therapeutic use include catalase, oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase. Human and animal nutritional uses of enzymes include nutraceuticals, nutritional sources of protein, chelation or controlled delivery of micronutrients, digestive aids and supplements; These may be derived from amylases, proteases, trypsin, lactases 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 in the production of biofuels. For example, ethanol for biofuels can be supported by enzymatic degradation of biomass feedstocks such as cellulosic and lignocellulosic materials. Treatment of the feedstock material with cellulases and ligninase converts biomass into substrates that can be fermented into fuel. In other commercial applications, enzymes are used as detergents, cleaners and stain removal aids for laundry, dishwashing, surface cleaning and equipment cleaning applications. Typical enzymes for this purpose include proteases, cellulases, amylases and lipases. In addition, non-therapeutic enzymes are used in a variety of commercial and industrial processes such as fabric softening with cellulase, leather processing, waste treatment, contaminated sediment treatment, water treatment, pulp bleaching, and pulp softening and debonding. . 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 manufacture 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 nutritional protein sources. In the cosmetic and personal care industries, collagen, elastin, keratin and hydrolyzed keratin are widely used as ingredients in skin care and hair care formulations. Another example of a non-therapeutic biopolymer is whey proteins such as beta-lactoglobulin, alpha-lactalbumin and serum albumin. These whey proteins are produced in large quantities as a by-product in the dairy industry and have been used in a variety of non-therapeutic applications.

2. Measure

In an embodiment, the protein-containing formulations described herein are resistant to monomer loss as measured by size exclusion chromatography (SEC) analysis. In SEC analysis as used herein, the main analyte peak is generally related to the target protein contained in the formulation, and this main peak of the protein is referred to as the monomer peak. The monomer peak represents the amount of the target protein, eg, protein active component, in the monomeric state as opposed to the aggregated (dimer, trimer, oligomer, etc.) or fragmented state. The monomer peak area can be compared to the total area of the monomer, aggregate and fragment peaks associated with the target protein. Thus, the stability of a protein-containing formulation can be observed by the relative amounts of monomers after elapsed time; Thus, an improvement in the stability of a protein-containing formulation of the present invention can be measured as a higher monomer percentage after a certain elapsed time compared to the monomer percentage of a control formulation containing no excipient.

In an embodiment, an ideal stability result is one with a 98-100% monomer peak as determined by SEC analysis. In an embodiment, the improvement in stability of a protein-containing formulation of the present invention is a higher monomer percentage after exposure to a stress condition compared to the monomer percentage of a control formulation containing no excipient when the control formulation is exposed to the same stress conditions. can be measured with In embodiments, the stress conditions 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 an embodiment, a protein containing formulation as described herein is resistant to an increase in protein particle size as measured by dynamic light scattering (DLS) analysis. In the DLS assay as used herein, the protein particle size of a protein-containing formulation can be viewed as the median particle diameter. Ideally, the median particle diameter of the target protein should remain relatively unchanged when applied to DLS analysis as the particle diameter represents the active ingredient in the monomeric state as opposed to the aggregated (dimer, trimer, oligomer, etc.) or fragmented state. . An increase in median particle diameter may indicate aggregated protein. Thus, the stability of a protein-containing formulation can be observed by the relative change in median particle diameter after elapsed time.

In an embodiment, a protein containing formulation as described herein is resistant to the formation of a polydisperse particle size distribution as measured by DLS analysis. In an embodiment, the protein containing formulation may contain a monodisperse particle size distribution of colloidal protein particles. In embodiments, the ideal stability result is a change of less than 10% in the median particle diameter compared to the initial median particle diameter of the formulation. In embodiments, an improvement in stability of a protein-containing formulation of the invention can be measured as a lower percent change in median particle diameter after a specified elapsed time compared to the median particle diameter of a control formulation containing no excipient. In an embodiment, the improvement in stability of a protein-containing formulation of the present invention is determined after exposure to a stress condition compared to the percent change in median particle diameter of a control formulation containing no excipient when the control formulation is exposed to the same stress conditions. It can be measured as a percent change in lower median particle diameter. In embodiments, the stress conditions 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 an embodiment, the improvement in stability of a protein-containing formulation therapeutic formulation of the present invention is determined by DLS as compared to the polydispersity of the particle size distribution of a control formulation containing no excipient when the control formulation is exposed to the same stress conditions. It can be measured as a less polydisperse particle size distribution when measured.

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. In turbidity, light scattering or particle counting analyses, lower values generally indicate a lower number of suspended particles in the formulation. An increase in turbidity, light scattering or particle count may indicate that the solution of the target protein is not stable. Thus, the stability of a protein containing formulation can be observed by the relative amount of turbidity, light scattering or particle count after elapsed time. In an embodiment, the ideal stability result is one with low and relatively constant turbidity, light scattering or particle count values. In an embodiment, the improvement in the stability of a protein containing formulation as described herein is a lower turbidity, lower light scattering after a specified elapsed time compared to the turbidity, light scattering or particle count values of a control formulation containing no excipient. or a lower particle count. In an embodiment, the improvement in stability of a protein-containing formulation as described herein is dependent on stress conditions as compared to the turbidity, light scattering, or particle count of a control formulation containing no excipient when exposed to the same stress conditions. lower turbidity, lower light scattering or lower particle count after exposure to In embodiments, the stress conditions 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 an embodiment, a protein containing formulation as disclosed herein retains a higher percentage of biological activity compared to a control formulation. Biological activity can be observed through binding assays or therapeutic effects in mammals.

3. Therapeutic Formulations

In one aspect, the formulations and methods disclosed herein provide improved or reduced viscosity stable liquid formulations comprising therapeutically effective amounts of a therapeutic protein and an excipient compound. In embodiments, formulations may improve stability while providing acceptable concentrations of active ingredients and acceptable viscosities. In embodiments, the formulation provides an improvement in stability when compared to a control formulation; For purposes of this disclosure, a control formulation is a formulation containing the same protein active ingredient on a dry weight basis in all respects as the therapeutic formulation except that it lacks an excipient compound. In embodiments, the formulation improves stability under stressful 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 lyophilization. provides In an embodiment, the improved stability of the protein containing formulation is in the form of a lower percentage of soluble aggregates, particulates, sub-visible particles or gel formation compared to a control formulation.

The viscosity of a liquid protein formulation depends on the properties of the protein itself (eg, enzyme, antibody, receptor, fusion protein, etc.); its size, three-dimensional structure, chemical composition and molecular weight; its concentration in the formulation; components of the formulation other than proteins; desired pH range; storage conditions for the formulation; and the method of administering the formulation to the patient, but is not limited thereto. Therapeutic proteins most suitable for use with the excipient compounds described herein are preferably essentially pure, ie, free of contaminating proteins. In an embodiment, an "essentially pure" Therapeutic protein comprises at least 90% by weight of the Therapeutic protein, or preferably at least 95% by weight, or more preferably at least A protein composition comprising 99% by weight of a therapeutic protein. For purposes of clarity, proteins added as excipients are not intended to be included in this definition. Therapeutic formulations described herein are used to treat mammals in pharmaceutical grade formulations, i.e., in a form that does not contain ingredients toxic to the mammal to which the formulation is administered and that allows the desired therapeutic efficacy of the protein active ingredient to be achieved. It is intended for use in formulations intended for

In an embodiment, the therapeutic formulation contains at least 25 mg/mL of the protein active ingredient. In another embodiment, the therapeutic formulation contains at least 100 mg/mL of the protein active ingredient. In another embodiment, the therapeutic formulation contains at least 10 mg/mL of the protein active ingredient. In another embodiment, the therapeutic formulation contains at least 50 mg/mL of the protein active ingredient. In another embodiment, the therapeutic formulation contains at least 200 mg/mL of the protein active ingredient. In another embodiment, the therapeutic formulation solution contains at least 300 mg/mL of the protein active ingredient. Generally, an excipient compound disclosed herein is added to a therapeutic formulation in an amount of about 5 to about 300 mg/mL. In embodiments, the excipient compound may be added in an amount of about 10 to about 200 mg/mL. In embodiments, the excipient compound may be added in an amount of about 20 to about 100 mg/mL. In embodiments, an excipient may be added in an amount of about 25 to about 75 mg/mL.

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

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

In embodiments, the viscosity reducing amount results in a therapeutic formulation having a viscosity of less than 100 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 50 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 20 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term "viscosity" refers to a dynamic viscosity value when measured by the methods described herein.

Therapeutic formulations according to the present disclosure have certain advantageous properties that improve the stability of the formulation. In embodiments, the therapeutic formulation is resistant to shear degradation, phase separation, turbidity, oxidation, deamidation, aggregation, precipitation and denaturation. In embodiments, the therapeutic formulation is processed, purified, stored, injected, administered, filtered, and centrifuged more effectively as compared to a control formulation.

In an embodiment, the therapeutic formulation is administered to the patient at a high concentration of the therapeutic protein. In embodiments, the therapeutic formulation is administered to the patient with a smaller injection volume and/or less discomfort than experienced with a similar formulation lacking therapeutic excipients. In embodiments, the therapeutic formulation is administered to the patient using a narrower gauge needle or less syringe force than required for a similar formulation lacking therapeutic excipients. In embodiments, the therapeutic formulation is administered as a depot injection. In an embodiment, the therapeutic formulation prolongs the half-life of a therapeutic protein in the body.

These characteristics of therapeutic formulations as disclosed herein are useful in clinical situations, i.e., where patient acceptance of intramuscular injections is typical for IM/SC purposes, the use of small bore needles and acceptable (e.g., 2-3 mL) injection volumes. will allow administration of the formulation by intramuscular or subcutaneous injection in situations where these conditions will result in administration of an effective amount of the formulation in a single injection at a single injection site. In contrast, injection of equivalent doses of therapeutic proteins using conventional formulation techniques would be limited by the higher viscosity of conventional formulations, and therefore SC/IM injections of conventional formulations would not be suitable for clinical situations.

Therapeutic formulations according to the present disclosure may have certain advantageous properties consistent with improved stability. In embodiments, the therapeutic formulation is resistant to shear degradation, phase separation, turbidity, precipitation, oxidation, deamidation, aggregation, and/or denaturation. In embodiments, the therapeutic formulation is processed, purified, stored, injected, administered, filtered, and/or centrifuged more effectively as compared to a control formulation.

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 usually associated with the active ingredient of the formulation, eg a therapeutic protein, and this main peak of the active ingredient is referred to as the monomer peak. The monomer peak represents the amount of active ingredient in the monomeric state as opposed to the aggregated (dimer, trimer, oligomer, etc.) state. High concentration solutions of therapeutic proteins formulated with excipient compounds described herein can be administered to a patient using a syringe or pre-filled syringe. Thus, the stability of a therapeutic formulation can be observed by the relative amounts of monomers after elapsed time. In embodiments, an improvement in the stability of a therapeutic formulation as disclosed herein can be measured as a higher percentage of monomer after a specified elapsed time compared to the percentage of monomer in a control formulation containing no excipient. In embodiments, an improvement in the stability of a therapeutic formulation as disclosed herein can be measured as a higher percentage monomer after exposure to stress conditions compared to the percentage monomers of a control formulation containing no excipient after exposure to stress conditions. . In embodiments, the stress conditions can be cold storage, high temperature storage, exposure to air, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.

In an embodiment, the therapeutic formulations of the invention are resistant to an increase in protein particle size as measured by dynamic light scattering (DLS) analysis. In DLS analysis, the particle size of a therapeutic protein can be viewed as the median particle diameter. Ideally, the median particle diameter of a therapeutic protein should be relatively unchanged. Thus, an increase in median particle diameter may indicate aggregated protein. Thus, the stability of a therapeutic formulation can be observed by the relative change in median particle diameter over time. In an embodiment, a therapeutic formulation as disclosed herein is resistant to forming a polydisperse particle size distribution as measured by dynamic light scattering (DLS) analysis. In embodiments, an improvement in stability of a therapeutic formulation of the present invention can be measured as a lower percent change in median particle diameter after a specified elapsed time compared to the median particle diameter of a control formulation containing no excipient. In embodiments, an improvement in the stability of a therapeutic formulation as disclosed herein can be measured as a lower percent change in median particle diameter after exposure to stress conditions compared to the percent change in median particle diameter of a control formulation containing no excipient. can In other words, in an embodiment, the improved stability prevents an increase in particle size as measured by light scattering. In embodiments, the stress conditions can be cold storage, high temperature storage, exposure to air, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles. In embodiments, an improvement in the stability of a therapeutic formulation as disclosed herein can be measured as a less polydisperse particle size distribution as measured by DLS compared to the polydispersity of the particle size distribution of a control formulation containing no excipient. .

In an embodiment, a therapeutic formulation as disclosed herein is resistant to sedimentation as measured by turbidity, light scattering or particle counting analysis. In embodiments, an improvement in the stability of a therapeutic formulation as disclosed herein is a result of lower turbidity, lower light scatter, or lower turbidity after a specified elapsed time compared to the turbidity, light scatter, or particle count values of a control formulation containing no excipient. A lower particle count can be measured. In an embodiment, the improvement in the stability of a therapeutic formulation as disclosed herein includes lower turbidity, lower light scatter after exposure to stress conditions compared to turbidity, light scatter or particle count of a control formulation containing no excipient. or a lower particle count. In embodiments, the stress conditions can be cold storage, high temperature storage, exposure to air, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.

In an embodiment, the therapeutic excipient has antioxidant properties that stabilize the therapeutic protein against oxidative damage to improve its stability. In an embodiment, the therapeutic formulation is stored for an extended period of time at ambient temperature or in refrigerator conditions without appreciable loss of efficacy of the therapeutic protein. In embodiments, the therapeutic formulation is dried for storage until needed; It is then reconstituted with a suitable solvent, eg water. Advantageously, formulations prepared as described herein may be stable over extended periods of time from several months to several years. If exceptionally long storage is required, the formulation can be stored in the refrigerator (and subsequently reactivated) without fear of protein denaturation. In embodiments, formulations may be prepared for long-term storage that do not require refrigeration.

In embodiments, an excipient compound disclosed herein is added to a therapeutic formulation in a stability enhancing amount. In an embodiment, the stability improvement amount is the amount of excipient compound that reduces degradation of the formulation by at least 10% when compared to a control formulation; For 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 except for lacking an excipient compound. In embodiments, a stability improvement amount is an amount of an excipient compound that reduces degradation of the formulation by at least 30% when compared to a control formulation. In embodiments, a stability improvement amount is an amount of an excipient compound that reduces degradation of the formulation by at least 50% when compared to a control formulation. In an embodiment, the stability improvement amount is an amount of excipient compound that reduces degradation of the formulation by at least 70% when compared to a control formulation. In embodiments, a stability improvement amount is an amount of an excipient compound that reduces degradation of the formulation by at least 90% when compared to a control formulation.

Methods for preparing therapeutic formulations may be familiar to the skilled artisan. Therapeutic formulations of the invention can be prepared, for example, by adding an excipient compound to the formulation before or after the therapeutic protein is added to the solution. For example, a therapeutic formulation can be created by combining a therapeutic protein and an excipient at a first (lower) concentration, followed by processing by filtration or centrifugation to produce a second (higher) concentration of the therapeutic protein. Therapeutic formulations can be formulated with one or more of the excipient compounds along with chaotropes, kosmotropes, hydrotropes and salts. Therapeutic formulations can be prepared with one or more of the excipient compounds using techniques such as encapsulation, dispersion, liposomes, vehicle formation, and the like. Methods for preparing therapeutic formulations comprising excipient compounds disclosed herein may include combinations of excipient compounds. In embodiments, combinations of excipients may result in benefits in lower viscosity, improved stability, or reduced injection site pain. Other additives may be incorporated into the therapeutic formulation during 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 a compound 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 improved or reduced viscosity stable liquid formulations comprising effective amounts of non-therapeutic proteins and excipient compounds. In embodiments, the formulation improves stability while providing an acceptable concentration of the active ingredient and an acceptable viscosity. In embodiments, the formulation provides an improvement in stability when compared to a control formulation; For purposes of this disclosure, a control formulation is a formulation containing the same protein active ingredient on a dry weight basis in all respects as the non-therapeutic formulation except that it lacks an excipient compound.

The viscosity of a liquid protein formulation depends on the nature of the protein itself (eg, enzyme, structural protein, degree of hydrolysis, etc.); its size, three-dimensional structure, chemical composition and molecular weight; its concentration in the formulation; components of the formulation other than proteins; desired pH range; and storage conditions for the formulation.

In an embodiment, the non-therapeutic formulation contains at least 25 mg/mL of the protein active ingredient. In another embodiment, the non-therapeutic formulation contains at least 100 mg/mL of the protein active ingredient. In another embodiment, the non-therapeutic formulation contains at least 200 mg/mL of the protein active ingredient. In another embodiment, the non-therapeutic formulation solution contains at least 300 mg/mL of the protein active ingredient. Generally, an excipient compound disclosed herein is added to a non-therapeutic formulation in an amount of about 5 to about 300 mg/mL. In embodiments, the excipient compound is added in an amount of about 10 to about 200 mg/mL. In an embodiment, the excipient compound is added in an amount of about 20 to about 100 mg/mL. In embodiments, the excipient is added in an amount of about 25 to about 75 mg/mL.

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

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

In embodiments, the viscosity reducing amount results in a non-therapeutic formulation having a viscosity of less than 100 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 50 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 20 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term “viscosity” refers to a dynamic viscosity value.

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

In an embodiment, the non-therapeutic excipient has antioxidant properties that stabilize the non-therapeutic protein against oxidative damage to improve its stability. In an embodiment, the non-therapeutic formulation is stored for an extended period of time at ambient temperature or in refrigerator conditions without appreciable loss of potency to the non-therapeutic protein. In embodiments, the non-therapeutic formulation is dried for storage until needed; It can then be reconstituted with a suitable solvent, eg water. Advantageously, formulations prepared as described herein are stable over extended periods of time from several months to several years. In exceptional cases where long-term storage is required, the formulation is stored in the refrigerator (and subsequently reactivated) without fear of protein denaturation. In embodiments, the formulation is prepared for long-term storage that does not require refrigeration.

In embodiments, an excipient compound disclosed herein is added to a non-therapeutic formulation in a stability enhancing amount. In an embodiment, the stability improvement amount is the amount of excipient compound that reduces degradation of the formulation by at least 10% when compared to a control formulation; For purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar on a dry weight basis to the therapeutic formulation except for lacking an excipient compound. In embodiments, a stability improvement amount is an amount of an excipient compound that reduces degradation of the formulation by at least 30% when compared to a control formulation. In embodiments, a stability improvement amount is an amount of an excipient compound that reduces degradation of the formulation by at least 50% when compared to a control formulation. In an embodiment, the stability improvement amount is an amount of excipient compound that reduces degradation of the formulation by at least 70% when compared to a control formulation. In embodiments, a stability improvement amount is an amount of an excipient compound that reduces degradation of the formulation by at least 90% when compared to a control formulation.

Methods for preparing non-therapeutic formulations comprising excipient compounds disclosed herein may be familiar to the skilled artisan. For example, an excipient compound may be added to the formulation before or after the non-therapeutic protein is added to the solution. A non-therapeutic formulation may be produced at a first (lower) concentration and then processed by filtration or centrifugation to produce a second (higher) concentration. Non-therapeutic formulations can be prepared with one or more of the excipient compounds along with chaotropes, cosmotropes, hydrotropes and salts. Non-therapeutic formulations can be prepared with one or more of the excipient compounds using techniques such as encapsulation, dispersion, liposomes, vehicle formation, and the like. Other additives may be incorporated into non-therapeutic formulations during manufacture, including preservatives, surfactants, stabilizers, and the like.

5. Excipient compounds

Several excipient compounds are described herein, each suitable for use with one or more therapeutic or non-therapeutic proteins, each allowing formulations to be constructed to contain high concentrations of the protein(s). Some of the categories of excipient compounds described below include (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) a clustering agent with hydrogen bonding elements. Without wishing to be bound by theory, it is believed that the excipient compounds described herein associate with specific fragments, sequences, structures or sections of therapeutic proteins that would otherwise be involved in interparticle (ie protein-protein) interactions. The association of these excipient compounds with therapeutic or non-therapeutic proteins can mask interactions between proteins so that the proteins can be formulated at high concentrations without causing excessive solution viscosity. In an embodiment, an excipient compound is capable of generating more stable protein-protein interactions; Protein-protein interactions can be measured by the protein diffusion parameter kD or the osmotic second virial coefficient B22, or other techniques familiar to the skilled person.

The excipient compounds may advantageously be water soluble and are therefore suitable for use with aqueous vehicles. In an embodiment, the excipient compound has a water solubility of >1 mg/mL. In an embodiment, the excipient compound has a water solubility of >10 mg/mL. In an embodiment, the excipient compound has a water solubility of >100 mg/mL. In an embodiment, the excipient compound has a water solubility of >500 mg/mL. In embodiments, a cosolute or hydrotrope may be added in combination with an 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 this solubility may be much lower under cold storage conditions. Co-solutes or hydrotropes may be added to increase the solubility of excipients in solution at cold storage conditions or ambient room temperature or elevated temperature conditions. Examples of co-solutes and hydrotropes include benzoate salts, benzyl alcohol, phenylalanine, nicotinamide, proline, procaine, 2,5-dihydroxybenzoate, tyramine and saccharin. In the case of therapeutic proteins, the excipient compounds can advantageously be derived from materials that are biologically acceptable, non-immunogenic and therefore suitable for pharmaceutical use. In therapeutic embodiments, an excipient compound may be metabolized in the body to produce by-products that are biologically compatible and non-immunogenic.

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 a small molecule containing at least one bulky or sterically hindered group consistent with the examples below. Hindered amines can be used in free base form, protonated form or a combination of the two. In protonated form, hindered amines can be associated with anionic counterions such as chloride, hydroxide, bromide, iodide, fluoride, acetate, formate, phosphate, sulfate or carboxylate. Hindered amine compounds useful as excipient compounds may contain secondary amine, tertiary amine, quaternary ammonium, pyridinium, pyrrolidone, pyrrolidine, piperidine, morpholine or guanidinium groups, so that the excipient compound It has a positive charge in aqueous solution at neutral pH. Hindered amine compounds also contain at least one bulky or sterically hindered group, such as a cyclic aromatic, cycloaliphatic, cyclohexyl or alkyl group. In an embodiment, the sterically hindered group may 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 sections of proteins such as phenylalanine, tryptophan and tyrosine by cationic pi interactions. In an embodiment, the cationic groups of the hindered amines may have an affinity for electron rich pi structures of aromatic amino acid residues of the protein, so that they may protect these sections of the protein, such that the protected protein associates and It can reduce the tendency to agglomerate.

In an embodiment, the hindered amine excipient compound is an imidazole, imidazoline, or imidazolidine group, or a salt 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- Contains imidazoline, 1-butyl-3-methylimidazolium chloride, uric acid, potassium urate, betazol, carnosine, aspartame, saccharin, acesulfame potassium, xanthine, theophylline, theobromine, caffeine, and anserine has a chemical structure that In an embodiment, the hindered amine excipient compound is dimethylethanolamine, dimethylaminopropylamine, triethanolamine, dimethylbenzylamine, dimethylcyclohexylamine, diethylcyclohexylamine, dicyclohexylmethylamine, hexamethylene biguanide, poly (hexamethylene biguanide), imidazole, lysine, methylglycine, sarcosine, dimethylglycine, agmatine, diazabicyclo[2.2.2]octane, sodium polynic acid salt, calcium salt of polynic acid, tetramethylethylenediamine, N,N-dimethylethanolamine, ethanolamine phosphate, glucosamine, choline chloride, phosphocholine, niacinamide, isonicotinamide, N,N-diethyl nicotinamide, nicotinic acid sodium salt, isonicotinic acid salt, tyramine, 3-amino Pyridine, 2,4,6-trimethylpyridine, 3-pyridine methanol, nicotinamide adenine dinucleotide, biotin, morpholine, N-methylpyrrolidone, 2-pyrrolidinone, dipyridamole, procaine, lidocaine, dicy Andiamide-taurine adduct, 2-pyridylethylamine, 6-hydroxypyridine-2-carboxylic acid, dicyandiamide-benzyl amine adduct, dicyandiamide-alkylamine adduct, dicyandiamide-cycloalkyl amine adducts, and dicyandiamide-aminomethanephosphonic acid adducts. In an embodiment, the hindered amine excipient compound is 1-(1-adamantyl)ethylamine, 1-aminobenzotriazole, 2-dimethylaminoethanol, 2-methyl-2-imidazoline, 2-methylimida Sol, 3-aminobenzamide, 3-indoleacetic acid, 4-aminopyridine, 6-amino-1,3-dimethyluracil, acetylcholine, agmatine sulfate, benzalkonium chloride, ethyl 3-aminobenzoate, sulfaceta Mead, butyl anthranilate, amino hippuric acid, benzamide oxime, benzethonium chloride, benzylamine, berberine chloride, castanospermine, clemisole, cycloserine, phenylserine, DL-3-phenylserine, cysteamine, Cytidine, diethanolamine, diphenhydramine, DL-norepinephrine, dopamine, emtricitabine, ethanolamine, guanfacine, isonicotinamide, lithium chloride, meglumine, methyl cytidine, myristyl gamma picolinium chloride , niacinamide, phenylethylamine, polyethyleneimine, pyridoxine, rasagiline mesylate, serotonin, synephrine, neamine, spermine, spermidine, 1,3-diaminopropane, adenosine, chloroquine phosphate, cystamine, pyridyl ethyl Amine, tetramethylethylenediamine, tryptamine, tyramine, 1-methylimidazole, spectinomycin, cyclohexane methylamine, N,N-dimethylphenethylamine, phenethylamine, tetraethylammonium, tetramethyl ammonium acetate, Dicyclomine, hordenine, methylaminoethylpyridine, nicotinamide riboside, 1-butylimidazole, 1-hexylimidazole, 1-methylimidazole, 2-ethylimidazole, 2-n-butyl imidazole, 2-methylimidazole, 1-dodecylimidazole, other imidazoles alkylated at the 1 or 2 position with a C ₁ to C ₁₂ hydrocarbon chain, pridinol methanesulfonate, hemin, N,N -It is selected from the group consisting of dimethylphenethylamine, voglibose, N-ethyl-L-glutamine, nicotine, piperazine, demeclocycline, and salts thereof. In an embodiment, the hindered amine excipient compound is a phenethylamine functional group such as phenethylamine, diphenhydramine, N-methylphenethylamine, N,N-dimethylphenethylamine, β,3-dihydroxy phenethylamine, β,3-dihydroxy-N-methylphenethylamine, 3-hydroxyphenethylamine, 4-hydroxyphenethylamine, tyrosinol, tyramine, N-methyltyramine, and hordenine can Preferably, the phenethylamine containing structure is a non-psychoactive compound.

Suitable salts of hindered amine structures may be chlorides, bromides, acetates, citrates, sulfates and phosphates. In an embodiment, a hindered amine compound consistent with the present disclosure is formulated as a protonated ammonium salt. In an embodiment, a hindered amine compound consistent with the present disclosure is formulated as a salt with an inorganic or organic anion as a counterion.

In an embodiment, the high concentration solution of the therapeutic or non-therapeutic protein is formulated with a combination of caffeine and benzoic acid, hydroxybenzoic acid or benzenesulfonic acid as an excipient compound. In an embodiment, the hindered amine excipient compound is metabolized in the body to produce biologically compatible by-products. In some embodiments, the hindered amine excipient compound is present in the formulation at a concentration of about 250 mg/mL or less. In a further embodiment, the hindered amine excipient compound is present in the formulation at a concentration of about 10 mg/mL to about 200 mg/mL. In a still further 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 class of hindered amines viscosity reducing excipients have typically been limited in their use due to their low water solubility, but may include methylxanthines such as caffeine and theophylline. In some applications, it may be advantageous to have highly concentrated solutions of these viscosity reducing excipients despite their low water solubility. For example, during processing, it may be advantageous to have a concentrated excipient solution that can be added to the concentrated protein solution since adding the excipient will not dilute the protein below the desired final concentration. In other cases, additional viscosity reducing excipients may be required to achieve the desired viscosity reduction, stability, tonicity, etc. of the final protein formulation despite its low aqueous solubility. In an embodiment, the highly concentrated excipient solution is (i) a viscosity reducing excipient at a concentration 1.5 to 50 times greater than the effective viscosity reducing amount, or (ii) its literature-reported solubility in pure water at 298 K (e.g., As reported in The Merck Index; Royal Society of Chemistry; Fifteenth Edition, (April 30, 2013)), 1.5 to 50 times higher concentration of viscosity reducing excipient, or both.

Certain co-solutes have been found to substantially increase the solubility limit of these low solubility viscosity reducing excipients, allowing excipient solutions with concentrations many times higher than the solubility values reported in the literature. These co-solutes can be classified into general hydrotrope categories. The co-solutes found to provide the greatest improvement in solubility in these applications were generally highly soluble in water (>0.25 M) at ambient temperature and physiological pH, and contained pyridine or benzene rings. Examples of compounds that may be useful as co-solutes are aniline HCl, isoniacinamide, niacinamide, n-methyltyramine HCl, phenol, procaine HCl, resorcinol, saccharin calcium salt, saccharin sodium salt, sodium aminobenzoic acid, sodium benzoate, sodium parahydroxybenzoate, sodium metahydroxybenzoate, sodium 2,5-dihydroxybenzoate, sodium salicylate, sodium sulfanilate, sodium parahydroxybenzene sulfonate, synephrine, and Contains tyramine HCl.

In embodiments, certain hindered amine excipient compounds may have other pharmacological properties. By way of example, xanthines, when absorbed systemically, are a class of hindered amines that generally have independent pharmacological properties, including stimulant properties and bronchodilator properties. Representative xanthines include caffeine, aminophylline, 3-isobutyl-1-methylxanthine, paraxanthin, pentoxifylline, theobromine, theophylline, and the like. Methylated xanthines are understood to affect the force of cardiac contraction, 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 topical injectable anesthetic compounds. Topical injectable anesthetic compounds are hindered amines having a three-component molecular structure of (a) a lipophilic aromatic ring, (b) an intermediate ester or amide linkage, and (c) a secondary or tertiary amine. This category of hindered amines is understood to interfere with nerve conduction by inhibiting the influx of sodium ions, thereby inducing local anesthesia. The lipophilic aromatic ring for the local anesthetic compound may be formed of carbon atoms (eg, a benzene ring) or it may include heteroatoms (eg, a thiophene ring). Representative local injectable anesthetic compounds are amylocaine, articaine, bupivicaine, butacaine, butanilicaine, chlorprocaine, cocaine ), cyclomethycaine, dimethocaine, editocaine, hexylcaine, isobucaine, levobupivacaine, lidocaine, metabute metabutoxycaine, mepivacaine, meprylcaine, propoxycaine, prilocaine, procaine, piperocaine (piperocaine), tetracaine (tetracaine), trimecaine (trimecaine), and the like, but are not limited thereto. Topically injectable anesthetic compounds can have a number of advantages, such as reduced viscosity in protein therapeutic formulations, improved stability, and reduced pain upon injection. In some embodiments, the local anesthetic compound is present in the formulation at a concentration of about 50 mg/mL or less.

In embodiments, hindered amines with independent pharmacological properties are used as excipient compounds according to the formulations and methods described herein. In some embodiments, excipient compounds with independent pharmacological properties are present in amounts that do not have pharmacological effects and/or are not therapeutically effective. In another embodiment, an excipient compound having independent pharmacological properties has a pharmacological effect and/or is present in a therapeutically effective amount. In certain embodiments, a hindered amine with independent pharmacological properties is used in combination with another selected excipient compound to reduce formulation viscosity, wherein the hindered amine with independent pharmacological properties reduces its pharmacological activity. Used to give advantage. For example, locally injectable anesthetic compounds can be used to reduce formulation viscosity and also reduce pain upon injection of the formulation. A reduction in injection pain may be caused by anesthetic properties; Also, a lower injection force may be required if the viscosity is reduced by the excipient. Alternatively, a topical injectable anesthetic compound may be used to impart the desired pharmacological benefit of reduced 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 aromatics

Solutions of therapeutic or non-therapeutic proteins can be formulated with an anionic aromatic small molecule as an excipient compound. Anionic aromatic excipient compounds may contain aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, phenol, hydroxyaryl, heteroaromatic groups or fused aromatic groups. Anionic aromatic excipient compounds may also contain anionic functional groups such as carboxylates, oxides, phenoxides, sulfonates, sulfates, phosphonates, phosphates or sulfides. Anionic aromatic excipients may be described as acids, sodium salts or otherwise, although it is understood that excipients may be used in a variety of salt forms. Without wishing to be bound by theory, it is believed that anionic aromatic excipient compounds are bulky sterically hindered molecules capable of associating with cationic segments of proteins, so that they protect these sections of proteins so that protein-containing formulations become viscous. or reduce interactions between protein molecules that 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, naphthalenesulfonic acid, 1, 5-naphthalenedisulfonic acid, 2,6-naphthalenedisulfonic acid, 2,7-naphthalenedisulfonic acid, hydroquinone sulfonic 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, indole acetic acid, potassium urate, furan dicarboxylic acid , furan-2-acrylic acid, 2-furanpropionic acid, sodium phenylpyruvate, sodium hydroxyphenylpyruvate, trimethoxybenzoic acid, dihydroxybenzoic acid, ferrocenecarboxylic acid, trihydroxybenzoic acid, pyrogallol, benzoic acid, and and compounds such as salts of these acids. In an embodiment, the anionic aromatic excipient compound is formulated in ionized salt form. In an embodiment, the anionic aromatic compound is formulated as a salt of a hindered amine such as dimethylcyclohexylammonium hydroxybenzoate. In embodiments, anionic aromatic excipient compounds are formulated with various counterions such as organic cations. In an embodiment, a high concentration solution of a therapeutic or non-therapeutic protein is formulated with an anionic aromatic excipient compound and caffeine. In an embodiment, the anionic aromatic excipient compound is metabolized in the body to produce biologically compatible by-products.

In embodiments, examples of aromatic excipient compounds include phenols and polyphenols. As used herein, the term “phenol” refers to an organic molecule composed of at least one aromatic group or fused aromatic groups bonded to at least one hydroxyl group, and the term “polyphenol” refers to an organic molecule composed of more than one phenolic group. indicate Such excipients may be advantageous in certain circumstances, for example when used in formulations with high concentration solutions of therapeutic or non-therapeutic PEGylated proteins to lower 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), benzenetrol 1,2,3 ,4-benzenetetrol and 1,2,3,5-benzenetetrol, and benzenepentol and benzenehexol. Non-limiting examples of polyphenols include tannic acid, ellagic acid, epigallocatechin gallate, catechin, 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, chalcones, 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 polyphenol compounds include alkylmethoxyphenols, alkylphenols, curcuminoids, hydroxybenzaldehydes, hydroxybenzoketones, hydroxycinnamaldehydes, hydroxycoumarins, hydroxyphenylpropenes, methoxyphenols, naphthoquinones, but is not limited to hydroquinone, phenol terpene, resveratrol and tyrosol. In an embodiment, the polyphenol is tannic acid. In an embodiment, the phenol is gallic acid. In an embodiment, the phenol is pyrogallol. In an embodiment, phenol is resorcinol. Without wishing to be bound by theory, for example, the hydroxyl groups of phenolic compounds such as gallic acid, pyrogallol and resorcinol form hydrogen bonds with ether oxygen atoms in the backbone of the PEG chain, thereby reducing solution viscosity. It forms a phenol/PEG complex that radically changes the structure of the PEG solution. Polyphenolic compounds such as tannic acid derive their viscosity reducing properties from their respective phenolic building blocks such as gallic acid, pyrogallol and resorcinol. The specific organization of phenolic groups in polyphenolic compounds can give rise to a complex behavior in which the viscosity reduction achieved by addition of phenol is enhanced by the addition of smaller amounts of each polyphenol.

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, wherein a single functionalized amino acid or an oligopeptide comprising one or more functionalized amino acids can be used as an excipient compound. In embodiments, functionalized amino acid compounds include molecules that can be hydrolyzed or metabolized to produce amino acids ("amino acid precursors"). In embodiments, functionalized amino acids may contain aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromatic groups or fused aromatic groups. In embodiments, functionalized amino acid compounds may contain esterified amino acids such as methyl, ethyl, propyl, butyl, benzyl, cycloalkyl, glyceryl, hydroxyethyl, hydroxypropyl, PEG and PPG esters. In an embodiment, the functionalized amino acid compound is selected from the group consisting of arginine ethyl ester, arginine methyl ester, arginine hydroxyethyl ester and arginine hydroxypropyl ester. In an embodiment, the functionalized amino acid compound is a charged ionic compound in an aqueous solution at neutral pH. For example, a single amino acid can be derivatized by forming an ester such as acetate or benzoate, and the product of hydrolysis together with the amino acid will be acetic acid or benzoic acid (both are natural substances). In an embodiment, the functionalized amino acid excipient compound is metabolized in the body to produce biocompatible by-products.

d. Excipient Compound Category 4: Oligopeptides

Solutions of therapeutic or non-therapeutic proteins can be formulated with oligopeptides as excipient compounds. In an embodiment, the oligopeptide is designed to have a structure with charged sections and bulky sections. In an embodiment, an oligopeptide is composed of 2 to 10 peptide subunits. An oligopeptide can be bifunctional, eg, a cationic amino acid coupled to a non-polar one, or an anionic amino acid coupled to a non-polar one. In an embodiment, an oligopeptide is composed of 2 to 5 peptide subunits. In an embodiment, the oligopeptide is a homopeptide such as polyglutamic acid, polyaspartic acid, poly-lysine, poly-arginine and poly-histidine. In an embodiment, the oligopeptide has a net cationic charge. In another embodiment, the oligopeptide is a heteropeptide such as Trp2Lys3. In an embodiment, an oligopeptide may have an alternating structure such as an ABA repeat pattern. In an embodiment, an oligopeptide comprises both anionic and cationic amino acids, e.g., Arg-Glu, Lys-Glu, His-Glu, Arg-Asp, Lys-Asp, His-Asp, Glu-Arg, Glu- Lys, Glu-His, Asp-Arg, Asp-Lys and Asp-His. Without wishing to be bound by theory, oligopeptides contain structures capable of associating with proteins in a manner that reduces intermolecular interactions that create high viscosity solutions and stability problems; For example, oligopeptide-protein associations can be charge-charge interactions, which disrupt hydrogen bonding in the hydration layer around the protein, leaving rather non-polar amino acids that lower viscosity or improve stability. 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 may be formulated with short chain organic acids as excipient compounds. As used herein, the term "short chain organic acid" refers to a C ₂ -C ₆ organic acid compound and its salt, ester, amide or lactone. This category includes saturated and unsaturated carboxylic acids, hydroxy functionalized carboxylic acids, amides, and linear, branched or cyclic carboxylic acids. In an embodiment, the acid group of the short chain organic acid is a carboxylic acid, sulfonic acid, phosphonic acid or a salt thereof.

Solutions of therapeutic or non-therapeutic proteins can be formulated as excipient compounds in the form of acids or salts of short chain organic acids such as sorbic acid, valeric acid, propionic acid, caproic acid and ascorbic acid. Examples of excipient compounds in this category are potassium sorbate, calcium gluconate, glucuronic acid, calcium lactate, 2-hydroxylactate, sodium glycolate, potassium glycolate, ammonium glycolate, sodium valproate, taurine, acetohydroxamic acid, acetone sodium bisulfite adduct, acetyl hydroxyproline, 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 can be formulated with specific excipient compounds that can lower solution viscosity or improve stability, and the excipient compounds are low molecular weight polyacids. Low molecular weight polyacids may include organic or inorganic polyacids. These low molecular weight polyacid excipients may also be used in combination with other excipients.

In an embodiment, the organic polyacid can be structured as a low molecular weight aliphatic polyacid. 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, wherein the acidic groups are understood to be proton-donating moieties. Non-limiting examples of acidic groups include carboxylate, phosphonate, phosphate, sulfonate, sulfate, nitrate and nitrite groups. The acidic groups of low molecular weight aliphatic polyacids can exist in the form of anionic salts such as carboxylates, phosphonates, phosphates, sulfonates, sulfates, nitrates and nitrites; Their counterions can be sodium, potassium, lithium and ammonium. Specific examples of low molecular weight aliphatic polyacids useful for interacting with PEGylated proteins as described herein include oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, succinic acid, maleic acid, tartaric acid, glu Taric acid, malonic acid, itaconic acid, methyl malonic acid, azelaic acid, citric acid, 3,6,9-trioxaundecanedioic acid, ethylenediaminetetraacetic acid (EDTA), aspartic acid, pyrrolidone carboxylic acid, pyroglutamic acid , glutamic acid, alendronic acid, medronic acid, etidronic acid and salts thereof.

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

In an embodiment, the low molecular weight aliphatic polyacid can also be an alpha hydroxy acid, wherein there is a hydroxyl group adjacent to the first acidic group, such as glycolic acid, lactic acid and gluconic acid and salts thereof. In an embodiment, the low molecular weight aliphatic polyacid is an oligomeric form having more than two acidic groups, such as polyacrylic acid, polyphosphate, polypeptide and salts thereof. In some embodiments, the low molecular weight aliphatic polyacid excipient is present in the composition at a concentration of about 50 mg/mL or less.

g. Excipient Compound Category 7: Diones and Sulfones

Effective viscosity reducing or stabilizing excipients can be molecules containing sulfone, sulfonamide or dione functional groups that are soluble in pure water at 298K to at least 1 g/L and have a net neutral charge at pH 7. 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 are not strong hydrogen bond donors. Without wishing to be bound by theory, the double bond nature may allow for weak pi-stacking interactions with proteins. In an embodiment, in high protein concentrations and proteins where only high viscosities occur at high concentrations, charged excipients are not effective as the regular interactions are longer range interactions. Solvated protein surfaces are primarily hydrophilic, making them water-soluble. Hydrophobic regions of proteins are normally protected within a three-dimensional structure, but the structure is constantly evolving, unfolding and refolding (sometimes referred to as “breathing”), and hydrophobic regions of adjacent proteins are They come into contact with each other and can cause aggregation by hydrophobic interactions. The pi-stacking nature of the dione and sulfone excipients can mask hydrophobic patches that may be exposed during the “breathing”. Another important role of excipients can be to disrupt hydrophobic interactions and hydrogen bonds between adjacent proteins, which will effectively reduce solution viscosity. Dione and sulfone compounds that fit this description include dimethylsulfone, ethyl methyl sulfone, ethyl methyl sulfonyl acetate, ethyl isopropyl sulfone, sodium cyclamate, bis(methylsulfonyl)methane, methane sulfonamide, methionine sulfone, 1,2 -Includes cyclopentanedione, 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 may be formulated with certain zwitterionic compounds as excipients to improve stability or reduce viscosity. As used herein, the term "zwitterionic" refers to compounds having positively charged sections and negatively charged sections. In an embodiment, the zwitterionic excipient compound is an amine oxide. In an embodiment, opposite charges are separated from each other by 2-8 chemical bonds. In an embodiment, the zwitterionic excipient compound can be a small molecule, such as having a molecular weight of about 50 to about 500 g/mol, or a medium molecular weight molecule, such as having a molecular weight of about 500 to about 2000 g/mol, It may be a high molecular weight molecule, such as a polymer having a molecular weight of about 2000 to about 100,000 g/mol.

Examples of zwitterionic excipient compounds are (3-carboxypropyl) trimethylammonium chloride, 1-aminocyclohexane carboxylic acid, homocycloleucine, 1-methyl-4-imidazoleacetic acid, 3-(1-pyridino)- 1-Propanesulfonate, 4-aminobenzoic acid, alendronate, aminoethyl sulfonic acid, aminohippuric acid, aspartame, aminotris (methylenephosphonic acid) (ATMP), calcobutrol, calteridol, cocamidopropyl betaine, Cocamidopropyl hydroxysulsteine, creatine, cytidine monophosphate, diaminopimelic acid, diethylenetriaminepentaacetic acid, dimethyl phenylalanine, methylglycine, sarcosine, dimethylglycine, zwitterionic dipeptides (e.g. Arg -Glu, Lys-Glu, His-Glu, Arg-Asp, Lys-Asp, His-Asp, Glu-Arg, Glu-Lys, Glu-His, Asp-Arg, Asp-Lys, Asp-His), diethylene Triamine penta(methylene phosphonic acid) (DTPMP), dipalmitoyl phosphatidylcholine, ectoin, ethylenediamine tetra(methylenephosphonic acid) (EDTMP), folate benzoate mixture, folate niacinamide mixture, gelatin, hydroxyproline, Iminodiacetic acid, isogubacin, lecithin, myristamine oxide, niconiamide adenine dinucleotide (NAD), N-methyl aspartic acid, N-methylproline, N-trimethyl lysine, ornithine, oxolinic acid, risendro nate, allyl cysteine, S-allyl-L-cysteine, somapacitan, taurine, theanine, trigonelline, vigabatrine, ectoin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonate, o -Octylphosphoryl choline, nicotinamide mononucleotide, triglycine, tetraglycine, β-guanidinopropionic acid, 5-aminolevulinic acid hydrochloride, picolinic acid, lidofenine, phosphocholine, 1-(5-carboxy Pentyl)-4-methylpyridin-1-ium bromide, L-Anserine nitrate, reduced L-glutathione, N-ethyl-L-glutamine, N-methyl proline, (Z)-1-[N-(2 -Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-dioleate (DETA-NONO ate), (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate (DPTA-NONate) and sol Contains redronic acid.

Without wishing to be bound by theory, the zwitterionic excipient compounds may interact with proteins, for example, by charge interactions, hydrophobic interactions and steric interactions that render proteins more resistant to aggregation, or by electrolytes. Viscosity reducing or stabilizing effects can be exerted by influencing the bulk properties of water in a protein formulation, such as distribution, surface tension reduction, change in the amount of unbound water available, or change in dielectric constant.

i. Excipient compound category 9: Flocculants with hydrogen bonding elements

Solutions of therapeutic or non-therapeutic proteins may be formulated with a flocculant having hydrogen bonding elements as excipients to improve stability or reduce viscosity. As used herein, the term "coagulant" refers to a formulation additive that increases the effective protein concentration by reducing the amount of water available to dissolve the protein in solution. In an embodiment, the flocculating agent can reduce protein particle size or reduce the amount of unfolded protein in solution. In an embodiment, the flocculant may act as a solvent modifier that causes structuring of water by hydrogen bonding and hydration effects. In an embodiment, the clustering agent can reduce the amount of intermolecular interactions between proteins in solution. In an embodiment, the clustering agent has a structure containing at least one hydrogen bond donor element, such as hydrogen attached to an oxygen, sulfur or nitrogen atom. In an embodiment, the clustering agent has a structure containing at least one weakly acidic hydrogen bond donor element having a pKa of from about 6 to about 11. In an embodiment, the clustering agent has a structure containing from about 2 to about 50 hydrogen bond donor elements. In an embodiment, the clustering agent has a structure containing at least one hydrogen bond acceptor element such as a Lewis base. In an embodiment, the clustering agent has a structure containing from about 2 to about 50 hydrogen bond acceptor elements. In an embodiment, the clustering agent has a molecular weight between about 50 and 500 g/mol. In an embodiment, the clustering agent has a molecular weight between about 100 and 350 g/mol. In another embodiment, the flocculant may have a molecular weight greater than 500 g/mol, such as raffinose, inulin, pullulan or sinistrin.

Examples of clustering excipients with hydrogen bonding elements are 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 acetone acetate, benzyl alcohol, chlorobutanol, cholestanol tetraacetyl-b-glucoside, cinnamaldehyde, cyclo Hexanone, deoxyribose, diethyl carbonate, dimethyl carbonate, dimethyl isosorbide, dimethylacetamide, dimethylformamide, dimethylol ethylene urea, dimethyluracil, epilactose, erythritol, erythrose, ethyl lac Tate, ethyl maltol, ethylene carbonate, formamide, fucose, galactose, genistein, gentisic acid ethanolamide, gluconolactone, glyceraldehyde, glycerol, glycerol carbonate, glycerol formal, glycerol urethane, glyceryl series acid, gosipine, harphagoside, hederacoside C, icodextrin, iditol, imidazolidone, inositol, inulin, isomaltitol, kojic acid, lactitol, lactobionic acid, lactose, lactulose, lyxose, Madecassoside, maltotriose, mangiferin, mannose, melgitose, methyl lactate, methylpyrrolidone, mogroside V, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, N -Methyl acetamide, N-methyl formamide, N-methyl propionamide, 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, nistose, kestose, turanose, acarbose, D-saccharic acid 1,4-lactone, thiodigalactoside, fucoidan, hydroxysaflor yellow A , shikimic acid, diosmin, pravastatin sodium salt, D-altrose, L-gulonic gamma-lactone, neomycin, rubusoside dihy droartemisinin, phloroglucinol, naringin, baicalein, hesperidin, apigenin, pyrogallol, morin, salsalate, kaempferol, myricetin, 3',4',7-trihydroxyisoflavone, (±)-taxifolin, silybin, perseitol diformal, 4-hydroxyphenylpyruvic acid, sulfacetamide, isopropyl β-D-1-thiogalactopyranoside, ethyl 2,5-dihydride Roxybenzoate, 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, verbenaloside, xylitol and xylose.

6. Protein/Excipient Solutions: Characterization and Process

In certain embodiments, the above-identified excipient compounds or combinations thereof (hereinafter "excipient additives"), such as hindered amines, anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids , solutions of therapeutic or non-therapeutic proteins formulated with diones and sulfones, zwitterionic excipients and crowding agents with hydrogen bonding elements are measured by the protein diffusion interaction parameter, k _D or the second virial coefficient B ₂₂ . Upon application, it results in improved protein-protein interaction characteristics. As used herein, an “improvement” in one or more protein-protein interaction parameters achieved by a test formulation using an excipient compound or combination thereof identified above is an “improvement” in which the test formulation does not contain the excipient compound or excipient additive. When compared under equivalent conditions using equivalent formulations that do not contain an equivalent formulation, a reduction in attractive protein-protein interactions can be demonstrated. The improvement may be ascertained by measuring a specific parameter applied to the overall process or aspect thereof, where a parameter is any metric pertaining to the process for which a change can be quantified and compared to a previous state or control. The parameter may relate to the process itself, for example its efficiency, cost, yield or speed. Improving the stability of protein-containing formulations during processing can have the benefits of improved yields, increased biological activity, and reduced presence of particulates in the formulation.

A parameter may also be a proxy parameter related to a feature or aspect of a larger process. As an example, parameters such as k _D or B ₂₂ parameters may be referred to as proxy parameters. Measurements of k _D and B ₂₂ can be made using standard techniques in the industry and can be indicative of process related parameters such as improved solution properties or stability of the protein in solution. While not wishing to be bound by theory, it is understood that highly negative k _D values may indicate that the protein has strong attractive interactions, which may result in aggregation, instability and rheological problems. When formulated in the presence of certain of the above-identified excipient compounds or combinations thereof, the same protein may have a less negative k _D value or an improved proxy parameter of a k _D value close to or greater than zero, such an improvement The modified proxy parameters relate to improvements in process-related parameters.

In an embodiment, certain of the above-described excipient compounds or combinations thereof, e.g., hindered amines, anionic fragrances, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic excipients and the flocculant having a hydrogen bond element is filtration, injection, transfer, pumping, mixing, heating or cooling by heat transfer, gas transfer, centrifugation, chromatography, membrane separation, centrifugal concentration, tangential flow filtration, radial flow filtration , used to improve protein-related processes such as preparation, processing, sterile filling, purification and analysis of protein-containing solutions using processing methods such as axial flow filtration, lyophilization and gel electrophoresis. In these and related protein-related processes, the protein of interest is dissolved in a solution that delivers it through a processing device. Such solutions, referred to herein as "carrier solutions," include cell culture medium (e.g., containing the secreted protein of interest), lysate solution after lysis of the host cells (if the protein of interest is present in the lysate), elution solution (chromatography containing the protein of interest after separation by chromatography), an electrophoresis solution, a transport solution for transporting the protein of interest through a conduit of a processing device, and the like. A carrier solution containing a protein of interest may also be referred to as a protein-containing solution or protein solution. As described in more detail below, one or more of the above-identified excipient compounds or combinations thereof may be added to the protein-containing solution to improve various aspects of processing. As used herein, the terms “improve,” “improvement,” and the like refer to a beneficial change in a parameter of interest in a carrier solution when the parameter is compared to the same parameter as measured in the control solution. As used herein, a “control solution” means a solution that lacks a viscosity reducing excipient, but is otherwise substantially similar to the carrier solution. As used herein, a "control process", eg, control filtration process, control chromatography process, etc., is a protein-related process that is substantially similar to the protein-related process of interest and is performed with a control solution instead of a carrier solution.

For example, in processes where a protein-containing solution is pumped through a conduit (eg flow chamber, piping or perfusion), it is advantageous to add the excipient compounds or combinations identified above to reduce the viscosity. Adding a viscosity lowering excipient to the protein solution as described above before or during the pumping process can substantially reduce the force and power required to pump the solution. It is understood that fluids generally exhibit resistance to flow, ie viscosity, and that a force must be applied to the fluid to overcome this viscosity in order to induce and propagate flow. The power P required for pumping scales with the head H and capacity Q as shown in the equation below:

P-HQ (Equation 1)

Viscous fluids tend to increase the power requirements for the pump, reduce pump efficiency, reduce pump head and capacity, and increase the frictional resistance of the piping. Adding the above-described viscosity lowering excipients to the protein solution before or during pumping can substantially lower processing costs by reducing head (H, Equation 1) or volume (Q, Equation 1) or both. The benefits of reduced viscosity may manifest, for example, in improved throughput, increased yield, or reduced processing time. Additionally, frictional losses from the transport of fluids through conduits can account for a significant portion of the costs associated with transporting such fluids. Addition of a viscosity lowering excipient as described above to the protein solution before or during pumping can substantially lower processing costs by reducing the friction involved in the pumping process. A measure of processing cost represents a processing parameter that can be improved using viscosity reducing excipients.

These processes and treatment methods for protein solutions can be effectively improved due to lower viscosity, improved solubility or improved stability of the protein in the solution during manufacturing, handling, purification and analysis steps. Measurements of processing efficiency or measurement of proxy parameters such as viscosity, solubility or stability of a protein in solution represent processing parameters that can be improved using viscosity reducing excipients. A number of different factors are understood that negatively affect protein viscosity, solubility and stability during processing. For example, protein-containing solutions undergo a variety of physical processes during manufacturing and purification, including significant shear stress induced by manipulating protein solutions through typical processing operations, including but not limited to pumping, mixing, centrifugation, and filtration. applied to stressors. Also, during these processing steps, air bubbles can be entrained in the fluid to which proteins can adsorb. This interfacial tension coupled with the typical shear stress encountered during processing can cause adsorbed protein molecules to unfold and aggregate. Significant protein unfolding can also occur during pump cavitation events and during exposure to solid surfaces during fabrication, such as ultrafiltration and diafiltration membranes. These events can impair protein folding and product quality.

, 및 유체의 점도

로 스케일링된다:For Newtonian fluids, the stress τ imposed by a given process is the shear rate as given in the equation

, and the viscosity of the fluid

is scaled by:

(방정식 2)

(Equation 2)

, 방정식 2 참조)의 증가 및 이에 따라 처리 시간의 감소와 관련된다. 또한, 상기 확인된 특정한 부형제 화합물 또는 이의 조합물의 첨가는 상이한 처리 단계 동안 단백질 용액의 안정성을 개선시킬 수 있다.By formulating a protein solution with one or more of the excipient compounds or combinations thereof described above, the solution viscosity can be reduced and thus the shear stress encountered by the protein solution can be reduced. Reduced shear stress can improve the stability of the processed formulation, as indicated, for example, by better or more desirable measures of processing parameters. Such improved processing parameters may include metrics such as reduced levels of protein aggregates, particles or invisible particles (visible to the naked eye, such as turbidity), reduced product loss, or improved overall yield. As another example of an improved processing parameter, reducing the viscosity of the protein-containing solution can reduce the processing time for the solution. Turnaround time for a given unit operation is generally inversely proportional to shear rate. Thus, for a given characteristic stress, the reduction in protein solution viscosity by addition of the above described excipient compounds or combinations thereof is the shear rate (

, see Equation 2) and thus a decrease in processing time. In addition, the addition of certain excipient compounds or combinations thereof identified above may improve the stability of the protein solution during different processing steps.

During treatment, it is understood that the protein in solution can be the desired protein active ingredient, eg, a therapeutic or non-therapeutic protein. Facilitating processing of the protein active ingredient using the excipients described herein may increase the yield or rate of production of the protein active ingredient, improve the efficiency of a particular process, reduce energy use, or the like; , none of these results show improved processing parameters by the use of viscosity reducing excipients. It is also understood that protein contaminants may be formed during certain processing techniques, such as during fermentation and purification steps of a bioprocess. Faster, more complete, or more efficient removal of contaminants can also improve processing of the desired protein, i.e., protein active ingredient; These results indicate improved processing parameters with the use of viscosity reducing excipient compounds or additives. As described herein, by lowering solution viscosity, improving protein stability, and/or increasing protein solubility, certain excipients as described herein can improve transport of a desired protein active ingredient, may improve the removal of undesirable protein contaminants; Both effects of improved processing parameters by the use of viscosity reducing excipients or additives indicate that these excipients or additives improve the overall process of protein manufacturing. Reduction of misfolded proteins, particulates, denatured proteins or other artifacts of minutely stabilized proteins in solution can be achieved by the use of stabilizing excipients during processing steps.

Specific platform unit operations for therapeutic protein production and purification provide additional examples of advantageous uses of the excipient compounds or combinations thereof identified above, and of improved processing parameters of these excipients or additives. For example, incorporating one or more of the above-identified excipient compounds or combinations thereof into these production and purification processes, as described below, can provide substantial improvements in molecular stability and recovery and reductions in 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 fermentation process followed by a series of steps for purification. Fermentation or upstream processing (USP) involves growing a therapeutic protein in a bioreactor, typically using bacterial or mammalian cell lines. The USP, in embodiments, may include steps as set forth in FIG. 4 . Purification or downstream processing (DSP) may, in an embodiment, include steps as shown in FIG. 5 .

As shown in Figure 4, the USP may begin with step 102 of thawing a vial from a master cell bank (MCB). The MCB can be expanded as shown in step 104 to form a working cell bank (not shown) and/or to create a working stock for further production. Cell culture takes place in a series of seed and production bioreactors as shown in steps 108 and 110 to produce bioreactor products 112 from which the desired therapeutic protein can be harvested as shown in step 114. After harvesting 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 stored at a temperature of approximately -80°C. It can be stored in bulk by

In an embodiment, protein production by cell culture techniques may be improved by use of the above-identified excipients as indicated by improvements in process-related parameters. In an embodiment, the desired excipient may be added in an amount effective to reduce the viscosity of the cell culture medium by at least 20% during USP. In another embodiment, the desired excipients can be added in an amount effective to reduce the viscosity of the cell culture medium by at least 30% during USP. In embodiments, the desired excipient may be added to the cell culture medium in an amount of about 1 mM to about 400 mM. In embodiments, the desired excipient may be added to the cell culture medium in an amount of about 20 mM to about 200 mM. In an embodiment, the desired excipient may be added to the cell culture medium in an amount of about 25 mM to about 100 mM. The desired excipient or combination of excipients can be added directly to the cell culture medium, or it can be added as a component of a more complex supplemental medium, e.g., as a nutrient-containing solution or "feed solution" formulated separately and added to the cell culture medium. may be added. In embodiments, a second excipient, eg, a viscosity reducing compound, may be added to the carrier solution either directly or via a supplemental medium, wherein the second viscosity reducing compound adds further improvement to a particular parameter of interest.

As described below, there are many process-related parameters during USP that can be improved by the use of one or more of the above-identified excipient compounds or combinations thereof. For example, in embodiments, the use of viscosity reducing excipients may improve parameters such as the rate and/or extent of cell growth during steps such as inoculum expansion (104) and cell culturing (108 and 110) and/or various It is possible to improve proxy parameters that are related to improvements in process parameters. For example, adding certain excipients identified above to the USP process at a stage such as production bioreactor stage 110 can reduce the viscosity of the cell culture medium, which in turn improves heat transfer efficiency and gas transfer efficiency. can Since cell culture processes require oxygen injection into cells to enable protein expression, diffusion of oxygen into cells can therefore be the rate-limiting step, improving gas transfer efficiency by reducing solution viscosity, thereby reducing the rate of oxygen uptake. Improving may improve the rate or amount of protein expression and/or its efficiency. In this context, parameters such as the ratio of oxygen uptake rate and gas transfer efficiency can be considered as proxy parameters whose improvements correlate with improvements in process parameters of improved protein expression or improved processing efficiency. As another example, the availability of viscosity reducing excipients can be improved during the inoculum expansion step 104 and during the cell culture steps 108 and 110 by improving proxy parameters such as, for example, the solubility of protein growth factors required for protein expression. treatment during treatment, and with improved growth factor solubility, these substances can become more available to cells to promote cell growth.

In embodiments, process parameters such as protein recovery or protein recovery during USP can be improved by reducing viscosity during USP by several mechanisms. For example, harvesting of the therapeutic protein at the end of the lysis step during harvest 114 from the completed cell culture may be more efficient or otherwise improved with the use of the excipients identified above. Without wishing to be bound by theory, by improving the viscosity of the expressed protein, these viscosity reducing excipients may increase the efficiency of diffusion of the therapeutic protein away from other lysate components. In addition, separation of membranes and other cell debris from protein-containing supernatants can be achieved with faster separation rates or higher degrees of supernatant purity with the use of viscosity reducing excipients, thereby improving process parameters of USP efficiency. . Furthermore, protein separation steps using centrifugation or filtration steps can be achieved more rapidly with the use of viscosity reducing excipients, since these 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 an embodiment, an excipient may improve stress tolerance of a protein during processing, which may reduce the amount of aggregation or denaturation of the protein during a processing step.

In an embodiment, as a further benefit, the use of the above described excipient compounds or combinations thereof, e.g., viscosity reducing excipients, in cell culture reduces process parameters such as protein yield during USP because protein misfolding and aggregation are reduced. can increase As cell culture is optimized to produce maximum yield of recombinant protein, the resulting protein is expressed in a highly concentrated manner, which can cause misfolding; Addition of the above-identified excipient compounds or combinations thereof, such as viscosity reducing excipients, can reduce attractive protein-protein interactions that result in misfolding and aggregation, thereby being utilized for harvesting (114). It is understood that one can increase the amount of intact recombinant protein available.

Downstream processing (DSP), shown in FIG. 5 in an exemplary embodiment, includes a series of steps that result in the recovery and purification of therapeutic proteins, such as monoclonal antibodies, biopharmaceuticals, vaccines, and other biologics. . At the end of the USP, the therapeutic protein of interest secreted from the host cells can be dissolved in the cell culture medium. Therapeutic proteins may also be dissolved in a fluid medium after lysis of the host cells at the end of the USP sequence. DSP is performed to recover a protein of interest from a lysed solution (eg, culture medium or host cell lysate medium) and purify it. During DSP, (i) various contaminants (e.g., insoluble cell debris and particulates) are removed from the medium, (ii) protein products are separated through techniques such as extraction, precipitation, adsorption or ultrafiltration, and (iii) ) the protein product is purified through techniques such as affinity chromatography, precipitation or crystallization, and (iv) the product is further polished and viruses are removed.

As shown in FIG. 5 , feedstock from cell culture harvest 200 (as also described in FIG. 4 ) is typically subjected to affinity chromatography (204), including protein-A chromatography or other similar chromatographic steps. ) is applied initially. Virus inactivation step 208 typically involves subjecting the feedstock to low pH maintenance. One or more abrasive 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 generally used as the initial polishing chromatography step 210, but may be accompanied by a preceding or subsequent second chromatography step 212. A second chromatography step 212 further removes host cell related impurities (eg, HCP or DNA) or product related impurities such as aggregates. Anion exchange (AEX) chromatography and hydrophobic interaction chromatography (HIC) may be used as the second chromatography step (212). Virus filtration 214 is performed to perform virus removal. A final purification step 218 may include ultrafiltration and diafiltration, and preparation for formulation.

As generally described above, the purification process or DSP following the fermentation process includes (1) harvesting the cell culture, (2) chromatography (e.g., Protein-A chromatography and ion exchange and hydrophobic interaction chromatography). chromatographic polishing step comprising), (3) virus inactivation and (4) filtration (e.g., virus filtration, sterile filtration, dialysis, and ultrafiltration to concentrate the protein and exchange the protein into a formulation buffer, and diafiltration step) may be included. Examples are provided below to illustrate the benefits from using the above-identified excipient compounds or combinations thereof, eg, viscosity reducing excipients, to improve process parameters associated with these purification processes. The above-identified excipient compounds or combinations thereof, e.g., viscosity-reducing excipients, can be added to a carrier solution or in any other way to manipulate the contact of the excipient, whether in soluble or stabilized form, with the protein of interest, thereby reducing the amount of the DSP. It is understood that it can be introduced at the stage of. In embodiments, a second excipient, eg, a viscosity reducing compound, may be added to the carrier solution during DSP, wherein the second compound adds further improvement to a particular parameter of interest.

(1) Cell culture harvesting : Cell culture harvesting generally involves centrifugation and depth filtration operations in which cell debris is physically removed from the protein-containing solution. The centrifugation step can provide a more complete separation of soluble proteins from cell debris with the benefit of viscosity reducing excipients. Centrifugal separation, whether performed by batch or continuous processing, requires a dense phase to incorporate as much as possible to maximize recovery of the target protein. In an embodiment, the addition of the above-identified excipients or combinations thereof increases the process parameters of protein yield, for example by increasing the yield of protein-containing centrates that flow away from the dense phase of a centrifuge separation process. can increase The depth filtration step is a viscosity limiting step and can therefore be made more efficient by using excipients that reduce solution viscosity. These processes can also introduce air bubbles into the protein solution, which coupled with the shear induced stress can destabilize the therapeutic protein molecule being purified. Addition of viscosity-reducing excipients to protein-containing solutions before and/or during cell culture harvest as described above can protect proteins from these stresses, thereby reducing the possibility of protein aggregation and improving process parameters for quantified product recovery. can make it

(2) Chromatography : After harvesting the cell culture by centrifugation or filtration, chromatography is typically used to separate therapeutic proteins from the fermentation broth. Protein A chromatography is used when the therapeutic protein is an antibody: Protein A is selective for IgG antibodies, which will bind dynamically at high flux and capacity. Cation exchange (CEX) chromatography can be used as a cost-effective alternative to Protein A chromatography. When CEX is used, the pH of the feed must be adjusted and its conductivity reduced before loading to the column to optimize the dynamic binding capacity. A mimetic resin can also be used as an alternative to Protein A chromatography. These resins provide ligands that bind immunoglobulins, eg Ig-binding proteins such as protein G or protein L, synthetic ligands or protein A-like porous polymers.

Other chromatographic processes may be used during DSP. Ion exchange chromatography (IEC) can be used to remove impurities introduced during previous processing, such as protein A, endotoxins or viruses leached from the cell line, remaining host cell proteins or DNA, or media components. IEC can be applied immediately after Protein A chromatography, whether CEX or anion exchange chromatography. Hydrophobic interaction chromatography (HIC) can complement IEC, which is commonly used as an abrasive step to remove aggregates. In an embodiment, the use of the above-identified excipients can increase the solubility of host cell proteins and decrease their viscosity during the chromatography column loading step. In embodiments, the use of the above-identified excipients can increase the solubility of the therapeutic protein and decrease its viscosity during the chromatography column loading and elution steps.

Chromatographic processes during protein purification include (a) low pH conditions during elution from the Protein-A chromatography column, (b) elevated local protein concentration within the pore-space of the chromatography resin (often around 300-400 mg/ml). mL), (c) elevated salt concentrations during ion exchange chromatography, and (d) elevated concentrations of salt-out agents during elution from HIC columns. Adding viscosity reducing excipients to protein-containing solutions before and/or during chromatography, as described above, promotes the migration of proteins through the chromatography column so that they are less exposed to potentially damaging conditions imposed by the chromatographic processing steps. make it possible In addition, elevated local protein concentrations within the column pore-space can create very viscous material within this space, which can exert significant back pressure on the column. To relieve this back pressure, media with relatively large pores are typically used. However, the resolution of large-pore media is lower than that of their small-pore counterparts. The incorporation of viscosity modifying excipients as described above may allow the use of smaller pores in the chromatography medium. In an embodiment, the elution step from Protein-A chromatography exposes the therapeutic protein to low pH conditions that can reduce solubility and increase aggregation of the target protein; Addition of an excipient can increase the solubility of the target protein to improve recovery yield from the Protein-A chromatography step. In another embodiment, the use of an excipient can enable elution of the target protein from the Protein-A resin at higher pH, which can reduce chemical stress on the target protein, resulting in the amount of proteolysis during processing. By reducing the protein yield process parameters can be improved.

(3) Virus inactivation : Virus inactivation processes typically involve maintaining the protein solution at a low pH, eg, a pH less than 4, for an extended period of time. However, these environments can destabilize therapeutic proteins. For example, formulating a protein in the presence of a viscosity reducing excipient by adding a viscosity reducing excipient before and/or during the virus inactivation process can improve a process parameter such as stability or solubility of the protein, or its net yield. .

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

(a) Viral filtration purifies the protein solution by removing viral particles that can be about twice the size of recombinant human monoclonal antibodies. Therefore, a filtration membrane for virus filtration may require nano-sized pores. As a result of the small pore size that proteins must pass through, this filtration step can introduce stress to the proteins, accompanied by significant levels of membrane fouling from protein aggregate particles. For example, as described above, the addition of viscosity reducing excipients before and/or during filtration can reduce a measurable parameter such as back pressure in a filtration system by increasing the collective diffusivity, and the protein-protein interactions that result in it. By mitigating, the tendency for membrane fouling can be reduced. The end result is an improvement in these parameters indicating improved performance of the virus filtration device during protein purification.

(b) The ultrafiltration and diafiltration (UF/DF) process concentrates the protein solution and exchanges the buffer system by passing the protein-containing solution through a filter membrane having a characteristic molecular weight cutoff smaller than the protein of interest. At this stage, the protein solution faces high shear stress within the filter device, elevated protein concentration, and adsorption of proteins to the hydrophobic membranes typically used during UF/DF processes, all of which can increase protein aggregation. . For example, the addition of viscosity reducing excipients before and/or during the UF/DF process as described above will reduce back pressure in the filtration system by increasing the collective diffusivity (eg, as measured by an increase in k _D ). can This not only reduces shear stress across the membrane but also promotes reverse diffusion away from the filter membrane, thereby lowering the effective protein concentration at the membrane interface and increasing the permeation flux. Consequently, the use of viscosity reducing excipients can improve parameters associated with higher throughput during these filtration processes, reducing product loss and increasing net yield. Additionally, passing viscous fluids through ultrafilters and diafilters can create large pressure drops across the filter unit, making separation inefficient. Formulating the protein solution in the presence of a viscosity reducing excipient as described above can substantially reduce the pressure drop across the filter device, thereby improving both by reducing the process parameters of operating cost and processing time. can make it

After upstream protein processing or downstream purification with an added excipient is completed, the excipient may remain part of the drug substance mixture or it may 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 excipients from protein active components. In addition to the beneficial effects on the protein purification process as described above, the use of the excipients identified above can protect and preserve the equipment used for protein preparation, processing and purification. For example, equipment-related processes such as cleanup, sterilization and maintenance of protein processing equipment can be facilitated by the use of the excipients identified above due to reduced contamination, reduced denaturation, lower viscosity and improved solubility of proteins; Parameters related to improvements in these processes are similarly improved.

While the use of excipient compounds to improve upstream and/or downstream processing has been extensively described herein, it is understood that combinations of excipients may be added together to achieve a desired effect, e.g., improvement of a parameter of interest. do. The term "excipient additive" can refer to a single excipient compound that produces a desired effect or improved parameter, or a combination of excipient compounds, the combination of which is responsible for a desired effect or improved parameter.

Example

ingredient:

Bovine gamma globulin (BGG), >99% purity, catalog #G5009, Sigma Aldrich

Histidine, Sigma Aldrich

· Other materials described in the examples below are from Sigma Aldrich unless otherwise specified.

Example 1: Preparation of Formulations Containing Excipient Compounds and Test Proteins

A formulation was prepared using an excipient compound and a test protein, wherein the test protein was intended to simulate either a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. The formulation was prepared in 50 mM histidine hydrochloride with different excipient compounds for viscosity measurement in the following manner. For histidine hydrochloride, first dissolve 1.94 g histidine in distilled water, adjust the pH to about 6.0 using 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO), and then add 250 mL of distilled water to a volumetric flask. Prepared by dilution to final volume. Then, excipient compounds were 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 prior to dissolution in 50 mM histidine HCl. In this case, the excipient compound was first dissolved at about 5 wt% in deionized water and the pH was adjusted to about 6.0 with hydrochloric acid or sodium hydroxide. Thereafter, the prepared salt solution was placed in a convection laboratory oven at about 65° C. to evaporate water and separate solid excipients. Once an excipient solution in 50 mM histidine HCl was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a rate of about 0.336 g BGG per 1 mL excipient solution. This resulted in a final protein concentration of about 280 mg/mL. A solution of BGG in 50 mM histidine HCl with excipients was formulated in a 20 mL vial and shaken at 100 rpm on an orbital shaker table overnight. 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 measurement.

Example 2: Viscosity measurement

Viscosity measurements of formulations prepared as described in Example 1 were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 for the sample.

Example 3: Protein concentration measurement

The protein concentration in the test solution was determined by measuring the light absorbance of the protein solution at a wavelength of 280 nm in a UV/VIS spectrometer (Perkin Elmer Lambda 35). First, the instrument was calibrated to zero absorbance using 50 mM histidine buffer at pH 6. Next, the protein solution was 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 Using Hindered Amine Excipient Compounds

Formulations containing 280 mg/mL BGG were prepared as described in Example 1, with some samples containing added excipient compounds. In these tests, the hydrochloride salts of dimethylcyclohexylamine (DMCHA), dicyclohexylmethylamine (DCHMA), dimethylaminopropylamine (DMAPA), triethanolamine (TEA), dimethylethanolamine (DMEA) and niacinamide were An example of a dud amine excipient compound was tested. In addition, hydroxybenzoic acid salts of DMCHA and taurine-dicyandiamide adducts were 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, which shows the benefit of the added excipient compound in reducing viscosity.

Table 1

Example 5: Formulations Using Anionic Aromatic Excipient Compounds

A formulation of 280 mg/mL BGG was prepared as described in Example 1, with some samples containing added excipient compounds. The viscosity of each solution was measured as described in Example 2 and the results are presented in Table 2 below, which shows the benefit of the added excipient compound in reducing viscosity.

Table 2

Example 6: Formulation using an oligopeptide excipient compound

Oligopeptides (n=5) were synthesized by NeoBioLab Inc. (Woburn, MA) in >95% purity with the N-terminus as a free amine and the C-terminus as a free acid. Dipeptides (n = 2) were synthesized by LifeTein LLC (Somerset, NJ) with 95% purity. Formulations of 280 mg/mL BGG were prepared as described in Example 1, and some samples contained 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, which shows the benefit of the added excipient compound in reducing viscosity.

Table 3

Example 7: Synthesis of Guanyl Taurine Excipients

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

Example 8: Protein formulations containing excipient compounds

A formulation was prepared using an excipient compound and a test protein, wherein the test protein was intended to simulate either a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. The formulation was prepared in a 50 mM histidine hydrochloride buffered aqueous solution with different excipient compounds for viscosity measurement in the following manner. Histidine hydrochloride buffer was prepared by first dissolving 1.94 g of histidine in distilled water, adjusting the pH to about 6.0 using 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO), and then using distilled water in a volumetric flask to adjust the pH to 250 mL. It was prepared by dilution to a final volume of Then, excipient compounds were dissolved in 50 mM histidine HCl buffer. A list of excipient compounds is provided in Table 4. In some cases, excipient compounds were dissolved in 50 mM histidine HCl buffer and the resulting solution pH was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. In some cases, excipient compounds were adjusted to pH 6 prior to dissolution in 50 mM histidine HCl. In this case, the excipient compound was first dissolved at about 5 wt% in deionized water and the pH was adjusted to about 6.0 with hydrochloric acid or sodium hydroxide. Thereafter, the prepared salt solution was placed in a convection laboratory oven at about 65° C. to evaporate water and separate solid excipients. Once an excipient solution in 50 mM histidine HCl was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a rate to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in 50 mM histidine HCl with excipients was formulated in a 20 mL vial and shaken at 100 rpm on an orbital shaker table overnight. 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 measurement.

Viscosity measurements of formulations prepared as described above were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 for the sample. The viscosity of the solution with excipients was normalized to that of the model protein solution without excipients. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipient to the viscosity of the model protein solution without excipient.

Table 4

Example 9: Preparation of Formulations Containing Excipient Combinations and Test Proteins

Formulations were prepared using a primary excipient compound, a secondary excipient compound and a test protein, wherein the test protein was intended to simulate either a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. Primary excipient compounds were selected from compounds having both anionic and aromatic functional groups as listed in Table 5 below. Secondary excipient compounds were selected from compounds having a nonionic or cationic charge and an imidazoline or benzene ring at pH 6 as listed in Table 5 below. Formulations of these excipients were prepared in 50 mM histidine hydrochloride buffer for viscosity measurements in the following manner. For histidine hydrochloride, first dissolve 1.94 g histidine in distilled water, adjust the pH to about 6.0 using 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO), and then add 250 mL of distilled water to a volumetric flask. Prepared by dilution to final volume. The 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 resulting solution pH was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of 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 proportions to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in 50 mM histidine HCl with excipients was formulated in a 20 mL vial and shaken at 100 rpm on an orbital shaker table overnight. 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 measurement.

Viscosity measurements of formulations prepared as described above were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 for the sample. The viscosity of the solutions with excipients was normalized to that of the model protein solution without excipients and is summarized in Table 5 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipient to the viscosity of the model protein solution without excipient. This example suggests that a combination of primary and secondary excipients may provide better results than a single excipient.

table 5

Example 10: Preparation of Formulations Containing Excipient Combinations and Test Proteins

Formulations were prepared using a primary excipient compound, a secondary excipient compound and a test protein, wherein the test protein was intended to simulate either a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. Primary excipient compounds were selected from compounds having both anionic and aromatic functional groups as listed in Table 6 below. Secondary excipient compounds were selected from compounds having a nonionic or cationic charge and an imidazoline or benzene ring at pH 6 as listed in Table 6 below. 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 resulting solution pH was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. Once an excipient solution in distilled water was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a rate to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in distilled water along with excipients was formulated in a 20 mL vial and shaken at 100 rpm on an orbital shaker table overnight. 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 measurement.

Viscosity measurements of formulations prepared as described above were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 for the sample. The viscosity of the solutions with excipients was normalized to that of the model protein solution without excipients and is summarized in Table 6 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipient to the viscosity of the model protein solution without excipient. This example suggests that a combination of primary and secondary excipients may provide better results than a single excipient.

table 6

Example 11: Preparation of Formulations Containing Excipient Compounds and PEG

Materials: All materials are from Sigma-Aldrich, St. It was purchased from St. Louis, MO. The formulation was prepared using an excipient compound and PEG, where the PEG is intended to simulate a therapeutic PEGylated protein to be used in a therapeutic formulation. The formulation was prepared by mixing equal volumes of a solution of PEG and a solution of an excipient. Both solutions were prepared in Tris buffer consisting of 10 mM Tris, 135 mM NaCl, 1 mM trans-cinnamic acid at pH 7.3. A PEG solution was prepared by mixing 3 g of poly(ethylene oxide) (Aldrich catalog # 372781) with an average Mw of about 1,000,000 and 97 g of Tris buffer. The mixture was stirred overnight for complete dissolution.

An example of excipient solution preparation is as follows: Prepare a solution of approximately 80 mg/mL citric acid in Tris buffer by dissolving 0.4 g of citric acid (Aldrich cat. # 251275) in 5 mL of Tris buffer, and a minimum amount of 10 M NaOH. The pH was adjusted to 7.3 with the solution. The PEG excipient solution was prepared by mixing 0.5 mL of the PEG solution with 0.5 mL of the excipient solution and mixed using a vortex for several seconds. A control sample was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of Tris buffer.

Example 12: Viscosity measurement of formulations containing excipient compounds and PEG

Viscosity measurements of the prepared formulations were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 for the sample.

The results presented in Table 7 suggest the effect of the added excipient compound on viscosity reduction.

table 7

Example 13: Preparation of PEGylated BSA with One PEG Chain per BSA Molecule

To the beaker was added 200 mL of phosphate buffered saline (Aldrich Cat. # P4417) and 4 g of BSA (Aldrich Cat. # A7906) and mixed with a magnetic bar. Next, 400 mg of methoxy polyethylene glycol maleimide, MW=5,000 (Aldrich Cat. # 63187) was added. The reaction mixture was allowed to react overnight at room temperature. The next day, the reaction was stopped by adding 20 drops of HCl 0.1 M. 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 molecular weight cutoff (MWCO) of 30 kDa and concentrated to several milliliters. Next, the sample was diluted 20-fold with 50 mM histidine buffer at a pH of approximately 6 and then concentrated until a high-viscosity fluid was obtained. The absorbance was measured at 280 nm and an extinction coefficient for BSA of 0.6678 was used to obtain the final concentration of the protein solution. Results indicated that the final concentration of BSA in the solution was 342 mg/mL.

Example 14: Preparation of PEGylated BSA with Multiple PEG Chains per BSA Molecule

A 5 mg/mL solution of BSA (Aldrich A7906) in 25 mM phosphate buffer, pH 7.2, was prepared by mixing 0.5 g of BSA with 100 mL of buffer. Next, 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 at pH=7.3) and concentrated using a 30 kDa Amicon centrifuge tube MWCO until a concentration of approximately 150 mg/mL was reached. .

Example 15: Preparation of PEGylated Lysozyme with Multiple PEG Chains per Lysozyme Molecule

A 5 mg/mL solution of lysozyme (Aldrich L6876) in 25 mM phosphate buffer, pH 7.2, was prepared by mixing 0.5 g of lysozyme with 100 mL of buffer. Next, 1 g of methoxy PEG propionaldehyde Mw = 5,000 (JenKem Technology, Plano, TX 75024) was added followed by 0.12 g of sodium cyanoborohydride (Aldrich 156159). The reaction was allowed to proceed overnight at room temperature. The next day, the reaction mixture was diluted 49-fold with 25 mM phosphate buffer at pH 7.2 and concentrated using a 30 kDa Amicon centrifuge tube MWCO. The absorbance was measured at 280 nm and an extinction coefficient for lysozyme of 2.63 was used to obtain the final concentration of the protein solution. The final concentration of lysozyme in the solution was 140 mg/mL.

Example 16: Effect of Excipients on the Viscosity of PEGylated BSA with One PEG Chain per BSA Molecule

Formulations of PEGylated BSA (from Example 13 above) with excipients were prepared by adding 6 or 12 milligrams of an excipient salt to 0.3 mL of PEGylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured at a shear rate of 500 s ⁻¹ with a RheoSense microVisc equipped with an A10 channel (100-micron depth). Viscometer measurements were completed at ambient temperature. The results presented in Table 8 suggest the effect of the added excipient compound on viscosity reduction.

Table 8

Example 17: Effect of Excipients on the Viscosity of PEGylated BSA with Multiple PEG Chains Per BSA Molecule

A formulation of PEGylated BSA (from Example 14 above) with sodium citrate salt as excipient was prepared by adding 8 milligrams of the excipient salt to 0.2 mL of PEGylated BSA solution. The solutions were mixed by gentle agitation and the viscosity was measured at a shear rate of 500 s ⁻¹ with a RheoSense microVisc equipped with an A10 channel (100 microns deep). Viscometer measurements were completed at ambient temperature. The results presented in Table 9 suggest the effect of the added excipient compound on viscosity reduction.

Table 9

Example 18: Effect of Excipients on the Viscosity of PEGylated Lysozyme with Multiple PEG Chains Per Lysozyme Molecule

A formulation of PEGylated lysozyme (from Example 15 above) with potassium acetate as excipient was prepared by adding 6 milligrams of an excipient salt to 0.3 mL of PEGylated lysozyme solution. The solutions were mixed by gentle agitation and the viscosity was measured at a shear rate of 500 s ⁻¹ with a RheoSense microVisc equipped with an A10 channel (100 microns deep). Viscometer measurements were completed at ambient temperature. The results presented in the following table (Table 10) suggest the benefit of the added excipient compound in viscosity reduction.

Table 10

Example 19: Protein Formulation Containing Excipient Combinations

Formulations were prepared using an excipient compound or a combination of two excipient compounds and a test protein, wherein the test compound was intended to simulate a therapeutic protein to be used in a therapeutic formulation. The formulation was prepared in 20 mM histidine buffer with different excipient compounds for viscosity measurement in the following manner. The excipient combination was dissolved in 20 mM histidine and the resulting solution pH was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. Excipient compounds for these examples are listed in Table 11 below. Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a rate to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in excipient solution was formulated in a 5 mL sterile polypropylene tube and shaken at 80-100 rpm on an orbital shaker table overnight. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for about 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 for the sample. The viscosity of the solution with excipients was normalized to that of the model protein solution without excipients, and the results are presented in Table 11 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipient to the viscosity of the model protein solution without excipient.

Table 11

Example 20: Protein formulations containing excipients to reduce viscosity and injection pain

Formulations were prepared using an excipient compound, a second excipient compound and a test protein, wherein the test compound was intended to simulate a therapeutic protein to be used in a therapeutic formulation. The first excipient compound, excipient A, was selected from the group of compounds having local anesthetic properties. The first excipient, Excipient A, and the second excipient, Excipient B, are listed in Table 12. These formulations were prepared in 20 mM histidine buffer using excipient A and excipient B in the following manner to allow their viscosity to be measured. The amounts of excipients listed in Table 12 were dissolved in 20 mM histidine, and the resulting solution was pH adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved in the excipient solution at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in excipient solution was formulated in a 5 mL sterile polypropylene tube and shaken at 80-100 rpm on an orbital shaker table overnight. The BGG-excipient solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for about 10 minutes to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 for the sample. The viscosity of the solutions with excipients was normalized to that of the model protein solution without excipients, and the results are presented in Table 12 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipient to the viscosity of the model protein solution without excipient.

Table 12

Example 21: Formulations containing excipient compounds and PEG

Formulations were prepared using an excipient compound and PEG, where the PEG was intended to simulate a therapeutic PEGylated protein to be used in a therapeutic formulation, and the excipient compound was provided in the amounts listed in Table 13. These formulations were prepared by mixing equal volumes of solutions of PEG and solutions of excipients. Both solutions were prepared in deionized (DI) water. A PEG solution was prepared by mixing 16.5 g of poly(ethylene oxide) (Aldrich catalog # 181986) with an average Mw of about 100,000 and 83.5 g of deionized water. The mixture was stirred overnight for complete dissolution.

An excipient solution was prepared by this general method, which is detailed in Table 13 below: A solution of potassium phosphate tribasic in deionized water at approximately 20 mg/mL (Aldrich catalog # P5629) was dissolved in 5 mL of deionized water at 0.05 g. It was prepared by dissolving potassium phosphate. The PEG excipient solution was prepared by mixing 0.5 mL of the PEG solution with 0.5 mL of the excipient solution and mixed using a vortex for several seconds. A control sample was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of deionized water. Viscosity was measured and the results are reported in Table 13 below.

Table 13

Example 22: Improved processing of protein solutions using 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: the buffer was 20 mM histidine buffer (pH=6) containing 15 mg/mL caffeine. Dissolution of solid BGG was performed by placing the sample in an orbital shaker set at 100 rpm. Buffer samples containing the caffeine excipient were observed to solubilize the protein more rapidly. For the sample with caffeine excipient (Sample B), complete dissolution of BGG was achieved within 15 minutes. For the sample without caffeine (Sample A), 35 minutes were required for dissolution. Next, the samples were placed in two separate Amicon Ultra 4 centrifugal filter devices with a 30 kDa molecular weight cutoff, and the samples were centrifuged at 2,500 rpm at 10 minute intervals. The volume of filtrate recovered after each 10 minute centrifugation run was recorded. The results in Table 14 suggest a faster recovery of filtrate for sample B. Also, while sample B continued to concentrate with each additional run, sample A reached the point of maximum concentration, and further centrifugation did not result in further sample concentration.

Table 14

Example 23: Protein formulation containing 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. 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). The viscosity of the solution was measured as described in the previous examples. The results suggest that caffeine, a hindered amine excipient, can be combined with known excipients such as arginine, and that the combination has better viscosity reducing properties than the individual excipients by themselves.

Table 15

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

Table 16

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

Table 17

Example 24: Caffeine effect during TFF concentration process

In this example, bovine gamma globulin (BGG) solutions were concentrated in the presence and absence of caffeine using tangential flow filtration (TFF). Experiments were performed using a Labscale TFF system produced by EMD Millipore (Billerica, MA). The system was equipped with a Pellicon XL TFF cassette (EMD Millipore, Billerica, Mass.) containing an Ultracel membrane with a 30 kDa molecular weight cutoff. The nominal membrane surface area was 50 cm ² . The supply pressure to the cassette was maintained at 30 psi, while the holding pressure was maintained at 10 psi. Filtrate flux was monitored throughout the course of the experiment by measuring 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 adjusted to pH 6. A control sample was prepared by dissolving 12 grams of BGG in 500 mL of a buffer containing 150 mM NaCl and 20 mM histidine adjusted to pH 6. Buffer components were purchased from Sigma-Aldrich. Both solutions were filtered through a 0.2 μm polyethersulfone (PES) filter (VWR, Radnor, PA) prior to TFF treatment. The performance of test and control samples during TFF was measured by mass transfer coefficient. The mass transfer coefficient was 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 As described in Memb. Sci. 508, 113-126 (2016)):

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

Equation 3 describes the filtrate flux, J, where k _c is the mass transfer coefficient, C _w is the protein concentration around the membrane, and C _b is the concentration in the bulk of the liquid, whereby Equation 3 gives the mass transfer coefficient k _c make calculation possible. A graph of the calculated flux J versus ln(C _b ) produces a linear plot with a slope of -kc. Here, flux J is calculated by taking the derivative of the filtrate mass with respect to time, and C _b is calculated using mass balance. The optimal mass transfer coefficients are listed in Table 18. Introduction of 15 mg/mL caffeine increased the mass transfer coefficient value by about 13% from 22.5 to 25.4 Lm ^-2 hr ^-1 (LMH).

Table 18

Example 25: Caffeine effect during TFF concentration process

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

Table 19

Example 26: Effect of Caffeine During Sterile Filtration of BGG Solutions

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

(방정식 4)

(Equation 4)

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

Table 20

Example 27: Excipients to improve Protein-A chromatographic elution

Four purified research grade biosimilar antibodies, ipilimumab, ustekinumab, omalizumab and tocilizumab were purchased from Bioceros (Utrecht, The Netherlands). They were provided in frozen aliquots at protein concentrations of 20, 26, 15 and 23 mg/mL, respectively, in aqueous 40 mM sodium acetate, 50 mM Tris-HCl buffer at pH 5.5. Protein solutions were thawed at room temperature prior to measurement and then filtered through a 0.2 μm polyethersulfone filter. The filtered protein stock solution was mixed in a 1:1 ratio of protein stock solution to binding buffer. The binding buffer used to promote binding of the antibody to the Protein-A resin consisted of 0.1 M sodium phosphate and 0.15 sodium chloride at pH 7.2 in deionized (DI) water. Deionized water was produced by purifying tap water using a Direct-Q 3 UV purification system from EMD Millipore (Billerica, MA). 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 of Protein-A resin. The resin was washed with binding buffer by adding 200 μL of binding buffer to each well, centrifuging the plate at 1000 x g for 1 minute, and discarding the flow-through. All subsequent centrifugation steps were performed at 1000 x g for 1 min. This washing procedure was repeated once. After these initial washing steps, diluted protein samples, i.e., 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 shaken at 260 rpm for 30 minutes, then the plate was centrifuged and the flow through was discarded. 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 plate using an elution buffer with different excipients added. For each elution, 50 μL of neutralization buffer consisting of 1 M sodium phosphate pH 7 was added to each well of the collection plate followed by 200 μL of elution buffer to each well of the plate. The plate was shaken at 260 rpm for 1 minute and then centrifuged. The flow through was recovered for analysis. This elution step was repeated once. Control buffer without excipients contained 20 mM citrate and had a pH of 2.6. Since Protein-A elution buffer often contains some amount of salt, an elution buffer of 100 mM NaCl in citrate buffer was prepared as a secondary control.

Table 21 lists the excipient solutions used in this example, their concentrations and the final pH of the elution buffer. Aspartame was purchased from Herb Store USA (Los Angeles, CA), trehalose was purchased from Cascade Analytical Reagents and Biochemicals (Corvallis, OR), and sucrose was purchased from Research Products International (Mt. Prospect, IL, product number S24060). All excipients were purchased from Sigma Aldrich (St. Louis, MO) except for Elution buffers containing all excipients were prepared by mixing appropriate amounts of excipients with approximately 10 mL of salt-free citrate buffer control. Elution buffer was prepared at approximately 100 mM excipient. However, not all excipients are soluble at this level; Table 21 therefore lists all excipient concentrations used. The pH of each elution buffer was adjusted to about 2.6±0.1 using hydrochloride or sodium hydroxide as needed.

For each protein sample, ASD high performance size-exclusion chromatography (SEC) using a TSKgel SuperSW3000 column (30 cm x 4.6 mm ID, Tosoh Bioscience, King of Prussia, PA) coupled to an HPLC workstation (Agilent HP 1100 system). ) analysis was performed. Separation was performed at room temperature with a flow of 0.35 mL/min. The mobile phase was an aqueous buffer of 100 mM sodium phosphate, 300 mM sodium chloride, pH 7. 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, i.e. ipilimumab, ustekinumab, omalizumab and tocilizumab, was estimated by integrating the chromatograms. The integrated peak areas for each protein, namely ipilimumab, ustekinumab, omalizumab and tocilizumab, are listed in Tables 22-25. Tables 22-25 also compare the experimental peak areas to those of the unsalted and salt-containing controls. Values greater than 100% indicate that the elution buffer recovered more protein from the Protein-A resin than the control, while values less than 100% indicate that the elution buffer recovered less protein from the Protein-A resin than the control.

Table 21

Table 22. Ipilimumab recovery from protein-A resin

Table 23. Ustekinumab recovery from Protein-A resin

Table 24. Omalizumab Recovery from Protein-A Resin

Table 25. Recovery of Tocilizumab from Protein-A Resin

Example 28: Excipients to improve Protein-A chromatographic elution

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

Table 26. Excipients used in Example 28

Table 27. Ipilimumab recovery from protein-A resin

Table 28. Ustekinumab recovery from Protein-A resin

Table 29. Omalizumab Recovery from Protein-A Resin

Table 30. Tocilizumab recovery from Protein-A resin

Example 29: Excipients that Improve Omalizumab Elution from Protein-A Chromatography Columns

Research grade omalizumab was purchased from Bioceros (Utrecht, The Netherlands) and supplied frozen at 15 mg/mL in aqueous 40 mM sodium acetate, 50 mM Tris-HCl buffer, pH 5.5. Proteins were thawed at room temperature before experiments and filtered through a 0.2 μm polyethersulfone filter. The filtered material was mixed in a 1:1 ratio with binding buffer consisting of 20 mM sodium phosphate, pH 7 in deionized water. Tap water was purified with EMD Millipore's Direct-Q 3 UV purification system (Billerica, MA) to produce deionized water. Protein-A purification was performed using a HiTrap Protein-A HP 1 mL column from GE Healthcare (Chicago, IL, catalog 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 of binding buffer. After washing the column, bound omalizumab was eluted from the column with one aliquot of the elution buffer containing the excipients listed in Table 31 below. Elution buffer was 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 regenerated 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, which was maintained by a Fusion 100 infusion pump (Chemyx, Stafford, TX). A 10-mL NormJect Luer Lok syringe was used (Henke Sass Wolf, Tuttlingen, Germany, reference number 4100-000V0).

Elution fractions E1, E2, E3, E4 and E5 were assayed 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) coupled to an HPLC workstation (Agilent HP 1100 system). Separation was performed at room temperature with a flow of 0.35 mL/min. The mobile phase was an aqueous buffer of 100 mM sodium phosphate, 300 mM sodium chloride, pH 7. 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.

Citrate is a common excipient used in Protein-A chromatography and was therefore used as a control herein. The eluate fraction for the control sample showed insoluble aggregates upon storage at 4°C overnight as evidenced by the formation of a precipitate phase. Thus, the peak areas reported in Table 31 below represent the total amount of soluble protein in the eluate fraction. We note that insoluble aggregates were observed only in the control sample and no other samples showed such aggregates. A peak area larger than that of the control (using the citrate excipient) indicates that the use of the test excipient may allow more efficient separation of the protein from the column.

Table 31

Example 30: BGG formulation with different amounts of caffeine excipient

Formulations were prepared using different molar concentrations of caffeine (concentrations listed in Table 32 below) and the test protein, wherein the test compound was intended to simulate the therapeutic protein to be used in the therapeutic formulation. Formulations for this example were prepared in 20 mM histidine buffer for viscosity measurement in the following manner. Stock solutions of 0 and 80 mM caffeine were prepared in 20 mM histidine, and the resulting solution pH was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. Additional solutions of various caffeine concentrations were prepared by blending the two stock solutions in various volume ratios to give a series of caffeine-containing solutions at the concentrations listed in Table 32 below. Once these excipient solutions are prepared, the test protein bovine gamma globulin (BGG) is prepared by adding 0.7 mL of each excipient solution to 0.25 g of lyophilized BGG powder at a rate to achieve a final protein concentration of approximately 280 mg/mL. dissolved in each test solution. Solutions containing BGG were formulated in 5 mL sterile polypropylene tubes and shaken at 100 rpm on an orbital shaker table overnight. These solutions were then transferred to 2 mL microcentrifuge tubes and centrifuged in an IEC MicroMax microcentrifuge at 2400 rpm for about 5 minutes to remove entrained air prior to viscosity measurement.

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

Table 32

Example 31: Preparation of solutions of co-solutes in deionized water

Compounds used as co-solutes to increase caffeine solubility in water were obtained from Sigma-Aldrich (St. Louis, MO), which include niacinamide, proline, procaine HCl, ascorbic acid, 2,5-dihydroxybenzoic acid , lidocaine, saccharin, acesulfame K, tyramine and aminobenzoic acid. The dry solid is dissolved in deionized water and the pH is adjusted to a value between a pH of about 6 and a pH of about 8 using 5 M hydrochloric acid or 5 M sodium hydroxide as needed in some cases, to separate each co-solute. A solution was prepared. The solution was then diluted to a final volume of 25 mL or 50 mL using a class A volumetric flask and the concentration recorded based on the mass of the dissolved compound and the final volume of the solution. The prepared solution was used either pure or diluted with deionized water.

Example 32: Caffeine solubility test

The effect of different co-solutes on the solubility of caffeine at ambient temperature (about 23° C.) was evaluated in the following way. Dry caffeine powder (Sigma-Aldrich, St. Louis, MO) was added to a 20 mL glass scintillation vial and the mass of caffeine was recorded. In certain cases, 10 mL of the co-solute solution prepared according to Example 31 was added to the caffeine powder; In other cases, a blend of the co-solute solution and deionized water was added to the caffeine powder to maintain a final addition volume of 10 mL. The volume contribution of dry caffeine powder was assumed to be negligible for any of these mixtures. A small magnetic stir bar was added to the vial and the solution was vigorously mixed on a stir plate for about 10 minutes. After about 10 minutes, the vial was observed for dissolution of the dry caffeine powder, the results are provided in Table 33 below. These observations suggest that niacinamide, procaine HCl, 2,5-dihydroxybenzoic acid sodium salt, saccharin sodium salt and tyramine chloride salt all exceed the reported caffeine solubility limit (approximately 16 mg/mL at room temperature according to Sigma-Aldrich). It has been shown to allow dissolution of caffeine by at least about 4-fold.

Table 33

Example 33: HUMIRA ^® profile of

HUMIRA ^® (AbbVie Inc., Chicago, IL) is used to reduce the inflammatory response of autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate to severe chronic psoriasis and juvenile idiopathic arthritis. A commercially available formulation of the therapeutic monoclonal antibody adalimumab, a TNF-alpha blocker typically prescribed for HUMIRA ^® silver 40 mg adalimumab, 4.93 mg sodium chloride, 0.69 mg sodium phosphate monobasic dihydrate, 1.22 mg sodium phosphate dibasic dihydrate, 0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 9.6 mg mannitol and 0.8 mg It is marketed as a 0.8 mL single use dose containing polysorbate 80. Viscosity versus concentration profiles of these formulations were generated in the following manner. An Amicon Ultra 15 centrifugal concentrator (EMD-Millipore, Billerica, MA) with a 30 kDa molecular weight cutoff was charged with approximately 15 mL of deionized water and centrifuged in a Sorvall Legend RT (ThermoFisher Scientific) at 4000 rpm for 10 minutes to remove the membrane. Rinse. Residual water was then removed, and 2.4 mL of HUMIRA ^® liquid formulation was added to the concentrator tube and centrifuged at 4000 rpm for 60 minutes at 25°C. The concentration of the residue was determined by diluting 10 μL of the residue with 1990 μL of deionized water, measuring the absorbance of the diluted sample at 280 nm, and calculating the concentration using a dilution factor and extinction coefficient of 1.39 mL/mg-cm. It was decided. The viscosity of the concentrated samples was measured with a microVisc viscometer equipped with an A05 chip (RheoSense, San Ramon, Calif.) at a shear rate of 250 s ^-1 at 23 °C. After measuring the viscosity, the sample was diluted with a small amount of filtrate and the concentration and viscosity measurements were repeated. This process was used to generate viscosity values at various adalimumab concentrations as described in Table 34 below.

Table 34

Example 34: HUMIRA with viscosity reducing excipients ^® Reformulation of

The example below describes a general process in which HUMIRA ^® is reformulated in a buffer with viscosity reducing excipients. A solution of the viscosity reducing excipient was prepared in 20 mM histidine by dissolving about 0.15 g histidine and 0.75 g caffeine (Sigma-Aldrich, St. Louis, Mo.) in deionized water. The pH of the resulting solution was adjusted to about 5 with 5 M hydrochloric acid. The solution was then diluted to a final volume of 50 mL in a volumetric flask with deionized water. HUMIRA ^® was then reformulated at high mAb concentrations using the resulting buffered viscosity reducing excipient solution. Next, about 0.8 mL of HUMIRA ^® was added to a rinsed Amicon Ultra 15 centrifugal concentrator tube with a 30 kDa molecular weight cutoff and centrifuged at 4000 rpm and 25 °C for 8-10 minutes in a Sorvall Legend RT. About 14 mL of the buffered viscosity reducing excipient solution prepared as described above was then added to the concentrated HUMIRA ^® in a centrifugal concentrator. After lightly mixing, the samples were centrifuged at 4000 rpm and 25°C for about 40-60 minutes. The residue was a concentrated sample of HUMIRA ^® reformulated in buffer with viscosity reducing excipients. The viscosity and concentration of the samples were measured and, in some cases, then diluted with a small amount of filtrate to measure the viscosity at lower concentrations. Viscosity measurements were completed with a microVisc viscometer in the same manner as for the concentrated HUMIRA ^® formulations in the previous examples. Concentrations were determined by Bradford assay using a standard curve generated from HUMIRA ^® stock solutions diluted in deionized water. Reformulation of HUMIRA ^® with viscosity reducing excipients provided a 30% to 60% reduction in viscosity compared to the viscosity values of HUMIRA ^® concentrated in a commercial buffer without reformulation as shown in Table 35 below.

Table 35

Example 35: Improved stability of adalimumab solutions using caffeine as excipient

The stability of adalimumab solutions with and without caffeine excipients was evaluated after exposing the samples to two different stress conditions, shaking and freeze-thaw. Adalimumab drug formulation HUMIRA® (AbbVie) was used, which has properties described in more detail in Example 33. HUMIRA® samples were concentrated to a concentration of 200 mg/mL adalimumab in the original buffer as described in Example 38; This enriched sample is designated "Sample 1". A second sample was prepared as described in Example 40 using about 200 mg/mL of adalimumab and 15 mg/mL of added caffeine; This concentrated sample with added caffeine is designated "Sample 2". Both samples were diluted to a final concentration of 1 mg/mL of adalimumab using the following diluents: sample 1 diluent was the original buffer, sample 2 diluent was 20 mM histidine, 15 mg/mL caffeine, pH=5. Both HUMIRA® dilutions were filtered through a 0.22 μm syringe filter. For every diluted sample, 3 batches of 300 μL each were prepared in 2 mL Eppendorf tubes in a laminar flow hood. The samples were subjected to the following stress conditions: for shaking, the samples were placed on an orbital shaker at 300 rpm for 91 hours; For freeze-thaw, samples were cycled 7 times from -17 to 30°C for an average of 6 hours per condition. Table 36 lists the samples prepared.

Table 36

Example 36: Evaluation of stability by dynamic light scattering (DLS)

The hydrodynamic radius of the adalimumab molecules was measured in samples from Example 35 using a Brookhaven Zeta Plus dynamic light scattering instrument, and evidence of aggregate population formation was found. Table 37 shows the DLS results for six samples prepared according to Example 35: some of them (1-A, 1-FT, 2-A and 2-FT) were exposed to stress conditions ("stress sample"), the rest (1-C and 2-C) were not stressed. The DLS data in Table 37 and accompanying Figures 1, 2 and 2 present the multimodal particle size distribution of the monoclonal antibodies in decaffeinated stress samples. In the absence of caffeine as excipient, stressed samples 1-A and 1-FT exhibited higher effective diameters than non-stressed sample 1-C, and they also exhibited a second population of significantly higher diameter particles; This new grouping of particles with larger diameters is evidence of aggregation into invisible particles. Stressed samples containing caffeine (Samples 2-A and 2-FT) display only one particle population at similar particle diameters to non-stressed Sample 2-C. These results demonstrate that adding caffeine to these samples reduced the formation of aggregates or invisible particles.

Table 37

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

Table 38A

Table 38B

Example 37: Evaluation of stability by size exclusion chromatography (SEC)

Size exclusion chromatography was used to detect invisible particulates less than about 0.1 micron in size from the stressed and non-stressed adalimumab samples described in Example 36. To perform SEC, a TSKgel SuperSW3000 column with a guard column (Tosoh Biosciences, Montgomeryville, PA) was used and elution was monitored at 280 nm. A total of 10 μL of each stressed and non-stressed sample from Example 36 was eluted isocratically with pH 6.2 buffer (100 mM phosphate, 325 mM NaCl) at a flow rate of 0.35 mL/min. The retention time of the adalimumab monomer was approximately 9 minutes. No detectable aggregates were identified in the sample containing the caffeine excipient, and the amount of monomer in all three samples remained constant.

Example 38: HERCEPTIN ^® Decreased formulation viscosity

The monoclonal antibody Trastuzumab ( ^HERCEPTIN® from Genentech) was received as a lyophilized powder and reconstituted at 21 mg/mL in deionized water. The resulting solution was concentrated as such in an Amicon Ultra 4 centrifugal concentrator tube (molecular weight cutoff, 30 kDa) by centrifugation at 3500 rpm for 1.5 hours. Samples were diluted 200-fold in an appropriate buffer and concentration was determined by measuring absorbance at 280 nm using an extinction coefficient of 1.48 mL/mg. Viscosity was measured using a RheoSense microVisc viscometer.

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

Table 39

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

Table 40

Buffer system 1 containing both salicylic acid and caffeine had a maximum viscosity reduction of 76% at 215 mg/mL compared to the control sample. Buffer system 2 containing only caffeine had a maximum viscosity reduction of 59% at 200 mg/mL.

Example 39: AVASTIN ^® Decreased formulation viscosity

^AVASTIN® (a formulation of the monoclonal antibody bevacizumab sold by Genentech) was received as a 25 mg/mL solution in histidine buffer. Samples were concentrated in Amicon Ultra 4 centrifugal concentrator 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). An excipient buffer was prepared by adding 10 mg/mL caffeine with 25 mM histidine HCl. The AVASTIN ^® stock solution was diluted with excipient buffer and then concentrated in an Amicon Ultra 15 centrifugal concentrator tube (MWCO 30 kDa). Concentrations of excipient samples were determined by Bradford assay, and viscosities were measured using a RheoSense microVisc. The results are presented in Table 41 below.

Table 41

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

Example 40: Preparation of Formulation Containing Caffeine, Secondary Excipients and Test Protein

Formulations were prepared using caffeine or a combination of caffeine and a second excipient compound as an excipient compound and a test protein, wherein the test protein was intended to simulate a therapeutic protein to be used in a therapeutic formulation. The formulation was prepared in 20 mM histidine buffer with different excipient compounds for viscosity measurement in the following manner. The excipient combination (excipients A and B as described in Table 42 below) was dissolved in 20 mM histidine and the resulting solution pH was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein did Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a rate to achieve a final protein concentration of approximately 280 mg/mL. A solution of BGG in excipient solution was formulated in a 20 mL glass scintillation vial and shaken at 80-100 rpm on an orbital shaker table overnight. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for about 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 reported as the viscosity for the sample in Table 42 below. The viscosity of the solution with excipients was normalized to that of the model protein solution without excipients. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipient to the viscosity of the model protein solution without excipient.

Table 42

Example 41: Preparation of Formulations Containing Dimethyl Sulfone and Test Proteins

Formulations were prepared using dimethyl sulfone (Jarrow Formulas, Los Angeles, Calif.) and a test protein as an excipient compound, wherein the test protein was intended to simulate a therapeutic protein to be used in a therapeutic formulation. The formulation was prepared in 20 mM histidine buffer for viscosity measurement in the following manner. Dimethyl sulfone was dissolved in 20 mM histidine, and the resulting solution pH was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve pH 6, then filtered through a 0.22 micron filter prior to dissolution of the model protein. Once the excipient solution was prepared, the test protein bovine gamma globulin (BGG) was dissolved at a concentration of about 280 mg/mL. A solution of BGG in excipient solution was formulated in a 20 mL glass scintillation vial and shaken at 80-100 rpm on an orbital shaker table overnight. The BGG solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for about 10 minutes in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Viscosity measurements of formulations prepared as described above were made 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 in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature, followed by a 20 second measurement collection period. 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 for the sample. The viscosity of the solution with excipients was normalized to that of the model protein solution without excipients. The normalized viscosity reported 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.

Table 43

Example 42: Preparation of buffer solution

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

20 mM Tris(hydroxymethyl)aminomethane (TRIS), 100 mM sodium chloride, 55 mM mannitol and A control buffer of 0.1 mM diethylenetriaminepentaacetic acid (DTPA) was prepared. After all contents were dissolved, Milli-Q ultrapure water was added to the volumetric flask to adjust the solution pH to 7.0 and the volume to 500 mL. The buffer was vacuum filtered through a 0.2 μm PES filter using a bottle top filter device.

Example 43: Ipilimumab formulation

Samples of the monoclonal antibody ipilimumab were obtained from Bioceros (The Netherlands) and used in Amicon Ultra 15 centrifugal concentrator tubes with a 30 kDa molecular weight cutoff (EMD Millipore, Billerica, Mass.) to prepare three preparations of Example 42. The buffer was exchanged with the prepared buffer. The target final protein concentration was 20 mg/mL, and the final concentration was determined 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 blank subtracted protein solution absorbance was divided by the reported extinction coefficient and then multiplied by the protein dilution factor (20x) to determine the final protein concentration. Since caffeine interferes with the absorbance measurement at 280 nm, the protein concentration of the solution containing caffeine was determined by mass balance for the A280 measured protein solution. This gave approximate concentrations close to A280 protein solutions measured on a mass basis.

Then, the prepared protein solution was added to a 384 microwell plate (Aurora Microplates, Whitefish, MT). Each solution was loaded into 3 wells at 35 μL per well. The microwell plate was then centrifuged at 400 x g in a Sorvall Legend RT centrifuge to remove any encapsulated air pockets. Pre-cut pressure sensitive sealing tape (Thermo Scientific) was applied on top of the microwell plate to prevent evaporation prior to placement into the DLS instrument (DynaPro II DLS plate reader, Wyatt Technology Corp., Goleta, Calif.). The DLS instrument sample compartment was maintained at 65° C. and the particle size of the protein solution was recorded for 9 hours. Table 44 presents the radius sizes for three different formulations of ipilimumab at the 1 hour measurement.

Table 44

Example 44: Stability test of protein formulation by urea denaturation

Urea is known to denature proteins in solution, causing them to unfold. The screening methodology of this Example involved adding a specific concentration of urea to a solution of a therapeutic protein such as Ustekinumab. This test example was based on the hypothesis that a protective excipient would prevent or reduce unfolding of a therapeutic protein in the presence of urea, and measuring the amount of unfolded protein would indicate which excipient is effective in stabilizing the protein in the presence of urea. will make it possible to check One way to track protein unfolding involves the use of exogenous fluorescent dyes such as Sypro orange. Sypro orange binds to the hydrophobic region of the unfolded protein structure, resulting in the observed increase in fluorescence signal. Therefore, measuring the difference in fluorescence intensity of the unfolded protein-Sypro orange complex in the presence of different excipients makes it possible to ascertain any 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, MI). Stock solutions of the 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 Milli-Q water and adjusting the pH to 6.0 with 1 M HCl. Then, excipient-containing protein formulations were prepared by combining each excipient preparation with Ustekinumab at a final concentration of 1 mg/mL at a final concentration of 5 mg/mL. A stock solution of 9 M urea for use in this example was prepared by dissolving 27 g of urea in the same histidine buffer and adjusting the pH to 6.0 with 1 M HCl. This urea stock solution was then added to a final concentration of 6M to create a test solution (excipient + protein + urea). Sypro orange dye from the stock solution (5000X) was then spiked to a final concentration of 20X for each. The pH of the mixture was rechecked and it was confirmed that the pH was 6.0. The test solution was incubated for 30 minutes at room temperature. 200 μL of the sample was transferred to a Greiner CellStar black well clear flat bottom 96 well plate and the fluorescence of each sample was measured using a BioTek Synergy HT plate reader with excitation at 485 nm and emission filters at 590/20 nm. The fluorescence intensities of the different test formulations were compared to those of the control formulations (protein and urea without any excipients) and test formulations exhibiting reduced fluorescence were considered to contain stabilizing excipients. As shown in Table 45 below, many of the excipients reflected increased stability compared to the control when their fluorescence was compared to that of the control. These conclusions were drawn as the ability of an excipient to stabilize a protein is related to its ability to reduce the fluorescence measured during the experiment: a stabilizing excipient will prevent or reduce protein unfolding, which reduces protein-Sypro orange interactions. will occur, which in turn will appear as reduced fluorescence intensity. The results of these tests are summarized in Table 45 below.

Table 45

Example 45: Stabilization of protein formulations at low pH

Therapeutic proteins, particularly antibodies, are exposed to low pH solution conditions during different stages of processing, particularly purification and viral clearance. This exposure to acidic pH conditions can cause conformational changes, which in turn can lead to protein unfolding and aggregation. A screening methodology to identify stabilizing excipients involved incubating a therapeutic protein such as omalizumab at acidic pH. A protective excipient will prevent or reduce unfolding of the therapeutic protein at low pH, thus measuring the amount of unfolded protein will make it possible to identify an excipient that is effective in stabilizing the protein in the presence of low pH. Unfolding of therapeutic proteins at acidic pH can be tracked using an exogenous fluorescent dye such as Sypro orange. Addition of Sypro orange (Thermo-Fisher, Waltham MA), as performed in this example, allows the dye to bind to hydrophobic regions within the unfolded protein, resulting in an increase in the fluorescence signal. Therefore, measuring the difference in fluorescence intensity of the unfolded protein-Sypro orange in the presence of different excipients makes it possible to identify the stabilizing agent.

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, MI). Stock solutions of the excipients were prepared by dissolving each excipient at a concentration of 100 mg/mL in 0.15 M glycine buffer pH 2.6. An acidification buffer was prepared by dissolving 1.65 g of histidine in 0.09 L Milli-Q water, adjusting the pH to 2.6 with 1 M HCl, and bringing the volume to 0.100 L. Each excipient-containing protein formulation was then prepared by combining each excipient preparation with a final concentration of 5 mg/mL with Ustekinumab at a final concentration of 1 mg/mL. Glycine acidification buffer was then added to the excipient-protein mixture and then spiked in Sypro orange dye from the stock solution (5000X) to a final concentration of 20X. 200 μL of the sample was transferred to a Greiner CellStar black well clear flat bottom 96 well plate 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 . Fluorescence intensities of the different test formulations were compared to those of the control formulations (protein and glycine buffer without any excipients) and those exhibiting reduced fluorescence were considered to contain stabilizing excipients. As shown in Table 46, many of the excipients reflected increased stability compared to the control when their fluorescence was compared to that of the control. These conclusions were drawn as the ability of an excipient to stabilize a protein is related to its ability to reduce the fluorescence measured during the experiment: a stabilizing excipient will prevent or reduce protein unfolding, which reduces protein-Sypro orange interactions. will occur, which in turn will appear as reduced fluorescence intensity. The results of these tests are summarized in Table 46 below.

Table 46

Example 46: Testing of excipients as heat stabilizers

Therapeutic proteins are frequently subjected to fluctuations in temperature that can lead to changes in tertiary and secondary structural elements. This can cause protein aggregation and reduce the amount of active native protein. Excipients that protect against heat stress are identified in this example by thermal 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, MI). Stock solutions of the excipients were prepared by dissolving each excipient at a concentration of 100 mg/mL in 20 mM histidine buffer at pH 6.0. Histidine buffer was prepared by dissolving 1.55 g of histidine in 0.500 L Milli-Q water and adjusting the pH to 6.0 with 1 M HCl. Then, excipient-containing protein formulations were prepared by combining each excipient preparation with Ustekinumab at a final concentration of 1 mg/mL at a final concentration of 5 mg/mL. The formulation was aliquoted into 0.2 mL microcentrifuge tubes and incubated at 65° C. in a heating block for 120 minutes. Aliquots were withdrawn at 0, 15, 30, 60, 90 and 120 minutes. The sample was quenched on ice for 5 minutes and spun at 5000 rpm for 10 minutes. Samples were then analyzed by size exclusion-HPLC with the supernatant 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. A mobile phase of 50 mM phosphate buffer at pH 6.5 and 100 mM NaCl 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 was related to the fraction of monomer remaining at the end of the 2 hour incubation.

Table 47

Example 47: Testing of excipients as stabilizers against mechanical shear stress

Therapeutic proteins are often subjected to mechanical stress by shaking, agitation, etc., and the applied shear stress can cause aggregation of the protein. Excipients that provide protection against shear stress were identified by shaking the therapeutic protein solution in the presence of the test excipients (listed in Table 48 below) and observing any change in the number of aggregated particles. 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, MI). Stock solutions of the excipients were prepared by dissolving each excipient at a concentration of 100 mg/mL in Milli-Q water. An excipient-containing protein formulation was then prepared by combining each excipient preparation with omalizumab at a final concentration of 2 mg/mL at a final concentration of 0.5 mg/mL. Samples were transferred to 0.5 mL frozen vials (ThermoFisher, Waltham MA) and placed on an orbital shaker. Then, they were incubated at 22° C. for 72 hours with shaking 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. Any increase in aggregation was measured by observing the change in light scatter at 350 nm and comparing the change to light scatter at 350 nm of a control formulation (prepared identically to the test sample, but without the addition of excipients). Inherently protective excipients were identified by their ability to slow aggregation and reduce A350 absorbance values after the shaking step.

Table 48

Example 48: Freeze-thaw stability of protein solutions using excipients

All excipients used in this example, listed in Table 49 below, were obtained from Sigma Aldrich (St. Louis, MO) or Cayman Chemical (Ann Arbor, MI). 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 Milli-Q water and adjusting the pH to 6.0 with 1 M HCl. Then, excipient-containing protein formulations were prepared by combining each excipient preparation 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, IL). Samples were frozen at −80° C. in the freezer for at least 5 cycles, then thawed at room temperature, then 100 μL of the sample was transferred to a Greiner CellStar black well clear flat-bottom 96-well plate. The stabilizing effect of the excipients was determined by measuring the formation of protein aggregates using light scattering analysis at 350 nm, and the change to light scattering at 350 nm of a control formulation (prepared identically to the test sample, but without the addition of excipients). Comparatively analyzed. Inherently protective excipients were identified by their ability to slow aggregation and reduce A350 absorbance values after the shaking step.

Table 49

Example 49: DLS diffusion interaction parameter k _D Excipient test by

A stock solution of 20 mM histidine hydrochloride (His HCl) buffer was prepared for use in formulating excipient and protein solutions by dissolving 3.1 g of histidine (Sigma-Aldrich, St. Louis, MO) in type 1 ultrapure water. . The resulting solution was titrated to pH 6 by dropwise addition of 1 M hydrochloric acid. After pH adjustment, the buffer was diluted to a final volume of 1 L in a volumetric flask with type 1 ultrapure water. 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, MI).

A series of six test protein solutions were prepared using the proteins listed in Table 50, with protein concentrations ranging from about 4 mg/mL to about 20 mg/mL, all prepared in 20 mM His HCl buffer at pH 6. In 384-well microplates (Aurora Microplates, Whitefish, MT), 15 μL of protein solution was combined with 15 μL of stock excipient solution prepared in 20 mM His HCl buffer at pH 6 using the excipients listed in Table 50, Each excipient was tested at 6 different protein concentrations. Microplates containing protein-excipient combinations were centrifuged at 400 xg in a Sorvall Legend RT centrifuge followed by shaking on a plate shaker to properly mix the samples. A second centrifugation step was completed to remove air bubbles. The diffusion interaction parameters (k _D ) of these protein-excipient formulations were measured by dynamic light scattering (DLS) in dilute solutions as a way to detect the effect of excipients on protein-protein interactions (PPI). To conduct the DLS study, the prepared microplate was 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-containing test solution, the measured diffusion coefficient was plotted as a function of protein concentration and the slope of the linear fit of the data was reported as k _D . A more negative k _D indicates a stronger net attraction PPI, and a more positive k _D indicates a stronger net repulsion PPI. Table 50 shows the k _D values of each excipient-containing test solution, where these test values for each excipient can be compared to the k _D values of a control solution (containing protein in histidine buffer but no excipient). .

Table 50

Example 50: Excipient test for viscosity reduction

Biosimilar monoclonal antibodies, omalizumab and ustekinumab, obtained from Bioceros (The Netherlands) were buffer exchanged into 20 mM His HCl buffer, pH 6, in Amicon Ultra 15 centrifugal concentrator tubes with a 30 kDa molecular weight cutoff. (EMD Millipore, Billerica, MA). The resulting concentrated formulation was prepared by serial dilutions of the concentrated formulation in 20 mM His HCl, and 100 μL of each dilution was loaded into a UV transparent 96 half-well microplate (Greiner Bio-One, Austria) and Synergy HT Protein concentration was assayed by absorbance at 280 nm by measuring absorbance at 280 nm with a plate reader (BioTek, Winooski, VT). The protein concentration was then determined by dividing the blanked path-length corrected absorbance measurements by their respective extinction coefficients and multiplying by the dilution coefficient. An excipient solution was prepared in 20 mM HisHCl pH 6 at 10 times the desired final concentration or solubility limit of the compound, and the pH was adjusted to 6 as necessary using concentrated hydrochloric acid or sodium hydroxide. The concentrated protein formulation was then combined with a 10-fold excipient solution (9 parts protein, 1 part excipient solution or buffer) of the excipients listed in Table 51 below in 384-well microplates (Aurora Microplates, Whitefish, MT). 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, MI). Since each sample was diluted to the same volume, the protein concentration of each sample is the same. The microplate was then centrifuged at 400 x g in a Sorvall Legend RT centrifuge and shaken on a plate shaker. After shaking, 2 μL of a 5-fold dilution of polyethylene glycol surface-modified gold nanoparticles (nanoComposix, San Diego, Calif.) in 20 mM His HCl was added to each sample well. After mixing the gold nanoparticles into the sample by shaking the microplate twice, the sample was placed in 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 viscosity of the protein formulation was determined according to the Stokes-Einstein equation using 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. In this example, the viscosity of the protein formulation in centipoise (cP) was calculated by multiplying the ratio of the apparent radius to the actual radius of the gold nanoparticles by the viscosity of water at 25°C. The results of these tests are summarized in Table 51 below.

Table 51

Example 51: Thermal degradation assay using infliximab

REMICADE ^® Infliximab was obtained from the Clinigen Group and reconstituted according to the instructions in the Janssen package insert to give 10 mg in 5 mM phosphate buffer with 50 mg/mL sucrose and 0.05 mg/mL polysorbate 80, pH about 7. /mL Infliximab solution was created. The reconstituted drug product was then combined in a 1:1 volume with 50 mM sodium acetate buffer at pH 5. The resulting solution was then injected into a small scale preparative cation exchange column (GE Healthcare, Chicago, IL). After loading 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 at pH 5. The eluted infliximab was then buffer exchanged into 20 mM phosphate buffer at pH 7 using an Amicon Ultra 15 (EMD Millipore, Billerica, Mass.) centrifugal concentrator with a 30 kDa molecular weight cutoff. Then, stock solutions of 4 or 8 mg/mL infliximab in 20 mM phosphate buffer at pH 7 were used in subsequent tests.

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, MI). 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 achieve a final infliximab concentration of about 2 mg/mL in each sample. mixed. Samples containing infliximab solution and stock excipient solutions were then aliquoted into PCR tubes in five 100 μL aliquots. Aliquots were incubated at 55° C. in a drying bath (Benchmark Scientific, Sayreville, NJ) for different periods ranging from 15 minutes to about 3 hours to thermally stress; The samples were then removed from the drying bath and placed on ice to quench thermal aggregation. These stress samples were then analyzed for monomer content by high performance size exclusion chromatography (HP-SEC) using an Agilent 1100 series HPLC equipped with a diode array detector monitoring absorbance at 280 nm. HPLC was performed at a column temperature of 25 °C with a mobile phase of 100 mM phosphate, 300 mM NaCl at pH 7 through a TSKgel SuperSW3000 4.6 mm x 30 cm column (Tosoh Bioscience, Tokyo, Japan) at a flow rate of 0.35 mL/min. For each sample, the percentage of monomer remaining after exposure to thermal stress was obtained by dividing the monomer peak area by the monomer peak area obtained in the same but unstressed sample. Monomer remaining as a percentage of non-stressed 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 reported as the monomer loss rate. Then, the monomer loss rate determined by dividing the monomer loss rate by the monomer loss rate of the buffer control without excipient was normalized, and the results are shown in Table 52 below.

Table 52

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 was purchased from Sigma-Aldrich (St. Louis, MO). These materials were used as excipients in the following experiments.

A biosimilar of the monoclonal antibody omalizumab obtained from Bioceros (The Netherlands) was buffer exchanged into 20 mM His HCl pH 6 buffer and placed in Amicon Ultra 15 centrifugal concentrator tubes with a 30 kDa molecular weight cutoff (EMD Millipore, Billerica, USA). MA) was used to concentrate. A buffer was prepared by dissolving 1.55 g of histidine in 0.5 L Milli-Q water and adjusting the pH to 6.0 with 1 M HCl. The resulting concentrated formulation was prepared by serial dilutions of the concentrated formulation in 20 mM His HCl, and 100 microliters of each dilution were loaded into a UV transparent 96 half-well microplate (Greiner Bio-One, Austria), Synergy Absorbance was measured at a wavelength of 280 nm with an HT plate reader (BioTek, Winooski, VT) and analyzed by A280 for protein concentration. The protein concentration was then determined by dividing the blanked path-length corrected A280 measurements for each sample by the respective extinction coefficient and multiplying by the dilution coefficient. Stock excipient solutions were prepared in 20 mM HisHCl pH 6 using the above-mentioned excipients at 1 M or at the solubility limit of the compounds, adjusting the pH to 6 with concentrated hydrochloric acid or sodium hydroxide as needed. The concentrated protein formulation was then combined at a ratio of 9 parts protein formulation: 1 part excipient solution or buffer (for control) with stock excipient solution or control, and aliquots were plated in 384 well microplates (Aurora Microplates, Whitefish, MT). was added to the wells of The microplate was then centrifuged at 400 x g 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 (nanoComposix, San Diego, Calif.) with a diameter of 100 nm in 2 microliters of 20 mM His HCl was added to each sample well. . After mixing the gold nanoparticles into the sample by shaking the microplate twice, the sample was placed in 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 viscosity of the protein formulation was determined according to the Stokes-Einstein equation using 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 buffer (no protein). In this example, the viscosity of the protein formulation in centipoise (cP) was calculated by multiplying the ratio of the apparent radius to the actual radius of the gold nanoparticles by the viscosity of water at 25°C. Results using the two different excipients are presented in Table 53 below.

Table 53

Example 53: DLS Viscosity Measurement of Concentrated Omalizumab

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), dicyclomine hydrochloride, predinol methanesulfonate, 1-butylimidazole and 1-hexylimidazole were purchased from Sigma Aldrich ( St. Louis, MO) and was used to prepare the excipient solution in this example. O-(Octylphosphoryl)choline was purchased from Sigma Aldrich (St. Louis, Mo.) as a 1 M solution and was used as a stock excipient solution in this example.

A concentrated formulation of a biosimilar 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 above-mentioned excipients were prepared in 1 M or 20 mM HisHCl pH 6 buffer at the solubility limit of the compounds (prepared as described in Example 52), using concentrated hydrochloric acid or sodium hydroxide. The pH was adjusted to 6 as needed. O-(octylphosphoryl)choline was used as a stock excipient without further preparation. The concentrated protein formulation was then combined at a ratio of 9 parts protein formulation: 1 part excipient solution or buffer (for control) with stock excipient solution or control, and aliquots were plated in 384 well microplates (Aurora Microplates, Whitefish, MT). was added to the wells of The microplate was then centrifuged at 400 x g 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 (nanoComposix, San Diego, Calif.) with a diameter of 100 nm in 2 microliters of 20 mM His HCl was added to each sample well. . After mixing the gold nanoparticles into the sample by shaking the microplate twice, the sample was placed in 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 viscosity of the protein formulation was determined according to the Stokes-Einstein equation using 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 buffer (no protein). In this example, the viscosity of the protein formulation in centipoise (cP) was calculated by multiplying the ratio of the apparent radius to the actual radius of the gold nanoparticles by the viscosity of water at 25°C. Results using five different excipients are presented in Table 54 below.

Table 54

Example 54: DLS Viscosity Measurement of Concentrated Omalizumab

Stock excipient solutions for this example were prepared using the following chemicals: tetraethylammonium chloride, tetramethylammonium acetate, 1-methylimidazole, 1-butylimidazole, 1-hexylimidazole, 2 -Ethylimidazole, 2-methylimidazole and spectinomycin were purchased from Sigma Aldrich (St. Louis, Mo.). Triglycine, tetraglycine and 2-butylimidazole were purchased from Chem-Impex (Wood Dale, IL). Hordenine HCl was purchased from Bulk Supplements (Henderson, NV).

A concentrated formulation of a biosimilar 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 above-mentioned excipients were prepared in 1 M or 20 mM HisHCl pH 6 buffer at the solubility limit of the compounds (prepared as described in Example 52), using concentrated hydrochloric acid or sodium hydroxide. The pH was adjusted to 6 as needed. The concentrated protein formulation was then combined with the stock excipient solution or control at a ratio of 9 parts protein:1 part excipient solution or buffer (for control), and aliquots were plated in 384 well microplates (Aurora Microplates, Whitefish, MT). was added to the well. The microplate was then centrifuged at 400 x g 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 (nanoComposix, San Diego, Calif.) with a diameter of 100 nm in 2 microliters of 20 mM His HCl was added to each sample well. . After mixing the gold nanoparticles into the sample by shaking the microplate twice, the sample was placed in 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 viscosity of the protein formulation was determined according to the Stokes-Einstein equation using 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 buffer (no protein). In this example, the viscosity of the protein formulation in centipoise (cP) was calculated by multiplying the ratio of the apparent radius to the actual radius of the gold nanoparticles by the viscosity of water at 25°C. Results using 12 different excipients are presented in Table 55 below.

Table 55

Example 55: Excipients that increase thermal stability

Therapeutic proteins are frequently subjected to fluctuations in temperature that can cause changes in their tertiary and secondary structural elements. This causes aggregation of the protein and a reduction of the active native species. Excipients that protect against heat stress were tested by thermal degradation studies in the presence or absence of excipients. 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 (prepared as described in Example 52). Each test sample was added to the buffer with the excipient stock to obtain a final concentration of 5 mg/mL of the excipient, and the protein from the 20 mg/mL Ustekinumab stock in histidine buffer (prepared as described in Example 51). was prepared by dilution to a final concentration of 1 mg/mL. The formulation was aliquoted into 0.2 mL microcentrifuge tubes and incubated at 65° C. in a heating block for 120 minutes. Aliquots were withdrawn at 0, 30, 60, 90 and 120 minutes. The samples were then quenched on ice for 5 minutes and spun at 9000 rpm for 10 minutes. A sample of the supernatant was then analyzed by SE-HPLC as follows: The supernatant was analyzed by Agilent 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. Loaded onto a 1100 HPLC system. 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. For each sample, the monomer fraction was calculated by integrating the peak area under the monomer peak and the change in the plotted integrated peak area as a function of time. Thermal stability was related to the fraction of monomer remaining at the end of the 2 hour incubation, and an increase in the percentage of monomer compared to the control (no excipient added) was recorded. Results for the seven tested excipients are summarized in Table 56 below.

Table 56

Example 56: Excipients that improve the thermal stability of ADCs

Antibody-drug conjugates (ADCs) are therapeutic proteins that are produced through the conjugation of small molecules to monoclonal antibodies via chemical linkers that allow site-specific delivery of small molecule drugs. Conjugated linker and small molecule combinations alter the chemical and physical properties (charge, hydrophobicity, etc.) of the ADC compared to its protein precursor and introduce additional stability issues. The compound Ustekinumab-FITC from Example 59 was used as a model ADC compound for these tests. Excipients that protect model ADCs against heat stress were tested by 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 (prepared as described in Example 52). Each test sample was prepared by adding excipient stock to buffer to obtain a final concentration of 5 mg/mL, and Ustekinumab-FITC (as described in Example 59 below) in histidine buffer was added to 1 mg/mL of Ustekinumab. It was prepared by adding the final concentration of Kinumab-FITC. The formulation was aliquoted into 0.2 mL microcentrifuge tubes and incubated at 65° C. in a heating block for 120 minutes. Aliquots were withdrawn at 0, 30, 60, 90 and 120 minutes. The samples were then quenched on ice for 5 minutes and spun at 9000 rpm for 10 minutes. Samples were analyzed by SE-HPLC loading the supernatant 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. 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 change in the peak area under the monomer peak and the integrated peak area plotted as a function of time, and the increase in monomer percentage compared to the control (no excipient added) was recorded. Thermal stability was related to the fraction of monomer remaining at the end of the 2 hour incubation.

Table 57

Example 57: Excipients that protect against freeze/thaw stress

Therapeutic proteins are often kept at low temperatures to improve their kinetic stability and to minimize structural perturbations that can lead to protein aggregation and reduction of active endogenous species. In certain 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 confirmed by thermal degradation studies in the presence or absence of excipients. Excipient stocks were prepared by dissolving the excipients listed in Table 58 below at a concentration of 1 M in 20 mM histidine buffer, pH 6.0 prepared as described in Example 52. Each test sample was prepared by adding an excipient stock to a final concentration of excipient of 100 mM and protein from a 20 mg/mL omalizumab stock in histidine buffer (prepared as described in Example 50) to a final concentration of 2 mg/mL. prepared by dilution to concentration; A control was prepared in the same manner but without the addition of excipient 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 maintained at room temperature. This freeze-thaw cycle was repeated 6 times, after which each sample was aliquoted into a 0.2 mL microcentrifuge tube and spun at 9000 rpm for 10 minutes. Samples were analyzed by SE-HPLC loading the supernatant 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. 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 plotted integrated peak area as a function of time.

Table 58

Example 58: Accelerated Aging Study

Excipients that protect against heat stress were tested by thermal degradation studies in the presence or absence of excipients. Excipient stocks were prepared by dissolving the excipients listed in Table 59 below at a concentration of 1 M in 20 mM histidine buffer, pH 6.0 (prepared as described in Example 52). 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; A control was prepared in the same manner but without the addition of excipient 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 withdrawn after 0, 1, 2, 3 and 4 weeks. Samples were analyzed by SE-HPLC loading the supernatant 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. 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 plotted integrated peak area as a function of time. Thermal stability was related to the fraction of monomer remaining at the end of the 4 week incubation.

Table 59

Example 59: Synthesis of Ustekinumab FITC

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

Example 60: Stability of Ustekinumab-FITC

The ADC model compound of Example 59 was diluted to a mAb concentration of 1 mg/mL in 20 mM histidine buffer (prepared as described in Example 52) at pH 6. Samples were prepared with the added excipients listed in Table 60 below and tested for their ability to protect the 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. After shearing, the control sample had a particle radius of 143 nm, indicating significant aggregation compared to the non-stressed sample (radius 5.5 nm). The excipient-containing samples did not show a significant increase in particle radius compared to the non-stressed control sample (radius 5.5 nm), demonstrating a protective effect against mechanical shear stress. The results are presented in Table 60 below.

table 60

Example 61: Effect of co-solutes on caffeine solubility in aqueous buffer during refrigerated storage

A 25 mM histidine buffer, pH 6, was prepared by dissolving 0.387 g of histidine in Milli-Q type 1 water, titrating to pH 6 with hydrochloric acid, and diluting to a final volume of 100 mL with Milli-Q water. . Thereafter, a 50 mM co-solute solution was prepared using the buffer solution as excipients of sodium benzoate, 1-methyl-2-pyrrolidone, proline, phenylalanine, arginine monohydrochloride, benzyl alcohol, and nicotinamide. About 0.1 g caffeine was dissolved in 5 mL aliquots of each resulting solution to achieve a caffeine concentration of 20 mg/mL. Corresponding solutions containing different volume ratios of 20 mg/mL caffeine and co-solute and excipient solutions, but without caffeine, were prepared in triplicate in 96 well microplates, maintaining a total well volume of 300 μL in all cases. Thereafter, the resulting microplate was sealed with microplate tape and stored in a refrigerator while maintaining the temperature in the range of 2 to 5°C. Throughout the storage process, the microplates were visually inspected for evidence of sediment in the wells. The first precipitate observed in three wells for each condition was recorded and the results are summarized in Table 61 below.

Table 61

Example 62: Testing of excipients to reduce the viscosity of antibody solutions

A stock buffer of 20 mM histidine-HCl pH 6.0 (His HCl) was prepared by dissolving 1.55 g of histidine (Sigma-Aldrich, St. Louis, MO) 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 final volume was up to 0.5 L in the volumetric flask. All excipients were dissolved in His HCl and prepared at 10x concentration (1 M) or the solubility limit of the compound. The excipient solution was pH measured and adjusted to pH 6.0 if necessary.

In this example, a protein solution having an excipient concentration of 0.1 M or less was measured for viscosity. The biosimilar monoclonal antibody omalizumab purchased from Bioceros (The Netherlands) was buffer exchanged with His HCl and pre-rinsed Amicon-15 centrifuges (EMD Millipore, Billerica, MA) with a 30 kDa molecular weight cutoff limit were used. was concentrated to approximately 200 mg/mL. A dispersion of polyethylene glycol surface-modified gold nanoparticles (nanoComposix, San Diego, Calif.) was thoroughly mixed and diluted 5-fold with His HCl. In a separate PCR tube, 2.1 μL of gold-nanoparticles, 5.3 μL of 10x excipient solution and 47.6 μL of concentrated omalizumab were combined and mixed thoroughly. Each solution was transferred twice in a volume of 25 μL to a 384-well plate (Aurora Microplates, Whitefish, MT) and centrifuged at 400 x g for 1 minute (Sorvall Legend RT). Tape sealing was used to prevent evaporation of the sample. Then, the plate was transferred to a DynaPro II DLS plate reader (Wyatt Technology Corp., Goleta, CA) and the apparent particle size of the gold nanoparticles was measured at 25°C. The viscosity of the protein formulation was determined according to the Stokes-Einstein equation by calculating the ratio of the measured apparent radius to the known radius particle size.

In this example, the diffusion interaction parameter (k _D ) of dilute protein solutions was measured by DLS in the presence of 0.1 M or less of excipient. From the previously prepared excipient stock solution, a 0.2 M excipient solution was prepared separately. k _D was determined 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 was combined with 15 μL of 0.2M excipient solution (1:1 mixture) 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. Upon mixing, the well plate was centrifuged at 400 xg in a Sorvall Legend RT for 1 minute to remove any air pockets. Then, the well plate was loaded into a DynaPro II DLS plate reader (Wyatt Technologies Corp., Goleta, Calif.), 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 reported as k _D . The results are summarized in Tables 62A and 62B below for two different series of tests.

Table 62A

Table 62B

equivalent

Although specific embodiments of the present invention have been disclosed herein, the above specification is illustrative and not restrictive. While this invention has been shown and described with particular reference to preferred embodiments thereof, it should be appreciated by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims. It will be understood. Upon review of this specification, many variations of this invention will become apparent to those skilled in the art. Unless otherwise indicated, 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." Accordingly, unless indicated to the contrary, the numerical parameters described herein are approximations that may vary depending on the desired properties sought to be obtained by the present invention.

Claims (32)

A stability-enhancing formulation comprising a therapeutic protein and a stability-enhancing amount of a stabilizing excipient, wherein the stability-enhancing formulation is otherwise identical to the stability-enhancing formulation, but is characterized by improved stability parameters compared to a control formulation lacking the stabilizing excipient. , stability-enhancing formulations. The stability enhancing formulation of claim 1 , wherein the therapeutic protein is an antibody. 3. The stability enhancing formulation of claim 2, wherein the antibody is an antibody-drug conjugate. The stability enhancing formulation of claim 1, wherein the stabilizing excipient is a hindered amine compound. The stability enhancing formulation of claim 1 wherein the stabilizing excipient is an anionic aromatic compound. The stability enhancing formulation of claim 1 wherein the stabilizing excipient is a functionalized amino acid compound. The stability enhancing formulation of claim 1, wherein the stabilizing excipient is an oligopeptide. The stability enhancing formulation of claim 1 wherein the stabilizing excipient is a short chain organic acid. The stability enhancing formulation of claim 1 wherein the stabilizing excipient is a low molecular weight polyacid. The stability enhancing formulation according to claim 1, wherein the stabilizing excipient is a dione compound or a sulfone compound. The stability enhancing formulation of claim 1, wherein the stabilizing excipient is a zwitterionic compound. The stability enhancing formulation according to claim 1, wherein the stabilizing excipient is a crowding agent having a hydrogen bonding component. The stability enhancing formulation of claim 1, wherein the stabilizing excipient is added in an amount of about 1 mM to about 500 mM. 14. The stability enhancing formulation of claim 13, wherein the stabilizing excipient is added in an amount of about 5 mM to about 250 mM. 15. The stability enhancing formulation of claim 14, wherein the stabilizing excipient is added in an amount of about 10 mM to about 100 mM. 16. The stability enhancing formulation of claim 15, wherein the stabilizing excipient is added in an amount of about 5 mg/mL to about 50 mg/mL. The stability enhancing formulation of claim 1 wherein the improved stability parameter is thermal storage stability. 18. The stability enhancing formulation of claim 17, wherein the heat storage stability is improved at a temperature of about 10°C to 30°C. The stability enhancing formulation of claim 1 wherein the improved stability parameter is improved freeze/thaw stability. The stability enhancing formulation of claim 1 wherein the improved stability parameter is improved shear stability. The stability enhancing formulation of claim 1 , wherein the formulation has a reduced number of particles compared to the control formulation. The stability enhancing formulation of claim 1 , wherein the formulation has improved biological activity compared to the control formulation. A method of improving the stability of a therapeutic formulation comprising adding a stability-enhancing amount of a stabilizing excipient to the therapeutic formulation, thereby improving the stability of the therapeutic formulation, wherein the stability of the therapeutic formulation is otherwise identical to the therapeutic formulation. as measured by comparison to the stability of a control formulation lacking a stabilizing excipient. 24. The method of claim 23, wherein the stabilizing excipient consists of hindered amines, anionic aromatic compounds, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight polyacids, diones, sulfones, zwitterionic compounds and flocculants with hydrogen bonding elements. How to be selected from the group. 24. The method of claim 23, wherein determining the stability of the therapeutic formulation compared to the stability of a control formulation comprises measuring a stability-related parameter. 26. The method of claim 25, wherein the stability-related parameter is selected from the group consisting of heat storage stability, freeze/thaw stability and shear stability. 24. The method of claim 23, wherein the therapeutic formulation comprises a therapeutic protein. 28. The method of claim 27, wherein the therapeutic protein is an antibody. 29. The method of claim 28, wherein the antibody is an antibody-drug conjugate. A method of improving a parameter of a protein-related process comprising adding a stability-enhancing amount of a stabilizing excipient to a carrier solution for the protein-related process, thereby improving the parameter, wherein the carrier solution contains the protein of interest. 31. The method of claim 30, wherein the parameter is selected from the group consisting of protein production cost, protein yield, protein production rate and protein production efficiency. 32. The method of claim 31, wherein the protein of interest is a Therapeutic protein.

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