CN112040931A - Excipient compounds for protein formulations - Google Patents

Excipient compounds for protein formulations Download PDF

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Publication number
CN112040931A
CN112040931A CN201980029042.9A CN201980029042A CN112040931A CN 112040931 A CN112040931 A CN 112040931A CN 201980029042 A CN201980029042 A CN 201980029042A CN 112040931 A CN112040931 A CN 112040931A
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protein
formulation
stability
excipient
therapeutic
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Inventor
D·S·索内
P·胡思里奇
R·P·马奥尼
S·奈克
T·坦瑞
R·C·波蒂拉
D·G·格林
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Improved Biological Products Co ltd
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Improved Biological Products Co ltd
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    • A61K39/39591—Stabilisation, fragmentation
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    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
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Abstract

Disclosed herein are enhanced stability formulations comprising a therapeutic protein and a stability-improving amount of a stability excipient, wherein the improved stability formulation is characterized by having an improved stability parameter as compared to a control formulation that is otherwise identical to the improved stability formulation except for the absence of the stability excipient. Further disclosed herein are methods of improving the stability of a therapeutic formulation or improving a protein-related process parameter.

Description

Excipient compounds for protein formulations
RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application serial No. 62/639,950 filed on 7/3/2018 and U.S. provisional application No. 62/679,647 filed on 1/6/2018. The entire contents of each of the above applications are incorporated herein by reference.
Technical Field
The present application relates generally to biopolymer formulations, such as protein formulations, with stabilizing excipients.
Background
Biopolymers can be used for therapeutic or non-therapeutic purposes. Biopolymer-based therapeutics, such as formulations including proteins, antibodies, or enzymes, are widely used in the treatment of diseases. Non-therapeutic biopolymers, such as preparations comprising enzymes, peptides or structural proteins, have utility in non-therapeutic applications, such as domestic, nutritional, commercial and industrial uses.
Therapeutic and non-therapeutic uses of protein biopolymers are of particular interest. Proteins are complex biopolymers, each with a unique folded 3D structure and surface energy profile (hydrophobic/hydrophilic regions and charge). In concentrated protein solutions, these macromolecules can strongly interact and even interlock in a complex manner, depending on their exact shape and surface energy distribution. "hot spots" with strong specific interactions lead to protein aggregation, thereby increasing solution viscosity. To address these problems, many excipient compounds have been used in biotherapeutic formulations aimed at reducing solution viscosity by preventing local interactions and aggregation. These jobs are typically customized empirically, sometimes guided by computer (in silico) simulations. Combinations of excipient compounds may be provided, but such combinations must be re-optimized empirically and on a case-by-case basis.
Biopolymers (such as proteins) for use in therapeutic applications must be formulated to allow their introduction into the body for the treatment of diseases. For example, it is advantageous in some cases to deliver antibody and protein/peptide biopolymer formulations via the Subcutaneous (SC) or Intramuscular (IM) routes, rather than administering these formulations by Intravenous (IV) injection. Although for better patient compliance and comfort using SC or IM injections, the liquid volume in the syringe is typically limited to 2 to 3mL, and the viscosity of the formulation is typically below about 20 centipoise (cP), so that the formulation can be delivered using conventional medical devices and small bore needles. These delivery parameters are not always very consistent with the dosage requirements of the formulation being delivered.
For example, the antibody may need to be delivered at high dosage levels to exert its intended therapeutic effect. Antibody formulations that use limited liquid volumes to deliver high dose levels may require higher antibody concentrations in the delivery vehicle, sometimes levels in excess of 150 mg/mL. At this dose level, the viscosity-versus-concentration curve of the protein solution exceeds its linear-nonlinear transition period, such that the viscosity of the formulation rises significantly as the concentration increases. However, the increased viscosity is not compatible with standard SC or IM delivery systems. Solutions of biopolymer-based or protein-based therapeutics are also prone to stability problems such as precipitation, fragmentation, oxidation, deamidation, clouding, opalescence, denaturation, and gel formation, i.e., reversible or irreversible aggregation. Stability issues limit the shelf life of the solution or require special handling.
One method of producing protein formulations for injection 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 produce protein powders for subsequent reconstitution. The viscosity of the powdered suspension prior to redissolution is low (compared to the solution at the same total dose) and therefore may be suitable for SC or IM injections, provided that the particles are small enough to fit through a needle. However, the presence of protein crystals in the powder has an inherent risk of triggering an immune response. The uncertain protein stability/activity after resolubilization poses additional concerns. There remains a need in the art for techniques to produce low viscosity protein formulations for therapeutic purposes while avoiding the limitations introduced by protein powder suspensions.
More complex antibody formulations, such as antibody-drug conjugates (ADCs), are particularly susceptible to viscosity and stability problems. ADC links small molecule drugs to monoclonal antibodies (mabs) via chemical linkers; the mAb targets a specific antigen on an abnormal "target cell" for which the small molecule drug has a specific effect. When a mAb comes into contact with a target cell antigen, it 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 broken down, releasing the drug to exert its biological effect on the cell. Typically, the drug is a chemotherapeutic agent that is too toxic to be released systemically. ADCs bring chemotherapy into direct contact with cancer cells as targets. This binding of small molecules to mabs exacerbates viscosity and stability issues affecting therapeutic protein formulations. The payload compound is typically a hydrophobic small molecule that can have a significant impact on the stability, solubility, and solution interaction characteristics of larger ADCs as the drug-antibody ratio is increased. High salt concentrations in the formulation may increase hydrophobic interactions between ADC complexes, thereby making the solubility of the ADC more sensitive to salt action (compared to unbound antibody). Handling or storage of ADC solutions can cause aggregation or precipitation of ADC species, especially where the drug-to-antibody ratio (DAR) is high. Drug binding may also affect the conformational stability of the mAb, particularly its Fc domain. In addition, drug binding may also reduce the net surface charge on the mAb, thereby affecting the solubility of the ADC.
In addition to the therapeutic applications of the above proteins, biopolymers (such as enzymes, peptides and structural proteins) can be used for non-therapeutic applications. These non-therapeutic biopolymers can be produced by many different routes, for example from plant sources, animal sources or from cell cultures.
The non-therapeutic protein may be produced, transported, stored and handled as a particulate or powder material or as a solution or dispersion (typically 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. These solution characteristics can present challenges to the formulation, handling, storage, pumping, and performance of non-therapeutic materials, and methods are needed to reduce viscosity and improve the solubility and stability of non-therapeutic protein solutions.
There remains a need in the art for a truly universal method to reduce viscosity and/or improve stability in protein formulations, particularly at high protein concentrations. There is a further need in the art to achieve such viscosity reduction while preserving the activity of the protein. It is also desirable to adapt the viscosity reduction system for use with formulations having a tunable and sustained release profile, and for use with formulations suitable for depot injections. In addition, improvements in methods for producing proteins and other biopolymers are desired.
Disclosure of Invention
In embodiments, disclosed herein are liquid formulations comprising a protein and an excipient compound selected from the group consisting of: hindered amines, anionic aromatic compounds, functionalized amino acids, oligopeptides, short chain organic acids, and low molecular weight aliphatic polyacids, wherein the excipient compound is added in a viscosity reducing amount. In embodiments, the protein is a pegylated protein and the excipient is a low molecular weight aliphatic polybasic acid. 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 embodiments, the formulation is a non-therapeutic formulation and the non-therapeutic formulation includes a non-therapeutic protein. In embodiments, the viscosity reducing amount reduces the viscosity of the formulation to a viscosity less than the viscosity of a control formulation. In embodiments, the viscosity of the formulation is at least about 10% less than the viscosity of the control formulation, or at least about 30% less than the viscosity of the control formulation, or at least about 50% less than the viscosity of the control formulation, or at least about 70% less than the viscosity of the control formulation, or at least about 90% less than the viscosity of the control formulation. In embodiments, the viscosity is less than about 100cP, alternatively less than about 50cP, alternatively less than about 20cP, alternatively less than about 10 cP. In embodiments, the excipient compound has a molecular weight of <5000Da, or <1500Da or <500 Da. In embodiments, the formulation contains at least about 25mg/mL of protein, or at least about 100mg/mL of protein, or at least about 200mg/mL of protein, or at least about 300mg/mL of protein. In embodiments, the formulation comprises between about 5mg/mL to about 300mg/mL of an excipient compound, alternatively between about 10mg/mL to about 200mg/mL of an excipient compound, alternatively between about 20mg/mL to about 100mg/mL, alternatively between about 25mg/mL to about 75mg/mL of an excipient compound. In embodiments, the formulation has improved stability when compared to a control formulation. In embodiments, the excipient compound is a hindered amine. In embodiments, the hindered amine is selected from the group consisting of: caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin, and potassium acesulfame. In embodiments, the hindered amine is caffeine. In embodiments, the hindered amine is an injectable local anesthetic compound. The hindered amine may have an independent pharmacological profile and the hindered amine may be present in the formulation in an amount to have an independent pharmacological effect. 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 a local anesthetic activity. In embodiments, the hindered amine with independent pharmacological activity is combined with a second excipient compound that further reduces the viscosity of the formulation. The second excipient compound may be selected from the group consisting of: caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin, and potassium acesulfame. In embodiments, the formulation may include an additive selected from the group consisting of: preservatives, surfactants, sugars, polysaccharides, arginine, proline, hyaluronidase, stabilizers, and buffers.
Also disclosed herein are methods of treating a disease or disorder in a mammal, comprising: administering to the mammal a liquid therapeutic formulation, wherein the therapeutic formulation comprises a therapeutically effective amount of a therapeutic protein, and wherein the formulation further comprises a pharmaceutically acceptable excipient compound selected from the group consisting of: hindered amines, anionic aromatic compounds, functionalized amino acids, oligopeptides, short chain organic acids, and low molecular weight aliphatic polybasic acids; and wherein the therapeutic agent is effective to treat the disease or the condition. In embodiments, the therapeutic protein is a pegylated protein and the excipient compound is a low molecular weight aliphatic polyacid. In embodiments, the excipient is a hindered amine. In embodiments, the hindered amine is a local anesthetic compound. In embodiments, the formulation is administered by subcutaneous injection or intramuscular injection 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 the viscosity of a control formulation. In embodiments, the therapeutic formulation has improved stability when compared to a control formulation. In embodiments, the excipient compound is substantially pure.
Also disclosed herein is a method of reducing pain at an injection site of a therapeutic protein in a mammal in need thereof, the method comprising: administering a liquid therapeutic formulation by injection, wherein the therapeutic formulation comprises a therapeutically effective amount of a therapeutic protein, wherein the formulation further comprises a pharmaceutically acceptable excipient compound selected from the group consisting of injectable local anesthetic compounds, wherein the pharmaceutically acceptable excipient compound is added to the formulation in a viscosity reducing amount; and wherein the mammal experiences less pain upon administration of the therapeutic formulation comprising the excipient compound compared to administration of a control therapeutic formulation, wherein the control therapeutic formulation is free of the excipient compound and is otherwise identical to the therapeutic formulation.
In embodiments, disclosed herein are methods of improving the stability of a liquid protein formulation, comprising: preparing a liquid protein formulation comprising a therapeutic protein and an excipient compound selected from the group consisting of: hindered amines, anionic aromatic compounds, functionalized amino acids, oligopeptides, and short chain organic acids and low molecular weight aliphatic polybasic acids, wherein the liquid protein formulation exhibits improved stability compared to a control liquid protein formulation, wherein the control liquid protein formulation does not contain the excipient compound and is otherwise identical to the liquid protein formulation. The stability of the liquid formulation may be refrigerated condition stability, room temperature stability or high temperature stability.
In embodiments, also disclosed herein are liquid formulations comprising a protein and an excipient compound selected from the group consisting of: hindered amines, anionic aromatic compounds, 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 an improved protein-protein interaction profile as measured by the protein diffusion interaction parameter kD or the second coefficient of force (visual coefficient) B22. In embodiments, the formulation is a therapeutic formulation and includes a therapeutic protein. In embodiments, the formulation is a non-therapeutic formulation and includes a non-therapeutic protein.
Also disclosed herein, in embodiments, are methods of improving a protein-related process, comprising: the liquid formulations described above are provided, and used in a treatment process. In an embodiment, the treatment method comprises: filtration, pumping, mixing, centrifugation, membrane separation, lyophilization, or chromatography. In embodiments, the treatment method is selected from the group consisting of: cell culture harvest, chromatography, virus inactivation, and filtration. In embodiments, the treatment process is a chromatographic process or a filtration process. In embodiments, the filtration process is a viral filtration process or an ultrafiltration/diafiltration process.
Also disclosed herein are methods of improving a parameter of a protein-related process, comprising providing 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, diketones and sulfones, and adding a viscosity reducing amount of the at least one excipient compound to a carrier solution for the protein-related process, wherein the carrier solution comprises a protein of interest, thereby improving a parameter thereof. In embodiments, the parameter may be selected from the group consisting of: protein production cost, protein production capacity, protein production rate, and protein production efficiency. The parameter may be a proxy parameter. In embodiments, the protein-related process is an upstream processing process. The carrier solution used in the upstream process may be a cell culture medium. In an embodiment, if the carrier solution is a cell culture medium, the step of adding the excipient additive to the carrier solution comprises a first sub-step of adding the excipient additive to a supplemental medium to form an excipient-containing supplemental medium, and a second sub-step of adding the excipient-containing supplemental medium to the cell culture medium. In other embodiments, the protein-related process is a downstream processing process. The downstream process may be a chromatography process, and the chromatography process may be a protein a chromatography process. In embodiments, the chromatographic process recovers a protein of interest, wherein the protein of interest is characterized by having an improved protein-related parameter compared to a control solution, the parameter selected from the group consisting of: increased purity, improved yield, fewer particles, fewer misfolds, or less aggregation. In embodiments, the improved protein-related parameter is an increased yield of the protein of interest from the chromatography process. In other embodiments, the protein-related process is selected from the group consisting of: filtration, injection, syringe injection (syringing), pumping, mixing, centrifugation, membrane separation and lyophilization, and the force required for the selected procedure was less compared to the control procedure. In embodiments, the protein-related process is selected from the group consisting of: a cell culture process, a cell culture harvest process, a chromatography process, a virus inactivation process and a filtration process. In embodiments, the protein-related process is a viral inactivation process, and the viral inactivation process is performed at a pH level of about 2.5 to about 5.0, or the viral inactivation process is performed at a higher pH than the control process. In other embodiments, the protein-related process is a filtration process. The filtration process may be a virus removal filtration process or an ultrafiltration/diafiltration process. The filtering process is characterized by having improved filtering related parameters. The improved filtration-related parameter may be a faster filtration rate than the filtration rate of the control solution. The improved filtration-related parameter may be the production of a smaller amount of aggregated protein than the amount of aggregated protein produced by a control filtration process. The improved filtration-related parameter may be a higher mass transfer efficiency than the mass transfer efficiency of the control filtration process. The improved filtration-related parameter can be a concentration or yield of the protein of interest that is higher than the concentration or yield of the protein of interest produced by a control filtration process.
Also disclosed herein are methods as described above, wherein the viscosity reducing excipient additive comprises two or more excipient compounds. In embodiments, the at least one excipient compound is a hindered amine. In embodiments, the at least one excipient compound is selected from the group consisting of: caffeine, saccharin, acesulfame potassium, aspartame, theophylline, taurine, 1-methyl-2-pyrrolidone, nicotinamide, and imidazole. In embodiments, the at least one excipient compound is selected from the group consisting of: caffeine, taurine, nicotinamide and imidazole. In embodiments, the at least one excipient compound is an anionic aromatic excipient, and in some embodiments, the anionic aromatic excipient may be 4-hydroxybenzenesulfonic acid. In embodiments, the at least one excipient compound in a viscosity reducing amount from about 1mg/mL to about 100mg/mL, or the at least one excipient compound in a viscosity reducing amount from about 1mM to about 400mM, or an amount in a viscosity reducing amount from about 2mM to about 150 mM. In embodiments, the carrier solution comprises another agent selected from the group consisting of: preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers, and buffers. In embodiments, the protein of interest is a therapeutic protein, and the therapeutic protein may be a recombinant protein, or may be selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, antibody fragments, fusion proteins, pegylated proteins, antibody-drug conjugates, synthetic polypeptides, protein fragments, lipoproteins, enzymes and structural peptides. In embodiments, the method further comprises the step of adding a second viscosity reducing excipient to the carrier solution, wherein the step of adding the second viscosity reducing compound adds additional improvements to the parameters.
Additionally, disclosed herein is a carrier solution comprising a liquid medium having a protein of interest dissolved therein and a viscosity reducing additive, wherein the viscosity of the carrier solution is lower than the viscosity of a control solution. The carrier solution may further comprise another agent selected from the group consisting of: preservatives, sugars, polysaccharides, arginine, proline, surfactants, stabilizers, and buffers.
Further, the present disclosure relates to a stability-improved formulation comprising a therapeutic protein and a stability-improving amount of a stability excipient, wherein the stability-improved formulation is characterized by having an improved stability parameter as compared to a control formulation that is otherwise identical to the stability-improved formulation except for the absence of the stability excipient. In embodiments, the therapeutic protein is an antibody, and the antibody may be an antibody-drug conjugate. The stabilizing excipient may be a hindered amine compound, an anionic aromatic compound, a functionalized amino acid compound, an oligopeptide, a short chain organic acid, a low molecular weight polyacid, a diketone compound or a sulfone compound, a zwitterionic compound or a crowding agent with hydrogen bonding elements. In embodiments, the stabilizing excipient may be added to the formulation in an amount of about 1mM to about 500mM, or in an amount of about 5mM to about 250mM, or in an amount of about 10mM to about 100mM, or in an amount of about 5mg/mL to about 50 mg/mL. The improved stability parameter may be thermal storage stability, for example, wherein thermal storage stability is improved at a temperature between about 10 ℃ and 30 ℃. In embodiments, the improved stability parameter is improved freeze/thaw stability or improved shear stability. In embodiments, the improved stability formulation has a reduced number of particles compared to a control. In embodiments, the improved stability formulation has improved biological activity over the control.
Also disclosed herein is a method of improving the stability of a therapeutic formulation, comprising adding a stability-improving amount of a stability excipient to a therapeutic formulation, thereby improving the stability of the therapeutic formulation, wherein the stability of the therapeutic formulation is measured relative to the stability of a control formulation that is otherwise identical to the therapeutic formulation except for the absence of the stability excipient. The stabilizing excipient may be a hindered amine compound, an anionic aromatic compound, a functionalized amino acid, an oligopeptide, a short chain organic acid, a low molecular weight polyacid, a diketone, a sulfone, a zwitterionic compound, or a crowding agent with hydrogen bonding elements. In embodiments, the step of measuring the stability of the therapeutic formulation may comprise measuring a stability-related parameter, for example a parameter selected from the group consisting of: thermal storage stability, freeze/thaw stability, and shear stability. In embodiments, the therapeutic agent comprises a therapeutic protein, which may be an antibody, and the antibody may be an antibody-drug conjugate. Also disclosed herein are methods of improving a parameter of a protein-related process, the method comprising adding a stability-improving amount of a stability excipient to a carrier solution of the protein-related process, wherein the carrier solution comprises a protein of interest, thereby improving the parameter, wherein the protein of interest can be a therapeutic protein. In embodiments, the parameter may be selected from the group consisting of: protein production cost, protein production capacity, protein production rate, and protein production efficiency.
Drawings
Figure 1 shows a particle size distribution plot of a monoclonal antibody solution under stressed and unstressed conditions as assessed by dynamic light scattering. The data curves in fig. 1 have baseline offsets for comparison: the plot for sample 1-A is shifted by 100 intensity units on the Y-axis, while the plot for sample 1-FT is shifted by 200 intensity units on the Y-axis.
Figure 2 shows a plot of measured sample diameter versus multimodal size distribution for several populations of molecules, assessed by dynamic light scattering. The data curves in fig. 2 have baseline offsets for comparison: the plot for sample 2-A is shifted by 100 intensity units on the Y-axis, while the plot for sample 2-FT is shifted by 200 intensity units on the Y-axis.
Figure 3 shows a size exclusion chromatogram of a monoclonal antibody solution with major monomer peaks at retention times of 8-10 minutes. The data curves in fig. 3 have baseline offsets for comparison: the curves for samples 2-C, 2-A and 2-FT are shifted in the Y-axis direction.
FIG. 4 shows a block diagram of the fermentation process ("upstream processing") steps for the production of therapeutic proteins, such as monoclonal antibodies.
Fig. 5 shows a block diagram of the purification process ("downstream processing") steps for the production of a therapeutic protein, such as a monoclonal antibody.
Detailed Description
Disclosed herein are formulations that allow for the delivery of concentrated protein solutions and methods of their production. In embodiments, the methods disclosed herein can result in lower viscosity liquid formulations or higher concentrations of therapeutic or non-therapeutic proteins in the liquid formulations as compared to traditional protein solutions.
In embodiments, the methods disclosed herein may result in liquid formulations with improved stability when compared to traditional protein solutions. In one aspect, a stable formulation is one in which the protein contained therein substantially retains its physical and chemical stability or integrity and its therapeutic or non-therapeutic efficacy upon exposure to stress conditions. In another aspect, a stable formulation is one in which the protein contained therein substantially retains its soluble, monomeric or non-aggregated state. As used herein, a stress condition is a physical or chemical condition that adversely affects the protein in the formulation. Advantageously, the stable formulation may also provide protection against aggregation or precipitation of the dissolved proteins therein.
Examples of physical stress conditions include physical perturbations such as mechanical shear under storage conditions (whether refrigerated, room temperature or high temperature storage conditions) or exposure to other denaturing conditions, contact with air/water interfaces, freeze-thaw cycling, extended storage. For example, refrigerated conditions may require storage in a refrigerator or freezer. In some examples, refrigerated conditions may require storage at temperatures of 10 ℃ or less. In further examples, the refrigerated conditions require storage at a temperature of about 2 ℃ to about 10 ℃. In other examples, refrigerated conditions require storage at a temperature of about 4 ℃. In further examples, refrigerated conditions require storage at freezing temperatures such as about-20 ℃ or less. In another example, refrigerated conditions require storage at temperatures of about-80 ℃ to about 0 ℃. Room temperature storage conditions may require storage at ambient temperatures, e.g., about 10 ℃ to about 30 ℃. High temperature storage conditions may require storage at greater than about 30 ℃. High temperature stability (e.g., at temperatures of about 30 ℃ to about 50 ℃) can be used as part of accelerated aging studies to predict long term storage under typical ambient (10-30 ℃) conditions. Stress conditions may also include chemical perturbations, such as pH changes, which may affect the stability or integrity of the protein in the formulation by, for example, 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 translational mobility of the entangled chains and interfere with the therapeutic or non-therapeutic efficacy of the protein. In embodiments, an excipient compound as disclosed herein can inhibit protein clustering due to specific interactions between the excipient compound and a target protein in solution. Excipient compounds as disclosed herein may be natural or synthetic and desirably are generally recognized as safe ("GRAS") by the FDA.
1. Definition of
For the purposes of this disclosure, the term "protein" refers to an amino acid sequence having a chain length long enough to produce a discrete tertiary structure, typically having a molecular weight between 1-3000 kDa. In some embodiments, the protein has a molecular weight between about 50-200 kDa; in other embodiments, the molecular weight of the protein is between about 20-1000kDa or between about 20-2000 kDa. In contrast to the term "protein", the term "peptide" refers to an amino acid sequence that does not have a discrete tertiary structure. A wide variety of biopolymers are included within the scope of the term "protein". For example, the term "protein" may refer to therapeutic or non-therapeutic proteins, including antibodies, aptamers, fusion proteins, pegylated proteins, synthetic polypeptides, protein fragments, lipoproteins, enzymes, structural peptides, and the like.
a. Therapeutic biopolymers and their related definitions
Biopolymers with a therapeutic effect, including proteins, may be referred to as "therapeutic biopolymers". Proteins having a therapeutic effect may be referred to as "therapeutic proteins".
As non-limiting examples, therapeutic proteins may include mammalian proteins such as hormones and prohormones (e.g., insulin and proinsulin, glucagon, calcitonin, thyroid hormone (T3 or T4 or thyroid stimulating hormone), parathyroid hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, and the like); coagulation factors and anti-coagulation factors (e.g., tissue factor, von Willebrand's factor, factor VIIIC, factor IX, protein C, plasminogen activator (urokinase, tissue plasminogen activator), thrombin); cytokines, chemokines, and inflammatory mediators; an interferon; a colony stimulating factor; interleukins (e.g., IL-1 to IL-10); growth factors (e.g., 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 (e.g., erythropoietin, thrombopoietin, etc.); osteoinductive factors (e.g., bone morphogenic proteins); receptors (e.g., integrins; cadherins, etc.); surface membrane proteins; a transporter protein; a regulatory protein; antigenic proteins (e.g., viral components that act as antigens); and antibodies.
In certain embodiments, the therapeutic protein may be an antibody. The term "antibody" is used herein in its broadest sense to include, as non-limiting examples, monoclonal antibodies (including, e.g., full-length antibodies having an immunoglobulin Fc region), single chain molecules, bispecific and multispecific antibodies, diabodies, antibody-drug conjugates, antibody compositions having polyepitopic specificity, polyclonal antibodies (such as polyclonal immunoglobulins used as therapy for immunocompromised patients), and antibody fragments (including, e.g., 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", being biologically important materials; administration of a therapeutically effective amount of the antibody to a patient can complex with the antigen, thereby altering its biological properties such that the patient experiences a therapeutic effect.
In embodiments, the antibody may be an antibody-drug conjugate (ADC). Antibody-drug conjugates are a class of therapeutic proteins that combine the highly specific targeting ability of antibodies with therapeutically active compounds (such as cytotoxic compounds): ADCs consist of an antibody linked to a therapeutically active agent by a biodegradable chemical linker. In more detail, the ADC may comprise a human or humanized mAb specific for an antigen expressed on abnormal "target" cells but with little or no expression on normal cells. The ADC further comprises an effective agent, such as a cytotoxic agent that can destroy a target cell; such agents are generally toxic to the whole body and are therefore not suitable for general systemic administration. The targeting ability of the mAb component of the ADC allows the agent to be specifically targeted to, taken up by, and act within target cells without systemic distribution. To form an ADC, a mAb is linked to an agent by an labile bond that is stable in the extracellular environment (e.g., in the venous and interstitial circulation), but which degrades when the ADC is internalized into a cell. When the linkage between the ADC and the drug degrades, the agent is released intracellularly to act on the cell. ADCs are particularly well suited for use with cytotoxic agents, especially when these compounds are too toxic to be used in therapy alone. For example, in cancer chemotherapy, certain agents selected for ADC are orders of magnitude more toxic than traditional anticancer agents. Examples include anti-microtubule agents, alkylating agents, and DNA minor groove binding agents, which may be too toxic to be successfully administered, but which can be targeted to cancer cells using the specificity of mabs that bind only to antigens expressed by cancer cells. Once the ADC is localized to the tumor and bound to the target cell antigen on the surface, the complex can be internalized into the cell as a vesicle. The internalized vesicles fuse to each other and enter the endosomal-lysosomal pathway where they encounter proteases that digest the mAb and/or linker molecule, thereby releasing the drug payload. The payload (e.g., cytotoxic agent) then passes through the lysosomal membrane into the cytoplasm and/or nucleus where it exerts its pharmacological effect (e.g., cytotoxicity). This concentrated delivery of highly potent pharmaceutical compounds maximizes their intended therapeutic effect while minimizing normal tissue exposure to these agents. Formulations comprising ADCs are suitable for intravenous or topical administration, such that the ADCs reach the target cells to be treated.
In certain embodiments, the proteins are pegylated, meaning that they comprise poly (ethylene glycol) ("PEG") and/or poly (propylene glycol) ("PPG") units. Pegylated proteins or PEG-protein conjugates have utility in therapeutic applications due to their beneficial properties such as solubility, pharmacokinetics, immunogenicity, renal clearance, and stability. Non-limiting examples of PEGylated proteins are PEGylated interferon (PEG-IFN), PEGylated anti-VEGF, PEG protein conjugate drugs, Adagen, pemetrexed (pegaspargese), PEGylated filgrastim (Pegfilgrastim), PEGylated recombinant uricase (Pegloticase), Pegvisomant (Pegvisomant), PEGylated recombinant human erythropoietin-beta (PEGylated erythropoietin-beta), and polyethylene glycol-conjugated Certolizumab (Certolizumab pegol).
Pegylated proteins can be synthesized by a variety of methods, such as the reaction of the protein with a PEG reagent having one or more reactive functional groups. Reactive functional groups on the PEG reagent can form linkages with the protein at target protein sites such as lysine, histidine, cysteine, and the N-terminus. Typical pegylation reagents have reactive functional groups such as aldehyde, maleimide or succinimide groups that are specifically reactive with targeted amino acid residues on the protein. The pegylation agent can have a PEG chain length of about 1 to about 1000 PEG and/or PPG repeat units. Other methods of pegylation include glycopegylation, in which a protein is first glycosylated and then the glycosylated residues are pegylated in a second step. Certain pegylation processes are facilitated by enzymes such as sialyltransferases and transglutaminases.
Although 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 a higher viscosity of the solution compared to the native protein, and this typically requires formulation of a pegylated protein solution at a lower concentration.
It is desirable to formulate protein therapeutics in stable, low viscosity solutions so that they can be administered to a patient with minimal injection volume. For example, Subcutaneous (SC) or Intramuscular (IM) injection of a drug typically requires a small injection volume, preferably less than 2 mL. The SC and IM routes of injection are well suited for self-administration care and are less costly and more readily available forms of treatment than Intravenous (IV) injections, which are performed under direct medical supervision only. Formulations for SC or IM injections require low solution viscosities, typically below 30cP, and preferably below 20cP, to allow the therapeutic solution to flow easily through narrow gauge needles. This combination of small injection volume and low viscosity requirements presents challenges for the use of pegylated protein therapeutics in SC or IM injection routes.
A formulation containing a therapeutically effective amount of a therapeutic protein may be referred to as a "therapeutic formulation. A therapeutic protein "included in a therapeutic formulation may also be referred to as its" protein active ingredient. "generally, a therapeutic formulation includes a therapeutically effective amount of a protein active ingredient and excipients, with or without other optional components. As used herein, the term "therapeutic" includes both treatment and prevention of an existing condition. For example, therapeutic proteins include proteins such as bevacizumab (bevacizumab), trastuzumab (trastuzumab), adalimumab (adalimumab), infliximab (infliximab), etanercept (etanercept), darbepoetin α (darbebepoetin alfa), epoetin α (epoetin alfa), cetuximab (cetuximab), filgrastim (filgrastim), and rituximab (rituximab). Other therapeutic proteins will be familiar to those of ordinary skill in the art.
"treating" includes the intent to cure, heal, alleviate, ameliorate, remedy, or otherwise favorably affect the condition, including preventing or delaying the onset of symptoms and/or alleviating or ameliorating the symptoms of the condition. Those in need of treatment include those already with the particular condition and those in whom prevention of the condition is desired. A disorder is any condition that alters the homeostatic health of a mammal, including an acute or chronic disease or a pathological condition that predisposes a mammal to an acute or chronic disease. Non-limiting examples of disorders include cancer, metabolic disorders (e.g., diabetes), allergic disorders (e.g., asthma), dermatological disorders, cardiovascular disorders, respiratory disorders, hematologic disorders, musculoskeletal disorders, inflammatory or rheumatological disorders, autoimmune disorders, gastrointestinal disorders, urinary disorders, sexual and reproductive disorders, neurological disorders, and the like. For therapeutic purposes, the term "mammal" may refer to any animal classified as a mammal, including humans, domestic animals, pet animals, farm animals, sport animals, work animals, and the like. Thus, "treatment" may include both veterinary and human treatment. For convenience, a mammal undergoing such "treatment" may be referred to as a "patient. "in certain embodiments, the patient may be of any age, including a fetal animal in utero.
In embodiments, the treatment involves providing a therapeutically effective amount of a therapeutic formulation to a mammal in need thereof. A "therapeutically effective amount" is at least the minimum concentration of a therapeutic protein administered to a mammal in need thereof to effect treatment of an existing condition or prevention of an intended condition (the treatment or the prevention being a "therapeutic intervention"). Therapeutically effective amounts of various therapeutic proteins that may be included as active ingredients in therapeutic formulations may be familiar to the art; alternatively, for therapeutic proteins found or hereinafter employed in therapeutic interventions, a therapeutically effective amount can be determined by standard techniques performed by those of ordinary skill in the art using only routine experimentation.
b. Non-therapeutic biopolymers and their related definitions
Those proteins that are used for non-therapeutic purposes (i.e., purposes not related to therapy), such as home, nutritional, commercial, and industrial applications, may be referred to as "non-therapeutic proteins". A "non-therapeutic protein-containing formulation" may be referred to as a "non-therapeutic formulation". The non-therapeutic protein may be derived from plant sources, animal sources, or produced by cell culture; they may also be enzymes or structural proteins. Non-therapeutic proteins are useful in domestic, nutritional, commercial, and industrial applications, such as catalysts, human and animal nutrition, processing aids, detergents, and waste treatment.
An important class of non-therapeutic biopolymers are enzymes. Enzymes have many non-therapeutic applications, for example as catalysts, human and animal nutritional ingredients, processing aids, detergents and waste treatment agents. Enzyme catalysts are used to accelerate various chemical reactions. Examples of enzyme catalysts for non-therapeutic use include catalase, oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase. Human and animal nutritional uses of enzymes include nutraceuticals, nutritional sources of protein, sequestration or controlled delivery of micronutrients, digestive aids, and supplements; these may be derived from amylases, proteases, trypsin, lactase, and the like. Enzyme processing aids are used to improve the production of food and beverage products in operations (baking, brewing, fermentation, juice processing and brewing). Examples of such food and beverage processing aids include amylases, cellulases, pectinases, glucanases, lipases, and lactases. Enzymes may also be used in the production of biofuels. For example, ethanol for biofuels can be assisted by enzymatic degradation of biomass feedstocks (such as cellulosic and lignocellulosic materials). Treatment of the feedstock material with cellulase and ligninase converts biomass into a substrate that can be fermented into fuel. In other commercial applications, enzymes are used as detergents, cleaners and dye lifting aids (stain lifting aids) for laundry, dishwashing, surface cleaning and device cleaning applications. Typical enzymes used 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 textile softening with cellulase enzymes, leather processing, waste treatment, treatment of contaminated precipitates, water treatment, pulp bleaching and pulp softening and debonding. Typical enzymes used for these purposes are amylases, xylanases, cellulases and ligninases.
Other examples of non-therapeutic biopolymers include fibrous or structural proteins such as keratin, collagen, gelatin, elastin, fibroin, actin, tubulin or hydrolyzed, degraded or derivatized forms thereof. These materials are used in the preparation and formulation of food ingredients such as gelatin, ice cream, yogurt and candy; they are also added to food as thickeners, rheology modifiers, mouthfeel improving agents and as a source of nutritional proteins. Collagen, elastin, keratin and hydrolyzed keratin are widely used as ingredients in skin care and hair care formulations in the cosmetic and personal care industries. Other examples of non-therapeutic biopolymers are whey proteins such as beta-lactoglobulin, alpha-lactalbumin and serum albumin. These whey proteins are produced on a large scale as a by-product from dairy operations and have been used in a variety of non-therapeutic applications.
2. Measuring
In embodiments, the protein-containing formulations described herein tolerate monomer loss as measured by Size Exclusion Chromatography (SEC) analysis. In the SEC analysis used herein, the major analyte peak is typically associated with the protein of interest contained in the formulation, and the major peak of the protein is referred to as the monomeric peak. The monomeric peak represents the amount of the protein of interest (e.g., protein active ingredient) in a monomeric state rather than in an aggregated (dimeric, trimeric, oligomeric, 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 protein of interest. Thus, the stability of a protein-containing formulation can be observed by the relative amounts of monomers over a period of time. Thus, the improvement in stability of the protein-containing formulations of the invention can be measured as a higher percentage of monomer after a period of time compared to the percentage of monomer of a control formulation that does not include an excipient.
In embodiments, the desired stability result is a peak having 98 to 100% monomer as determined by SEC analysis. In embodiments, the improvement in stability of a protein-containing formulation of the invention may be measured as a higher percentage of monomer under stress exposure compared to the percentage of monomer of a control formulation that does not include excipients and is exposed to the same stress conditions. In embodiments, the stress condition may be low temperature storage, high temperature storage, exposure to air, exposure to light, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.
In embodiments, the protein-containing formulations described herein are resistant to an increase in protein particle size as measured by Dynamic Light Scattering (DLS) analysis. In the DLS analysis used herein, the particle size of the protein in the protein-containing formulation can be observed as the median particle size. Ideally, the median particle size of the protein of interest should be relatively invariant when subjected to DLS analysis, since the particle size represents the active component in the monomeric state rather than in the aggregated (dimeric, trimeric, oligomeric, etc.) or fragmented state. An increase in median particle size may be indicative of aggregated protein. Thus, the stability of the protein-containing formulation can be observed by the relative change in median particle size over time.
In embodiments, the protein-containing formulations described herein are tolerant to forming a polydisperse particle size distribution as measured by DLS analysis. In embodiments, the protein-containing formulation may comprise a monodisperse particle size distribution of colloidal protein particles. In embodiments, a desirable stability result is a median particle size that varies by less than 10% from the initial median particle size of the formulation. In embodiments, the improvement in stability of the protein-containing formulation of the invention may be measured as a lower percentage change in median particle size over time as compared to the median particle size in a control formulation that does not include an excipient. In embodiments, the improvement in stability of the protein-containing formulation of the invention may be measured as a lower percent change in median particle size under stress exposure than the percent change in median particle size in a control formulation not including an excipient and exposed to the same stress conditions. In embodiments, the stress condition may be low temperature storage, high temperature storage, exposure to air, exposure to light, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles. In embodiments, the improvement in stability of the protein containing formulation therapeutic formulation of the present invention may be measured as a particle size distribution of less polydispersity as measured by DLS compared to the polydispersity of the particle size distribution in a control formulation that does not include excipients and is exposed to the same stress conditions.
In embodiments, the protein-containing formulations disclosed herein are resistant to particle formation, denaturation, or precipitation as measured by turbidity, light scattering, and/or particle count analysis. Lower values generally indicate a lower number of suspended particles in the formulation in turbidity, light scattering and/or particle count assays. An increase in turbidity, light scattering or particle count may indicate that the solution of the protein of interest is not stable. Thus, the stability of the protein-containing formulation can be observed by relative turbidity, light scattering or particle count over time. In embodiments, the desired stability results are of low and relatively constant turbidity, light scattering or particle count values. In embodiments, the improvement in stability of a protein-containing formulation described herein can be measured as lower turbidity, lower light scattering, or lower particle count over a period of time as compared to the turbidity, light scattering, or particle count value in a control formulation without excipients. In embodiments, the improvement in stability of a protein-containing formulation described herein can be measured as lower turbidity, lower light scattering, or lower particle count under stress exposure compared to turbidity, light scattering, or particle count in a control formulation without an excipient and exposed to the same stress conditions. In embodiments, the stress condition may be low temperature storage, high temperature storage, exposure to air, exposure to light, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles. In embodiments, the protein-containing formulations disclosed herein retain a higher percentage of biological activity compared to a control formulation. The biological activity can be observed by binding assays or by therapeutic effects in mammals.
3. Therapeutic formulations
In one aspect, the formulations and methods disclosed herein provide stable liquid formulations with improved or reduced viscosity comprising a therapeutically effective amount of a therapeutic protein and an excipient compound. In embodiments, the formulations may improve stability while providing acceptable active ingredient concentrations and acceptable viscosities. In embodiments, the formulations provide an improvement in stability when compared to a control formulation; for the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is identical in all respects to the therapeutic formulation on a dry weight basis except for the absence of an excipient compound. In embodiments, the formulations provide improved stability under stress conditions, long term storage, high temperatures (such as 25-45 ℃), freeze/thaw conditions, shear or mixing, syringe injection (syringing), dilution, bubble exposure, oxygen exposure, light exposure, and lyophilization. In embodiments, the protein-containing formulation with improved stability is in the form of a lower percentage of soluble aggregates, particulates, sub-visible particles, or gel formation than a control formulation.
It is understood that the viscosity of the liquid protein formulation may be affected by a variety of factors including, but not limited to: the nature of the protein itself (e.g., enzyme, antibody, receptor, fusion protein, etc.); its size, three-dimensional structure, chemical composition and molecular weight; its concentration in the formulation; a component of the formulation other than a protein; the desired pH range; storage conditions of the formulation; and methods of administering the formulations to patients. The therapeutic proteins most suitable for use with the excipient compounds described herein are preferably substantially pure, i.e., free of contaminating proteins. In embodiments, a "substantially pure" therapeutic protein is a protein composition that includes at least 90 wt.%, or preferably at least 95 wt.%, or more preferably at least 99 wt.% of the therapeutic protein, all based on the total weight of the therapeutic protein and contaminating protein in the composition. For purposes of clarity, protein added as an excipient is not intended to be included in this definition. The therapeutic formulations described herein are intended for use as pharmaceutical grade formulations, i.e., formulations intended for use in the treatment of mammals, in a form such that the desired therapeutic efficacy of the protein active ingredient is achieved, and which is free of components which are toxic to the mammal to which the formulation is to be administered.
In an embodiment, the therapeutic formulation contains at least 25mg/mL of the protein active ingredient. In other embodiments, the therapeutic formulation contains at least 100mg/mL of the protein active ingredient. In an embodiment, the therapeutic formulation contains at least 10mg/mL of the protein active ingredient. In other embodiments, the therapeutic formulation contains at least 50mg/mL of the protein active ingredient. In other embodiments, the therapeutic formulation contains at least 200mg/mL of the protein active ingredient. In other embodiments, the therapeutic formulation solution contains at least 300mg/mL of the protein active ingredient. Typically, the excipient compounds disclosed herein are added to the therapeutic formulation in an amount between about 5 and 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, the excipient may be added in an amount of about 25 to about 75 mg/mL.
When combined with the protein active ingredient in the formulation, excipient compounds having various molecular weights are selected for specific advantageous properties. Examples of therapeutic formulations that include excipient compounds are provided below. In embodiments, the excipient compound has a molecular weight <5000 Da. In embodiments, the excipient compound has a molecular weight <1000 Da. In embodiments, the excipient compound has a molecular weight <500 Da.
In embodiments, the excipient compounds disclosed herein are added to the 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 the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is identical in all respects to the therapeutic formulation on a dry weight basis except for the absence of an excipient compound. In embodiments, the viscosity reducing amount is an 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 an 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 an 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 an amount of excipient compound that reduces the viscosity of the formulation by at least 90% when compared to a control formulation.
In embodiments, the amount reduced in viscosity produces a therapeutic formulation having a viscosity of less than 100 cP. In other embodiments, the viscosity of the therapeutic formulation is less than 50 cP. In other embodiments, the viscosity of the therapeutic formulation is less than 20 cP. In other embodiments, the viscosity of the therapeutic formulation is less than 10 cP. The term "viscosity" as used herein refers to the dynamic viscosity value when measured by the methods disclosed 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 more efficiently processed, purified, stored, injected, administered, filtered, and centrifuged as compared to the control formulation.
In embodiments, the therapeutic formulation is administered to the patient at a high concentration of the therapeutic protein. In embodiments, the therapeutic formulation is administered to a patient in a smaller injection volume and/or the patient has less discomfort that will be experienced as compared to administration of a similar formulation lacking the therapeutic excipient. In embodiments, the therapeutic formulation is administered to the patient with a narrower gauge needle or less syringe force than would be used to administer a similar formulation lacking the therapeutic excipient. In embodiments, the therapeutic formulation is administered as a depot injection. In embodiments, the therapeutic formulation extends the half-life of the therapeutic protein in vivo.
These features of the therapeutic formulations as disclosed herein will allow the formulations to be administered by intramuscular or subcutaneous injection in a clinical setting, namely the following: wherein the patient receiving an intramuscular injection will comprise the use of a small bore needle typically used for IM/SC purposes and the use of a tolerable (e.g., 2-3mL) injection volume, and wherein the conditions are such that an effective amount of the formulation is administered in a single injection at the site of the single injection. In contrast, injection of comparable doses of therapeutic proteins using conventional formulation techniques will be limited by the higher viscosity of conventional formulations, such that SC/IM injection of conventional formulations will 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 more efficiently processed, purified, stored, injected, administered, filtered, and/or centrifuged as compared to the control formulation.
In embodiments, the therapeutic formulations disclosed herein tolerate monomer loss as measured by Size Exclusion Chromatography (SEC) analysis. In SEC analysis, the major analyte peak is typically associated with an active ingredient of a formulation (such as a therapeutic protein), and the major peak of the active ingredient is referred to as the monomeric peak. The monomer peak indicates the amount of active component in a monomeric state, not in an aggregated state (dimeric, trimeric, oligomeric, etc.). A high concentration therapeutic protein solution formulated with the 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 over a period of time. In embodiments, the improvement in stability of a therapeutic formulation disclosed herein can be measured as a higher percentage of monomer after a period of time as compared to the percentage of monomer of a control formulation that does not include an excipient. In embodiments, the improvement in stability of a therapeutic formulation disclosed herein can be measured as a higher percentage monomer under stress exposure compared to the percentage monomer of a control formulation that does not include an excipient and is exposed to the stress condition. In embodiments, the stress condition may be low temperature storage, high temperature storage, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.
In embodiments, the therapeutic formulations of the present invention tolerate an increase in protein particle size as measured by Dynamic Light Scattering (DLS) analysis. In DLS analysis, the particle size of the therapeutic formulation can be observed as the median particle size. Ideally, the median particle size of the therapeutic protein should be relatively constant. Thus, an increase in median particle size may be indicative of aggregated protein. Thus, the stability of the therapeutic formulation can be observed by the relative change in median particle size over time. In embodiments, the therapeutic formulations disclosed herein are resistant to forming a polydisperse particle size distribution as measured by Dynamic Light Scattering (DLS) analysis. In embodiments, the improvement in stability of the therapeutic formulation of the present invention may be measured as a lower percentage change in median particle size over time as compared to the median particle size in a control formulation that does not include an excipient. In embodiments, the improvement in stability of the therapeutic formulations disclosed herein can be measured as a lower percent change in median particle size under exposure to stress conditions as compared to the percent change in median particle size in a control formulation that does not include an excipient. In other words, in embodiments, improved stability prevents an increase in particle size as measured by light scattering. In embodiments, the stress condition may be low temperature storage, high temperature storage, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles. In embodiments, the improvement in stability of the therapeutic formulations disclosed herein can be measured as a particle size distribution of less polydispersity as measured by DLS compared to the polydispersity of the particle size distribution in a control formulation that does not include an excipient.
In embodiments, the therapeutic formulations disclosed herein are resistant to precipitation as measured by turbidity, light scattering or particle counting analysis. In embodiments, the improvement in stability of a therapeutic formulation disclosed herein can be measured as lower turbidity, lower light scattering, or lower particle count over a period of time as compared to turbidity, light scattering, or particle count in a control formulation without an excipient. In embodiments, the improvement in stability of a therapeutic formulation disclosed herein can be measured as lower turbidity, lower light scattering, or lower particle count under exposure to stress conditions as compared to turbidity, light scattering, or particle count in a control formulation without an excipient and exposed to the same stress conditions. In embodiments, the stress condition may be low temperature storage, high temperature storage, exposure to air bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.
In embodiments, the therapeutic excipient has antioxidant properties that stabilize the therapeutic protein against oxidative damage, thereby improving its stability. In embodiments, the therapeutic formulation is stored at ambient temperature, or stored under refrigerator conditions for extended periods of time without significant loss of efficacy of the therapeutic protein. In embodiments, the therapeutic formulation is stored dry until it is needed; it is then reconstituted with a suitable solvent, such as water. Advantageously, formulations prepared as described herein can be stable over extended periods of time, months to years. When a particularly long shelf life is required, the formulation can be stored in a freezer (and later reactivated) without fear of protein denaturation. In embodiments, the formulations can be prepared for long-term storage without refrigeration.
In embodiments, the excipient compounds disclosed herein are added to the therapeutic formulation in an amount that improves stability. In embodiments, the stability-improving amount is an amount of an excipient compound that reduces degradation of the formulation by at least 10% when compared to a control formulation; for the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar in all respects to the therapeutic formulation on a dry weight basis, except for the absence of an excipient compound. In embodiments, the stability-improving 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, the stability-improving 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 embodiments, the stability-improving amount is an amount of the excipient compound that reduces degradation of the formulation by at least 70% when compared to a control formulation. In embodiments, the stability-improving 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 those skilled in the art. Therapeutic formulations of the invention may be prepared, for example, by adding an excipient compound to the formulation before or after the therapeutic protein is added to the solution. Therapeutic formulations can be produced, for example, 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 may be formulated with one or more excipient compounds having chaotropic agents, ordering agents (kosmotropes), hydrotropes and salts. Therapeutic formulations may be prepared with one or more excipient compounds using techniques such as encapsulation, dispersion, liposomes, vesicle formation, and the like. Methods for preparing therapeutic formulations comprising the excipient compounds disclosed herein may comprise combining the excipient compounds. In embodiments, the combination excipients may produce the benefits of lower viscosity, improved stability, or reduced pain at the injection site. Other additives may be introduced during the manufacture of the therapeutic formulation, including preservatives, surfactants, sugars, sucrose, trehalose, polysaccharides, arginine, proline, hyaluronidase, stabilizers, buffers, and the like. As used herein, a pharmaceutically acceptable excipient compound is one that is non-toxic and suitable for animal and/or human administration.
4. Non-therapeutic formulations
In one aspect, the formulations and methods disclosed herein provide stable liquid formulations with improved or reduced viscosity comprising an effective amount of a non-therapeutic protein and an excipient compound. In embodiments, the formulations have improved stability while providing acceptable active ingredient concentrations and acceptable viscosities. In embodiments, the formulations provide an improvement in stability when compared to a control formulation; for the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is identical in all respects to the non-therapeutic formulation on a dry weight basis, except for the absence of an excipient compound.
It is understood that the viscosity of the liquid protein formulation may be affected by a variety of factors including, but not limited to: the nature of the protein itself (e.g., enzyme, structural protein, degree of hydrolysis, etc.); its size, three-dimensional structure, chemical composition and molecular weight; its concentration in the formulation; a component of the formulation other than a protein; the desired pH range; storage conditions for the formulation.
In embodiments, the non-therapeutic formulation contains at least 25mg/mL of the protein active ingredient. In other embodiments, the non-therapeutic formulation contains at least 100mg/mL of the protein active ingredient. In other embodiments, the non-therapeutic formulation contains at least 200mg/mL of the protein active ingredient. In other embodiments, the non-therapeutic formulation solution contains at least 300mg/mL of the protein active ingredient. Typically, the excipient compounds disclosed herein are added to the non-therapeutic formulation in an amount between about 5 and about 300 mg/mL. In embodiments, the excipient compound is added in an amount of about 10 to about 200 mg/mL. In embodiments, 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.
When combined with the protein active ingredient in the formulation, excipient compounds having various molecular weights are selected for specific advantageous properties. Examples of non-therapeutic formulations that include excipient compounds are provided below. In embodiments, the excipient compound has a molecular weight <5000 Da. In embodiments, the excipient compound has a molecular weight <1000 Da. In embodiments, the excipient compound has a molecular weight <500 Da.
In embodiments, the excipient compounds disclosed herein are added to the 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 the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is identical in all respects to the therapeutic formulation on a dry weight basis except for the absence of an excipient compound. In embodiments, the viscosity reducing amount is an 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 an 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 an 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 an amount of excipient compound that reduces the viscosity of the formulation by at least 90% when compared to a control formulation.
In embodiments, the amount of viscosity reduction produces a non-therapeutic formulation having a viscosity of less than 100 cP. In other embodiments, the viscosity of the non-therapeutic formulation is less than 50 cP. In other embodiments, the viscosity of the non-therapeutic formulation is less than 20 cP. In other embodiments, the viscosity of the non-therapeutic formulation is less than 10 cP. The term "viscosity" as used herein refers to the dynamic viscosity value.
Non-therapeutic formulations according to the present disclosure 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, therapeutic formulations can be processed, purified, stored, pumped, filtered, and centrifuged more efficiently than control formulations.
In embodiments, the non-therapeutic excipient has antioxidant properties that stabilize the non-therapeutic protein against oxidative damage, thereby improving its stability. In embodiments, the non-therapeutic formulation is stored at ambient temperature, or stored under refrigerator conditions for extended periods of time without significant loss of efficacy of the non-therapeutic protein. In embodiments, the non-therapeutic formulation is stored dry until it is needed; which can then be reconstituted with a suitable solvent such as water. Advantageously, the formulations prepared as described herein are stable over extended periods of time, months to years. When a particularly long shelf life is required, the formulation is stored in a freezer (and later reactivated) without fear of protein denaturation. In embodiments, the formulation is prepared for long-term storage without refrigeration.
In embodiments, the excipient compounds disclosed herein are added to the non-therapeutic formulation in an amount that improves stability. In embodiments, the stability-improving amount is an amount of an excipient compound that reduces degradation of the formulation by at least 10% when compared to a control formulation; for the purposes of this disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar in all respects to the therapeutic formulation on a dry weight basis, except for the absence of an excipient compound. In embodiments, the stability-improving 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, the stability-improving 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 embodiments, the stability-improving amount is an amount of the excipient compound that reduces degradation of the formulation by at least 70% when compared to a control formulation. In embodiments, the stability-improving 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 the 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. The non-therapeutic agent may be produced at a first (lower) concentration and then processed by filtration or centrifugation to produce a second (higher) concentration. Non-therapeutic formulations may be formulated with one or more excipient compounds having chaotropic agents, ordering agents (kosmotropes), hydrotropes and salts. Non-therapeutic formulations may be prepared with one or more excipient compounds using techniques such as encapsulation, dispersion, liposomes, vesicle formation, and the like. Other additives may be introduced during manufacture of the non-therapeutic formulation, including preservatives, surfactants, stabilizers, and the like.
5. Excipient compounds
Described herein are several excipient compounds, each suitable for use with one or more therapeutic or non-therapeutic proteins, and each allowing a formulation to be made such that the formulation contains a high concentration of the protein. Some classes of excipient compounds described below are: (1) a hindered amine; (2) an anionic aromatic compound; (3) a functionalized amino acid; (4) oligopeptides; (5) short chain organic acids; (6) a low molecular weight aliphatic polybasic acid; (7) diketones and sulfones; (8) a zwitterionic excipient; (9) crowding agents with hydrogen bonding elements. Without being bound by theory, the excipient compounds described herein are believed to associate with certain fragments, sequences, structures, or portions of the therapeutic protein that are otherwise involved in interparticle (i.e., protein-protein) interactions. The association of these excipient compounds with therapeutic or non-therapeutic proteins can mask protein-protein interactions, allowing the proteins to be formulated in high concentrations without causing excessive solution viscosity. In embodiments, the excipient compound may result in a more stable protein-protein interaction; protein-protein interactions can be measured by the protein diffusion parameter kD or the osmotic second force coefficient (viral coefficient) B22 or by other techniques familiar to those skilled in the art.
Advantageously, the excipient compound may be water soluble and therefore suitable for use with an aqueous vehicle (vehicle). In embodiments, the excipient compound has a solubility in water of >1 mg/mL. In embodiments, the excipient compound has a water solubility of >10 mg/mL. In embodiments, the excipient compound has an aqueous solubility of >100 mg/mL. In embodiments, the excipient compound has an aqueous solubility of >500 mg/mL. In embodiments, co-solutes or hydrotropes may be added in combination with the excipient compound to increase the solubility of the excipient compound. For example, the solubility of certain excipients in aqueous solutions containing therapeutic proteins may be limited, and may be even lower under refrigerated conditions. Co-solutes or hydrotropes can be added to increase the solubility of the excipient in solution under refrigeration conditions or under room or high temperature conditions. Examples of co-solutes and hydrotropes include benzoate, benzyl alcohol, phenylalanine, nicotinamide, proline, procaine, 2, 5-dihydroxybenzoate, tyramine, and saccharin. Advantageously, for therapeutic proteins, the excipient compound may be derived from a material that is biologically acceptable and non-immunogenic and therefore suitable for pharmaceutical use. In therapeutic embodiments, the excipient compound may be metabolized in vivo to produce biocompatible and non-immunogenic byproducts.
a. Excipient Compound class 1 hindered Amines
Solutions of therapeutic or non-therapeutic proteins can be formulated with small molecules of hindered amines 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. The hindered amine may be used in the free base form, the protonated form, or a combination of both. In the protonated form, the hindered amine may be associated with an anionic counterion such as chloride, hydroxide, bromide, iodide, fluoride, acetate, formate, phosphate, sulfate, or carboxylate. Hindered amine compounds useful as excipient compounds can contain secondary, tertiary, quaternary ammonium, pyridinium, pyrrolidone, pyrrolidine, piperidine, morpholine, or guanidinium groups such that the excipient compound has a cationic charge in aqueous solution at neutral pH. The hindered amine compound also contains at least one bulky or sterically hindered group such as a cyclic aryl, cycloaliphatic, cyclohexyl, or alkyl group. In embodiments, the sterically hindered group may itself be an amine group, such as a dialkylamine, trialkylamine, guanidinium, pyridinium, or quaternary ammonium group. Without being bound by theory, hindered amine compounds are believed to associate with aromatic moieties of proteins such as phenylalanine, tryptophan, and tyrosine through cationic pi interactions. In embodiments, the cationic groups of the hindered amines may have an affinity for electron-rich pi structures of aromatic amino acid residues in proteins, such that they may shield these portions of the protein, thereby reducing the tendency of such shielded proteins to associate and aggregate.
In embodiments, the hindered amine excipient compound has a chemical structure comprising: 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, α -methylhistamine, betahistine (betahistine), β -alanine, 2-methyl-2-imidazoline, 1-butyl-3-methylimidazolium chloride, uric acid, potassium urate, betazole (betazole), carnosine, aspartame, saccharin, potassium acetamido sulfonate, xanthine, theophylline, theobromine, caffeine, and anserine. In embodiments, the hindered amine excipient compound is selected from the group consisting of: dimethylethanolamine, dimethylaminopropylamine, triethanolamine, dimethylbenzylamine, dimethylcyclohexylamine, diethylcyclohexylamine, dicyclohexylmethylamine, hexamethylenebiguanide, poly (hexamethylenebiguanide), imidazole, lysine, methylGlycine, sarcosine, dimethylglycine, agmatine, diazabicyclo [2.2.2]Octane, sodium folinate, calcium folinate, tetramethylethylenediamine, N-dimethylethanolamine, ethanolamine phosphate, glucosamine, choline chloride, choline phosphate, nicotinamide, isonicotinamide, N, n-diethylnicotinamide, nicotinic acid sodium salt, isonicotinate, tyramine, 3-aminopyridine, 2,4, 6-trimethylpyridine, 3-pyridinemethanol, nicotinamide adenine dinucleotide, biotin, morpholine, N-methylpyrrolidone, 2-pyrrolidone, dipyridamole, procaine, lidocaine, dicyandiamide-taurine adduct, 2-pyridylethylamine, 6-hydroxypyridine-2-carboxylic acid, dicyandiamide-benzylamine adduct, dicyandiamide-alkylamine adduct, dicyandiamide-cycloalkylamine adduct and dicyandiamide-aminomethylphosphonic acid adduct. In embodiments, the hindered amine excipient compound is selected from the group consisting of: 1- (1-adamantyl) ethylamine, 1-aminobenzotriazole, 2-dimethylaminoethanol, 2-methyl-2-imidazoline, 2-methylimidazole, 3-aminobenzamide, 3-indoleacetic acid, 4-aminopyridine, 6-amino-1, 3-dimethyluracil, acetylcholine, agmatine sulfate (agmatine sulfate), benzalkonium chloride, ethyl 3-aminobenzoate, sulfacetamide, butyl anthranilate, aminomauric acid, benzamidoxime, benzethonium chloride, benzylamine, berberine chloride (berberine chloride), castanospermine (castanospermine), clemizole, cycloserine, phenylserine, DL-3-phenylserine, mercaptoethylamine, cytidine, diethanolamine, diphenhydramine, DL-noradrenaline, and benzethonium, Dopamine, emtricitabine, ethanolamine, guanfacine, isonicotinamide, lithium chloride, meglumine, methylcytidine, myristyl gamma-methylpyridine chloride (mysterityl gamma picolinium chloride), niacinamide, phenylethylamine, polyethyleneimine, pyridoxine, rasagiline mesylate, serotonin, synephrine, neomycin amine, spermine, spermidine, l, 3-diaminopropane, adenosine, chloroquine phosphate, cystamine, pyridylethylamine, tetramethylethylenediamine, tryptamine, tyramine, 1-methylimidazole, spectinomycin, cyclohexanemethanamine, N-dimethylphenylethylamine, phenethylamine, tetraethylammonium, tetramethylammonium acetate, bicycloamine, hordenine, methylaminoethylpyridine, nicotinamide riboside, 1-butyltinum, spectinomycin, cyclohexylamine, N-dimethylphenylethylamine, phenethylamine, tetraethylammonium acetate, bicycloamine, hordenine, methylaminoethylpyridine, nicotinamide riboside, and 1-butylimine Methylimidazole, 1-hexylimidazole, 1-methylimidazole, 2-ethylimidazole, 2-n-butylimidazole, 2-methylimidazole, 1-dodecylimidazole, substituted in the 1-or 2-position with C1To C12Other imidazoles with alkylated hydrocarbon chains, pridinol mesylate, heme, N-dimethyl phenethylamine, voglibose, N-ethyl-L-glutamine, nicotine, piperazine, demeclocycline, and salts thereof. In embodiments, the hindered amine excipient compound may have phenylethylamine functionality such as phenylethylamine, diphenhydramine, N-methylphenylethylamine, N-dimethylphenylethylamine, β, 3-dihydroxyphenylethylamine, β, 3-dihydroxy-N-methylphenylethylamine, 3-hydroxyphenylethylamine, 4-hydroxyphenylethylamine, tyrosine, tyramine, N-methyltyramine, and hordenine. Preferably, the structure containing phenethylamine is a non-psychoactive compound.
Suitable salts of hindered amine structures may be chloride, bromide, acetate, citrate, sulfate, and phosphate. In embodiments, hindered amine compounds consistent with the present disclosure are formulated as protonated ammonium salts. In embodiments, hindered amine compounds consistent with the present disclosure are formulated as salts with inorganic or organic anions as counterions.
In embodiments, a high concentration solution of a therapeutic or non-therapeutic protein is formulated with caffeine in combination with benzoic acid, hydroxybenzoic acid or benzenesulfonic acid as an excipient compound. In embodiments, the hindered amine excipient compound is metabolized in vivo to produce biocompatible byproducts. In some embodiments, the hindered amine excipient compound is present in the formulation at a concentration of about 250mg/mL or less. In further embodiments, the hindered amine excipient compound is present in the formulation at a concentration of from about 10mg/mL to about 200 mg/mL. In other aspects, the hindered amine excipient compound is present in the formulation at a concentration of about 20 to about 120 mg/mL.
In embodiments, the viscosity reducing excipients in this class of hindered amines may include methylxanthines such as caffeine and theophylline, although their use is generally limited due to their low water solubility. In some applications, higher concentration solutions of these viscosity-reducing excipients may be advantageous despite their low aqueous solubility. For example, in processing, it is advantageous to have a concentrated excipient solution that can be added to a concentrated protein solution, such that the addition of the excipient does not dilute the protein below the desired final concentration. In other cases, despite its low water solubility, additional viscosity reducing excipients may be required to achieve the viscosity reduction, stability, tonicity, etc., required for the final protein formulation. In embodiments, a high concentration excipient solution may be formulated as (i) a viscosity reducing excipient at a concentration 1.5 to 50 times higher than the effective viscosity reducing amount, or (ii) a viscosity reducing excipient at a concentration 1.5 to 50 times higher than its literature reported solubility in pure water at 298K (e.g., as reported in the merck index; the royal chemical society; 15 th edition, (2013, 4, 30), or both.
It has been found that certain co-solutes substantially increase the solubility limit of these low solubility viscosity reducing excipients, such that the concentration of the excipient solution is several times higher than the solubility values reported in the literature. These co-solutes can be classified under the general class of hydrotropes. Co-solutes that have been found to provide the greatest solubility improvement for this application are typically highly soluble (>0.25M) in water at ambient temperature and physiological pH and contain pyridine or benzene rings. Examples of compounds that can be used as co-solutes include aniline HCl, isonicotinamide, nicotinamide, n-methyltyramine HCl, phenol, procaine HCl, resorcinol, saccharin calcium salts, saccharin sodium salts, sodium aminobenzoate, sodium benzoate, sodium p-hydroxybenzoate, sodium m-hydroxybenzoate, sodium 2, 5-dihydroxybenzoate, sodium salicylate, sodium sulfadiazine, sodium p-hydroxybenzoate, synephrine, and tyramine HCl.
In embodiments, certain hindered amine excipient compounds may have other pharmacological properties. As an example, xanthines are a class of hindered amines that generally have independent pharmacological properties including stimulatory properties upon systemic absorption and bronchodilator properties. Representative xanthines include caffeine, aminophylline, 3-isobutyl-1-methylxanthine, accessory xanthines, pentoxifylline, theobromine, theophylline, and the like. Methylated xanthines are thought to affect cardiac contractility, heart rate, and bronchiectasis. In some embodiments, the xanthine excipient compound is present in the formulation at a concentration of about 30mg/mL or less.
Another class of hindered amines with independent pharmacological properties are injectable local anesthetic compounds. Injectable local 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. Such hindered amines are believed to interrupt nerve conduction by inhibiting the influx of sodium ions, thereby inducing local anesthesia. The lipophilic aromatic ring of the local anesthetic compound may be formed from carbon atoms (e.g., benzene ring), or it may include heteroatoms (e.g., thiophene ring). Representative local anesthetic compounds that can be injected include, but are not limited to, amethocaine, articaine, bupivacaine, butacaine, chloroprocaine, cocaine, cyclomethicaine (cyclomethicaine), dicaine, etidocaine (etidocaine), hecaine (hexylcaine), isobucaine (isobucaine), levobupivacaine (levobupivacaine), lidocaine, metabutamine (methabutethamine), mepivacaine (methabutoxycaine), carbocaine (mepivacaine), mepivacaine, propoxycaine, prilocaine, procaine, perocaine, tetracaine, trimecaine, and the like. Injectable local anesthetic compounds can have multiple benefits in protein therapeutic formulations, such as reduced viscosity, improved stability, and reduced pain after injection. In some embodiments, the local anesthetic compound is present in the formulation at a concentration of about 50mg/mL or less.
In embodiments, hindered amines having independent pharmacological properties are used as excipient compounds for formulations and methods according to the present disclosure. In some embodiments, excipient compounds having independent pharmacological properties are present in an amount that is pharmacologically ineffective and/or non-therapeutically effective. In other embodiments, excipient compounds having independent pharmacological properties are present in an amount that does have a pharmacological effect and/or is therapeutically effective. In certain embodiments, hindered amines having independent pharmacological properties are used in combination with another excipient compound that has been selected to reduce the viscosity of the formulation, wherein hindered amines having independent pharmacological properties are used to impart a pharmacological activity benefit thereto. For example, injectable local anesthetic compounds can be used to reduce formulation viscosity and also to reduce pain after injection of the formulation. Reduction in injection pain can be caused by anesthetic properties; lower injection forces may also be required when the viscosity is reduced by excipients. Alternatively, injectable local anesthetic compounds can be used to impart a desired pharmacological benefit of reduced local perception during injection of the formulation when combined with another excipient compound that reduces the viscosity of the formulation.
b. Excipient compound class 2: anionic aromatic compounds
Solutions of therapeutic or non-therapeutic proteins may be formulated with anionic aromatic small molecule compounds as excipient compounds. The anionic aromatic excipient compound may contain aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, phenolic, hydroxyaryl, heteroaryl, or fused aryl groups. The anionic aromatic excipient compound may also contain anionic functional groups such as carboxylate, oxide, phenoxide, sulfonate, sulfate, phosphonate, phosphate or sulfide. Although the anionic aromatic excipients may be described as acids, sodium salts or otherwise, it is understood that the excipients may be used in a variety of salt forms. Without being bound by theory, anionic aromatic excipient compounds are believed to be bulky, sterically hindered molecules that can associate with cationic segments of proteins so that they can shield these portions of the protein, thereby reducing interactions between protein molecules that make protein-containing formulations viscous or cause stability problems.
In embodiments, examples of anionic aromatic excipient compounds include compounds such as: salicylic acid, aminosalicylic acid, hydroxybenzoic acid, aminobenzoic acid, p-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 (hydroquinone sulfonic acid), sulfanilic acid, vanillic acid, homovanillic acid, vanillin-taurine adduct, aminophenol, anthranilic acid, cinnamic acid, menadione sodium bisulfite, 4-hydroxy-3-methoxycinnamic acid, caffeic acid, chlorogenic acid, gentisic acid, coumaric acid, adenosine monophosphate, indoleacetic acid, potassium urate, furandicarboxylic acid, furan-2-acrylic acid, 2-furanpropionic acid, sodium phenylpropionate, sodium hydroxyphenylpyruvate, trimethoxybenzoic acid, dihydroxybenzoic acid, dihydrobenzoic acid, dihydrogenbutyric acid, cinnamic acid, indolylamine, indolacetic acid, potassium urate, fur, Ferrocenecarboxylic acid, trihydroxybenzoic acid, pyrogallol, benzoic acid and salts of the above acids. In embodiments, the anionic aromatic excipient compound is formulated in the form of an ionized salt. In embodiments, the anionic aromatic compound is formulated as a salt of a hindered amine, such as dimethylcyclohexylammonium hydroxybenzoate. In embodiments, anionic aromatic excipient compounds are formulated with various counterions such as organic cations. In embodiments, a high concentration solution of a therapeutic or non-therapeutic protein is formulated with an anionic aromatic excipient compound and caffeine. In embodiments, the anionic aromatic excipient compound is metabolized in vivo to produce biocompatible byproducts.
In embodiments, examples of aromatic excipient compounds include phenols and polyphenols. As used herein, the term "phenol" refers to an organic molecule consisting of at least one aromatic group or fused aromatic group bonded to at least one hydroxyl group, and the term "polyphenol" refers to an organic molecule consisting of more than one phenol group. Such excipients may be advantageous in certain situations, for example when used in formulations having a high concentration of a therapeutic or non-therapeutic pegylated protein to reduce the viscosity of the solution. Non-limiting examples of phenols include resorcinol (1, 3-benzenediol), catechol (1, 2-benzenediol) and hydroquinone (1, 4-benzenediol), benzenetriol trimellitic acid (1,2, 4-benzenetriol), pyrogallol (1,2, 3-benzenetriol) and phloroglucinol (1,3, 5-benzenetriol), pyromellitic acid 1,2,3, 4-benzenetetraol and 1,2,3, 5-benzenetetraol, and benzenepentanol and benzenehexanol. Non-limiting examples of polyphenols include tannic acid, ellagic acid, epigallocatechin gallate, catechin, tannin, ellagitannin, and gallotannin. 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 isoflavones. Phenolic acids include, but are not limited to, hydroxybenzoic acid, hydroxycinnamic acid, hydroxyphenylacetic acid, hydroxyphenylpropionic acid, and hydroxyphenylvaleric acid. Other polyphenolic compounds include, but are not limited to, alkyl methoxyphenols, alkyl phenols, curcumin, hydroxybenzaldehydes, hydroxybenzophenones, hydroxycinnamals, hydroxycoumarins, hydroxyphenylpropenes, methoxyphenols, naphthoquinones, hydroquinones, phenolic terpenes, resveratrol and tyrosols. In embodiments, the polyphenol is tannic acid. In embodiments, the phenol is gallic acid. In embodiments, the phenol is pyrogallol. In embodiments, the phenol is resorcinol. Without being bound by theory, 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, thus forming a phenol/PEG complex that fundamentally alters the structure of the PEG solution, thereby reducing the solution viscosity. Polyphenol compounds (such as tannins) acquire viscosity reducing properties from their respective phenolic building blocks (such as gallic acid, pyrogallol and resorcinol). The specific organization of the phenolic groups in the polyphenolic compounds can lead to complex behavior, wherein the viscosity reduction obtained by adding phenols is enhanced by adding smaller amounts of the corresponding polyphenols.
c. Excipient compound class 3: functionalized amino acids
Solutions of therapeutic or non-therapeutic proteins may 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 may be used as an excipient compound. In embodiments, functionalized amino acid compounds include molecules that can be hydrolyzed or metabolized to produce an amino acid ("amino acid precursors"). In embodiments, the functionalized amino acid may contain an aromatic functional group, such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaryl, or fused aryl. In embodiments, the functionalized amino acid compounds may contain esterified amino acids such as methyl esters, ethyl esters, propyl esters, butyl esters, benzyl esters, cycloalkyl esters, glyceryl esters, hydroxyethyl esters, hydroxypropyl esters, PEG esters, and PPG esters. In embodiments, the functionalized amino acid compound is selected from the group consisting of: arginine ethyl ester, arginine methyl ester, arginine hydroxyethyl ester, and arginine hydroxypropyl ester. In embodiments, the functionalized amino acid compound is a charged ionic compound in an aqueous solution at neutral pH. For example, a single amino acid may be derivatized by forming an ester (e.g., an acetate or benzoate), and the hydrolysis product will be acetic acid or benzoic acid, both of natural materials, plus the amino acid. In embodiments, the functionalized amino acid excipient compound is metabolized in vivo to produce a biocompatible byproduct.
d. Excipient compound class 4: oligopeptides
Solutions of therapeutic or non-therapeutic proteins may be formulated with the oligopeptides as excipient compounds. In embodiments, the oligopeptide is designed such that the structure has a charged portion and a bulky portion. In embodiments, the oligopeptide consists of 2 to 10 peptide subunits. The oligopeptide may be bifunctional, e.g. a cationic amino acid coupled to a non-polar amino acid, or an anionic amino acid coupled to a non-polar amino acid. In embodiments, the oligopeptide is composed of 2 to 5 peptide subunits. In embodiments, the oligopeptide is a homologous peptide, such as polyglutamic acid, polyaspartic acid, polylysine, polyarginine, and polyhistidine. In embodiments, the oligopeptide has a net cationic charge. In other embodiments, the oligopeptide is a heteropeptide, such as Trp2Lys 3. In embodiments, the oligopeptides may have an alternating structure, such as an ABA repeating pattern. In embodiments, the oligopeptide may contain anionic amino acids and cationic amino acids, such as Arg-Glu, Lys-Glu, His-Glu, Arg-Asp, Lys-Asp, His-Asp, Glu-Arg, Glu-Lys, Glu-His, Asp-Arg, Asp-Lys, and Asp-His. Without being bound by theory, oligopeptides include structures that associate with proteins in such a way that intermolecular interactions that lead to high viscosity solutions and stability problems can be reduced; for example, oligopeptide-protein associations can be charge-charge interactions, leaving slightly non-polar amino acids to break hydrogen bonds of the hydration layer around the protein, thereby reducing viscosity or improving stability. In some embodiments, the oligopeptide excipient is present in the composition at a concentration of about 50mg/mL or less.
e. Excipient compound class 5: short chain organic acids
Solutions of therapeutic or non-therapeutic proteins can be formulated with short chain organic acids as excipient compounds. As used herein, the term "short chain organic acid" refers to C2-C6An organic acid compound and a salt, ester, amide or lactone thereof. This category includes saturated and unsaturated carboxylic acids, hydroxy-functionalized carboxylic acids, amides, and straight chain, branched chain, or cyclic carboxylic acids. In embodiments, the acid group in the short chain organic acid is a carboxylic acid, sulfonic acid, phosphonic acid, or salts thereof.
Solutions of therapeutic or non-therapeutic proteins can be formulated with short chain organic acids (e.g., acid or salt forms of sorbic, valeric, propionic, caproic, and ascorbic acids) as excipient compounds. Examples of excipient compounds in this class include potassium sorbate, calcium gluconate, glucuronic acid, calcium lactate, 2-hydroxy lactic acid, sodium glycolate, potassium glycolate, ammonium glycolate, sodium valproate, taurine, acetohydroxamic acid, acetone sodium bisulfite adduct, acetylhydroxyproline, calcium propionate, magnesium propionate, sodium ascorbate, and salts thereof.
f. Excipient compound class 6: low molecular weight polybasic acid
Solutions of therapeutic or non-therapeutic proteins or pegylated proteins may be formulated with certain excipient compounds capable of reducing the viscosity or improving the stability of the solution, wherein the excipient compound is a low molecular weight polyacid. The low molecular weight polyacid may include an organic polyacid or an inorganic polyacid. These low molecular weight polyacid excipients may also be used in combination with other excipients.
In embodiments, the organic polyacid may be configured as a low molecular weight aliphatic polyacid. As used herein, the term "low molecular weight aliphatic polybasic acid" refers to an organic aliphatic polybasic acid having a molecular weight of less than about 1500Da and having at least two acid groups, wherein the acid groups are considered to be proton donating moieties. Non-limiting examples of acid groups include carboxylic acid groups, phosphonic acid groups, phosphoric acid groups, sulfonic acid groups, sulfuric acid groups, nitric acid groups, and nitrous acid groups. The acid groups on the low molecular weight aliphatic polyacid may be 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 polybasic acids useful for interacting with the pegylated proteins as described herein include oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, succinic acid, maleic acid, tartaric acid, glutaric acid, malonic acid, itaconic acid, methylmalonic acid, azelaic acid, citric acid, 3,6, 9-trioxaundecanedioic acid, ethylenediaminetetraacetic acid (EDTA), aspartic acid, pyrrolidone carboxylic acid, pyroglutamic acid, glutamic acid, alendronic acid, methylenephosphoric acid, etidronic acid, and salts thereof.
In other embodiments, the low molecular weight polyacid is an inorganic polyacid. Other examples of low molecular weight polyacids in the form of anionic salts include Phosphate (PO)4 3-) Hydrogen Phosphate (HPO)4 2-) Dihydrogen phosphate (H)2PO4 -) Sulfates, Hydrogen Sulfates (HSO)4 -) Pyrophosphate (P)2O7 4-) Hexametaphosphate, borate, Carbonate (CO)3 2-) And bicarbonate (HCO)3 -). The counter ion of the anion salt may be Na, Li, K or ammonium ion.
In embodiments, the low molecular weight aliphatic polybasic acid may also be an alpha-hydroxy acid in which a hydroxyl group adjacent to the first acid group is present, such as glycolic, lactic and gluconic acids and salts thereof. In embodiments, the low molecular weight aliphatic polybasic acids are in oligomeric form with more than two acid groups, such as polyacrylic acids, polyphosphates, polypeptides and salts thereof. In some embodiments, the low molecular weight aliphatic polyacid excipient is present in the composition at a concentration of about 50mg/mL or less.
g. Excipient compound class 7: diketones and sulfones
An effective viscosity reducing or stabilizing excipient may be a molecule containing sulfone, sulfonamide, or diketone functionality that is soluble in pure water at 298K at least 1g/L and has a net neutral charge at pH 7. Preferably, the molecular weight of the molecule is less than 1000g/mol, more preferably less than 500 g/mol. Diketones and sulfones effective in reducing viscosity and/or improving stability have multiple double bonds, are soluble in water, have no net or anionic charge at pH7, and are not strong hydrogen bond donors. Without being bound by theory, the double bond nature may allow for weak pi-stacking interactions with proteins. In embodiments, charged excipients are ineffective at high protein concentrations and in proteins that produce only high viscosities at high concentrations because electrostatic interactions are longer range interactions. The surfaces of the solvated proteins are predominantly hydrophilic, making them water soluble. Hydrophobic regions of proteins are typically shielded within a three-dimensional structure, but the structure is constantly evolving, unfolding and refolding (sometimes referred to as "respiration"), and hydrophobic regions of adjacent proteins may come into contact with each other, leading to aggregation through hydrophobic interactions. The pi-stacking properties of the diketone and sulfone excipients may mask hydrophobic patches (hydrophic patches) that may be exposed during this "breathing" process. Another important role of excipients can be to disrupt hydrophobic interactions and hydrogen bonding between adjacent proteins, effectively reducing solution viscosity. Diketones and sulfone compounds suitable for this description include dimethyl sulfone, ethyl methyl sulfonyl acetate, ethyl isopropyl sulfone, sodium cyclamate, bis (methylsulfonyl) methane, methane sulfonamide, methionine sulfone, 1, 2-cyclopentanedione, 1, 3-cyclopentanedione, 1, 4-cyclopentanedione, and butane-2, 3-dione.
h. Excipient compound class 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 a compound having a cationically charged moiety and an anionically charged moiety. In embodiments, the zwitterionic excipient compound is an amine oxide. In embodiments, the opposite charges are separated from each other by 2-8 chemical bonds. In embodiments, the zwitterionic excipient compound may be a small molecule, such as a molecule having a molecular weight of about 50 to about 500g/mol, or may be a medium molecular weight molecule, such as a molecule having a molecular weight of about 500 to about 2000g/mol, or 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 include (3-carboxypropyl) trimethylammonium chloride, 1-aminocyclohexanecarboxylic acid, homocyclic leucine, 1-methyl-4-imidazoleacetic acid, 3- (1-pyridyl) -1-propanesulfonate, 4-aminobenzoic acid, alendronate, aminoethanesulfonic acid, aminomauric acid, aspartame, aminotri (methylenephosphonic Acid) (ATMP), cobutrol calcium (calcobutrol), calcinol (calteridol), cocamidopropyl betaine, cocamidopropyl hydroxysultaine, creatine, cytidine monophosphate, diaminopimelic acid, diethylenetriaminepentaacetic acid, dimethylphenylalanine, methylglycine, sarcosine, dimethylglycine, dipeptide amphiphatic (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), diethylenetriamine penta (methylenephosphonic acid) (DTPMP), dipalmitoylphosphatidylcholine, tetrahydropyrimidine, ethylenediaminetetra (methylenephosphonic acid) (EDTMP), folate benzoate mixtures, folate nicotinamide mixtures, gelatin, hydroxyproline, iminodiacetic acid, isoguanidinocaine, lecithin, myristamine oxide, Nicotinamide Adenine Dinucleotide (NAD), N-methylaspartic acid, N-methylproline, N-trimethyllysine, ornithine, oxolinic acid, risedronate (risedronate), allylcysteine, S-allyl-L-cysteine, somapatatin (somapatatin), taurine, Theanine, trigonelline, vigabatrin, tetrahydropyrimidine, 4- (2-hydroxyethyl) -1-piperazineethanesulfonate, o-octylphosphorylcholine, nicotinamide mononucleotide, triglycine, tetraglycine, beta-guanidinopropionic acid, 5-aminolevulinic acid hydrochloride, picolinic acid, lidonine, phosphorylcholine, 1- (5-carboxyphenyl) -4-methylpyridin-1-ium bromide, L-anserine nitrate, L-glutathione reduced, N-ethyl-L-glutamine, N-methylproline, (Z) -1- [ N- (2-aminoethylethyl) -N- (2-aminomethylethyl) amino ] diazen-1-ium-1, 2-iolate (DETA-NONONONONONONAte), (Z) -1- [ N- (3-aminopropyL) -N- (3-one- amoniopropyl) amino ] diazen-1-ium-1,2-diolate (DPTA-NONate) and zoledronic acid.
Without being bound by theory, the zwitterionic excipient compound may exert viscosity-lowering or stabilizing effects by interacting with the protein (e.g., through charge, hydrophobic, and steric interactions) to make the protein more resistant to aggregation, or by affecting the bulk properties of the water in the protein formulation, such as electrolyte contribution, reduction in surface tension, changes in the amount of unbound available water, or changes in dielectric constant.
i. Excipient compound class 9: crowding agents with hydrogen bonding elements
Solutions of therapeutic or non-therapeutic proteins may be formulated with crowding agents with hydrogen bonding elements as excipients to improve stability or reduce viscosity. As used herein, the term "crowding agent" refers to a formulation additive that reduces the amount of available water to dissolve protein in solution, thereby increasing the effective protein concentration. In embodiments, the crowding agent may reduce the protein particle size or reduce the amount of protein that is spread out in solution. In embodiments, the crowding agent may act as a solvent modifier that causes the structuring of water through hydrogen bonding and hydration effects. In embodiments, the crowding agent may reduce the amount of intermolecular interactions between proteins in solution. In embodiments, the crowding agent has a structure comprising at least one hydrogen bond donor element (such as hydrogen attached to an oxygen, sulfur, or nitrogen atom). In an embodiment, the crowding agent has a structure comprising at least one weakly acidic hydrogen bond donor element having a pKa of about 6 to about 11. In embodiments, the crowding agent has a structure comprising from about 2 to about 50 hydrogen bond donor elements. In embodiments, the crowding agent has a structure comprising at least one hydrogen bond acceptor element (such as a lewis base). In embodiments, the crowding agent has a structure comprising from about 2 to about 50 hydrogen bond acceptor elements. In embodiments, the molecular weight of the crowding agent is about 50 to 500 g/mol. In embodiments, the crowding agent has a molecular weight of about 100 to 350 g/mol. In other embodiments, the molecular weight of the crowding agent may be 500g/mol or more, such as raffinose, inulin, amylopectin, or allium sugar (sinistrin).
Examples of crowding agent excipients having hydrogen bonding elements include 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2 (1H) -pyrimidinone, 15-crown-5, 18-crown-6, 2-butanol, 2-butanone, 2-phenoxyethanol, acetaminophen, allantoin, arabinose, arabitol, benzyl acetoacetate (benzyl acetate), benzyl alcohol, chlorobutanol, cholesterol tetraacetyl-b-glucoside, cinnamaldehyde, cyclohexanone, deoxyribose, diethyl carbonate, dimethyl isosorbide, dimethylacetamide, dimethylformamide, dimethylol urea, dimethyluracil, epi-lactose, erythritol, ethyl lactate, ethyl maltol, ethylene carbonate, formamide, methyl acetate, and the like, Fucose, galactose, genistein, gentisic acid ethanolamide, gluconolactone, glyceraldehyde, glycerol carbonate, glycerol formal, glycerol carbamate, glycyrrhizic acid, cotton cellulose (gossypin), harpagoside (harpagoside), hederacoside C (hederacoside C), icodextrin (icodextrin), iditol, imidazolone, inositol, inulin, isomalt, kojic acid, lactitol, lactobionic acid, lactulose, lyxose, madecassoside (madecassoside), maltotriose, mangiferin, mannose, melezitose, methyl lactate, methylpyrrolidone, mogroside V, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, N-methylacetamide, N-methylformamide, N-methylpropionamide, pentaerythritol, pinoresinol diglucoside, glycitose, mogroside, diglucoside, glycofuroxanide, diglucoside, xylosylglucoside, xylosylamide, xylosylglucoside, xylo, Piracetam, propyl gallate, propylene carbonate, psicose, pullulan, pyrogallol, quinic acid, raffinose, rebaudioside A, rhamnose, ribitol, ribose, ribulose, saccharin, sedoheptulose, eleutherol (sinistran), solketal (solketal), stachyose, sucralose (sucralose), tagatose, tert-butanol, tetraethylene glycol, triacetin, N-acetyl-D-mannosamine, fructotetraose, kestose, turanose, acarbose, D-glucaric acid, 4-lactone, thiodigalactoside (thiodigalactoside), fucoidan (fucoidan), hydroxysafflor yellow A, shikimic acid, diosmin, pravastatin sodium salt, D-altrose sugar, L-gulonic acid-gamma-lactone, neomycin, rubusoside, dihydrothearubiside, phloroglucinol, artemisinin, Naringin, baicalin, hesperidin, apigenin, pyrogallol, morin, salsalate, kaempferol, myricetin, 3 ', 4', 7-trihydroxyisoflavone, (+ -) -taxifolin ((+ -) -taxifolin), silybin, avocadol glyoxal (perseitol diformal), 4-hydroxyphenylpyruvic acid, sulfanilamide, isopropyl beta-D-1-thiogalactopyranoside, ethyl 2, 5-dihydroxybenzoate, spectinomycin, resveratrol, quercetin, kanamycin sulfate, 1- (2-pyrimidinyl) piperazine, 2- (2-pyridyl) ethylamine, 2-imidazolidinone, DL-1, 2-isopropylideneglycerin, metformin, m-xylylenediamine, meclocycline, tripropylene glycol, tuberin methyl (tubeimoside I), Verbascoside, xylitol and xylose.
6. Protein/excipient solution: characteristics and procedures
In certain embodiments, a solution of a therapeutic or non-therapeutic protein is formulated with excipient compounds identified above, such as hindered amines, anionic aromatic compounds, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diketones and sulfones, zwitterionic excipients, and crowding agents with hydrogen bonding elements, or combinations thereof (hereinafter "excipients"), such that the interaction parameter, k, is as determined by protein diffusion, kDOr a second coefficient of gravity coefficient (B)22The measured protein-protein interaction profile improves. As used herein, "improvement" in one or more protein-protein interaction parameters achieved by a test formulation using an excipient compound identified above, or a combination thereof, means a reduction in the mutually attractive protein-protein interaction when the test formulation is compared under comparable conditions to a comparative reagent that does not include the excipient compound or excipient additive. Such improvement may be determined by measuring certain parameters applicable to the entire process or an aspect thereof, where a parameter is any metric related to the process, where changes may be quantified and compared to previous states or controls. The parameters may be related to the process itself, such as efficiency, cost, yield, or rate. Improving stability of protein-containing formulations during processing May have the advantages of improved yield, increased biological activity and reduced presence of particles in the formulation.
The parameter may also be a proxy parameter related to a feature or aspect of a larger process. E.g. such as kDOr B22Such parameters may be referred to as proxy parameters. k is a radical ofDAnd B22Can be performed using standard techniques in the industry and can be an indicator of a process-related parameter, such as improved solution properties or stability of the protein in solution. Without being bound by theory, it is understood that high negative kDValues may indicate that the proteins have strongly attractive interactions, and this may lead to 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 lower negative kDK having a value of either close to or higher than zeroDAn improved proxy parameter of value, the improved proxy parameter being associated with an improvement in a process-related parameter.
In embodiments, certain of the above-described excipient compounds such as hindered amines, anionic aromatic compounds, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diketones and sulfones, zwitterionic excipients, and crowding agents with hydrogen bonding elements, or combinations thereof, are used to improve protein related processes such as the manufacture, handling, sterile filling, purification and analysis of protein containing solutions using the following processing methods: such as 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, axial flow filtration, lyophilization, and gel electrophoresis. In these and related protein-related processes, the protein of interest is dissolved in a solution that is transported through the processing equipment. Such solutions, referred to herein as "carrier solutions," may include cell culture media (e.g., containing secreted proteins of interest), lysate solutions after lysis of host cells (where the proteins of interest reside in the lysate), elution solutions (which contain the proteins of interest after chromatographic separation), electrophoresis solutions, transport solutions for carrying the proteins of interest through a catheter in a processing device, and the like. The carrier solution comprising the protein of interest may also be referred to as a protein-containing solution or a 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 the process. As used herein, the terms "improve," "improving," and the like refer to a favorable change in a parameter of interest in a carrier solution when compared to the same parameter measured in a control solution. As used herein, a "control solution" refers to a solution that lacks viscosity-reducing excipients but is otherwise substantially similar to the carrier solution. As used herein, a "control process," e.g., a control filtration process, a control chromatography process, etc., is a protein-related process that is substantially similar to a protein-related process of interest and is performed using a control solution rather than a carrier solution.
For example, during pumping of a protein-containing solution through a conduit (e.g., a flow cell, pipe, or tube), it may be advantageous to add the excipient compounds identified above or combinations thereof to reduce viscosity. As described above, the addition of viscosity reducing excipients to a protein solution prior to or during the pumping process can greatly reduce the force and power required to pump the solution. It will be appreciated that fluids typically exhibit resistance to flow, i.e. viscosity, and that a force must be applied to the fluid to overcome this viscosity in order to induce and conduct flow. The power P required for pumping is measured in terms of head H and capacity Q as shown in the following equation:
P-HQ (formula 1)
Viscous fluids tend to increase the power requirements of the pump, reduce the efficiency of the pump, reduce the pump head and capacity, and increase frictional drag in the piping. The addition of the viscosity reducing excipient described above to the protein solution before or during pumping can significantly reduce processing costs by reducing the head (H, equation 1) or the volume (Q, equation 1), or both. The benefits of reduced viscosity can be manifested by, for example, improved throughput, increased yield, or reduced processing time. Furthermore, frictional losses due to the transport of fluids through the conduits can represent a significant portion of the costs associated with transporting such fluids. The addition of viscosity reducing excipients as described above to the protein solution prior to or during pumping can significantly reduce processing costs by reducing friction associated with the pumping process. The measurement of treatment cost represents a treatment parameter that can be improved by using viscosity reducing excipients.
These processes and methods of processing protein solutions may have improved efficiency due to lower viscosity, improved solubility or improved stability of the proteins in solution during the manufacturing, processing, purification and analysis steps. The measurement of treatment efficiency or the measurement of a proxy parameter (such as viscosity, solubility or stability of the protein in solution) represents a treatment parameter that can be improved by using viscosity reducing excipients. Several different factors are believed to adversely affect protein viscosity, solubility and stability during processing. For example, protein-containing solutions can be subjected to various physical pressures during manufacture and purification, including significant shear stresses that can be generated by manipulation of the protein solution through typical processing operations, including but not limited to pumping, mixing, centrifugation, and filtration. Additionally, during these processing steps, gas bubbles may become entrained in the fluid to which the protein may adsorb. This interfacial tension, coupled with the typical shear stresses encountered during processing, causes the adsorbed protein molecules to unfold and aggregate. In addition, significant protein unfolding can occur during pump cavitation events (pump cavitation events) and during exposure to solid surfaces during manufacturing, such as ultrafiltration and diafiltration membranes. Such events may impair protein folding and product quality.
As shown in the following equation, for Newtonian fluids, the stress applied by a given process, τ, is the shear rate
Figure BDA0002748917970000502
And viscosity η of the fluid:
Figure BDA0002748917970000501
by using one or more than oneFormulating a protein solution with the identified excipient compound or combination thereof can reduce the viscosity of the solution, thereby reducing the shear stress encountered by the protein solution. Reduced shear stress may improve the stability of the treated formulation, for example, as indicated by measurements of better or more desirable treatment parameters. Such improved processing parameters may include metrics such as reduced levels of protein aggregates, particulates or sub-visible particulates (macroscopically manifested as turbidity), reduced product loss or increased overall yield. As another example of improved processing parameters, reducing the viscosity of a protein-containing solution may reduce the processing time of the solution. The processing time for a given unit operation is generally inversely proportional to the shear rate. Thus, for a given characteristic stress, the viscosity reduction and shear rate of a protein solution achieved by the addition of an excipient compound identified above or a combination thereof (
Figure BDA0002748917970000503
See equation 2) and thus to a reduction in processing time. Furthermore, the addition of certain of the above-identified excipient compounds or combinations thereof may improve the stability of the protein solution at different stages of processing.
During processing, it will be appreciated that the protein in solution may be the desired protein active ingredient, for example a therapeutic or non-therapeutic protein. The use of the excipients described herein to facilitate the treatment of such protein active ingredients can increase the yield or productivity of the protein active ingredient, or improve the efficiency of a particular process, or reduce energy usage, etc., any of which results represent improved treatment parameters through the use of viscosity reducing excipients. It is also understood that protein contaminants may form during certain treatment processes, such as during fermentation and purification steps of biological processes. Faster, more thorough or more efficient removal of contaminants may also improve the processing of the desired protein (i.e., protein active ingredient); these results represent improved processing parameters through the use of viscosity reducing excipient compounds or additives. As described herein, certain excipients described herein can improve the transport of a desired protein active ingredient, and can improve the removal of undesirable protein contaminants, by reducing solution viscosity, improving protein stability, and/or increasing protein solubility. Both of these effects, which represent improved processing parameters through the use of viscosity reducing excipients or additives, indicate that these excipients or additives improve the overall process of protein production. Misfolded proteins, microparticles, denatured proteins, or other artifacts of unstable proteins in solution may be reduced by using stabilizing excipients in the processing steps.
Specific platform unit operations for therapeutic protein production and purification provide further examples of advantageous uses of the excipient compounds identified above, or combinations thereof, as well as further examples of the processing parameters that these excipients or additives improve. For example, as described below, the introduction of one or more of the above identified excipient compounds, or combinations thereof, into the production and purification processes described below can significantly improve the stability and recovery of the molecule and reduce the cost of the procedure.
It is understood in the art that widely practiced techniques for producing and purifying therapeutic proteins, such as monoclonal antibodies, typically consist of a fermentation process followed by a series of purification process steps. Fermentation or upstream processing (USP) includes those steps that typically use bacterial or mammalian cell lines to grow therapeutic proteins in bioreactors. In an embodiment, the USP may include steps such as those shown in figure 4. In embodiments, purification or downstream processing (DSP) may include steps such as those shown in fig. 5.
As shown in FIG. 4, USP may begin with a step 102 of thawing vials from a Master Cell Bank (MCB). The MCB may be expanded as shown in step 104 to form a working cell bank (not shown) and/or to generate working stock for further production. As shown in steps 108 and 110, cell cultures are performed in a series of seed and production bioreactors to produce bioreactor product 112 from which the desired therapeutic protein is harvested as shown in step 114. 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 the products may be stored in bulk, typically by freezing and storage at a temperature of about-80 ℃.
In embodiments, proteins produced by cell culture techniques may be improved by the use of the above-identified excipients, as evidenced by improvements in process-related parameters. In embodiments, the desired excipient may be added during USP in an amount effective to reduce the viscosity of the cell culture medium by at least 20%. In other embodiments, the desired excipient may be added during USP in an amount effective to reduce the viscosity of the cell culture medium by at least 30%. In embodiments, the desired excipient may be added to the cell culture medium in an amount of about 1mM to about 400 mM. In embodiments, the desired excipient may be added to the cell culture medium in an amount of about 20mM to about 200 mM. In embodiments, the desired excipient may be added to the cell culture medium in an amount of about 25mM to about 100 mM. The required excipient or combination of excipients may be added directly to the cell culture medium or may be added as a component of a more complex supplemental medium, such as a nutrient solution or "feed solution" formulated separately and added to the cell culture medium. In embodiments, a second excipient (e.g., a viscosity reducing compound) may be added to the carrier solution, either directly or through a supplemental medium, where the second viscosity reducing compound adds additional improvements to the particular parameter of interest.
As described below, there are a number of process-related parameters during USP that can be improved by using 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, such as during the inoculum expansion 104 and cell culture 108 and 110 steps, and/or may improve surrogate parameters related to the improvement of various process parameters. For example, the addition of certain of the above-identified excipients to the USP process in a step such as production bioreactor step 110 can reduce the viscosity of the cell culture medium, which can subsequently improve heat transfer efficiency and gas transfer efficiency. Since the cell culture process requires the injection of oxygen into the cells to achieve protein expression, diffusion of oxygen into the cells may be a rate limiting step, and increasing the efficiency of gas transport by decreasing the solution viscosity may increase the rate of oxygen uptake and thus improve the rate or amount of protein expression and/or its efficiency. In this case, parameters such as oxygen uptake rate and gas transfer efficiency may be considered as proxy parameters, the improvement of which correlates with an improvement in process parameters for improved protein expression or improved processing efficiency. As another example, the presence of viscosity reducing excipients may improve processing during, for example, the inoculum expansion step 104 and during the cell culture steps 108 and 110 by improving surrogate parameters, such as the solubility of protein growth factors required for protein expression. For protein expression; by improving the solubility of growth factors, these substances can be more readily utilized by cells, thereby promoting cell growth.
In embodiments, process parameters such as the amount of protein recovered or the rate of protein recovery during USP can be improved by reducing the viscosity during USP by several mechanisms. For example, harvesting the therapeutic protein at the end of the lysis step during harvesting 114 from the finished cell culture may be more efficient, or may be improved by the use of excipients as described above, for example. Without being bound by theory, these viscosity reducing excipients may increase the efficiency of diffusion of the therapeutic protein away from other lysate components by reducing the viscosity of the expressed protein. In addition, by using viscosity reducing excipients, membranes and other cell debris can be separated from the protein-containing supernatant at a faster separation rate or a higher degree of supernatant purity, thereby improving the process parameters of USP efficiency. In addition, the protein separation step using the centrifugation or filtration step can be accomplished more quickly by using a viscosity reducing excipient because the excipient reduces the viscosity of the medium. Upstream and downstream processing of proteins may benefit from the use of these excipients, as excipients may also improve the stability of therapeutic protein solutions. In embodiments, the excipient may improve the stress tolerance of the protein during processing, and this may reduce the amount of aggregation or denaturation of the protein during the processing step.
In embodiments, as an additional benefit, the use of the above excipient compounds or combinations thereof (e.g., viscosity reducing excipients) in cell culture can increase process parameters such as protein yield during USP, as protein misfolding and aggregation are reduced. It will be appreciated that, as the cell culture is optimized to produce the maximum yield of recombinant protein, the resulting protein is expressed in a highly concentrated manner, which may lead to misfolding. The addition of the excipient compounds identified above or combinations thereof (e.g., viscosity reducing excipients) can reduce the attractive protein-protein interactions that lead to misfolding and aggregation, thereby increasing the amount of intact recombinant protein available for harvest 114.
In one illustrative embodiment, as shown in fig. 5, downstream processing (DSP) involves a series of steps that result in the recovery and purification of therapeutic proteins (e.g., monoclonal antibodies, biopharmaceuticals, vaccines and other biologicals). At the conclusion of USP, the therapeutic protein of interest can be secreted from the host cell and then solubilized in the cell culture medium. Following lysis of the host cells at the end of the USP sequence, the therapeutic protein may also be dissolved in liquid medium. DSP can be performed to recover and purify the protein of interest from a solution (e.g., culture medium or host cell lysate medium) in which the protein is dissolved. During DSP, (i) various contaminants, such as insoluble cell debris and particulates, are removed from the culture medium, (ii) the protein product is isolated by techniques such as extraction, precipitation, adsorption or ultrafiltration, (iii) the protein product is purified by techniques such as affinity chromatography, precipitation or crystallization, (iv) the product is further polished and the virus is removed.
As shown in fig. 5, the feedstock from cell culture harvest 200 (also shown in fig. 4) is subjected to initial affinity chromatography 204, typically involving protein a chromatography or other similar chromatography steps. The virus inactivation step 208 typically requires subjecting the feedstock to a low pH hold. One or more polishing chromatography steps 210 and 212 are performed to remove impurities such as Host Cell Proteins (HCPs), DNA, charge variants, and aggregates. Cation Exchange (CEX) chromatography is typically used as the initial polishing chromatography step 210, but it may be accompanied by a second chromatography step 212, either before or after it. The second chromatography step 212 further removes host cell-related impurities (e.g., 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 filtering 214 is performed to achieve virus removal. The final purification step 218 may include ultrafiltration and diafiltration, and preparation of the formulation.
As described above, the purification process or DSP after the fermentation process may include (1) cell culture harvest, (2) chromatography (e.g., protein a chromatography and chromatography polishing steps, including ion exchange and hydrophobic interaction chromatography), (3) virus inactivation, and (4) filtration (e.g., virus filtration, sterile filtration, dialysis, and ultrafiltration and diafiltration steps to concentrate and exchange proteins into the formulation buffer). Examples are provided below to illustrate the advantages of using the above-described excipient compounds or combinations thereof (e.g., viscosity reducing excipients) in improving the process parameters associated with these purification processes. It will be appreciated that the excipient compounds identified above, or combinations thereof (e.g., viscosity reducing excipients), may be introduced at any stage of the DSP by adding them to the carrier solution or by engineering in any other way the contact of the protein of interest with the soluble or stabilized form of the excipient. In embodiments, a second excipient (e.g., a viscosity reducing compound) may be added to the carrier solution during DSP, where the second compound adds additional improvement to the particular parameter of interest.
(1)Cell culture harvest: cell culture harvesting typically involves centrifugation and depth filtration operations in which cell debris is physically removed from a protein-containing solution. The centrifugation step may provide for a more complete separation of soluble proteins from cell debris thanks to the viscosity reducing excipient. Whether batch or continuous, centrifugation requires that the dense phases be combined as much as possible in order to maximize recovery of the protein of interest. In embodiments, the addition of the above-identified excipients or combinations thereof may increase the process parameters of protein yield, for example, by increasing the yield of the protein-containing centrate that flows away from the dense phase of the centrifugation process. The depth filtration step is a viscosity limiting step and can therefore be carried out by using a viscosity-reducing excipient to reduce the viscosity of the solutionA shaping agent to make it more effective. These processes may also introduce gas bubbles into the protein solution that may combine with shear-induced stress to destabilize the therapeutic protein molecule being purified. As described above, addition of viscosity reducing excipients to the protein-containing solution prior to and/or during cell culture harvest can protect the protein from these stresses, thereby reducing the likelihood of protein aggregation and improving the process parameters for quantitative product recovery.
(2)Chromatography method: after harvesting the cell culture by centrifugation or filtration, the therapeutic protein is typically isolated from the fermentation broth using chromatography. When the therapeutic protein is an antibody, protein a chromatography can be used: protein a is selective for IgG antibodies and will bind to it dynamically at high flow rates and volumes. Cation Exchange (CEX) chromatography is useful as an economical and efficient alternative to protein a chromatography. If CEX is used, the pH of the feed must be adjusted and its conductivity reduced before loading onto the column to optimize dynamic binding capacity. Simulated resins may also be used as a substitute for protein a chromatography. These resins provide immunoglobulin-binding ligands, for example Ig-binding proteins like protein G or protein L, synthetic ligands or protein a-like porous polymers.
Other chromatographic processes may be employed during the DSP. Ion Exchange Chromatography (IEC) can be used to remove impurities introduced in previous processes, such as leached protein a, endotoxins or viruses in cell lines, residual host cell proteins or DNA, or media components. IEC, either CEX or anion exchange chromatography, can be applied directly after protein C chromatography. Hydrophobic Interaction Chromatography (HIC) can complement IEC, and is typically used as a polishing step to remove aggregates. In embodiments, the use of the above-identified excipients in a chromatography column loading step may increase the solubility and decrease the viscosity of the host cell protein. In embodiments, the use of the above-identified excipients may increase the solubility and decrease the viscosity of the therapeutic protein during the column loading step and the elution step.
The chromatographic process in the protein purification process imposes harsh conditions on the protein preparation, such as (a) low pH conditions upon elution from the protein a chromatography column, (b) local protein concentration increases in the pore space of the chromatography resin (typically around 300-400 mg/mL), (c) salt concentration increases in ion exchange chromatography, and (d) salting-out agent concentration increases upon elution from the HIC column. As noted above, the addition of viscosity reducing excipients to the protein-containing solution prior to and/or during chromatography may facilitate the transport of proteins through the chromatography column, thereby exposing them less to potentially damaging conditions imposed by the chromatography processing step. Furthermore, the local increase in protein concentration in the pore space of the column results in a very high viscosity of the material in this space, thus giving a huge back pressure to the column. To alleviate this back pressure, media having relatively large pores are typically used. However, macroporous media have lower resolution than microporous media. Incorporation of viscosity modifying excipients as described above allows the use of smaller pores in the chromatographic medium. In embodiments, the elution step from protein a chromatography exposes the therapeutic protein to low pH conditions that can reduce solubility and increase aggregation of the protein of interest. The addition of excipients can increase the solubility of the protein of interest, thereby improving the recovery of the protein a chromatography step. In other embodiments, the use of excipients may elute the protein of interest from the protein a resin at a higher pH, and this may reduce the chemical stress on the protein of interest, thereby improving the process parameters of protein yield by reducing the amount of protein degradation during processing.
(3)Inactivation of viruses: the virus inactivation process typically involves maintaining the protein solution at a low pH, e.g., a pH below 4, for a substantial period of time. However, such an environment may destabilize the therapeutic protein. Formulating the protein in the presence of a viscosity reducing excipient, for example, by adding the viscosity reducing excipient prior to and/or during the virus inactivation process, can improve process parameters such as the stability or solubility of the protein, or the net yield thereof.
(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 replace the buffer system。
(a) Viral filtration the protein solution was purified by removing viral particles, approximately twice the size of the recombinant human monoclonal antibodies. Therefore, filtration membranes for virus filtration may require nanometer-sized pores. Since the protein must pass through a small pore size, this filtration step can put stress on the protein with significant fouling by aggregated particles of the protein. As described above, the addition of viscosity-reducing excipients, e.g., before and/or during filtration, can reduce measurable parameters in the filtration system (such as back pressure) by increasing collective diffusivity, and can reduce the tendency of membrane fouling by mitigating protein-protein interactions that cause membrane fouling. The end result is an improvement in these parameters, indicating an improvement in the performance of the viral filtration unit during protein purification.
(b) The ultrafiltration and diafiltration (UF/DF) process concentrates the protein solution and replaces the buffer system by passing the protein-containing solution through a filter membrane having a molecular weight cut-off less than the protein of interest. In this step, the protein solution is subjected to high shear stress within the filter unit, the protein concentration increases and the protein adsorbs on the hydrophobic membranes typically used in UF/DF processes, all of which increase protein aggregation. As described above, addition of viscosity-reducing excipients, e.g., before and/or during UF/DF procedures, can increase collective diffusivity (e.g., by k)DMeasured as an increase in pressure) to reduce back pressure in the filtration system. This not only reduces the shear stress across the membrane, but also promotes back diffusion from the filter membrane, thereby reducing the effective protein concentration at the membrane interface and increasing the permeate flux. Thus, the use of viscosity reducing excipients in these filtration processes may improve parameters associated with greater throughput, thereby reducing product loss and increasing net yield. In addition, passing viscous fluids through the ultrafilter and the diafilter creates a significant pressure drop across the filter apparatus, thereby making the separation inefficient. As described above, formulating a protein solution in the presence of viscosity reducing excipients can significantly reduce the pressure drop across the filter device, thereby improving the process parameters of both by reducing operating costs and processing time.
After upstream protein processing or downstream purification with added excipients, the excipients may remain part of the bulk drug mixture or may be separated from the protein active ingredient. Typical small molecule separation methods can be used to separate excipients from the protein active ingredient, such as buffer exchange, ion exchange, ultrafiltration, and dialysis. In addition to the beneficial effects on the protein purification process outlined above, the use of the excipients identified above may also protect and preserve equipment used for protein manufacture, processing and purification. For example, equipment-related processes, such as cleaning, sterilization and maintenance of protein processing equipment, may be facilitated by the use of the above-identified excipients due to reduced fouling, reduced denaturation, lower viscosity and improved solubility of the proteins, and parameters associated with improved modification of these processes are similarly improved.
Although the methods of using excipient compounds to improve upstream and/or downstream processing have been broadly described herein, it is understood that combinations of excipients may be added together to achieve a desired effect, such as improving a parameter of interest. The term "excipient additive" may refer to a single excipient compound that results in a desired effect or improved parameter, or to a combination of excipient compounds wherein the combination results in a desired effect or improved parameter.
Examples
Materials:
bovine Gamma Globulin (BGG), purity > 99%, Cat G5009, Sigma Aldrich
Histidine, Sigma Aldrich
Unless otherwise stated, other materials described in the examples below were from Sigma Aldrich.
Example 1: preparation of formulations containing excipient Compounds and test proteinsFormulations are prepared using an excipient compound and a test protein, where the test protein is intended to mimic a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. Preparation of the preparation with different excipient Compounds in 50mM histidine HClThe agent was used for viscosity measurement in the following manner. Histidine hydrochloride was first prepared by the following method: 1.94g of histidine was dissolved in distilled water and the pH was adjusted to about 6.0 with 1M hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.), and then diluted with distilled water in a volumetric flask to a final volume of 250 mL. The excipient compound was then dissolved in 50mM histidine HCl. A list of excipients is provided in examples 4, 5, 6 and 7 below. In some cases, the excipient compound was adjusted to pH6 prior to dissolution in 50mM histidine HCl. In this case, the excipient compound is first dissolved at about 5% by weight in deionized water and the pH is adjusted to about 6.0 with hydrochloric acid or sodium hydroxide. The prepared salt solution was then placed in a convection laboratory oven at about 65 ℃ to evaporate the water and isolate the solid excipients. Once the excipient solution has been prepared in 50mM histidine HCl, the test protein (bovine gamma globulin (BGG)) is solubilized at a rate of about 0.336g BGG per 1mL of excipient solution. This resulted in a final protein concentration of about 280 mg/mL. A solution of BGG in 50mM histidine HCl with vehicle was prepared in 20mL vials and allowed to shake overnight at 100rpm on an orbital shaker table. The BGG solution was then transferred to a 2mL microcentrifuge tube and centrifuged in an IEC micromamax microcentrifuge at 2300rpm for 10 minutes to remove entrapped air before viscosity measurements were taken.
Example 2: viscosity measurement
Viscosity measurements of the formulations prepared as described in example 1 were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample.
Example 3: protein concentration measurement
The concentration of the protein in the test solution was determined by measuring the absorbance of the protein solution at a wavelength of 280nm in a UV/VIS spectrometer (Perkin Elmer Lambda 35). First, the instrument was calibrated to zero absorbance with 50mM histidine buffer at pH 6. Next, the protein solution was diluted 300-fold with the same histidine buffer, and the absorbance at 280nm was recorded. The final concentration of protein in the solution was calculated by using an extinction coefficient value of 1.264mL/(mg x cm).
Example 4: formulations with hindered amine excipient compounds
Formulations containing 280mg/mL BGG were prepared as described in example 1, with some samples containing added excipient compounds. Among these tests, the hydrochloride salts of Dimethylcyclohexylamine (DMCHA), Dicyclohexylmethylamine (DCHMA), Dimethylaminopropylamine (DMAPA), Triethanolamine (TEA), trimethylethanolamine (DMEA), and nicotinamide were tested as examples of hindered amine excipient compounds. The hydroxybenzoates of DMCHA and taurine-dicyandiamide adducts were also tested as examples of hindered amine excipient compounds. The viscosity of each protein solution was measured as described in example 2 and the results are presented in table 1 below, showing the benefit of the added excipient compound in reducing viscosity.
TABLE 1
Figure BDA0002748917970000611
Figure BDA0002748917970000621
Example 5: formulations with anionic aromatic excipient compounds
Formulations with 280mg/mL BGG were 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, showing the benefit of the added excipient compound in reducing viscosity.
TABLE 2
Excipient Concentration of excipients Viscosity of the oil Amount of viscosity reduction
Is free of 0 79 0％
Amino benzoic acid sodium salt 43 48 39％
Sodium hydroxy benzoate 26 50 37％
Sulphamic acid sodium salt 44 49 38％
Sulphamic acid sodium salt 96 42 47％
Indole sodium acetate 52 58 27％
Indole sodium acetate 27 78 1％
Vanillic acid, sodium salt 25 56 29％
Vanillic acid, sodium salt 50 50 37％
Salicylic acid sodium salt 25 57 28％
Salicylic acid sodium salt 50 52 34％
Adenosine monophosphate 26 47 41％
Adenosine monophosphate
50 66 16％
Sodium benzoate 31 61 23％
Sodium benzoate 56 62 22％
Example 6: formulations with oligopeptide excipient compounds
Oligopeptides (N ═ 5) were synthesized by NeoBioLab Inc (Woburn, MA) with a purity > 95% with the N-terminus as free amine and the C-terminus as free acid. Dipeptide (n ═ 2) was synthesized by LifeTein LLC (Somerset, NJ) with a purity of 95%. Formulations with 280mg/mL BGG were prepared as described in example 1, with some samples containing synthetic oligopeptides as added excipient compounds. The viscosity of each solution was measured as described in example 2 and the results are presented in table 3 below, showing the benefit of the added excipient compound in reducing viscosity.
TABLE 3
Added excipients Concentration of excipients Viscosity of the oil Amount of viscosity reduction
Is free of 0 79 0％
ArgX5
100 55 30％
ArgX5
50 54 32％
HisX5
100 62 22％
HisX5
50 51 35％
HisX5 25 60 24％
Trp2Lys3
100 59 25％
Trp2Lys3
50 60 24％
AspX5
100 102 -29％
AspX5
50 82 -4％
Dipeptide LE
50 72 9％
Dipeptide YE
50 55 30％
Dipeptide RP
50 51 35％
Dipeptide RK
50 53 33％
Dipeptide RH
50 52 34％
Dipeptide RR
50 57 28％
Dipeptide RE
50 50 37％
Dipeptide LE
100 87 -10％
Dipeptide YE
100 68 14％
Dipeptide RP
100 53 33％
Dipeptide RK
100 64 19％
Dipeptide RH
100 72 9％
DipeptidesRR
100 62 22％
Dipeptide RE
100 66 16％
Example 7: synthesis of guanyltaurine excipients
Guanyltaurine was prepared according to the method described in U.S. patent No. 2,230,965. 3.53 parts taurine (Sigma-Aldrich, St.Louis, Mo.) are mixed with 1.42 parts dicyandiamide (Sigma-Aldrich, St.Louis, Mo.) and ground in a mortar and pestle until a homogeneous mixture is obtained. Next, the mixture was placed in a flask and heated at 200 ℃ for 4 hours. The product was used without further purification.
Example 8: protein formulations containing excipient compounds
Formulations are prepared using an excipient compound and a test protein, where the test protein is intended to mimic a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. The formulations were prepared with different excipient compounds in 50mM aqueous histidine hydrochloride buffer solution for viscosity measurements performed in the following manner. A histidine hydrochloride buffer solution was first prepared by: 1.94g of histidine was dissolved in distilled water and the pH was adjusted to about 6.0 with 1M hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.), and then diluted with distilled water in a volumetric flask to a final volume of 250 mL. The excipient compound was then dissolved in 50mM histidine HCl buffer solution. A list of excipient compounds is provided in table 4. In some cases, the excipient compound is dissolved in 50mM histidine HCl buffer solution, and the pH of the resulting solution is adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve pH6, before dissolution of the model protein is performed. In some cases, the excipient compound was adjusted to pH6 prior to dissolution in 50mM histidine HCl. In this case, the excipient compound is first dissolved at about 5% by weight in deionized water and the pH is adjusted to about 6.0 with hydrochloric acid or sodium hydroxide. The prepared salt solution was then placed in a convection laboratory oven at about 65 ℃ to evaporate the water and isolate the solid excipients. Once the excipient solution had been prepared in 50mM histidine HCl, the test protein Bovine Gamma Globulin (BGG) was solubilized at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in 50mM histidine HCl with vehicle was prepared in 20mL vials and allowed to shake overnight at 100rpm on an orbital shaker table. The BGG solution was then transferred to a 2mL microcentrifuge tube and centrifuged in an IEC micromamax microcentrifuge at 2300rpm for 10 minutes to remove entrapped air before viscosity measurements were taken.
Viscosity measurements of the formulations prepared as described above were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient. 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
Figure BDA0002748917970000661
Figure BDA0002748917970000671
Example 9: preparation of formulations containing excipient combinations and test proteins
Formulations are prepared using a primary excipient compound, a secondary excipient compound, and a test protein, wherein the test protein is intended to mimic a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. The main excipient compounds are selected from the compounds with anionic and aromatic functionalities as listed in table 5 below. The secondary excipient compound is selected from compounds having a non-ionic or cationic charge at pH6 and having an imidazoline or benzene ring, as listed in table 5 below. Formulations of these excipients were prepared in 50mM histidine hydrochloride buffer solution for viscosity measurements performed in the following manner. Histidine hydrochloride was first prepared by the following method: 1.94g of histidine was dissolved in distilled water and the pH was adjusted to about 6.0 with 1M hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.), and then diluted with distilled water in a volumetric flask to a final volume of 250 mL. The individual primary or secondary excipient compounds were then dissolved in 50mM histidine HCl. The combination of primary and secondary excipients was dissolved in 50mM histidine HCl and the pH of the resulting solution was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve pH6 before solubilization of the model protein. Once the excipient solutions have been prepared as described above, the test protein (bovine gamma globulin (BGG)) is dissolved into each test solution at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in 50mM histidine HCl with vehicle was prepared in 20mL vials and allowed to shake overnight at 100rpm on an orbital shaker table. The BGG solution was then transferred to a 2mL microcentrifuge tube and centrifuged in an IEC micromamax microcentrifuge at 2300rpm for 10 minutes to remove entrapped air before viscosity measurements were taken.
Viscosity measurements of the formulations prepared as described above were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of the solutions with excipients were normalized to the viscosity of the model protein solution without excipients and are 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. The examples show that the combination of a primary excipient and a secondary excipient may produce better results than a single excipient.
TABLE 5
Figure BDA0002748917970000681
Figure BDA0002748917970000691
Example 10: preparation of formulations containing excipient combinations and test proteins
Formulations are prepared using a primary excipient compound, a secondary excipient compound, and a test protein, wherein the test protein is intended to mimic a therapeutic protein to be used in a therapeutic formulation or a non-therapeutic protein to be used in a non-therapeutic formulation. The main excipient compounds are selected from the compounds with anionic and aromatic functionalities as listed in table 6 below. The secondary excipient compounds were selected from compounds having a non-ionic or cationic charge at pH6 and having an imidazoline or benzene ring, as listed in table 6 below. Formulations of these excipients were prepared in distilled water for viscosity measurement in the following manner. The combination of primary and secondary excipients was dissolved in distilled water, and the pH of the resulting solution was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve pH6, followed by dissolution of the model protein. Once the excipient solution has been prepared in distilled water, the test protein (bovine gamma globulin (BGG)) is solubilized at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in distilled water with excipients was formulated in a 20mL vial and allowed to shake overnight at 100rpm on an orbital shaker table. The BGG solution was then transferred to a 2mL microcentrifuge tube and centrifuged in an IEC micromamax microcentrifuge at 2300rpm for 10 minutes to remove entrapped air before viscosity measurements were taken.
Viscosity measurements of the formulations prepared as described above were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of the solutions with excipients were normalized to the viscosity of the model protein solution without excipients and are 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. The examples show that the combination of a primary excipient and a secondary excipient may produce better results than a single excipient.
TABLE 6
Figure BDA0002748917970000701
Example 11: preparation of formulations containing excipient Compounds and PEG
Materials: all materials were purchased from Sigma-Aldrich (st. louis, MO). Formulations were prepared using the excipient compound and PEG, which was intended to mimic the therapeutic pegylated protein to be used in the therapeutic formulation. The formulation is prepared by mixing equal volumes of PEG solution with excipient solution. Both solutions were prepared in Tris buffer pH 7.3 consisting of 10mM Tris, 135mM NaCl, 1MM trans-cinnamic acid. PEG solutions were prepared by mixing 3g of poly (ethylene oxide) (Aldrich catalog number 372781) with 97g of Tris buffer, with an average Mw of 1,000,000. The mixture was stirred overnight to completely dissolve.
Examples of excipient solutions prepared are as follows: a solution of approximately 80mg/mL citric acid in Tris buffer was prepared by dissolving 0.4g citric acid (Aldrich Cat. No. 251275) in 5mL Tris buffer and adjusting the pH to 7.3 with a minimal amount of 10M NaOH solution. The PEG excipient solution was prepared by mixing 0.5mL of PEG solution with 0.5mL of excipient solution and by mixing for several seconds using vortex. Control samples were prepared by mixing 0.5mL of PEG solution with 0.5mL of Tris buffer solution.
Example 12: viscosity measurement of formulations containing excipient compounds and PEG
Viscosity measurements of the prepared formulations were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample.
The results are presented in table 7, showing the effect of the added excipient compound in reducing viscosity.
TABLE 7
Figure BDA0002748917970000711
Figure BDA0002748917970000721
Example 13: preparation of Pegylated BSA with 1 PEG chain/BSA molecule
To the beaker were added 200mL of phosphate buffered saline (Aldrich Cat. No. P4417) and 4g of BSA (Aldrich Cat. No. A7906) and mixed with a magnetic bar. Next, 400mg of methoxypolyethylene glycol maleimide, MW 5,000, (Aldrich catalog No. 63187) was added. The reaction mixture was allowed to react at room temperature overnight. The next day, 20 drops of HCl 0.1M were added to stop the reaction. The reaction product was characterized by SDS-Page and SEC which clearly showed pegylated BSA. The reaction mixture was placed in Amicon centrifuge tubes with a molecular weight cut-off (MWCO) of 30kDa and concentrated to a few milliliters. Next, the sample was diluted 20-fold with 50mM histidine buffer at pH of about 6, followed by concentration until a high viscosity fluid was obtained. The final concentration of the protein solution was obtained by measuring the absorbance at 280nm and using the extinction coefficient of 0.6678 against BSA. The results indicated that the final concentration of BSA in solution was 342 mg/mL.
Example 14: preparation of Pegylated BSA with multiple PEG chains/BSA molecule
A5 mg/mL solution of BSA (Aldrich A7906) in phosphate buffer (25mM, pH 7.2) was prepared by mixing 0.5g of BSA with 100mL of buffer. Next, 1g methoxy PEG propionaldehyde (Mw ═ 20,000) (jenkemttechnology, plato, TX75024) was added followed by 0.12g 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 (10mM Tris, 135mM NaCl, pH 7.3) and concentrated using Amicon centrifuge tubes with MWCO of 30kDa until a concentration of approximately 150mg/mL was reached.
Example 15: preparation of Pegylated Lysozyme with multiple PEG chains/Lysozyme molecule
A5 mg/mL solution of lysozyme (Aldrich L6876) in phosphate buffer (25mM, pH 7.2) was prepared by mixing 0.5g of lysozyme with 100mL of buffer. Next, 1g methoxy PEG propionaldehyde (Mw ═ 5,000) (JenKem Technology, plato, TX75024) was added followed by 0.12g sodium cyanoborohydride (Aldrich 156159). The reaction was allowed to proceed overnight at room temperature. The next day, the reaction mixture was diluted 49-fold with phosphate buffer (25mM, pH 7.2) and concentrated using Amicon centrifuge tubes with MWCO of 30 kDa. The final concentration of the protein solution was obtained by measuring the absorbance at 280nm and using an extinction coefficient of 2.63 for lysozyme. The final concentration of lysozyme in the solution was 140 mg/mL.
Example 16: effect of excipients on the viscosity of Pegylated BSA with 1 PEG chain/BSA molecule
Formulations of pegylated BSA (from example 13 above) with vehicle were prepared by adding 6 or 12 mg of vehicle salt to 0.3mL of the pegylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured at a shear rate of 500/s by a RheoSense microVisc equipped with a10 channel (100 micron depth). Viscometer measurements were done at ambient temperature. The results are presented in table 8, showing the effect of the added excipient compound in reducing viscosity.
TABLE 8
Figure BDA0002748917970000741
Example 17: effect of excipients on the viscosity of Pegylated BSA with multiple PEG chains/BSA molecules
A formulation of pegylated BSA (from example 14 above) with Na citrate salt as an excipient was prepared by adding 8 mg of the excipient salt to 0.2mL of the pegylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured at a shear rate of 500/s by a RheoSense microVisc equipped with a10 channel (100 micron depth). Viscometer measurements were done at ambient temperature. The results are presented in table 9, showing the effect of the added excipient compound in reducing viscosity.
TABLE 9
Figure BDA0002748917970000751
Example 18: effect of excipients on the viscosity of Pegylated Lysozyme with multiple PEG chains/Lysozyme molecule Sound box
A formulation of pegylated lysozyme (from example 15 above) with potassium acetate as a vehicle was prepared by adding 6 mg of the vehicle salt to 0.3mL of a solution of pegylated lysozyme. The solutions were mixed by gentle shaking and the viscosity was measured at a shear rate of 500/s by a RheoSense microVisc equipped with a10 channel (100 micron depth). Viscometer measurements were done at ambient temperature. The results are presented in the following table (table 10), showing the benefit of the added excipient compound in reducing viscosity.
Watch 10
Figure BDA0002748917970000752
Example 19: protein formulations containing excipient combinations
Formulations are prepared using an excipient compound or a combination of two excipient compounds and a test protein, wherein the test protein is intended to mimic a therapeutic protein to be used in a therapeutic formulation. These formulations were prepared with different excipient compounds in 20mM histidine buffer for viscosity measurements performed in the following manner. The excipient combination was dissolved in 20mM histamine (Sigma-Aldrich, st. louis, MO) and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve pH6 before dissolution of the model protein. The excipient compounds for this example are listed in table 11 below. Once the excipient solution has been prepared, the test protein (bovine gamma globulin (BGG)) is solubilized at a rate to achieve a final protein concentration of about 280 mg/mL. Solutions of BGG in vehicle solutions were prepared in 5mL sterile polypropylene tubes and shaken overnight at 80-100rpm on an orbital shaker table. The BGG solution was then transferred to a 2mL microcentrifuge tube and centrifuged in an IEC micromamax microcentrifuge at 2300rpm for 10 minutes to remove entrapped air before viscosity measurements were taken.
Viscosity measurements of the formulations prepared as described above were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of the solutions with excipients were normalized to the viscosity of the model protein solution without excipients, and these results are shown 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
Figure BDA0002748917970000771
Example 20: protein formulations containing excipients for reducing viscosity and reducing injection pain
The formulation is prepared using an excipient compound, a second excipient compound, and a test protein, wherein the test protein is intended to mimic a therapeutic protein to be used in a therapeutic formulation. First excipient compound excipient a is selected from the group of compounds with local anesthetic properties. First excipient a and second excipient B are listed in table 12. These formulations were prepared in 20mM histidine buffer using excipient a and excipient B in such a way that their viscosity could be measured. The amount of excipient disclosed in table 12 was dissolved in 20mM histamine, and the pH of the resulting solution was adjusted with a small amount of sodium hydroxide or hydrochloric acid to achieve pH6, followed by dissolution of the model protein. Once the excipient solution has been prepared, the test protein (bovine gamma globulin (BGG)) is dissolved in the excipient solution at a rate to achieve a final protein concentration of about 280 mg/mL. Solutions of BGG in vehicle solutions were prepared in 5mL sterile polypropylene tubes and shaken overnight at 80-100rpm on an orbital shaker table. The BGG-excipient solution was then transferred to a 2mL microcentrifuge tube and centrifuged in an IEC micromamax microcentrifuge at 2300rpm for about 10 minutes to remove entrapped air before viscosity measurements were taken.
Viscosity measurements of the formulations prepared as described above were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosities of the solutions with excipients were normalized to the viscosity of the model protein solution without excipients, and these results are shown 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
Figure BDA0002748917970000781
Example 21: formulations containing excipient compounds and PEG
Formulations were prepared using an excipient compound and PEG, where the PEG was intended to mimic a therapeutic pegylated protein to be used in a therapeutic formulation, and where the excipient compound was provided in an amount as listed in table 13. These formulations were prepared by mixing equal volumes of PEG solution with excipient solution. Both solutions were prepared in Deionized (DI) water. PEG solutions were prepared by mixing 16.5g of poly (ethylene oxide) (Aldrich catalog number 181986) with an average Mw of 100,000 with 83.5g of DI water. The mixture was stirred overnight to completely dissolve.
Excipient solutions were prepared by this general method and as detailed in table 13 below: a solution of tripotassium phosphate (Aldrich catalog number P5629) at about 20mg/mL in DI water was prepared by dissolving 0.05g of potassium phosphate in 5mL of DI water. The PEG excipient solution was prepared by mixing 0.5mL of PEG solution with 0.5mL of excipient solution and by mixing for several seconds using vortex. Control samples were prepared by mixing 0.5mL of PEG solution with 0.5mL of DI water. The viscosity was measured and the results are recorded in table 13 below.
Watch 13
Figure BDA0002748917970000791
Example 22: improved handling of protein solutions with excipients
Two BGG solutions were prepared by mixing 0.25g of solid BGG with 4mL of buffer solution. For sample a: the buffer solution was 20mM histidine buffer (pH 6.0). For sample B: the buffer solution was 20mM histidine buffer containing 15mg/mL caffeine (pH 6). Dissolution of solid BGG was performed by placing the sample in an orbital shaker set at 100 rpm. It was observed that the buffered sample containing the caffeine excipient dissolved 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 sample was placed in 2 separate Amicon Ultra 4 centrifugal filter devices with a 30kDa molecular weight cut-off and the sample was centrifuged at 2,500rpm for 10 minute intervals. The volume of filtrate recovered after every 10 minutes of centrifugation was recorded. The results in table 14 show faster recovery of filtrate for sample B. In addition, sample B remained concentrated with each additional run, but sample a reached the point of maximum concentration and further centrifugation produced further sample concentrations.
TABLE 14
Figure BDA0002748917970000801
Example 23: protein formulations 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 0.18g solid BGG with 0.5mL 20mM histidine buffer at pH 6. Each buffer solution contained a different excipient or combination of excipients as described in the following table (table 15). The viscosity of the solution was measured as described in the previous examples. The results show that the hindered amine excipient caffeine can be combined with known excipients such as arginine and that the combination has better viscosity reducing properties than the excipient alone.
Watch 15
Figure BDA0002748917970000811
Arginine was added to a 280mg/mL solution of BGG in histidine buffer at pH 6. As shown in table 16, at levels above 50mg/mL, the addition of more arginine did not further reduce the viscosity.
TABLE 16
Figure BDA0002748917970000812
Figure BDA0002748917970000821
Caffeine was added to a 280mg/mL solution of BGG in histidine buffer at pH 6. As shown in table 17, at levels above 10mg/mL, the addition of more caffeine did not further reduce the viscosity.
TABLE 17
Figure BDA0002748917970000822
Example 24: caffeine Effect in TFF concentration Process
In this example, a Bovine Gamma Globulin (BGG) solution was concentrated in the presence and absence of caffeine using Tangential Flow Filtration (TFF). The experiment was performed using a Labscale TFF system produced by EMD Milbpore (Billerica, MA). The system was equipped with a Pellicon XL TFF cassette comprising an Ultracel membrane (EMD Millipore, Billerica, MA) with a molecular weight cut-off of 30 kDa. Nominal surface area of the film was 50cm 2. The feed pressure to the cassette was maintained at 30psi and the retentate pressure at 10 psi. During the experiment, the filtrate flux was monitored by measuring the filtrate mass as a function of time. About 12 grams of BGG was dissolved in 500mL of buffer comprising 15mg/mL caffeine, 150mM NaCl and 20mM histidine, pH adjusted to 6. A control sample was prepared by dissolving 12 g of BGG in 500mL of a buffer containing 150mM NaCl and 20mM histidine, pH adjusted to 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 the test and control samples during TFF was measured by mass transfer coefficient. The mass transfer coefficient of the sample was determined using the following formula (as described in J.Hung, A.U.Borwankar, B.J.Dear, T.M.Truskett, K.P.Johnston, High concentration permanent flow amplification of stable monoclonal antibody solutions with low viscosity viscosities.J.Memb.Sci.508, 113-126 (2016)):
J＝kcln(Cw/Cb) (formula 3)
Equation 3 describes the filtrate flux J, where kcIs the mass transfer coefficient, CwIs the protein concentration in the vicinity of the membrane, CbIs the concentration in the bulk of the liquid. Thus, the curve in equation 3 allows the mass transfer coefficient k to be calculated c. Relative to ln (C)b) The graph of the calculated flux J yields a linear plot with a slope of-kc. Here, the flux J is determined by taking the mass of the filtrateThe derivative with respect to time is calculated, and CbIs calculated using mass balance. Table 18 lists the mass transfer coefficients for the best fit. The introduction of caffeine at 15mg/mL increases the value of the mass transfer coefficient by-13%, from 22.5 to 25.4Lm-2/h。
Watch 18
Sample (I) Mass transfer coefficient kc(LMH)
Control 22.5±0.1
Caffeine 15mg/mL 25.4±0.1
Example 25: caffeine Effect in TFF concentration Process
In this example, a Bovine Gamma Globulin (BGG) solution was concentrated in the presence and absence of caffeine using Tangential Flow Filtration (TFF). The experiment was performed using a Labscale TFF system produced by EMD Milbpore (Billerica, MA). The system was equipped with a Pellicon XL TFF cassette comprising an Ultracel membrane (EMD Millipore, Billerica, MA) with a molecular weight cut-off of 30 kDa. Nominal surface area of the film was 50cm2. Control samples were prepared by dissolving 14.6 grams of BGG in 582mL of buffer containing 150mM NaCl and 20mM histidine, pH adjusted to 6, such that the initial BGG concentration was nominally 25.1 mg/mL. The material was filtered through a 0.2 μm PES filter (VWR, Radnor, PA) and then processed in a TFF apparatus. The pump speed was adjusted to give an initial feed pressure of 30psi and the retentate valve was adjusted to give an initial retentate pressure of 10 psi. Concentrating the material for 4.1 hr without adjusting Pump speed or retentate valve. As shown in Table 19 below, the initial and final concentrations were 25.4. + -. 0.6 and 159. + -. 6mg/mL, respectively, as determined by the Bradford assay. Caffeine containing samples were prepared by dissolving 14.2 grams of BGG in 566mL of buffer containing 15mg/mL caffeine, 150mM NaCl and 20mM histidine, pH adjusted to 6, such that the initial BGG concentration was nominally 25.1 mg/mL. The material was filtered through a 0.2 μm PES filter (VWR, Radnor, PA) and then processed in a TFF apparatus. The pump speed and retentate valve were set to the same levels as before. As previously described, the feed pressure and retentate pressure were determined to be 30psi and 10psi, respectively. The material was concentrated for 4.1 hours without adjusting the pump speed or retentate valve. As shown in Table 19 below, the initial and final concentrations were 24.4. + -. 0.5 and 225. + -. 10mg/mL, respectively, as determined by the Bradford assay. The use of caffeine during the TFF treatment increased the final protein concentration by about 42%, from 159 to 225mg/mL compared to the control.
Watch 19
Figure BDA0002748917970000841
Example 26: caffeine action 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 produced from tap water using the Direct-Q3 UV purification system of EMD Millipore (Billerica, MA). A25 mM Polyethersulfone (PES) filter with 0.2 μm pores was purchased from GE Healthcare (Chicago, IL, Cat. No. 6780-2502). A1 mL Luer-Lok syringe was purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, reference 309628). A20 mM histidine buffer, pH 6.0, was prepared using L-histidine and deionized water and titrated to pH 6.0 with 1M HCl. A15 mg/mL caffeine solution was prepared using histidine buffer. The BGG was reconstituted with caffeine-free and caffeine-containing buffers to a final concentration of about 280 mg/mL. Protein concentration c was calculated using the following formula:
Figure BDA0002748917970000851
Wherein m ispFor protein mass, b is the volume of buffer added, v is the BGG partial specific volume, taken here as 0.74 mL/g. The viscosity of each sample was measured using a microVisc rheometer (RheoSense, San Ramon, CA) at a temperature of 23 ℃ and a shear rate of 250/s. The energy required to pass the BGG solution through the sterile filter was measured using a tension Compression Tester (TCT, Instron, needleham, MA, part No. 3343) equipped with a 100N load cell (Instron, needleham, MA, part No. 2519-103). The syringe plunger was depressed 50mM at a rate of 159 mM/min. The energy requirements were calculated by integrating the load versus extension curves measured by TCT and the results are summarized in table 20 below.
Watch 20
Figure BDA0002748917970000852
Example 27: excipients for improved chromatographic elution of protein A
Four purified, research-grade, biosimilar antibodies ipilimumab (ipilimumab), ustekinumab (usekinumab), omalizumab (omalizumab), and tocilizumab (tocilizumab) were purchased from Bioceros (urtrecht, The Netherlands). They were provided as frozen aliquots having protein concentrations of 20, 26, 15 and 23mg/mL in 40mM aqueous sodium acetate, 50mM tris-HCl buffer, pH5.5, respectively. Prior to the measurement, the protein solution was thawed at room temperature and then filtered through a 0.2 μm polyethersulfone filter. The filtered protein stock solution was mixed at a ratio of 1:1 protein stock solution to binding buffer. The binding buffer used to facilitate binding of the antibody to the protein a resin consisted of 0.1M sodium phosphate and 0.15 sodium chloride in Deionized (DI) water at ph 7.2. Deionized water was produced by purifying tap water using the Direct-Q3UV purification system of EMD Millipore (Billerica, MA). These solutions are used to use PIERCE TMProtein A binding and elution studies performed by Protein-A Spin Plate for IgG Screening (ThermoFisher, scientific catalog number 45202). The plateThere were 96 wells, each well containing 50. mu.L of protein A resin. The resin was washed with binding buffer, which included adding 200. mu.L of binding buffer to each well and centrifuging the plate at 1000Xg for 1 min and discarding the flow-through (flow-through). All subsequent centrifugation steps were performed at 1000Xg for 1 minute. The washing procedure was repeated once. After these initial washing steps, diluted protein samples, i.e. samples containing ipilimumab (ipilimumab), ustekumab (ustekinumab), omalizumab (omalizumab) and tocilizumab (tocilizumab), were added to the wells of the plate (200 μ L per well). The plate was then placed on an orbital shaker of Daigger Scientific (Vernon Hills, IL) Labgenius and stirred at 260rpm for 30 minutes, after which the plate was centrifuged and the flow-through (flow-through) discarded. The wells were then washed by adding 500 μ L binding buffer to each well, centrifuging the plate, and discarding the flow-through (flow-through). The washing step was repeated twice. After these washing steps, the proteins were eluted from the plate using elution buffers with different excipients added. For each elution, 50 μ L of neutralization buffer at pH7 consisting of 1M sodium phosphate was added to each well of the collection plate, followed by 200 μ L of elution buffer added to each well of the plate. The plate was stirred at 260rpm for 1 minute and then centrifuged. The flow-through was recovered for analysis. The elution step was repeated once. Control buffer without excipients contained 20mM citrate, pH 2.6. Since protein a elution buffers typically include a certain amount of salt, an elution buffer of 100mM 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. All excipients were purchased from Sigma Aldrich (st. louis, MO), except, aspartame from Herb Store USA (Los Angeles, CA), trehalose from Cascade Analytical Reagents and Biochemicals (Corvallis, OR), and sucrose from Research Products International (mt. prospect, IL, product No. S24060). All elution buffers containing excipients were prepared by mixing the appropriate amount of excipient with about 10mL of a no-salt citrate buffer control. Elution buffer was prepared with about 100mM excipient. However, not all excipients are soluble at this level. Thus, table 21 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 exclusion chromatography (SEC) analysis was performed using a TSKgel SuperSW3000 column (30cm x 4.6mm ID, Tosoh Bioscience, King of Prussia, PA) connected to an HPLC workstation (Agilent HP1100 system). The separation was carried out at room temperature at a flow rate of 0.35 mL/min. The mobile phase was 100mM sodium phosphate, 300mM sodium chloride in aqueous buffer, pH 7. Protein concentration was monitored by absorbance at 280nm using an Agilent1100 series G1315B diode array detector. The total amount of protein eluted from the protein a resin was estimated for each protein (i.e., ipilimumab (ipilimumab), ustekumab (ustekinumab), omalizumab (omalizumab), and tocilizumab (tocilizumab)) by integrating the chromatogram. Tables 22-25 list the complete peak areas for each protein (i.e., ipilimumab (ipilimumab), ustekumab (ustekinumab), omalizumab (omalizumab), and toclizumab (tocilizumab)). Tables 22-25 also compare the experimental peak areas to the peak areas of the no salt and salt containing controls. Values greater than 100% indicate that more protein was recovered from the protein a resin by the elution buffer than the control, while values less than 100% indicate that less protein was recovered from the protein a resin by the elution buffer than the control.
TABLE 21
Figure BDA0002748917970000881
TABLE 22 recovery of ipilimumab (ipilimumab) from protein A resin
Figure BDA0002748917970000882
Figure BDA0002748917970000891
TABLE 23 recovery of Ultecumab from protein A resin (ustekinumab)
Figure BDA0002748917970000892
Figure BDA0002748917970000901
TABLE 24 recovery of omalizumab from protein A resin
Figure BDA0002748917970000902
TABLE 25 recovery of Tolizumab (tocilizumab) from protein A resin
Figure BDA0002748917970000903
Figure BDA0002748917970000911
Example 28: excipients for improved elution by protein A chromatography
The test proteins used in this example were the same as in example 27, i.e. ipilimumab (ipilimumab), ustekumab (usekinumab), omalizumab (omalizumab) and tocilizumab (tocilizumab). Protein a binding and elution studies were performed using the same plates as in example 27. The method of 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 elutions were performed. However, in the present example, washing was performed only once. Elution buffer was prepared as in example 27 from 20mM citrate, ph2.6 control buffer. The 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 protein recovery results for each protein (i.e., ipilimumab (ipilimumab), ustekumab (ustekinumab), omalizumab (omalizumab), and toclizumab (tocilizumab)) are recorded in the following tables 26 to 30.
TABLE 26 excipients used in example 28
Figure BDA0002748917970000912
Figure BDA0002748917970000921
TABLE 27 recovery of ipilimumab (ipilimumab) from protein A resin
Figure BDA0002748917970000922
TABLE 28 recovery of Ultekemab (Ustekinumab) from protein A resin
Figure BDA0002748917970000923
Figure BDA0002748917970000931
TABLE 29 recovery of tositumomab (tocilizumab) from protein A resin
Figure BDA0002748917970000932
TABLE 30 recovery of omalizumab from protein A resin
Figure BDA0002748917970000933
Example 29: excipients for improved elution of omalizumab from protein a chromatography columns
Study-grade omalizumab was purchased from Bioceros (urtrecht, The Netherlands) and provided as 15mg/mL frozen in 40mM aqueous sodium acetate, 50mM tris-HCl buffer, ph 5.5. Proteins were thawed at room temperature prior to the experiment and filtered through a 0.2 μm polyethersulfone filter. The filtered material was mixed with a binding buffer consisting of 20mM sodium phosphate at pH7 in DI water in a ratio of 1: 1. Tap water was purified using the Direct-Q3 UV purification system of EMD Millipore (Billerica, MA) to produce deionized water. Protein A purification was performed using a HiTrap Protein-A HP 1mL chromatography column from GE Healthcare (Chicago, IL, product number 29048576). For each experiment, the column was first equilibrated with 10mL of binding buffer. After equilibration, 30mg of protein was loaded onto the protein a column. The column was then washed with 5mL of binding buffer. After washing the column, bound omalizumab was eluted from the column using a fraction of one of the elution buffers containing the excipients listed in table 31 below. The elution buffer was prepared by dissolving the indicated excipients in 20mM citrate buffer at ph 4.0. All elution buffers were adjusted to ph 4.0. Five l-mL fractions were collected. Finally, protein A was regenerated by washing the column with 5mL of 100mM citrate buffer, pH 3.0. The flow rate for each step was 1mL/min, maintained by a Fusion 100 infusion pump (Chemyx, Stafford, TX). A10 mL NormJect Luer Lok syringe (Henke Sass Wolf, Tuttlingen, Germany, reference 4100-000V0) was used.
The total protein content of the elution fractions E1, E2, E3, E4 and E5 was determined by high performance Size Exclusion Chromatography (SEC) analysis. SEC analysis was performed using a TSKgel SuperSW3000 column (30 cm. times.4.6 mm ID, Tosoh Bioscience, King of Prussia, Pa.) attached to an HPLC workstation (Agilent HP1100 System). The separation was carried out at room temperature at a flow rate of 0.35 mL/min. The mobile phase was 100mM sodium phosphate, 300mM sodium chloride in aqueous buffer, pH 7. Protein concentration was monitored by absorbance at 280nm using an Agilent1100 series G1315B diode array detector. The total amount of protein eluted from the protein a resin was estimated by integrating the chromatogram.
Citrate is a commonly used excipient in protein a chromatography and therefore is used here as a control. The eluate fraction of the control sample stored overnight at 4 ℃ showed insoluble aggregates, as evidenced by the formation of a precipitated phase. Thus, the peak areas reported in table 31 below represent the total soluble protein mass in the eluate fractions. We note that insoluble aggregates were only observed in the control sample, and none of the other samples showed such aggregates. The peak area was greater than that of the control (using citrate excipient) indicating that the use of the test excipient allows more efficient separation of the protein from the column.
Watch 31
Figure BDA0002748917970000951
Example 30: BGG formulations with varying amounts of caffeine excipients
Formulations were prepared with varying molar concentrations of caffeine (concentrations listed in table 32 below) and test proteins intended to mimic the therapeutic proteins that would be used in a therapeutic formulation. The formulation used in this example was prepared in 20mM histidine buffer for viscosity measurement in the following manner. Stock solutions of 0 and 80mM caffeine were prepared in 20mM histidine and the pH of the resulting solution was adjusted with a small amount of sodium hydroxide or hydrochloric acid to reach pH6 prior to dissolution of the model protein. Additional solutions of various caffeine concentrations were provided by mixing the two stock solutions in different volume ratios to provide a range of caffeine-containing solutions, the concentrations of which are listed in table 32 below. Once these excipient solutions were prepared, the test protein (bovine gamma globulin (BGG)) was dissolved in each test solution at a rate to achieve a final protein concentration of about 280mg/mL, which was accomplished by adding 0.7mL of each excipient solution to 0.25g of lyophilized BGG powder. The BGG containing solution was prepared in a 5mL sterile polypropylene tube and shaken overnight at 100rpm on an orbital shaker table. These solutions were then transferred to 2mL microcentrifuge tubes and centrifuged in an IEC MicroMax microcentrifuge at 2400rpm for 5 minutes to remove entrapped air before viscosity measurements were taken.
Viscosity measurements of the formulations prepared as described above were performed with a microVisc viscometer (RheoSense, San Ramon, CA). The viscometer was equipped with an A-10 chip with a channel depth of 100 microns and operated at a shear rate of 250/s and 25 ℃. To measure viscosity, the test formulation was loaded into the viscometer, taking care to remove all air bubbles on the pipette. The pipette with the loaded sample formulation was placed in the instrument and allowed to incubate at the measurement temperature for about 5 minutes. The instrument was then run until the channel was fully equilibrated with the test fluid (indicated by a stable viscosity reading), and the viscosity was then recorded in centipoise. The viscosity results obtained are shown in table 32 below.
Watch 32
Figure BDA0002748917970000961
Figure BDA0002748917970000971
Compounds used as co-solutes that increase the solubility of caffeine in water are available from Sigma-Aldrich (st. louis, MO) and include nicotinamide, proline, procaine HCl, ascorbic acid, 2, 5-dihydroxybenzoic acid, lidocaine, saccharin, potassium acetamidosulfonate, tyramine, and aminobenzoic acid. Solutions of each co-solute were prepared by dissolving the dried solid in deionized water and adjusting the pH to a value between about pH 6 and about pH 8 with 5M hydrochloric acid or 5M sodium hydroxide as needed in some cases. The solution was then diluted to a final volume of 25mL or 50mL using a class a volumetric flask and the concentration was recorded according to the mass of dissolved compound and the final volume of the solution. The prepared solution can be used directly or after being 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 ℃) was evaluated by the following method. Caffeine dry powder (Sigma-Aldrich, st. louis, MO) was added to a 20mL glass scintillation vial and the quality of caffeine was recorded. In some cases, 10mL of the co-solute solution prepared according to example 31 was added to the caffeine powder. In other cases, a mixture of co-dissolved solution and deionized water was added to the caffeine powder, maintaining a final addition of 10 mL. The volume contribution of the caffeine dry powder is negligible in 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 vials were observed for dissolution of the caffeine dry powder and the results are listed in table 33 below. These observations indicate that niacinamide, procaine HCl, sodium 2, 5-dihydroxybenzoate, saccharin sodium salt, and tyramine chloride salt all gave caffeine solubility at least four times the reported caffeine solubility limit (-16 mg/mL at room temperature as described by Sigma-Aldrich).
Watch 33
Figure BDA0002748917970000981
Figure BDA0002748917970000991
CD is completely dissolved; most of MD is dissolved; DND ═ undissolved
Example 33:
Figure BDA0002748917970000992
is characterized by
Figure BDA0002748917970000993
(AbbVie Inc., Chicago, IL) is a commercially available formulation of the therapeutic monoclonal antibody adalimumab (adalimumab), a TNF- α blocker, commonly used to reduce autoimmune diseaseInflammatory responses to diseases such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, crohn's disease, ulcerative colitis, moderate to severe chronic psoriasis and juvenile idiopathic arthritis.
Figure BDA0002748917970000994
Sold in a single use dose of 0.8mL, which includes 40mg adalimumab, 4.93mg sodium chloride, 0.69mg sodium dihydrogen phosphate monohydrate (sodium dihydrogen monobasic dihydrate), 1.22mg sodium dihydrogen phosphate dihydrate (sodium dihydrogen monobasic dihydrate), 0.24mg sodium citrate, 1.04mg citric acid monohydrate, 9.6mg mannitol, and 0.8mg polysorbate 80. The viscosity versus concentration profile of this formulation is generated in the following manner. An Amicon Ultra 15 centrifugal concentrator with a molecular weight cut-off of 30kDa (EMD-Millipore, Billerica, MA) was filled with approximately 15mL of deionized water and the membranes were rinsed by centrifugation at 4000rpm for 10 minutes in a Sorvall Legend RT (ThermoFisher Scientific). Thereafter, the remaining water was removed, and 2.4mL of
Figure BDA0002748917970000995
The liquid formulation was added to the concentrator tube and centrifuged at 4000rpm for 60 minutes at 25 ℃. The concentration of the retentate was determined by diluting 10. mu.L of the retentate with 1990. mu.L of deionized water, measuring the absorbance of the diluted sample at 280nm, and calculating the concentration using a dilution factor of 1.39mL/mg-cm and an extinction factor. The viscosity of the concentrated samples was measured at 23 ℃ with a microVisc viscometer equipped with an A05 chip (RheoSense, San Ramon, Calif.) at a shear rate of 250/s. After measuring the viscosity, the sample was diluted with a small amount of filtrate and the measurement of concentration and viscosity was repeated. As shown in table 34 below, this method was used to generate viscosity values at different adalimumab concentrations.
Watch 34
Figure BDA0002748917970001001
Example 35: improved stability of adalimumab solutions with coffee as an excipient
The stability of adalimumab solutions with and without caffeine excipients was evaluated after exposing the samples to two different stress conditions (agitation and freeze/thaw). Use of adalimumab pharmaceutical formulations
Figure BDA0002748917970001002
(AbbVie) having the characteristics described in more detail in example 33. As described in example 38, in the original buffer solution
Figure BDA0002748917970001003
The sample was concentrated to a concentration of 200mg/mL adalimumab. This concentrated sample was designated "sample 1". A second sample was prepared as described in example 40 with-200 mg/mL adalimumab and 15mg/mL caffeine; the concentrated sample with caffeine added is referred to as "sample 2". Both samples were diluted with diluent as described below to a final adalimumab concentration of 1 mg/mL: the sample 1 diluent was the original buffer solution and the sample 2 diluent was 20mM histidine at pH 5, 15mg/mL caffeine. Two kinds of
Figure BDA0002748917970001004
The dilutions were filtered through a 0.22 μm syringe filter. For each diluted sample, 3 batches of 300 μ L of each sample were prepared in 2mL Eppendorf tubes in a laminar flow cabinet. The samples were placed under the following stress conditions: for agitation, the samples were placed in an orbital shaker at 300rpm for 91 hours; for freeze/thaw, samples were cycled 7 times at-17 to 30 ℃ for an average of 6 hours each time. Table 36 describes the samples prepared.
Watch 36
Sample # Added excipients Stress condition
1-C Is free of Is free of
1-A Is free of Agitation
1-FT Is free of Freeze/thaw
2-C Caffeine 15mg/mL Is free of
2-A Caffeine 15mg/mL Agitation
2-FT Caffeine 15mg/mL Freeze/thaw
Example 36: evaluation of stability by Dynamic Light Scattering (DLS)
The hydrodynamic radius of adalimumab molecules in the samples of example 35 were measured using a Brookhaven Zeta Plus dynamic light scattering instrument and looked for evidence of aggregate formation. Table 37 shows the DLS results for 6 samples prepared according to example 35: some of them (1-A, 1-FT, 2-A and 2-FT) had been exposed to stress conditions ("stress samples"), while others (l-C and 2-C) had not. The DLS data in table 37 and figures 1, 2 and 2 show the multimodal particle size distribution of monoclonal antibodies in the stress sample without caffeine. In the absence of caffeine as an excipient, stressed samples 1-A and 1-FT showed a higher effective diameter than unstressed sample 1-C, and in addition, they showed a second population of particles with significantly higher diameters. This new grouping of particles with larger diameters is evidence of aggregation as sub-visible particles. The stressed samples containing caffeine (samples 2-A and 2-FT) showed only one population of particles with particle sizes similar to those of the unstressed sample 2-C. These results indicate that the addition of caffeine to these samples can reduce the formation of aggregates or sub-visible particles.
Watch 37
Figure BDA0002748917970001021
Tables 38A and 38B show the DLS raw data for adalimumab samples from example 36, which shows the particle size distribution. In these tables, G (d) is the intensity weighted differential particle size distribution. C (d) is the cumulative intensity weighted differential particle size distribution.
TABLE 38A
Figure BDA0002748917970001022
TABLE 38B
Figure BDA0002748917970001031
Example 37: evaluation of stability by Size Exclusion Chromatography (SEC)
Sub-visible particles having a size of less than about 0.1 micron were detected from the stressed and unstressed adalimumab samples described in example 36 using size exclusion chromatography. For 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. mu.L of each stressed and unstressed sample from example 36 was isocratically eluted with pH6.2 buffer (100mM phosphate, 325mM NaCl) at a flow rate of 0.35 mL/min. The retention time of adalimumab monomer was about 9 minutes. No detectable aggregates were found in the samples containing the caffeine excipient and the amount of monomers remained constant in all three samples.
Example 38:
Figure BDA0002748917970001032
viscosity reduction of formulations
Obtaining the monoclonal antibody trastuzumab in the form of a lyophilized powder
Figure BDA0002748917970001033
From Genentech) and reconstituted to 21mg/mL in deionized water. The resulting solution was concentrated as such in an Amicon Ultra4 centrifuge concentrator tube (molecular weight cut-off, 30kDa) by centrifugation at 3500rpm for 1.5 hours. The concentration was measured by diluting the sample 200-fold in the appropriate buffer and measuring the absorbance at 280nm using an extinction coefficient of 1.48 mL/mg. The viscosity was measured using a RheoSense microVisc viscometer.
An excipient buffer containing one or a combination of salicylic acid and caffeine is prepared by dissolving histidine and the excipient in distilled water and then adjusting the pH to an appropriate level. The conditions of buffer systems 1 and 2 are summarized in table 39.
Watch 39
Figure BDA0002748917970001041
Will be provided with
Figure BDA0002748917970001042
The solution was diluted in excipient buffer at a ratio of about 1:1 and concentrated in an Amicon Ultra 15(MWCO 30kDa) concentration tube. The concentration was determined using the Bradford assay and compared to a standard calibration curve extracted from the sample. The viscosity was measured using a RheoSense microVisc viscometer. Various kinds of
Figure BDA0002748917970001043
The concentration and viscosity measurements of the solutions are shown in table 40 below, where buffer systems 1 and 2 refer to those buffers described in table 39.
Watch 40
Figure BDA0002748917970001044
The maximum viscosity reduction at 215mg/mL for buffer system 1 containing both salicylic acid and caffeine was 76% compared to the control sample. Buffer system 2 with caffeine alone showed up to a 59% reduction in viscosity at 200 mg/mL.
Example 39:
Figure BDA0002748917970001051
viscosity reduction of formulations
Obtained as a 25mg/mL solution in histidine buffer
Figure BDA0002748917970001052
(monoclonal antibody bevacizumab formulation, sold by Genentech). The sample was concentrated in Amicon Ultra 4 centrifugal concentrator tube (MWCO 30kDa) at 3500 rpm. The viscosity was measured by RheoSense microVisc and the concentration was determined by the absorbance at 280nm (extinction coefficient, 1.605 mL/mg). The excipient buffer was prepared by adding 10mg/mL caffeine and 25mM histidine HCl. Will be provided with
Figure BDA0002748917970001053
The stock solution was diluted with excipient buffer and then concentrated in Amicon Ultra 15 spin-concentration tubes (MWCO 30 kDa). The concentration of the excipient samples was determined by Bradford analysis and the viscosity was measured using a RheoSense microVisc. The results are shown in table 41 below.
Table 41
Figure BDA0002748917970001054
And contrast with
Figure BDA0002748917970001055
Compared with the sample, the sample has the advantages that,
Figure BDA0002748917970001056
a maximum viscosity reduction of 73% was shown when concentrated to 213mg/mL with 10mg/mL caffeine.
Example 40: preparation of formulations containing caffeine, minor excipients and test proteins
Formulations are prepared using caffeine as an excipient compound or a combination of caffeine and a second excipient compound, and a test protein intended to mimic a therapeutic protein to be used in a therapeutic formulation. Formulations were prepared for viscosity measurements in 20mM histidine buffer with different excipient compounds in the following manner. The excipient combination (excipients a and B, as described in table 42 below) was dissolved in 20mM histidine and the pH of the resulting solution was adjusted to pH 6 with a small amount of sodium hydroxide or hydrochloric acid before dissolving the model protein. Once the excipient solution was prepared, the test protein (bovine gamma globulin (BGG)) was solubilized at a rate to achieve a final protein concentration of about 280 mg/mL. The BGG solution in vehicle solution was prepared in 20mL glass scintillation vials and shaken overnight at 80-100rpm on an orbital shaker table. The BGG solution was then transferred to a 2mL microcentrifuge tube and centrifuged in an IEC micromamax microcentrifuge at 2300rpm for about 10 minutes to remove entrapped air before viscosity measurements were taken.
Viscosity measurements of the formulations prepared as described above were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient. 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.
Watch 42
Figure BDA0002748917970001071
Example 41: preparation of formulations containing dimethyl sulfone and test protein
Formulations were prepared using dimethyl sulfone as an excipient compound (Jarrow Formulas, Los Angeles, CA) and a test protein intended to mimic the therapeutic protein to be used in a therapeutic formulation. Formulations were prepared in 20mM histidine buffer for viscosity measurement in the following manner. Dimethyl sulfone was dissolved in 20mM histidine and the pH of the resulting solution was adjusted with a small amount of sodium hydroxide or hydrochloric acid to reach pH 6 before dissolving the model protein and then filtered through a 0.22 micron filter. Once the excipient solution was prepared, the test protein (bovine gamma globulin (BGG)) was solubilized at a protein concentration of about 280 mg/mL. The BGG solution in vehicle solution was prepared in 20mL glass scintillation vials and shaken overnight at 80-100rpm on an orbital shaker table. The BGG solution was then transferred to a 2mL microcentrifuge tube and centrifuged in an IEC micromamax microcentrifuge at 2300rpm for about 10 minutes to remove entrapped air before viscosity measurements were taken.
Viscosity measurements of the formulations prepared as described above were performed with a DV-IIT LV cone-plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3rpm and 25 ℃. The formulation was loaded into the viscometer in a volume of 0.5mL and allowed to incubate at the given shear rate and temperature for 3 minutes followed by a measurement collection period of twenty seconds. Then, 2 additional steps were followed, including a 1 minute shear incubation followed by a twenty second measurement collection period. The three data points collected were then averaged and recorded as the viscosity of the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient. 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.
Watch 43
Dimethyl sulfone concentration (mg/mL) Normalized viscosity
0 1.00
15 0.92
30 0.71
50 0.71
30 0.72
Example 42: preparation of buffer solution
A buffer of 20mM 2- (N-morpholine) ethanesulfonic acid (MES), 50mM glycine and 35MM caffeine was prepared by dissolving 0.392g MES monohydrate, 0.374g glycine and 0.682g caffeine in 90mL Milli-Q ultrapure water. After all the contents were dissolved, the solution pH was adjusted to 5.5 by adding Milli-Q ultrapure water in a volumetric flask to a final volume of 100 mL. The buffer solution was then vacuum filtered through a 0.2 μm PES filter using a bottle top filter set. Similar buffers containing 20mM histidine, 50mM glycine and 35mM caffeine were also prepared in the same manner.
A control buffer of 20mM TRIS (hydroxymethyl) aminomethane (TRIS), 100mM sodium chloride, 55MM mannitol, and 0.1MM diethylenetriaminepentaacetic acid (DTPA) was prepared by dissolving 1.211g TRIS, 2.938g sodium chloride, 2.098g mannitol, and 0.019g DTPA in 450mL Milli-Q ultrapure water. After all the contents were dissolved, the solution pH was adjusted to 7.0 by adding Milli-Q ultrapure water in the volumetric flask and the volume was adjusted to 500 mL. The buffer solution was vacuum filtered through a 0.2 μm PES filter using a bottle top filter set.
Example 43: ipimumab preparation
Samples of monoclonal antibody ipilimumab (ipilimumab) were obtained from Bioceros (The Netherlands) and The buffers were exchanged for The three prepared buffers of example 42 using Amicon Ultra 15 centrifugal concentrator tubes (EMD Millipore, Billerica, MA) with a molecular weight cutoff of 30 kDa. The target final protein concentration was 20mg/mL and was measured by absorbance at 280nm (a280) read using a Synergy HT plate reader (BioTek, Winooski, VT). The absorbance of the protein solution was subtracted from the absorbance of the blank buffer solution. The absorbance of the blank protein solution was subtracted, divided by the reported extinction coefficient, and then multiplied by a protein dilution factor (20 ×) to determine the final protein concentration. Since caffeine interferes with the absorbance measurement at 280nm, the protein concentration of the caffeine-containing solution is determined by mass balance relative to the measured a280 protein solution. This gives an approximate concentration close to the a280 protein solution measured on a mass basis.
The prepared protein solution was then added to 384 microwell plates (Aurora Microplates, whitefhish, MT). Each solution was loaded into three wells at a concentration of 35 μ Ι _ per well. The plates were then centrifuged at 400x g in a Sorvall Legend RT centrifuge to remove all encapsulated air pockets. A precut pressure sensitive sealing tape (Thermo Scientific) was applied on top of the microplate to prevent evaporation and then placed into a DLS instrument (DynaPro II DLS plate reader, Wyatt Technology corp. The DLS instrument sample chamber was maintained at 65 ℃ and the particle size of the protein solution was recorded for 9 hours. Table 44 shows the radius size of ipilimumab (ipilimumab) in three different formulations over a 1 hour measurement.
Watch 44
Figure BDA0002748917970001101
Example 44: testing of protein formulation stability by Urea denaturation
Urea is known to denature proteins in solution and to unfold proteins. The screening method in this example involves adding urea at a specific concentration to a therapeutic protein solution, such as ustekinumab. The test example is based on the following assumptions: protective excipients will prevent or reduce unfolding of the therapeutic protein in the presence of urea, and measuring the amount of unfolded protein will allow one to identify excipients that are effective in stabilizing the protein in the presence of urea. One method of tracking protein unfolding involves the use of an extrinsic fluorescent dye (such as Sypro orange). Sypro orange binds to a hydrophobic region in the unfolded protein structure, resulting in an increase in the observed fluorescence signal. Thus, measuring the difference in fluorescence intensity of the unfolded protein-Sypro orange complex in the presence of different excipients allows one to identify any stabilization 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 excipients were prepared by dissolving each excipient at a concentration of 100mg/mL in 20mM histidine buffer, ph 6.0. Histidine buffer was prepared by dissolving 1.55g histidine in 0.500L Milli-Q water and adjusting the pH to 6.0 using 1M HCl. Then, a protein formulation containing excipients was prepared by combining each excipient formulation (final concentration of 5mg/mL) with Ultekumab (ustekinumab) (final concentration of 1 mg/mL). A stock solution of 9M urea for this example was prepared by dissolving 27g of urea in the same histidine buffer and adjusting the pH to 6.0 using 1M HCl. This urea stock solution was then added to a final concentration of 6M to produce the test solution (excipient plus protein plus urea). Sypro orange dye (5000X) was then incorporated into the stock solution to a final concentration of 20X each. The pH of the mixture was rechecked and confirmed to be pH 6.0. The test solution was incubated at room temperature for 30 minutes. 200 μ L of samples were transferred to Greiner CellStar black well clear flat bottom 96-well plates and the fluorescence of each sample was measured using a BioTek Synergy HT plate reader with 485nm excitation and 590/20nm emission filter. The fluorescence intensity of the different test formulations was compared to the fluorescence intensity of the control formulations (protein and urea without any excipients), and those test formulations that exhibited reduced fluorescence were considered to include stabilizing excipients. As shown in table 45 below, many excipients reflected increased stability compared to the control. These conclusions were drawn because the ability of the excipient to stabilize the protein correlates with its ability to reduce fluorescence as measured during the experiment: the stabilizing excipient prevents or reduces protein unfolding, resulting in a reduction in protein-Sypro orange interaction, which manifests itself in a decrease in fluorescence intensity. The results of these tests are summarized in table 45 below.
TABLE 45
Test number Excipient Increased stability%
1 Castanospermine (Castanospermine) 7.0
2 Theanine 6.1
3 4-Phenylbutyric acid 9.0
4 Para aminobenzoic acid 2.7
5 Arabinol 2.7
6 Sedoheptulose 3.7
7 Nicotinamide 2.7
8 Xylitol, its preparation method and use 6.3
9 Isonicotinic acid 4.4
10 Spermine 16.8
11 Spermidine 12.9
12 Cystamine 8.8
13 Neomycylamine 2.8
14 Tryptamine 11.6
15 Cytidine 1.6
16 Methylcytidine 4.0
17 Benzamide oximes 1.4
18 Nicotinamide adenine dinucleotide 32.5
19 Adenosine (I) 53.6
20 Melezitose 4.7
21 Cotton seed candy 1.7
Example 45: stabilization of protein formulations at low pH
Therapeutic proteins (particularly antibodies) are exposed to low pH solutions during various stages of processing, particularly during purification and virus clearance. Exposure to acidic pH conditions can result in conformational changes that in turn lead to protein unfolding and aggregation. Screening methods to identify stable excipients involve incubating a therapeutic protein, such as omalizumab, at acidic pH. Protective excipients will prevent or reduce unfolding of the therapeutic protein at low pH, and therefore, measuring the amount of unfolded protein will allow one to identify excipients that are effective in stabilizing the protein in the presence of low pH. The unfolding of therapeutic proteins at acidic pH can be followed using an external fluorescent dye (such as Sypro orange). As performed in this example, the addition of Sypro orange (Thermo-Fisher, Waltham MA) allows the dye to bind to hydrophobic regions in the unfolded protein, resulting in an increase in the fluorescence signal. Thus, measuring the difference in fluorescence intensity of the unfolded protein, Sypro orange, in the presence of different excipients allows one to identify the stabilizer.
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 excipients were prepared by dissolving each excipient at a concentration of 100mg/mL in 0.15M glycine buffer, pH 2.6. The acidification buffer was prepared by dissolving 1.65g of histidine in 0.09L Milli-Q water, adjusting the pH to 2.6 using 1M HCl, and bringing the volume to 0.100L. Then, a protein formulation containing excipients was prepared by combining each excipient formulation (final concentration of 5mg/mL) with Ultekumab (ustekinumab) (final concentration of 1 mg/mL). Glycine acidification buffer was then added to the excipient-protein mixture before incorporation of Sypro orange dye (5000X) in the stock solution to a final concentration of 20X. 200 μ L of the samples were 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 485nm and an emission filter wavelength of 590/20 nm. The fluorescence intensity of the different test formulations was compared to the fluorescence intensity of the control formulation (protein and glycine buffer without any excipients), and those test formulations that exhibited reduced fluorescence were considered to include a stabilizing excipient. As shown in table 46, many excipients reflected increased stability when their fluorescence was compared to that of the control, as compared to the control. These conclusions were drawn because the ability of the excipient to stabilize the protein correlates with its ability to reduce fluorescence as measured during the experiment: the stabilizing excipient prevents or reduces protein unfolding, resulting in a reduction in protein-Sypro orange interaction, which manifests itself in a decrease in fluorescence intensity. The results of these tests are summarized in table 46 below.
TABLE 46
Figure BDA0002748917970001141
Figure BDA0002748917970001151
Example 46: excipient as heat stabilizerTest (2)
Therapeutic proteins are often subject to temperature fluctuations that can lead to changes in tertiary and secondary structural elements. This can lead to protein aggregation and reduce the amount of active native protein. Excipients that resisted thermal stress were identified in this example by thermal degradation studies, with or without 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 excipients were prepared by dissolving each excipient at a concentration of 100mg/mL in 20mM histidine buffer, ph 6.0. Histidine buffer was prepared by dissolving 1.55g histidine in 0.500L Milli-Q water and adjusting the pH to 6.0 using 1M HCl. Then, a protein formulation containing excipients was prepared by combining each excipient formulation (final concentration of 5mg/mL) with Ultekumab (ustekinumab) (final concentration of 1 mg/mL). The formulation was aliquoted into 0.2mL microcentrifuge tubes and incubated in a heat block at 65 ℃ for 120 minutes. Aliquots were taken at 0, 15, 30, 60, 90 and 120 minutes. The samples were quenched on ice for 5 minutes and centrifuged at 5000rpm for 10 minutes. The samples were then analyzed by size exclusion-HPLC, where the supernatant was loaded onto an Agilent 1100HPLC system equipped with a TSKgel SW3000 column (30cm x 4.8mm ID) and an Agilent G1351B diode array detector set at 280 nm. A mobile phase of 50mM phosphate buffer, 100mM NaCl, pH 6.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the monomer peak area and the change in the integrated peak area was plotted as a function of time. Thermostability was correlated with the fraction of monomer remaining at the end of the 2 hour incubation.
Watch 47
Added excipients % change in monomer fraction from control
Sedoheptulose 3.4
Melezitose 2.0
Arabinol 6.0
Pullulan polysaccharide 1.5
Adenosine (I) 8.4
Nicotinamide adenine dinucleotide 9.1
Example 47: testing of excipients as stabilizers against mechanical shear stress
Therapeutic proteins are often mechanically stressed by agitation, stirring, etc., and the applied shear stress can cause the protein to aggregate. Excipients that provide protection against shear stress were determined by agitating 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 excipients were prepared by dissolving each excipient in Milli-Q water at a concentration of 100 mg/mL. Then, an excipient-containing protein formulation was prepared by combining each excipient formulation (final concentration of 0.5mg/mL) with omalizumab (final concentration of 2 mg/mL). The samples were transferred to 0.5mL cryovials (ThermoFisher, Waltham MA) and fixed on an orbital shaker. Then, they were incubated at 22 ℃ for 72 hours with stirring at 300 rpm. At the end of incubation, 100 μ L 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 changes in light scattering at 350nm and comparing these changes to the light scattering at 350nm of a control formulation (prepared identically to the test sample, but without the addition of excipients). Protective excipients were identified by their ability to slow the rate of aggregation and reduce the a350 absorbance value after the agitation step.
Watch 48
Figure BDA0002748917970001171
Example 48: freeze/thaw stability of protein solutions with 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 the test excipients (as shown in table 49) were prepared by dissolving each excipient at a concentration of 100mg/mL in 20mM histidine buffer, pH 6.0. The buffer was prepared by dissolving 1.55g of histidine in 0.500L Milli-Q water and adjusting the pH to 6.0 using 1M HCl. Then, an excipient-containing protein formulation was prepared by combining each excipient formulation (final concentration of 5mg/mL) with omalizumab (final concentration of 5 mg/mL). 0.4mL of each formulation was transferred to a well in a 96-well polypropylene plate (Advanene, IL). Samples were frozen to-80 ℃ in a freezer and then thawed at room temperature for at least 5 cycles before transferring 100 μ Ι _ of the samples to Greiner CellStar black well clear flat bottom 96-well plates. The stability effect of the excipients was analyzed by measuring the formation of protein aggregates using light scattering analysis at 350nm and then comparing these changes with the light scattering at 350nm of the control formulation (prepared identically to the test sample, but without the addition of excipients). Protective excipients were identified by their ability to slow the rate of aggregation and reduce the a350 absorbance value after the agitation step.
Watch 49
Excipient Light scattering at 350nm relative to control
Arabinogalactan 15.3
Cotton seed candy 3.7
Melezitose 7.4
Pullulan polysaccharide 4.0
Sedoheptulose 16.0
Arabinol 17.8
Iditol 11.7
Psicose 15.3
Meglumine 15.3
DTPA 17.2
Allyl cysteine 4.9
Isonicotinamides 4.8
Xylitol, its preparation method and use 10.7
Mannitol 17.8
DExample 49: excipient testing by DLS diffusion interaction parameter k
Stock solutions of 20mM histidine hydrochloride (His HCl) buffer were prepared by dissolving 3.1g histidine (Sigma-Aldrich, St. Louis, Mo.) in type 1 ultra pure water for formulation of excipients and protein solutions. The resulting solution was titrated to pH 6 by dropwise addition of 1M hydrochloric acid. After adjusting the pH, the buffer was diluted to a final volume of 1L 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 Sigma Aldrich (st. louis, MO) or Cayman Chemical (Ann Arbor, MI).
A series of six test protein solutions were prepared using the proteins described in Table 50, with protein concentrations ranging from about 4mg/mL to about 20mg/mL, all in 20mM His HCl buffer at pH 6. In 384-well Microplates (Aurora Microplates, whitefly, MT), 15 μ L of the protein solution was combined with 15 μ L of a stock excipient solution (prepared in 20mM His HCl buffer at pH 6 using the excipients described in table 50) such that each excipient was tested at 6 different protein concentrations. Microwell plates containing protein-excipient combinations were placed in The Sorvall Legend RT centrifuge was centrifuged at 400x g and then shaken on a shaker to mix the samples thoroughly. The second centrifugation step was completed to remove air bubbles. Diffusion interaction parameters (k) for these protein-excipient formulationsD) The effect of excipients on protein-protein interactions (PPIs) was explored by Dynamic Light Scattering (DLS) in dilute solutions. For DLS studies, the microplates prepared above were loaded into a DynaPro II DLS plate reader (Wyatt Technologies corp., Goleta, CA) and the diffusion coefficient of each sample was measured at 25 ℃. For each test solution containing excipients, the measured diffusion coefficients were plotted as a function of protein concentration and the slope of the linear fit of the data was recorded as kD. Negative kDA larger value indicates a stronger net attractive PPI, while a positive kDThe larger the value, the stronger the net repulsive PPI. Table 50 lists k for each test solution containing excipientsDValues where the test value for each excipient can be compared to the k of a control solution (containing the protein in histidine buffer, but no excipient)DThe values are compared.
Watch 50
Figure BDA0002748917970001201
Figure BDA0002748917970001211
Example 50: viscosity reducing excipient testing
Biomock monoclonal antibodies omalizumab (omalizumab) and Ultecumab (usekinumab) purchased from Bioceros (The Netherlands) were buffer exchanged to 20mM His HC1 buffer at pH 6 and concentrated using Amicon Ultra 15 centrifugal concentration tubes (EMD Millipore, Billerica, Mass.) with a molecular weight cut-off of 30 kDa. The protein concentration of the resulting concentrated formulation was analyzed by absorbance at 280nm, by preparing serial dilutions of the concentrated formulation in 20mM His HCl, loading 100 μ Ι _ of each dilution in a UV transparent 96 half-well microplate (Greiner Bio-One, Austria), and then measuring the absorbance at 280nm using a Synergy HT plate reader (BioTek, Winooski, VT). The blank, path length corrected absorbance measurements were then divided by the respective extinction coefficients and multiplied by the dilution factor to determine protein concentration. The excipient solution was prepared in 20mM HisHCl at pH 6 at 1OX at the desired final concentration or solubility limit of the compound, and the pH was adjusted to 6 with concentrated hydrochloric acid or sodium hydroxide as needed. The concentrated protein formulation was then combined with a 10X excipient solution of the excipients listed in table 51 below (9 parts protein, 1 part excipient solution or buffer) in 384 well Microplates (Aurora Microplates, whitefhish, 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). The concentration of protein in each sample was the same because each sample was diluted to the same volume. The plates were then centrifuged at 400x g in a Sorvall Legend RT centrifuge and shaken on a plate shaker. After shaking, 2 μ L of 5-fold diluted polyethylene glycol surface-modified gold nanoparticles (nanoComposix, San Diego, CA) in 20mM His HCl was added to each sample well. The microplate was again shaken to mix the gold nanoparticles into the sample, which was then placed into a DynaPro II DLS plate reader (Wyatt Technology corp., Goleta, CA) to measure the apparent particle size of the gold nanoparticles at 25 ℃. The ratio of the apparent particle size of gold nanoparticles in a protein formulation to the known particle size of gold nanoparticles in water was used to determine the viscosity of the protein formulation according to the Stokes-Einstein equation. 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 ℃. The results of these tests are summarized in table 51 below.
Watch 51
Figure BDA0002748917970001221
Figure BDA0002748917970001231
Example 51: thermal degradation test of infliximab (infliximab)
Figure BDA0002748917970001232
Infliximab (infliximab) was obtained from Clinigen Group and reconstituted as described in the Janssen package insert to form a 10mg/mL solution of infliximab (infliximab) in 5mM phosphate buffer, pH of about 7, containing 50mg/mL sucrose and 0.05mg/mL polysorbate 80. The reconstituted drug product is then mixed with 50mM sodium acetate buffer at pH 5 in a volume ratio of 1: 1. The resulting solution was then injected onto a mini preparative cation exchange column (GE Healthcare, Chicago, IL). After infliximab (infliximab) was loaded onto the column, the column was washed with 10 column volumes of 50mM sodium acetate buffer, pH 5. Then, infliximab (infliximab) was eluted from the column with 5 column volumes of 250mM sodium chloride, 50mM sodium acetate buffer, pH 500. The eluted infliximab (infliximab) buffer was then exchanged with 20mM phosphate buffer at pH 7 using an Amicon Ultra 15(EMD Millipore, Billerica, MA) centrifugal concentrator with a molecular weight cutoff of 30 kDa. Stock solutions of 4 or 8mg/mL infliximab (infliximab) in 20mM phosphate buffer at pH 7 were then 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). Stock infliximab (infliximab) solutions were mixed with stock excipient solutions including excipients listed in table 52, formulated in 20mM phosphate buffer at pH 7, to give a final concentration of infliximab (infliximab) in each sample of about 2 mg/mL. Then, samples comprising infliximab (infliximab) solution and stock excipient solution were aliquoted into 5 100 μ L aliquots in PCR tubes. Aliquots were incubated in a dry bath (Benchmark Scientific, Sayreville, NJ) at 55 ℃ for various times, varying from 15 minutes to about 3 hours, to be subjected to thermal stress. Then, once removed from the dry bath, the sample was placed on ice to quench the thermal aggregation. These stressed 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 that monitors absorbance at 280 nm. The HPLC was operated at a column temperature of 25 ℃ with a mobile phase of 100mM phosphate, 300mM NaCl, pH 7, passing through a TSKgel SuperSW30004.6 mM X30cm column (Tosoh Bioscience, Tokyo, Japan) at a flow rate of 0.35 mL/min. For each sample, the monomer peak area was divided by the monomer peak area obtained from the same but unstressed sample to obtain the percentage of monomer remaining after exposure to thermal stress. The percentage of residual monomer to unstressed sample was then plotted as a function of incubation time and the absolute value of the slope linearly fitted to the data was recorded as the monomer loss rate. The monomer loss rates determined were then normalized by dividing the monomer loss rate by the monomer loss rate of the buffer control without excipient, and the results are shown in table 52 below.
Table 52
Figure BDA0002748917970001251
Example 52: DLS of enriched omalizumab (omalizumab) with nicotinamide mononucleotide and itaconic acid Viscosity measurement
Nicotinamide Mononucleotide (NMN) was collected from nutritional supplement capsules purchased from Genex Formulas (Orlando, FL). These substances were used as excipients in the following experiments.
Biomimic buffer of monoclonal antibody omalizumab (omalizumab) obtained from Bioceros (The Netherlands) was exchanged for 20mM His HC1 buffer at pH 6 and concentrated using Amicon Ultra 15 centrifugal concentration tubes (EMD Millipore, Billerica, Mass.) with a molecular weight cutoff of 30 kDa. The buffer was prepared by dissolving 1.55g histidine in 0.5L Milli-Q water and adjusting the pH to 6.0 using 1M HCl. The protein concentration of the resulting concentrated formulation was analyzed by a280, by preparing serial dilutions of the concentrated formulation in 20mM His HCl, loading 100 μ L of each dilution in a UV transparent 96 half-well microplate (Greiner Bio-One, Austria), and then measuring the absorbance at 280nm using a Synergy HT plate reader (BioTek, Winooski, VT). The blank, path length corrected a280 measurements for each sample were then divided by the respective extinction coefficient and multiplied by the dilution factor to determine the protein concentration. Stock excipient solutions were prepared using the above excipients in 20mM HisHCl at pH 6 at the solubility limit of 1M or compound, and pH adjusted to 6 with concentrated hydrochloric acid or sodium hydroxide as required. Then, following 9 parts of protein formulation: ratio of 1 part excipient solution or buffer (for control) the concentrated protein formulation was combined with the stock excipient solution or control before aliquots were added to the wells of 384-well Microplates (Aurora Microplates, whitefh, MT). The plates were then centrifuged at 400x g in a Sorvall Legend RT and shaken on a plate shaker. After shaking the microplate, 2 microliters of 5-fold diluted polyethylene glycol surface-modified gold nanoparticles (nanoginosix, San Diego, CA) with a diameter of 100nm in 20mM His HCl were added to each sample well. The microplate was again shaken to mix the gold nanoparticles into the sample, which was then placed into a DynaPro II DLS plate reader (Wyatt Technology corp., Goleta, CA) to measure the apparent particle size of the gold nanoparticles at 25 ℃. The ratio of the apparent particle size of gold nanoparticles in the protein formulation to the apparent particle size of gold nanoparticles in the buffer (no protein) was used to determine the viscosity of the protein formulation according to the Stokes-Einstein equation. In this example, the 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 ℃. The results using two different excipients are shown in table 53 below.
Watch 53
Figure BDA0002748917970001271
Example 53: DLS viscometry of concentrated omalizumabMeasurement of
4- (2-hydroxyethyl) piperazine-1-ethanesulfonate (HEPES), bicyclic amine hydrochloride, pridinol mesylate, 1-butylimidazole, and 1-hexanimidazole were purchased from Sigma Aldrich (St. Louis, Mo.) and used to prepare the excipient solutions in this example. O- (octylphosphoryl) choline was purchased from Sigma Aldrich (st. louis, MO) as a 1M solution and used as a stock excipient solution in this example.
Biomimic concentrated formulations of The monoclonal antibody omalizumab (omalizumab) obtained from Bioceros (The Netherlands) were prepared as described in example 52 and analyzed for protein concentration as described in example 52. Stock excipient solutions were prepared using the above excipients in 20mM HisHCl pH 6 buffer (prepared as described in example 52) at the solubility limit of 1M or compound, and pH adjusted to 6 with concentrated hydrochloric acid or sodium hydroxide as required. O- (octylphosphoryl) choline was used as a stock excipient without additional preparation. Then, the concentrated protein formulation was mixed with a stock excipient solution or control at a ratio of 9 parts protein formulation; 1 part of the excipient solution or buffer (for control) and aliquots were added to the wells of 384-well Microplates (Aurora Microplates, whitefhish, MT). Then, following 9 parts of protein formulation: ratio of 1 part excipient solution or buffer (for control) the concentrated protein formulation was combined with the stock excipient solution or control before aliquots were added to the wells of 384-well Microplates (Aurora Microplates, whitefh, MT). The plates were then centrifuged at 400x g in a Sorvall Legend RT and shaken on a plate shaker. After shaking the microplate, 2 microliters of 5-fold diluted polyethylene glycol surface-modified gold nanoparticles (nanoginosix, San Diego, CA) with a diameter of 100nm in 20mM His HCl were added to each sample well. The microplate was again shaken to mix the gold nanoparticles into the sample, which was then placed into a DynaPro II DLS plate reader (Wyatt Technology corp., Goleta, CA) to measure the apparent particle size of the gold nanoparticles at 25 ℃. The ratio of the apparent particle size of gold nanoparticles in the protein formulation to the apparent particle size of gold nanoparticles in the buffer (no protein) was used to determine the viscosity of the protein formulation according to the Stokes-Einstein equation. In this example, the 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 ℃. The results using five different excipients are shown in table 54 below.
Watch 54
Figure BDA0002748917970001281
Binding of excipients to proteins leads to reversible precipitation
Example 54: DLS viscosity measurement of concentrated omalizumab
The stock excipient solution of this example was prepared with the following chemicals: tetraethylammonium chloride, tetramethylammonium acetate, 1-methylimidazole, 1-butylimidazole, 1-hexylimidazole, 2-ethylimidazole, 2-methylimidazole and spectinomycin, purchased from Sigma Aldrich (st. Triglycine, tetraglycine and 2-butylimidazole were purchased from Chem-Impex (Wood Dale, IL). Hordenine HC1 was purchased from Bulk Supplements (Henderson, NV).
Biomimic of The monoclonal antibody omalizumab (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 were prepared using the above excipients in 20mM HisHCl pH 6 buffer (prepared as described in example 52) at the solubility limit of 1M or compound, and pH adjusted to 6 with concentrated hydrochloric acid or sodium hydroxide as required. Then, following 9 parts of protein formulation: ratio of 1 part excipient solution or buffer (for control) the concentrated protein formulation was combined with the stock excipient solution or control before aliquots were added to the wells of 384-well Microplates (Aurora Microplates, whitefh, MT). The plates were then centrifuged at 400x g in a Sorvall Legend RT and shaken on a plate shaker. After shaking the microplate, 2 microliters of 5-fold diluted polyethylene glycol surface-modified gold nanoparticles (nano composix, San Diego, CA) with a diameter of 100nm were added to each sample well. The microplate was again shaken to mix the gold nanoparticles into the sample, which was then placed into a DynaPro II DLS plate reader (Wyatt Technology corp., Goleta, CA) to measure the apparent particle size of the gold nanoparticles at 25 ℃. The ratio of the apparent particle size of gold nanoparticles in the protein formulation to the apparent particle size of gold nanoparticles in the buffer (no protein) was used to determine the viscosity of the protein formulation according to the Stokes-Einstein equation. In this example, the 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 ℃. The results using 12 different excipients are shown in table 55 below. Tetraethylammonium chloride, tetramethylammonium acetate, 1-methylimidazole, 1-butylimidazole, 1-hexylimidazole, 2-ethylimidazole, 2-methylimidazole and spectinomycin, purchased from Sigma Aldrich (st. Triglycine, tetraglycine and 2-butylimidazole were purchased from Chem-Impex (Wood Dale, IL). Hordenine HC1 was purchased from Bulk Supplements (Henderson, NV).
Watch 55
Figure BDA0002748917970001301
Example 55: excipients for increasing thermal stability
Therapeutic proteins are often subject to temperature fluctuations that may result in changes in their tertiary and secondary structural elements. This results in aggregation of the protein and a reduction in active native species. Excipients that resist thermal stress were tested by thermal degradation studies in the presence or absence of excipients. An excipient stock solution (prepared as described in example 52) was prepared by dissolving the excipients listed in table 56 below at a concentration of 100mg/mL in 20mM histidine buffer at pH 6. Each test sample was prepared by adding the excipient stock to a buffer to reach a final concentration of 5mg/mL of excipient and diluting the protein from a 20mg/mL Ultezumab (usekinumab) stock to a final concentration of 1mg/mL in histidine buffer (prepared as described in example 51). The formulation was aliquoted into 0.2mL microcentrifuge tubes and incubated in a heating block for 120 minutes at 65 ℃. Aliquots were taken at 0 min, 30 min, 60 min, 90 min and 120 min. The samples were then quenched on ice for 5 minutes and centrifuged at 9000rpm for 10 minutes. Thereafter, the samples were analyzed by size exclusion-HPLC as follows: the supernatant was loaded onto an Agilent 1100HPLC system equipped with a TSKgel SW3000 size exclusion chromatography column (30cm x 4.8mm ID) and an Agilent G1351B diode array detector set to detect at 280 nm. A mobile phase of 0.5% phosphoric acid, 150mM 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 plotting the change in the integrated peak area as a function of time. The thermostability was correlated with the fraction of monomer remaining at the end of the 2h incubation and the increase in the percentage of monomer compared to the control (no excipient added) was recorded. The results for the seven excipients tested are summarized in table 56 below.
Watch 56
Figure BDA0002748917970001311
Figure BDA0002748917970001321
Antibody-drug conjugates (ADCs) are therapeutic proteins that are produced by linking small molecules to monoclonal antibodies through chemical linkers, which enable site-specific delivery of small molecule drugs. The conjugated linker and small molecule combination changes the chemical and physical properties (charge, hydrophobicity, etc.) of the ADC compared to the protein precursor and introduces additional stability problems. The compound of example 59, Ultecorzumab (ustekinumab) -FITC, was used as a model ADC compound for these tests. Excipients that protect model ADCs against thermal 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 100mg/mL in 20mM histidine buffer (prepared as described in example 52) at pH 6.0. Each test sample was prepared by adding the excipient stock to buffer to reach a final concentration of 5mg/mL, and adding Ultekumab (Ultekumab) -FITC (prepared as described in example 59 below) to histidine buffer to reach a final concentration of 1mg/mL Ultekumab-FITC. The formulation was aliquoted into 0.2mL microcentrifuge tubes and incubated in a heating block for 120 minutes at 65 ℃. Aliquots were taken at 0 min, 30 min, 60 min, 90 min and 120 min. The samples were then quenched on ice for 5 minutes and centrifuged at 9000rpm for 10 minutes. Samples were analyzed by SE-HPLC, where the supernatant was loaded onto an Agilent 1100HPLC system equipped with a TSKgel SW3000 size exclusion chromatography column (30cm x 4.8mm ID) and an Agilent G1351B diode array detector set to detect at 280 nm. A mobile phase of 0.5% phosphoric acid, 150mM NaCl, pH 3.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the peak area under the monomer peak and plotting the change in the integrated peak area as a function of time and recording the increase in the percentage of monomer compared to the control (no excipient added). Thermostability was correlated with the fraction of monomer remaining at the end of the 2 hour incubation.
Watch 57
Figure BDA0002748917970001331
Example 57: excipient against freezing/melting stress
Therapeutic proteins are often maintained at low temperatures to improve their kinetic stability and minimize structural perturbations that may lead to protein aggregation and reduction of active native species. In some cases, this may be accomplished by freezing the formulation until use. However, during repeated freezing and thawing, low temperatures, concentration gradients, and ice formation can stress proteins. Excipients that prevent thermal stress were tested and identified 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 1M in 20mM histidine buffer (prepared as described in example 52) at pH 6.0. Each test sample was prepared by adding the excipient stock to the excipient at a final concentration of 100mM and diluting the protein from a 20mg/mL ustekumab (usekinumab) stock to a final concentration of 2mg/mL in histidine buffer (prepared as described in example 50). Controls were prepared in the same manner, but without addition of the excipient stock. Then, the formulation was aliquoted into 0.5mL cryovials and frozen at-80 ℃ for 120 minutes. The samples were then thawed using a water bath maintained at room temperature. The freeze-thaw cycle was repeated 6 times, then each sample was aliquoted into 0.2mL microcentrifuge tubes and centrifuged at 9000rpm for 10 minutes. Samples were analyzed by SE-HPLC and supernatants were loaded onto an Agilent 1100HPLC system equipped with a TSKgel SW3000 size exclusion chromatography column (30cm x 4.8mm ID) and an Agilent G1351B diode array detector set to detect at 280 nm. A mobile phase of 0.5% phosphoric acid, 150mM NaCl, pH 3.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the peak area under the monomer peak and plotting the change in the integrated peak area as a function of time.
Watch 58
Figure BDA0002748917970001341
Example 58: accelerated aging study
Excipients that prevent thermal 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 1M in 20mM histidine buffer (prepared as described in example 52) at pH 6.0. Each test sample was prepared by adding the excipient stock to a final concentration of 100mM and diluting the protein from a 20mg/mL stock of ustekumab (usekinumab) to a final concentration of 2mg/mL in histidine buffer; controls were prepared in the same manner, but without addition of the excipient stock. 1mL of each sample was aliquoted into 2mL glass vials (West Pharmaceutical services, Pa.) and incubated at 40 ℃ for 4 weeks. Aliquots were taken after 0, 1, 2, 3 and 4 weeks. Samples were analyzed by SE-HPLC, where the supernatant was loaded onto an Agilent 1100HPLC system with TSKgel SW3000 size exclusion chromatography column (30cm x 4.8mm ID) and Agilent G1351B diode array detector set to detect at 280 nm. A mobile phase of 0.5% phosphoric acid, 150mM NaCl, pH 3.5 was used at a flow rate of 0.35 mL/min. The monomer fraction was calculated by integrating the peak area under the monomer peak and plotting the change in the integrated peak area as a function of time. Thermostability was correlated with the fraction of monomer remaining at the end of 4 weeks incubation.
Watch 59
Figure BDA0002748917970001351
Example 59: synthesis of Ultezumab (ustekinumab) FITC
Model compounds representing Antibody Drug Conjugates (ADCs) were synthesized in the following manner. Ultekumab (Ustekinumab) was purchased as a lyophilized aliquot from Bioceros (Utrecht, The Netherlands) at a mAb concentration of 26mg/mL in 40mM sodium acetate, 50mM tris-HCl aqueous buffer, pH 5.5. The sample buffer was 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 a spiked 1.6 equivalents of FITC per equivalent of ustekinumab (usekinumab). The average molar ratio of FITC to Ultekumab (dustekinumab) was determined by measuring the absorbance at 280nm (representing protein + FITC) and at 495nm (representing FITC). The calculation used 1.61L/g.cm as the extinction coefficient of the mAb at 280nm, 148,600 as the MW of the mAb, and 68,000L/g.cm as the extinction coefficient of FITC at 495 nm. Excess unreacted FITC was removed by dialysis against 20mM histidine buffer pH 6. Next, the sample was concentrated to 15mg/mL using Amicon 30kDa MWCO centrifuge tubes.
Example 60: ultekemab (ustekinumab) -FITC stability
The ADC model compound of example 59 was diluted to a mAb concentration of 1mg/mL in 20mM histidine buffer (prepared as described in example 52) at pH 6. Samples were prepared using the added excipients listed in table 60 below and tested for their ability to protect model ADC compounds from mechanical shear stress. The samples were mechanically stressed by placing on a shaker table at 300rpm, 72h, 23 ℃. After stressing the solution, the particle size of the ADC complex was determined by dynamic light scattering. The particle radius of the sheared control sample was 143nm, indicating significant agglomeration compared to the unstressed sample (radius 5.5 nm). The sample containing the excipient showed no significant increase in particle radius compared to the unstressed control sample (radius 5.5nm), indicating protection against mechanical shear stress. The results are shown in table 60 below.
Watch 60
Figure BDA0002748917970001361
Example 61: effect of Co-solutes on the solubility of caffeine in aqueous buffers during refrigeration
25mM histidine buffer, pH 6, was prepared by dissolving 0.387g histidine in Milli-Q I type water, titrating to pH 6 with hydrochloric acid, and diluting to a final volume of 100mL with Milli-Q water. The buffer was then used to prepare a 50mM solute solution, using the following excipients: sodium benzoate, 1-methyl-2-pyrrolidone, proline, phenylalanine, arginine monohydrochloride, benzyl alcohol, and nicotinamide. Approximately 0.1g of caffeine was dissolved in a 5mL aliquot of each of the resulting solutions to achieve a caffeine concentration of 20 mg/mL. Different volume ratios of 20mg/mL caffeine to solute solutions and the corresponding excipient solution but no caffeine containing solutions were prepared in triplicate in 96-well microplates, with the total well volume being maintained at 300 μ L in all cases. The resulting microplates were then sealed with a microplate strip and stored in a refrigerator, with the temperature maintained in the range of 2 to 5 ℃. During storage, the microplates were visually observed to find precipitates in the wells. The earliest observed precipitate was recorded for the three wells under each condition and the results are summarized in table 61 below.
Watch 61
Figure BDA0002748917970001371
Figure BDA0002748917970001381
Figure BDA0002748917970001391
(PPT) ═ precipitation was observed
Clear solution was observed
Example 62: testing excipients to reduce the viscosity of antibody solutions
Stock buffer solutions of 20mM histidine HCl pH 6.0(His HCl) were prepared by dissolving 1.55g histidine (Sigma-Aldrich, St. Louis, Mo.) in class I ultra pure water. The contents were completely dissolved and the pH was adjusted to 6.0 using hydrochloric acid solution. After adjusting the pH, the final volume was adjusted to 0.5L in a volumetric flask. All excipients were dissolved in His HCl and prepared at 10x concentration (1M) or the solubility limit of the compound. The pH of the excipient solution was measured and adjusted to 6.0 if necessary.
In this example, the viscosity of a protein solution at an excipient concentration of 0.1M or less was measured. Biomock monoclonal antibody omalizumab (omalizumab) buffer purchased from Bioceros (The Netherlands) was exchanged for His HC1 and concentrated to about 200mg/mL using a pre-washed Amicon-15 centrifuge device (EMD Millipore, Billerica, Mass.) with a molecular weight cutoff of 30 kDa. A dispersion of polyethylene glycol surface modified gold nanoparticles (nanoComposix, San Diego, CA) was mixed well and diluted 5-fold into His HCl. In a separate PCR tube, 2.1. mu.L of gold nanoparticles, 5.3. mu.L of 10 × excipient solution and 47.6. mu.L of concentrated omalizumab (omalizumab) were combined and mixed well. Each solution was transferred twice in 25. mu.L volumes to 384-well plates (Aurora Microplates, Whitedish, MT) and centrifuged (Sorvall Legend RT) at 400x g for 1 min. Tape sealing was used to prevent evaporation of the sample. The plate was then transferred to a DynaPro II DLS plate reader (Wyatt Technology corp., Goleta, CA) to measure the apparent particle size of the gold nanoparticles at 25 ℃. The ratio of the measured apparent radius to the known radius particle size is calculated to determine the viscosity of the protein formulation according to the Stokes-Einstein equation.
In this example, the diffusion interaction parameter (k) of a dilute protein solution was measured by DLS in the presence of 0.1M or less excipientD). From the previously prepared excipient stock solutions, 0.2M excipient solutions were prepared, respectively. K was measured using 5 different protein concentrations (ranging from 10mg/mL to 0.6mg/mL) in the presence of 0.1M excipientD. mu.L of the protein solution was combined with 15. mu.L of a 0.2M excipient solution (1:1 mix) onto 384-well plates (Aurora Microplates, Whitedish, MT). After loading the sample, the well plate was shaken on a plate shaker to mix the contents for 5 minutes. After mixing, the well plate was centrifuged at 400x g for 1 min in a Sorvall Legend RT to clear all air pockets. The well plates were then loaded into a DynaPro II DLS plate reader (Wyatt Technologies corp., Goleta, CA) and the diffusion coefficient of each sample was measured at 25 ℃. For each excipient, the measured diffusion coefficient was plotted as a function of protein concentration and the slope of the linear fit of the data was recorded as kD. The results are summarized in tables 62A and 62B below for two different series of tests.
TABLE 62A
Figure BDA0002748917970001411
Data error, no k for this testDInformation
TABLE 62B
Figure BDA0002748917970001412
Figure BDA0002748917970001421
Equivalent scheme
While specific embodiments of the invention have been disclosed herein, the foregoing description is illustrative and not restrictive. While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. Many variations of the invention will become apparent to those skilled in the art upon reading the specification. Unless otherwise indicated, all numbers expressing reaction conditions, amounts of ingredients, and so forth, 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 set forth herein are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

Claims (32)

1. An enhanced stability formulation comprising a therapeutic protein and a stability-improving amount of a stability excipient, wherein the enhanced stability formulation is characterized by having an improved stability parameter as compared to a control formulation that is otherwise identical to the enhanced stability formulation except for the absence of the stability excipient.
2. The enhanced stability formulation of claim 1, wherein the therapeutic protein is an antibody.
3. The enhanced-stability formulation of claim 2, wherein the antibody is an antibody-drug conjugate.
4. The enhanced stability formulation of claim 1, wherein the stability excipient is a hindered amine compound.
5. The enhanced stability formulation of claim 1, wherein the stabilizing excipient is an anionic aromatic compound.
6. The enhanced stability formulation of claim 1, wherein the stability excipient is a functionalized amino acid compound.
7. The enhanced stability formulation of claim 1, wherein the stability excipient is an oligopeptide.
8. The enhanced stability formulation of claim 1, wherein the stabilizing excipient is a short chain organic acid.
9. The enhanced stability formulation of claim 1, wherein the stability excipient is a low molecular weight polyacid.
10. The enhanced-stability formulation of claim 1, wherein the stability excipient is a diketone compound or a sulfone compound.
11. The enhanced stability formulation of claim 1, wherein the stability excipient is a zwitterionic compound.
12. The enhanced stability formulation of claim 1, wherein the stabilizing excipient is a crowding agent with hydrogen bonding elements.
13. The enhanced stability formulation of claim 1, wherein the stability excipient is added in an amount of about 1mM to about 500 mM.
14. The enhanced stability formulation of claim 13, wherein the stability excipient is added in an amount of about 5mM to about 250 mM.
15. The enhanced stability formulation of claim 14, wherein the stability excipient is added in an amount of about 10mM to about 100 mM.
16. The enhanced stability formulation of claim 15, wherein the stability excipient is added in an amount from about 5mg/mL to about 50 mg/mL.
17. The enhanced stability formulation of claim 1, wherein the improved stability parameter is thermal storage stability.
18. The enhanced stability formulation of claim 17, wherein said heat storage is improved at a temperature between about 10 ℃ and 30 ℃.
19. The enhanced stability formulation of claim 1, wherein the improved stability parameter is improved freeze/thaw stability.
20. The enhanced stability formulation of claim 1, wherein the improved stability parameter is improved shear stability.
21. The enhanced-stability formulation of claim 1, wherein the formulation has a reduced number of particles compared to a control formulation.
22. The enhanced-stability formulation of claim 1, wherein the formulation has improved biological activity compared to a control formulation.
23. A method of improving the stability of a therapeutic formulation comprising adding a stability-improving amount of a stability excipient to a therapeutic formulation, thereby improving the stability of the therapeutic formulation, wherein the stability of the therapeutic formulation is measured relative to the stability of a control formulation that is otherwise identical to the therapeutic formulation except for the absence of the stability excipient.
24. The method of claim 23, wherein the stabilizing excipient is selected from the group consisting of: hindered amine compounds, anionic aromatic compounds, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight polyacids, diketones, sulfones, zwitterionic compounds, or crowding agents with hydrogen bonding elements.
25. The method of claim 23, wherein the step of measuring the stability of the therapeutic formulation compared to the stability of the 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: thermal storage stability, freeze/thaw stability, and shear stability.
27. The method of claim 23, wherein the therapeutic agent 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.
30. A method of improving a parameter of a protein-related process, comprising adding a stability-improving amount of an excipient to a carrier solution of the protein-related process, wherein the carrier solution comprises a protein of interest, thereby improving the parameter.
31. The method of claim 30, wherein the parameter is selected from the group consisting of: protein production cost, protein production capacity, 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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