Camphorsulfonic acid and its combination with cationic excipients as viscosity-reducing agents in highly concentrated protein formulations

Abstract

The present invention relates to highly concentrated protein compositions that exhibit reduced viscosity, which is induced by the addition of at least camphorsulfonic acid. The proteins contained in the prepared formulations are stabilized against aggregation and denaturation and are therefore sufficiently storage-stable until administration to patients.

Description

[Technical Field]

[0001] The present invention relates to a composition of a highly concentrated protein formulation exhibiting reduced viscosity, which is induced by the addition of at least camphorsulfonic acid. The protein contained in the prepared formulation is stabilized against aggregation and denaturation, and is therefore sufficiently storage-stable until administration to a patient. [Background technology]

[0002] technical level Most biotherapeutic protein products under development are in related formats such as monoclonal antibodies (mAbs) or bispecific antibodies or antibody fragments. The therapeutic doses of such products are often high across a wide range of clinically important indications.

[0003] However, peptide and protein molecules are larger and more complex (i.e., they have multiple functional groups in addition to complex three-dimensional structures) than existing organic and inorganic drug molecules. Formulation of such proteins presents specific problems for formulators. One of these problems is the increased viscosity of protein formulations, especially at high concentrations.

[0004] However, the latter is a particular problem because, from the standpoint of patient convenience, compliance, and overall healthcare costs, it is highly desirable that the resulting product be delivered via low-volume subcutaneous injection.

[0005] However, the combination of high therapeutic doses and highly desirable low injection volumes often leads to the need for extremely concentrated formulations of the active ingredient. It is well known that achieving stable aqueous formulations of biotherapeutics at high concentrations can be exceptionally difficult, often leading to high rates of aggregation, particle formation, and a substantial increase in viscosity. High viscosity is unacceptable, as it significantly limits the injectability of the product.

[0006] Antibodies and other protein therapeutics can be administered parenterally, such as by intravenous (IV), intramuscular (IM), or subcutaneous (SC) routes. Subcutaneous injection has attracted increasing attention for the delivery of protein therapeutics due to its potential to simplify patient administration (fast, low-volume injections) and reduce treatment costs (shorter medical support). To ensure patient compliance, subcutaneous injection dosage forms are desirably isotonic and contain small injection volumes (<2.0 ml per injection site). To reduce injection volume, proteins are often administered in the range of 1 mg / ml to 150 mg / ml.

[0007] Thus, the development of protein formulations primarily for subcutaneous administration is often associated with viscosity issues. Volume limitations (<2 ml) and dose requirements (usually >100 mg administered) often require highly concentrated protein formulations. However, at high concentrations, as already mentioned, proteins tend to form highly viscous solutions, and stability can be difficult to resolve due to the formation of soluble and insoluble protein-protein aggregates. Therefore, viscosity is a key factor in determining the viscosity of a protein formulation. a) the manufacturing process, and b) Administration to patients This is a significant challenge.

[0008] In manufacturing processes, highly viscous, highly concentrated protein preparations present difficulties in processing, particularly in ultrafiltration and sterile filtration. Furthermore, the increased viscosity creates increased shear stress on the protein, which frequently results in product loss.

[0009] mAb-based treatments are often administered repeatedly over extended periods and require several mg / kg of dosing. Antibody solutions or suspensions can be administered parenterally, such as by intravenous (IV) infusion, subcutaneous (SC) or intramuscular (IM) injection. Here, high viscosity is a problem in injection solutions. To solve this problem and improve solution stability, high concentrations of additives and excipients are often added as well. At desired protein concentrations for formulations intended for intramuscular or subcutaneous administration, high concentrations of stabilizers (such as sucrose and sodium chloride) are required to achieve long-term protein stability. The resulting solutions often cause injection pain due to high injection force and tissue damage.

[0010] Therefore, it is important to balance the required amount of stabilizer for stability and osmolality of high protein concentration formulations. As a result, technical difficulties due to viscosity often lead to failure in developing protein formulations for subcutaneous delivery.

[0011] To increase the success rate in developing subcutaneous formulations, reducing and controlling viscosity by chemical methods has received considerable attention in recent years. Numerous publications and patent applications refer to excipients from the family of salts (mostly NaCl) and special amino acids (preferably arginine, histidine and proline), which have been shown to be efficient in reducing the viscosity of certain highly concentrated protein therapeutics.

[0012] Unfortunately, these well-known approaches to reduce viscosity are not universally applicable, due to the fact that the viscosity of protein formulations is the result of various intermolecular forces.Depending on protein molecules and their formulation conditions, different interactions can affect viscosity, such as molecular crowding, dipole-dipole or dipole-charge interaction, or the interaction between hydrophobic or charged groups.As a result, the pharmaceutical industry has a strong need for viscosity-reducing excipients, especially as an alternative when the standard solution based on NaCl and amino acids mentioned above fails.

[0013] A large number of viscosity-lowering additives and excipients have been investigated in the past, however, currently not all therapeutic protein solutions that exhibit viscosity problems at high concentrations can be adequately addressed by known viscosity-lowering excipients.

[0014] During bioprocessing, solutions need to be pumped through tubing and chromatography columns. At high viscosities, the flow rate through such columns is limited by the viscosity, which can lead to longer processing times, significant protein loss during chromatography, or complete unprocessability of the protein solution. Furthermore, shear stresses can be generated when passing through connectors from narrow tubing to less narrow columns. Shear stresses are a typical reason why proteins denature and potentially aggregate, thereby reducing process yield. Clearly, such aggregation-induced shear stresses have a negative impact on process economics. Furthermore, the gel bed within the chromatography column can be damaged by high pressure.

[0015] In addition, some proteins are formulated to high concentrations through tangential flow filtration (FFF). When the viscosity of the solution becomes significant, a gel-like layer may form near the membrane. In particular, the membrane flux is significantly reduced, resulting in increased processing time and therefore significantly higher production costs. As previously discussed, shear stress also occurs during TFF, which may result in insoluble protein aggregates and reduced yield.

[0016] It has generally been observed that highly viscous solutions develop a certain stickiness that makes it difficult to completely recover the solution from a vessel, from a tube, or to remove all of the material from a processing system. This loss of material has obvious adverse effects on process economics and results in significantly reduced product yields. Summary of the Invention [Problem to be solved by the invention]

[0017] Object of the invention Protein formulations intended for pharmaceutical use (e.g., monoclonal antibodies, fusion proteins, etc.) often require stabilizers to protect against undesired aggregation and to prevent physical or chemical degradation. These problems are exacerbated at high protein concentrations, which, however, are often desirable for therapeutic administration of this class of molecules.

[0018] At high concentrations, proteins tend to self-associate, resulting in highly viscous formulations and complicating administration of these protein solutions, for example, by injection, but also complicating the manufacturing process, in which tangential flow filtration is often used for buffer exchange and to increase protein concentration. By increasing backpressure and shear stress during filtration and injection, therapeutic proteins are potentially destabilized or the processing time is excessively extended. Consequently, there is a high need within the biopharmaceutical industry for formulation additives and excipients, or combinations thereof, with viscosity-reducing properties. However, formulating proteins such as monoclonal antibodies requires careful selection of formulation additives and / or excipients to avoid protein denaturation and loss of biological activity.

[0019] However, the large number of emerging new antibodies and antibody formats still requires the development of suitable innovative viscosity-lowering additives and / or excipients, or specific additive / excipient combinations, or targeted formulation strategies. Because protein formulations are administered parenterally (including intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous routes), these additives / excipients need to be pharmaceutically safe. Consequently, additives that can be used in these formulations must be physiologically compatible and must not have undesirable side effects and must not, under any circumstances, lead to allergic reactions; in particular, they must not cause any anaphylactic-like side effects.

[0020] Furthermore, a problem to be solved is to provide a combination of excipients that effectively reduces the viscosity of a protein solution.

[0021] Another problem to be solved is that many viscosity-reducing excipients used at relevant concentrations can adversely affect protein stability. Therefore, a further problem to be solved is the provision of an excipient combination that effectively reduces the viscosity of a protein solution and shows improved protein stability compared to one viscosity-reducing excipient used alone at a higher concentration that results in a similar viscosity reduction compared to the combination.

[0022] Highly viscous protein solutions cause numerous difficulties in bioprocessing. Since the known additives used so far to reduce the viscosity of corresponding protein solutions often do not lead to a sufficient viscosity-reducing effect, the object of the present invention is to find new possibilities, by which the corresponding viscosity-reducing effect can be improved and the adverse effects on process economics can be reduced. [Means for solving the problem]

[0023] Subject of the Invention Unexpectedly, in experiments for formulating highly concentrated protein preparations, excipients were found to be suitable, alone or in combination with other excipients, for substantially reducing viscosity.

[0024] The present invention refers to a method for reducing the viscosity of a liquid composition containing a protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, comprising combining the liquid composition with at least camphorsulfonic acid as an excipient at a concentration having a viscosity-reducing effect.

[0025] In accordance with the present invention, an excipient or combination of excipients selected from the group of molecules listed below is added to a highly concentrated protein liquid formulation at an optimized concentration, thereby substantially reducing the viscosity of the protein-containing liquid composition.

[0026] Preferably, the liquid protein composition is combined with camphorsulfonic acid and at least one cationic excipient. Good viscosity reduction is achieved when camphorsulfonic acid is added to a liquid protein composition in combination with at least one cationic excipient selected from the group consisting of arginine, meglumine, ornithine, and carnithine.

[0027] Surprisingly, the excipient combination of the present invention can synergistically reduce the viscosity of a liquid protein composition and, optionally, simultaneously increase protein stability. Surprisingly, protein stability is less negatively affected in relation to viscosity-reducing potential when using the excipient combinations of the present invention compared to when only one viscosity-reducing excipient is used.

[0028] The protein may be a therapeutically or pharmaceutically active protein and may be a protein selected from the group consisting of antibodies, antibody fragments, minibodies, nanobodies, modified antibodies, antibody-like molecules, antibody-drug conjugates, and fusion proteins, whereby the viscosity of the formulation is reduced by at least 12%, preferably by at least 50%. Consequently, an object of the present invention is a liquid protein composition or liquid pharmaceutical formulation having reduced viscosity compared to the same formulation without excipients or without excipients produced by this method.

[0029] Good viscosity reduction is achieved using a liquid pharmaceutical formulation containing a protein, preferably a therapeutic protein, at a concentration of at least 40 mg / ml to 250 mg / ml, preferably at least 90 mg / ml to 250 mg / ml, and when at least camphorsulfonic acid is added as a viscosity-reducing excipient. Particularly good viscosity-reducing effects are achieved when the excipient concentration in the liquid pharmaceutical composition is less than about 500 mM, particularly less than 200 mM, and the liquid pharmaceutical composition has a pH of about 3 to about 8, preferably 4.5 to about 8.0, more preferably about 4.7 to about 7.5, and particularly preferably about 5 to about 7.2, and includes a buffer. The formulation may contain a phosphate buffer or an acetate buffer. Furthermore, the formulation may contain a stabilizer, which may be a sugar or a surfactant, such as sucrose, a resorbate, preferably polysorbate 80, or a poloxamer.

[0030] The reduced-viscosity pharmaceutical formulations prepared according to the present invention can be prepared as lyophilized powders. Such lyophilized powders contain a therapeutic protein and camphorsulfonic acid, where camphorsulfonic acid or a combination of camphorsulfonic acid and a cationic excipient is present in an amount sufficient to yield a concentration of less than 500 mM, preferably less than 200 mM, upon reconstitution, and the protein yields a concentration of at least 40 mg / ml to 250 mg / ml, preferably at least 90 mg / ml to 250 mg / ml. Reconstitution of the powder involves adding a sterile aqueous diluent.

[0031] An object of the present invention is therefore the method as characterized herein, wherein a formulation is prepared, wherein the therapeutic protein is selected from the group of antibodies, antibody fragments, minibodies, nanobodies, modified antibodies, antibody-like molecules and fusion proteins, preferably the therapeutic protein is selected from the group of antibodies, antibody fragments, minibodies, nanobodies, modified antibodies, antibody-like molecules and fusion proteins, and particularly preferably the therapeutic protein is selected from the group of antibodies, antibody fragments, minibodies, nanobodies, modified antibodies, antibody-like molecules and fusion proteins.

[0032] Another object of the present invention is a method for reducing the viscosity of a liquid protein composition in a bioprocess, comprising the step of combining the liquid protein composition with at least camphorsulfonic acid as an excipient at a concentration that has a viscosity-reducing effect in the liquid protein composition.Preferably, the liquid protein composition is combined with camphorsulfonic acid and at least one cationic excipient.Successful viscosity reduction is achieved when camphorsulfonic acid is added to the liquid protein composition in combination with at least one cationic excipient selected from the group consisting of arginine, mercury, ornithine, and carnitine. The subject of the present invention is also a kit containing the previously characterized pharmaceutical formulation or a lyophilized powder of a certain embodiment. The kit may contain a lyophilized or spray-dried preparation of the pharmaceutical composition obtained by the method described above and which can be converted into a solution preparation prior to use. Thus, the corresponding kit may contain a ready-to-use lyophilized or spray-dried formulation in a 96-well plate. The kit may also contain a container, a syringe and / or other administration device with or without a needle, such as an infusion pump, a jet injector, a pen device, a transdermal injector, or other needleless injector, and instructions depending on the application needs of the kit. [Brief explanation of the drawings]

[0033] [Figure 1]FIG. 1 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine, arginine, and camphorsulfonic acid on mAb C formulated in phosphate buffer pH 7.2. [Figure 2] FIG. 2 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine, arginine, and camphorsulfonic acid on mAbD formulated in acetate buffer pH 5.0. [Figure 3] FIG. 3 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine, arginine, and camphorsulfonic acid on mAbE formulated in acetate buffer pH 5.5. [Figure 4] FIG. 4 shows the viscosity-reducing effect of meglumine, L-ornithine, and L-carnitine in combination with camphorsulfonic acid for mAbC formulated in phosphate buffer pH 7.2. [Figure 5] FIG. 5 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine when combined with camphorsulfonic acid for mAbD formulated in acetate buffer pH 5.0. [Figure 6] FIG. 6 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine when combined with camphorsulfonic acid for mAbE formulated in acetate buffer pH 5.5. [Figure 7] FIG. 7 shows the synergistic viscosity-reducing effect of L-arginine in combination with camphorsulfonic acid for mAbC formulated in phosphate buffer pH 7.2. [Figure 8] FIG. 8 shows the residual monomer content of solutions containing approximately 80 mg / mL mAbC in phosphate buffer pH 7.2, with or without excipients, stored at 40° C. / 75% rH for 28 days. [Figure 9] Figures 9-11 highlight the process improvement indicated by the increased permeate flux. [Figure 10] Figures 9-11 highlight the process improvement indicated by the increased permeate flux. [Figure 11]Figures 9-11 highlight the process improvement indicated by the increased permeate flux. [Figure 12] Figures 12-14 show the reduction in processing time that can be achieved using 75 mM cationic or anionic excipients, respectively. [Figure 13] Figures 12-14 show the reduction in processing time that can be achieved using 75 mM cationic or anionic excipients, respectively. [Figure 14] Figures 12-14 show the reduction in processing time that can be achieved using 75 mM cationic or anionic excipients, respectively. [Figure 15] Figures 15 and 16 show the effect of 75 mM cationic and / or anionic excipients, respectively, on protein recovery. [Figure 16] Figures 15 and 16 show the effect of 75 mM cationic and / or anionic excipients, respectively, on protein recovery. [Figure 17] FIG. 17 shows the effect of formulation on average permeation flux. [Figure 18] Figure 18 shows the results of measuring the time it takes for the volume of the formulation to be reduced to 10 ml. [Figure 19] FIG. 19 shows the process yield in the form of protein recovery from stirred cells. [Figure 20] Figure 20 shows the processing time to reach a volume of approximately 0.5 ml. [Figure 21] Figure 21 shows the processing time to reach a volume of approximately 0.5 ml. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description of the Invention As outlined above, high protein concentration presents challenges for the physical and chemical stability of protein in liquid formulations, and difficulties in the manufacture, storage and administration of said protein formulations.The main problem is that protein tends to aggregate and form particles during processing and / or storage, which makes handling difficult during further processing and / or administration.Concentration-dependent degradation and / or aggregation is the main challenge when developing liquid protein formulations with higher concentrations.In addition to the potential for non-native protein aggregation and particle formation, reversible self-association can occur in aqueous solution, which contributes to the increase in viscosity, which complicates delivery by injection, among other things.

[0035] definition The term "protein," as generally used herein, refers to a polymer of amino acids linked together by peptide bonds to form a peptide of sufficient chain length to produce at least detectable tertiary structure. Proteins with a molecular weight greater than about 100 kDa (expressed in kDa, where "Da" stands for "Dalton" and 1 kDa = 1,000 Da) can be designated "high molecular weight proteins," whereas proteins with a molecular weight less than about 100 kDa can be designated "low molecular weight proteins." The term "low molecular weight proteins" excludes small peptides that lack the requisite three-dimensional structure necessary to be considered proteins. Protein molecular weight can be determined using standard methods known to those skilled in the art, including, but not limited to, mass spectrometry (e.g., ESI, MALDI) or calculation from known amino acid sequence and glycosylation. Proteins can be naturally occurring or non-naturally occurring, synthetic, or semi-synthetic.

[0036] "Essentially pure protein(s)" and "substantially pure protein(s)" are used interchangeably herein and refer to a composition comprising at least about 90% by weight pure protein, preferably at least about 95% by weight pure protein. "Essentially homogeneous" and "substantially homogeneous" are used interchangeably herein and refer to a composition in which at least about 90% by weight, preferably at least about 95% by weight, of the protein present is a combination of monomers and reversible di- and oligomeric associates (as opposed to irreversible associates).

[0037] The term "antibody", as generally used herein, broadly covers mAbs (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with epitope specificity, diabodies, bispecific antibodies, and single-chain antibody molecules, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv), single domain antibodies, multivalent single domain antibodies, Fab fusion proteins, and fusions thereof.

[0038] The term "monoclonal antibody" or "mAb," as generally used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies that comprise the population that are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. They are typically synthesized by culturing hybridoma cells as described by Kohler et al. (Nature 256: 495, 1975), or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), or may be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al. (Nature 352: 624-628, 1991) and Marks et al. (J. Mol. Biol. 222: 581-597, 1991).

[0039] As used herein, "mAb" specifically includes derivatized antibodies, antibody-drug conjugates, and "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984).

[0040] "Antibody fragments" include portions of intact antibodies that include the antigen-binding and / or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments; bispecific antibodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8:1057-1062, 1995); single-chain antibody molecules; multivalent single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0041] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) of mostly human sequence, which contain minimal sequence derived from non-human immunoglobulin (e.g., Jones et al., Nature 321:522-525, 1986; Reichmann et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992).

[0042] In this context, the term "therapeutically active protein," "pharmaceutically active protein," or "therapeutic protein" refers to a protein or peptide that is administered to a subject for the purpose of treating or preventing a disease or medical condition, as defined above. In particular, the subject may be a mammal or a human. Therapeutic proteins may be administered for different purposes, such as to replace a missing or abnormal protein, to enhance an existing pathway, to provide a new function or activity, to interact with a molecule or organism, and to deliver other compounds or proteins (such as radionuclides, cytotoxic drugs, or effector proteins).

[0043] Therapeutic proteins include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, modified scaffolding proteins, enzymes, growth factors, hormones, interferons, interleukins, antibody-drug conjugates (ADCs), and thrombolytic drugs. Therapeutic proteins can be naturally occurring proteins or recombinant proteins. Their sequences can be natural or modified.

[0044] "Rheology" refers to the study of the deformation and flow of matter, and "viscosity" refers to the resistance to flow of a substance (typically a liquid). Viscosity is related to the concept of shear stress; it can be understood as the effect of different layers of a liquid exerting shear stress against each other or against another surface as they move against each other. Several viscosity standards exist. The unit of viscosity is Ns / m, also known as the Pascal-second (Pa-s). 2 Viscosity may be "kinematic" or "absolute." Kinematic viscosity is a measure of the rate at which momentum is transmitted through a fluid. It is measured in Stokes (St). Kinematic viscosity is a measure of the resistance of a fluid to flow under the influence of gravity.

[0045] When two fluids of equal volume and different viscosities are placed in the same capillary viscometer and allowed to flow by gravity, the more viscous fluid will take longer to flow through the capillary than the less viscous fluid. If, for example, one fluid takes 200 seconds (s) to complete its flow and another takes 400 s, the second fluid is twice as viscous as the first on the kinematic viscosity scale. The dimension of kinematic viscosity is the length 2 / hour. Kinematic viscosity is generally expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is equal to 1 cSt. "Absolute viscosity" (sometimes called "dynamic viscosity" or "simple viscosity") is the product of kinematic viscosity and fluid density. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is millipascal-second (mPa-s), where 1 cP = 1 mPa-s.

[0046] Viscosity can be measured, for example, using a viscometer at a given shear rate or multiple shear rates. The "estimated zero shear" viscosity can be determined by constructing a best-fit line of the four highest shear points on a plot of absolute viscosity versus shear rate and linearly extrapolating the viscosity to zero shear. Alternatively, for Newtonian flow, viscosity can be determined by averaging viscosity values ​​at multiple shear rates. Viscosity can also be measured using a microfluidic viscometer at single or multiple shear rates (also called flow rates), where absolute viscosity is derived from the change in pressure as the liquid flows through the channel. Viscosity is equal to the shear stress versus the shear rate.

[0047] In some embodiments, the viscosity measured using a microfluidic viscometer can be directly compared to the extrapolated zero-shear viscosity (e.g., extrapolated from viscosities measured at multiple shear rates using a cone-and-plate viscometer). The viscosity of compositions and formulations according to the present invention is reduced when at least one of the above methods demonstrates a viscosity-reducing effect. Preferably, the viscosity is measured using mVROC™ technology. More preferably, the viscosity is measured at 20°C using mVROC™ technology. Most preferably, the viscosity is measured at 20°C using mVROC™ technology and using a 500 μl syringe, a shear rate of 3000 s-1 or 2000 s-1, and a volume of 200 μl. Those skilled in the art are familiar with viscosity measurements using mVROC™ technology, particularly selecting the above parameters. Detailed specifications, methods, and settings can be found in 901003.5.1-mVROC_User's_Manual.

[0048] "Shear rate" refers to the rate of change of velocity at which one layer of fluid passes over an adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plate. This simple case shows a uniform velocity gradient with a shear rate (v1-v2) / h, with units of (CM / SEC) / (CM)=1 / sec. Thus, shear rate units are reciprocal seconds, or more commonly, reciprocal hours. For microflow viscometers, changes in pressure and flow velocity are related to shear rate. "Shear rate" is the speed at which a material deforms. Formulations containing proteins and viscosity-lowering agents typically exhibit a shear rate of about 0.5 s when measured using a cone and plate viscometer and spindle appropriately selected by one of skill in the art to accurately measure the viscosity of the sample in the viscosity range of interest. -1 From about 200s -1 when measured using a micro flow viscometer, shear rates ranging from 0.01 to 1.00 (i.e., a 20 cP sample is most accurately measured with a CPE40 spindle mounted on a DV2T viscometer (Brookfield)); -1 ~about 3,000s -1 is greater than.

[0049] For classical "Newtonian" fluids, as that term is generally used herein, viscosity is essentially independent of shear rate. However, for "non-Newtonian fluids," viscosity either decreases or increases with increasing shear rate (e.g., the fluids are "shear-thinning" or "shear-thickening," respectively). In the case of concentrated (i.e., highly concentrated) protein solutions, this can manifest as pseudoplastic shear-thinning behavior (i.e., a decrease in velocity with shear rate).

[0050] The term "chemical stability," as generally used herein, refers to the ability of protein components in a formulation to resist degradation via chemical pathways, such as oxidation, deamidation, or hydrolysis. A protein formulation is typically considered chemically stable when less than about 5% of the components degrade after 24 months at 4°C.

[0051] The term "physical stability," as generally used herein, refers to the ability of a protein formulation to resist physical deterioration, such as aggregation. A physically stable formulation forms only an acceptable percentage of irreversible aggregates (e.g., dimers, trimers, or other aggregates) of the bioactive protein agent. The presence of aggregates can be assessed in several ways, including measuring the average particle size of the protein in the formulation using dynamic light scattering. A formulation is considered physically stable if less than about 5% irreversible aggregates form after 24 months at 4°C. An acceptable level of aggregated contaminants would ideally be less than about 2%. While levels as low as about 0.2% are achievable, approximately 1% is more typical.

[0052] The term "stable formulation," as generally used herein, means that the formulation is chemically and physically stable. A stable formulation may be one in which approximately 95% of the biologically active protein molecules retain their biological activity after 24 months of storage at 4°C or in an equivalent solution state at a high temperature, such as 1 month of storage at 40°C. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, Ed., Marcel Dekker, Inc., New York, NY (1991) and Jones, A., Adv. Drug Delivery Revs. 10:29-90, 1993. Stability can be measured for a period of time at a selected temperature. For rapid screening, for example, the formulation can be maintained at 40°C for 2 weeks to 1 month, at which point the residual biological activity is measured and compared with the initial conditions to assess stability.

[0053] When a formulation is to be stored at 2-8°C, it should generally be stable for at least one month at 30°C or 40°C, and / or for at least two years at 2°C-8°C. When a formulation is to be stored at room temperature, about 25°C, it should generally be stable for at least two years at about 25°C, and / or for at least about six months at 40°C. The degree of aggregation after lyophilization and storage can be used as an indicator of protein stability. In some embodiments, stability is assessed by measuring the particle size of the protein in the formulation. In some embodiments, stability can be assessed by measuring the activity of the formulation using standard biological activity or binding assays well within the capabilities of one of ordinary skill in the art.

[0054] The term "particle size" as generally used herein refers to the mean diameter of the predominant population of bioactive molecule particles in a formulation, or their particle size distribution, as determined using well-known particle size classification instruments, such as dynamic light scattering, SEC (size exclusion chromatography), or other methods known to those skilled in the art.

[0055] The terms "concentrated" or "highly concentrated," as generally used herein, describe liquid protein formulations having a final protein concentration of at least 1 mg / ml, particularly greater than about 10 mg / mL, preferably greater than about 50 mg / mL, more preferably greater than about 100 mg / mL, even more preferably greater than about 200 mg / mL, or most preferably greater than about 250 mg / mL.

[0056] "Reconstituted formulation," as generally used herein, refers to a formulation prepared by dissolving a dry powder, lyophilized, spray-dried, or solvent-precipitated protein in a diluent such that the protein is dissolved or dispersed in an aqueous solution for administration.

[0057] A "lyoprotectant" is a substance that, when combined with a protein, significantly reduces the chemical and / or physical instability of the protein during lyophilization and / or subsequent storage. Protectants are generally added to the formulation before lyophilization in a "protecting amount." This means that, following lyophilization of the protein in the presence of a protecting amount of the protectant, the protein essentially retains its physical and chemical stability and integrity.

[0058] A "diluent" or "carrier," as generally used herein, is a pharmaceutically acceptable (i.e., safe and non-toxic for administration to humans or another mammal) and useful ingredient for preparing a liquid formulation, such as an aqueous formulation to be reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffer (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution, and combinations thereof.

[0059] "Preservatives" are compounds that can be added to the formulations herein to reduce contamination and / or the action of bacteria, fungi, or other infectious agents. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0060] A "bulking agent," as generally used herein, is a compound added to a lyophilized mixture to increase its mass and contribute to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains a porous structure).

[0061] A "therapeutically effective amount" is the minimum concentration required to effect a measurable improvement or prevention of any symptom or specific condition or disorder, to effect a measurable increase in life expectancy, or to generally improve the quality of life of a patient. The therapeutically effective amount depends on the particular biologically active molecule and the particular condition or disorder being treated. Therapeutically effective amounts of many proteins (such as the mAbs described herein) are well known in the art. The therapeutically effective amount of a protein for treating a particular disorder using a known protein (such as a mAb) that has not yet been established or that is clinically applied to treat additional disorders can be determined by standard techniques that are well within the skill of a physician or other skilled artisan.

[0062] The terms "injectability" or "syringeability," as generally used herein, refer to the ability to inject a pharmaceutical formulation through an 18- to 32-gauge needle, optionally a thin-walled syringe. Injectability depends on factors such as the pressure or force required for injection, uniformity of flow, aspiration performance, and freedom from clogging. The injectability of a liquid pharmaceutical formulation can be assessed by comparing the injection force of the reduced-viscosity formulation with a standard formulation without added viscosity-lowering agent. A reduced injection force for a formulation containing a viscosity-lowering agent reflects the improved injectability of that formulation.

[0063] A reduced-viscosity formulation has improved injectability when the injection force is reduced by at least 10%, preferably at least 30%, more preferably at least 50%, and most preferably at least 75%, compared to a standard formulation having the same concentration of protein under otherwise identical conditions, except that the viscosity-lowering agent is replaced with an appropriate buffer of approximately the same concentration. Alternatively, the injectability of liquid pharmaceutical formulations can be assessed by comparing the time required to inject the same volume (such as 0.5 mL, or more preferably about 1 mL) of different liquid protein formulations when the syringe is constrained with the same force.

[0064] The term "injection force," as generally used herein, refers to the force required to push a given liquid formulation through a given syringe with a given needle gauge at a given injection speed. Injection force is typically reported in Newtons. For example, injection force can be measured as the force required to push a liquid formulation through a 1 mL plastic syringe with a 0.25-inch inner diameter and a 0.50-inch 27-gauge needle at a 250 mm / min injection speed. Testing equipment can be used to measure injection force. When measured under the same conditions, formulations with lower viscosities generally require lower overall injection forces.

[0065] The term "reduced viscosity formulation," as generally used herein, refers to a liquid formulation having a high concentration of a high molecular weight protein (such as a mAb) or a low molecular weight protein that is modified by the presence of one or more additives that reduce the viscosity compared to a corresponding formulation that does not contain the viscosity-lowering additive(s) or agent(s).

[0066] The term "viscosity-lowering agent," as used herein, refers to a compound that acts to reduce the viscosity of a solution compared to the viscosity of the solution in the absence of the viscosity-lowering agent. The viscosity-lowering agent may be a single compound or a mixture of one or more compounds. When the viscosity-lowering agent is a mixture of two or more compounds, the concentrations listed refer to the concentrations of each individual agent unless otherwise specified. As an example, a formulation containing about 0.25 M meglumine benzenesulfonate as the viscosity-lowering agent is a solution having a 0.25 M concentration of benzenesulfonic acid and a 0.25 M concentration of meglumine.

[0067] Some viscosity-lowering agents contain acidic or basic functional groups and may exhibit hydrophilic and hydrophobic regions, which together affect their interaction characteristics with proteins in solution. Whether the functional groups are fully or partially ionized depends on the pH of the formulation in which they are present. Unless otherwise specified, reference to a formulation containing a viscosity-lowering agent with an ionizable functional group encompasses both the parent compound and any viable ionized state.

[0068] The term "liquid formulation" or "formulation," as used herein, is a protein that is either provided in an acceptable pharmaceutical diluent or that is reconstituted in an acceptable pharmaceutical diluent prior to administration to a patient.

[0069] Biosimilars can be produced in microbial cells (prokaryotic, eukaryotic), cell lines of human or animal origin (e.g., mammalian, avian, or insect), or tissues derived from animals or plants. The expression construct for a proposed biosimilar product will generally encode the same primary amino acid sequence as the reference product. Minor modifications, such as N- or C-terminal truncations, that would not affect safety, purity, or potency may be present.

[0070] Biosimilar mAbs are physiochemically or biologically similar to the reference mAb in terms of both safety and efficacy. Biosimilar mAbs can be evaluated against the reference mAb using one or more in vitro studies, including assays detailing binding to target antigen(s); binding to Fc gamma receptor isoforms (FcγRI, FcγRII, and FcγRIII), FcRn, and complement (C1q); Fab-related functions (e.g., neutralization of soluble ligands, receptor activation, or blockade); or Fc-related functions (e.g., antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, complement activation). In vitro comparisons can be combined with in vivo data demonstrating pharmacokinetic, pharmacological, and / or safety similarities.

[0071] Clinical evaluation of a biosimilar mAb against a reference mAb involves evaluating its pharmacokinetic properties (e.g., AUC 0-inf , AUC 0-t , C max , t max , C troughpharmacodynamic endpoints; or similarity of clinical efficacy (e.g., using randomized, parallel-group, controlled clinical trials). Comparative properties between a biosimilar mAb and a reference mAb can be assessed using established procedures, including those described in the "Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: Quality issues" (EMEA / CHMP / BWP / 49348 / 2005) and the "Guideline on development, production, characterization, and specifications for monoclonal antibodies and related substances" (EMEA / CHMP / BWP / 157653 / 2007).

[0072] Differences between a biosimilar mAb and a reference mAb may include post-translational modifications (e.g., by attaching other biochemical groups such as phosphate, various lipids, and carbohydrates to the mAb; by post-translational proteolytic cleavage; by altering the chemical nature of amino acids (e.g., formylation); or by many other mechanisms). Other post-translational modifications may be the result of manufacturing process manipulations—for example, glycation may occur upon exposure of the product to reducing sugars. In other cases, storage conditions may tolerate certain degradation pathways, such as oxidation, deamidation, or aggregation, as all of these product variants may be encompassed in a biosimilar mAb.

[0073] As used herein, the term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids and bases, and organic acids and bases.

[0074] As used herein, the term "alkyl group" refers to straight-chain, branched-chain, and cyclic hydrocarbon groups. Unless otherwise specified, the term alkyl group encompasses hydrocarbon groups containing one or more double or triple bonds. An alkyl group containing at least one ring system is a "cycloalkyl" group. An alkyl group containing at least one double bond is an "alkenyl group," and an alkyl group containing at least one triple bond is an "alkynyl group."

[0075] As used herein, the term "aryl" refers to an aromatic carbocyclic ring system, including fused ring systems. In an "aryl" group, each of the atoms forming the ring is a carbon atom.

[0076] As used herein, "heteroaryl" refers to aromatic ring systems, including fused ring systems, where at least one of the atoms forming the ring is a heteroatom. Furthermore, as used herein, the term "heterocycle" refers to ring systems, including fused ring systems, that are not aromatic, where at least one of the atoms forming the ring is a heteroatom.

[0077] As used herein, the term "heteroatom" is any non-carbon or non-hydrogen atom. Preferred heteroatoms include oxygen, sulfur, and nitrogen.

[0078] The term "bioprocessing" refers to the therapeutic cell manufacturing process, which can be divided into upstream and downstream processes. Upstream processing is defined as the entire process prior to the separation of proteins from cellular compounds. Upstream processing includes cell banking and cell growth, from initial cell isolation and culture to final harvest. The downstream portion of the bioprocess refers to the portion where the target protein is purified from the upstream feed and processed to meet purity and quality requirements. Some types of cells need to be disrupted before entering the downstream process. Still other cells can secrete the target protein into the medium and need to be removed via filtration. Further downstream processing is often divided into two main sections: purification and polishing. Bioprocessing can be a batch process or a semi-continuous or continuous process.

[0079] The term "permeate flux" refers to the volume passing through a given filter in a period of time, typically on the order of minutes.

[0080] The term "filtration step" refers to a process in which a liquid passes through a material with a predetermined pore size, which allows for the separation of materials based on size. For some filters, the pore size is specified in nanometers. For other filters, the pore size is not directly specified, but is given by the weight of the molecules to be retained. The filtration material can be arranged in a manner that blocks the cross section of the filtration device (dead-end filtration). Furthermore, the filtration material can be arranged in a manner that the solution to be filtered flows tangentially through the surface of the material (for example, tangential flow filtration). The filtration material can be a membrane, a glass filter, a metal filter, or a resin. The resin can be held in a chromatography column. The resin can be a cation or anion exchange resin, an affinity resin (such as protein A or glutathione resin), or a hydrophobic or hydrophilic resin.

[0081] The term "protein recovery" after buffer exchange and volume reduction refers to the fraction of protein recovered after the process steps.

[0082] The term "tangential flow filtration" or "TFF" refers to a filtration method in which a solution passes tangentially through a given filter. Materials smaller than the filter pores are forced out of the solution passing through the filter by pressures resulting from solution flow rate, viscosity, temperature, and other factors.

[0083] formulation Biocompatible, low-viscosity protein solutions (such as those of mAbs) can be used to deliver therapeutically effective amounts of protein in volumes useful for subcutaneous (SC) and intramuscular (IM) injection, typically about 2 ml or less for SC and about 5 ml or less for IM, more preferably about 1 ml or less for SC and about 3 ml or less for IM. Proteins generally have any molecular weight, although in some embodiments high molecular weight proteins are preferred. In other embodiments, the protein is a low molecular weight protein.

[0084] The present invention provides a method for reducing the viscosity and optionally improving the stability of a liquid composition containing a protein, comprising combining the liquid composition with a viscosity-reducing amount of camphorsulfonic acid as an excipient, which may be combined with at least one suitable cationic excipient. The cationic excipient may be selected from the group consisting of arginine, meglumine, ornithine, and carnitine, or a mixture thereof. These excipients are added in amounts suitable for the formulation. Preferably, they are added to the liquid protein composition in equimolar amounts. The viscosity-reducing effect varies depending on the pH value of the solution, the concentration of the liquid protein composition, the properties of the protein, and the resulting concentration and chemical properties of the added excipient(s). In a specific embodiment, the liquid composition is a liquid pharmaceutical formulation, and the protein is a therapeutic protein.

[0085] In particular, particularly good viscosity reduction is achieved when camphorsulfonic acid and at least one cationic excipient are added in equimolar amounts to a concentrated liquid protein composition, for example, to a solution of (mAbC, mAbD, and mAbE). According to the present invention, mAbC represents the monoclonal antibody infliximab, mAbD represents the monoclonal antibody evolocumab, and mAbE represents the monoclonal antibody reslizumab.

[0086] Unexpectedly, experiments have shown that a mixture of camphorsulfonic acid in combination with a cationic acid selected from the group of arginine, meglumine, ornithine, and carnitine or mixtures thereof (as a specific equimolar mixture) significantly reduces the viscosity of highly concentrated liquid protein formulations of monoclonal antibodies or fusion proteins.

[0087] Furthermore, it has been found that a mixture of camphorsulfonic acid and a cationic acid selected from the group consisting of arginine, meglumine, ornithine, and ornithine can synergistically reduce viscosity and / or increase stability in protein-containing compositions and formulations. As defined herein, "synergistically" refers to an effect in which the effect of the combination of components is greater than the sum of the effects of each of the components alone.

[0088] The present invention further provides a method for synergistically reducing the viscosity of a liquid composition containing a protein or a liquid formulation containing a pharmaceutically active protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, comprising administering the liquid protein composition in combination with at least camphorsulfonic acid as an excipient and a cationic acid selected from the group consisting of arginine, meglumine, ornithine, and carnitine at a viscosity-reducing concentration. In a specific embodiment, the viscosity is synergistically reduced by the combination of camphorsulfonic acid and arginine. Preferably, the viscosity is synergistically reduced by the combination of camphorsulfonic acid and arginine at a concentration ratio of 1:1.

[0089] Furthermore, it has surprisingly been found that the addition of a cationic acid selected from the group of arginine, meglumine, ornithine, and carnitine to camphorsulfonic acid results in improved protein stability compared to camphorsulfonic acid alone.

[0090] The present invention further provides a method for stabilizing a protein in a liquid composition or a pharmaceutically active protein in a liquid formulation at a concentration ranging from at least 50 mg / ml to 300 mg / ml, comprising combining the liquid protein composition with at least camphorsulfonic acid as an excipient in combination with a cationic acid selected from the group consisting of arginine, meglumine, ornithine, and carnitine at a concentration having a viscosity-reducing effect. In a specific embodiment, the protein is stabilized by a combination of camphorsulfonic acid and arginine or camphorsulfonic acid and ornithine.

[0091] Preferably, the protein is stabilized by a combination of camphorsulfonic acid and arginine or camphorsulfonic acid and ornithine at a concentration ratio of 1:1. Preferably, the stability of the pharmaceutically active protein is improved by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to a liquid composition comprising the protein or a liquid formulation comprising the pharmaceutically active protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, and includes combining a liquid protein composition with camphorsulfonic acid as an excipient.

[0092] The present invention further provides a method for synergistically reducing the viscosity of a liquid composition comprising a protein or a liquid formulation comprising a pharmaceutically active protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, comprising combining a liquid protein composition having camphorsulfonic acid as an excipient in combination with arginine at a concentration having a viscosity-reducing effect, wherein the stability of the pharmaceutically active protein is improved by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to a liquid composition comprising a protein or a liquid formulation comprising a pharmaceutically active protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, comprising combining the liquid protein composition with camphorsulfonic acid as an excipient.

[0093] In exemplary embodiments, the protein or therapeutic protein is present at a high protein concentration as described above. In some embodiments, the viscosity reduction is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% compared to a control formulation in which the same volume of buffer is added to the liquid protein composition instead of the viscosity-reducing agent solution.

[0094] In exemplary embodiments, the protein or therapeutic protein is present at a high protein concentration, as described above, of at least 50 mg / ml, preferably greater than 75 mg / ml, and more preferably greater than 100 mg / ml. Formulations tested and disclosed herein have protein concentrations ranging from 150 to 280 mg / ml. In some embodiments, the viscosity reduction is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% or more compared to a control formulation.

[0095] In another aspect, the present invention provides a liquid solution comprising a therapeutic protein and camphorsulfonic acid as an excipient, and at least one additional cationic excipient selected from the group consisting of arginine, meglumine, ornithine, and carnitine, or a mixture thereof, wherein the formulation exhibits reduced viscosity compared to a control formulation. In exemplary embodiments, the therapeutic protein is present at a high protein concentration, as described above, and the excipient(s) described herein are present at viscosity-reducing concentrations. Camphorsulfonic acid and the excipient(s) may be used in concentrations up to their solubility limits. Such solutions may further contain other additives in amounts effective to improve stability, reduce aggregation, and / or render the formulation isotonic, without significantly increasing viscosity.

[0096] In further embodiments, the concentration of camphorsulfonic acid as an excipient is less than about 500 mM, particularly less than 200 mM. Preferably, the concentration of camphorsulfonic acid is at least about 50 mM to about 300 mM, or at least about 100 mM to about 250 mM, or at least about 140 mM to about 200 mM. In exemplary embodiments, the concentration of the excipient is at least about 50, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 250, or 300 mM or more. The concentration of at least one cationic excipient selected from the group consisting of arginine, meglumine, ornithine, and carnitine or mixtures thereof is at least about 50 mM to about 300 mM, or at least about 100 mM to about 250 mM, or at least about 140 mM to about 200 mM.

[0097] In exemplary embodiments, the concentration of the at least one cationic excipient is at least about 50, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 250, or 300 mM or more. Preferred concentrations of camphorsulfonic acid, when used alone or in combination with at least one cationic excipient, are between 25 and 250 mM, more preferably between 50 and 200 mM, and most preferably between 75 and 150 mM. When used in combination, the concentration of the at least one cationic excipient is preferably between 25 mM and 250 mM, more preferably between 50 mM and 200 mM, and most preferably between 75 mM and 150 mM.When camphorsulfonic acid is used in combination with a cationic excipient selected from the group consisting of arginine, carnitine, meglumine, and ornithine, the molar ratio between camphorsulfonic acid and the cationic excipient is preferably 1:3 to 3:1, more preferably 1:2 to 2:1, and most preferably 1:1.

[0098] For example, a combination of 25 mM carnitine and 50 mM camphorsulfonic acid results in an excipient concentration of 75 mM in a 1:2 mixture of these excipients. Other exemplary embodiments include excipient concentrations effective to render the formulation isotonic without significantly increasing viscosity. Exemplary concentrations include those at about 150 mM or greater, and in further embodiments, the amount is at least about 170 mM or greater. However, the concentration selected must be one that results in an effective reduction in viscosity in the liquid protein composition, and the selected concentration must remain safe and tolerable to living organisms.

[0099] In another aspect, the present invention provides a lyophilized protein formulation comprising a therapeutic protein, camphorsulfonic acid as an excipient, and at least one cationic excipient selected from the group consisting of arginine, meglumine, ornithine, and carnitine, or a mixture thereof, wherein upon reconstitution with a recommended amount of diluent, the formulation exhibits reduced viscosity compared to a control formulation. In exemplary embodiments, the therapeutic protein is present at a high protein concentration, as described above. In some embodiments, the excipient is present in an amount effective to reduce viscosity upon reconstitution with diluent, e.g., 98 mg mAbC:150 mM excipient. Such formulations may contain additional additives in amounts that further improve stability, reduce aggregation, and / or render the formulation isotonic without significantly increasing viscosity.

[0100] In exemplary embodiments of the invention, the concentration of the excipient(s) and selected excipients is at least about 1 μg per mg of therapeutic protein to about 1.0 mg per mg of therapeutic protein. In some embodiments, the concentration of the excipient is at least about 1, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 μg per mg of therapeutic protein. In other exemplary embodiments, the concentration of the excipient is up to about 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μg per mg of therapeutic protein.

[0101] In another aspect, the present invention provides a method of preventing protein self-association in a liquid formulation by using camphorsulfonic acid in combination with at least one cationic excipient selected from the group of arginine, meglumine, ornithine, and carnitine or mixtures thereof as excipient(s) in any of the amounts or concentrations described herein.

[0102] The present invention also provides kits comprising a liquid protein formulation of the present invention and instructions for its administration, optionally with a container, syringe, and / or other administration device. The present invention further provides kits comprising a lyophilized protein formulation of the present invention (optionally in a container) and instructions for its reconstitution and administration (optionally with a vial of sterile diluent, and optionally with a syringe or other administration device). Exemplary containers include vials, tubes, bottles, single- or multi-chamber pre-filled syringes, or cartridges, as well as 96-well plates containing ready-to-use lyophilized or spray-dried formulations disposed in the wells. Exemplary administration devices include syringes (with or without needles), infusion pumps, jet injectors, pen devices, transdermal injectors, or other needleless injectors.

[0103] Another aspect of the present invention provides a method for screening viscosity-reducing concentrations of excipients, the method comprising the steps of: (1) assessing the viscosity of a first solution containing a first concentration of camphorsulfonic acid as an excipient and a suitable excipient selected from the group consisting of arginine, meglumine, ornithine, and carnitine, or a mixture thereof, and a therapeutic protein (such as an antibody); (2) assessing the viscosity of a second solution containing a different second concentration of the excipient and the therapeutic protein; and (3) determining that the first concentration of the excipient is more viscosity-reducing than the second concentration of the excipient when the viscosity of the first solution is lower. Viscosity can be determined, for example, using an m-VROC™ technology rheometer (RheoSense, San Ramon, California, USA), an Aries ARG2 rheometer, or a Brookfield RV-DVIII rheometer.

[0104] Similar methods are provided for screening aggregation-reducing or stabilizing concentrations of excipients.

[0105] Stability can be assessed in a number of ways, including monitoring conformational changes over a wide range of temperatures (thermal stability) and / or time periods (shelf life) and / or after exposure to stressful handling events (e.g., physical shaking). The stability of formulations containing varying concentrations of formulation components can be measured using a variety of methods. For example, the amount of protein aggregation can be measured by visual observation of turbidity, by measuring absorbance at specific wavelengths, by size exclusion chromatography (where protein aggregates elute in different fractions compared to the protein in its native state), HPLC, or other chromatographic methods. Other methods of measuring conformational changes can be used, including using differential scanning calorimetry (DSC) (e.g., to determine the temperature of denaturation) or circular dichroism (CD), which measures the molar ellipticity of a protein.

[0106] Fluorescence can also be used to analyze compositions. Fluorescence involves the emission of light followed by its absorption (which requires a suitable wavelength). Potential readouts are changes in the polarity characteristics, light intensity, or emission wavelength of the light. Fluorescence emission may be intrinsic to the protein or may result from a fluorescent reporter molecule that binds, for example, to the hydrophobic pocket of a partially unfolded protein. Increased binding of the reporter molecule can be monitored by detecting the fluorescent signal of the protein sample. Other means for measuring stability can be used and are well known to those skilled in the art. The stability of compositions and formulations according to the present invention is increased when at least one of the methods described above shows a stabilizing effect.

[0107] In the experiments performed, the viscosity-lowering potential of camphorsulfonic acid was first tested alone and in combination with antibodies (mAbC, mAbD, mAbE). Protein concentrations were adjusted as given in the examples below to create high viscosity levels.

[0108] As already mentioned above, the pH value of these formulations is particularly important for their effectiveness and the usefulness of each pharmaceutically active protein. Therefore, it is desirable to adjust the pH of the protein formulation to be investigated in the range between about 3 and about 8, preferably between 4.5 and about 8.0. Depending on the nature of the contained protein or peptide, the pH value is preferably adjusted to between about 3 and about 8, preferably between 4.5 and about 5.5, or between about 5.0 and about 8.0. The buffer used to adjust the pH is preferably acetate buffer (25 mM) at pH 5.0 and phosphate buffered saline (10 mM) at pH 7.2 or 7.5. However, if necessary, another buffer can be used, which is compatible with the contained pharmaceutically active protein and physiologically acceptable.

[0109] The viscosity-reducing agent concentration was adjusted in the range of 50 mM to 500 mM. Dynamic viscosity was measured using a chip-based (microelectromechanical systems) capillary rheometer, m-VROC™ (RheoSence, San Ramon, CA). Generally, dynamic viscosity (also called absolute viscosity) (the absolute viscosity coefficient) is a measure of the internal resistance that can be determined by the self-association of protein molecules in highly concentrated solutions.

[0110] The determined viscosities clearly demonstrate that applying a concentration of camphorsulfonic acid together with a specific antibody in solution results in a measurably significant reduction in the viscosity of a highly concentrated liquid protein composition.

[0111] However, experiments have shown that, particularly and unexpectedly, the addition of a combination of camphorsulfonic acid and an excipient selected from the group of arginine, meglumine, ornithine, and carnitine or mixtures thereof leads to a significantly higher viscosity reduction.

[0112] As already pointed out, particularly good viscosity reduction is achieved when a mixture of camphorsulfonic acid and an excipient selected from the group of arginine, meglumine, ornithine, and carnitine or a mixture thereof is added in equimolar amounts to a concentrated liquid protein composition (e.g., a solution of mAbC, mAbD, and mAbE).

[0113] In further experiments, the potential of mixtures of any one of the tested cationic excipients in combination with camphorsulfonic acid to reduce the viscosity of highly concentrated antibody solutions (mAbC, mAbD, and mAbE) was investigated. For each of these studies, equimolar amounts of the mixture of these excipients were added. Equimolar amounts of these excipients are preferred. Particularly good results were found here for a 150 mM concentration of added excipient.

[0114] All model antibodies were formulated in acetate buffer at pH 5 or pH 5.5, or phosphate buffer at pH 7.2, at fairly high concentrations of approximately 100 mg / ml, some at approximately 150 mg / ml, especially above 200 mg / ml, and especially 220 mg / ml (mAbE). Viscosity was measured at 20°C using a chip-based (microelectromechanical systems) capillary rheometer, m-VROC™ (RheoSence, San Ramon, CA).

[0115] In all cases, the particular equimolar mixture of cationic excipients at a concentration of 150 mM shows a significant reduction in the viscosity measured in the highly concentrated antibody solutions.

[0116] In further experiments, a combination of camphorsulfonic acid and at least one of the cationic excipients mentioned above, when each added to a liquid protein composition at a total concentration of 150 mM, can significantly reduce the viscosity of an antibody formulation.

[0117] Also, in solutions containing mAbD as a protein, the addition of 150 mM each of camphorsulfonic acid and cationic excipients as excipients causes a significant reduction in viscosity.

[0118] In addition, as shown by the experiments performed, various other combinations of the adjuvants mentioned herein reduce the viscosity of highly concentrated antibody solutions. Thus, the combinations of excipients mentioned herein are not exclusive, and other viable combinations exist that lead to corresponding results.

[0119] In this regard, attempts to reduce the viscosity of various liquid protein compositions have shown that, depending on the protein contained in the particular solution, different additives result in the best stabilization and viscosity reduction.

[0120] In this regard, the best formulation for the protein mAbC is a composition comprising phosphate buffer, polysorbate 80, 75 mM camphorsulfonic acid, and 75 mM arginine dissolved in Milli-Q water and adjusted to pH 7.2.

[0121] Next, for mAbE, the best formulation is a composition containing acetate buffer, 0.1 g / L polysorbate 80, 75 mM camphorsulfonic acid, and 75 mM arginine dissolved in Milli-Q water and adjusted to pH 5.5.

[0122] Therefore, a particularly preferred embodiment of the present invention consists in adding camphorsulfonic acid as an excipient, either alone or in combination with a cationic excipient selected from the group consisting of arginine, meglumine, ornithine, and carnitine, to a highly concentrated liquid protein composition to reduce the viscosity as described above. Particularly preferably, the addition of camphorsulfonic acid in combination with meglumine, ornithine, or carnitine leads to a good viscosity reduction. In another preferred embodiment of the present invention, the combination of camphorsulfonic acid and arginine as an excipient leads to a particularly good viscosity reduction in a solution containing an acetate buffer at pH 5.0, as well as in a solution containing a phosphate buffer at pH 7.2.

[0123] Formulation and lyophilization of the solution preparation can be carried out by methods as described above.

[0124] In summary, viscosity, an important physiological property of highly concentrated liquid protein compositions, can be advantageously reduced by adding the excipient camphorsulfonic acid. A particularly good viscosity-reducing effect can be achieved when camphorsulfonic acid is combined with at least one cationic excipient selected from the group consisting of arginine, meglumine, ornithine, and carnitine. Thus, a particularly good viscosity-reducing effect is achieved by adding the following combinations: meglumine and camphorsulfonic acid, ornithine and camphorsulfonic acid, carnitine and camphorsulfonic acid, and arginine and camphorsulfonic acid.

[0125] In one aspect, the present invention provides a method for reducing the viscosity of a liquid formulation comprising a pharmaceutically active protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, comprising combining the protein solution with at least camphorsulfonic acid as an excipient at a concentration that has a viscosity-reducing effect on the protein solution.

[0126] In another aspect, the invention provides the above-referenced method, comprising combining the protein solution with camphorsulfonic acid and at least one cationic excipient.

[0127] In another aspect, the present invention provides the above-mentioned method, wherein camphorsulfonic acid is added in combination with at least one cationic excipient selected from the group of L-arginine, meglumine, L-ornithine, and L-carnitine.

[0128] In another aspect, the present invention provides the above-mentioned method, wherein the therapeutic protein is selected from the group of antibodies, antibody fragments, minibodies, nanobodies, modified antibodies, antibody-like molecules and fusion proteins.

[0129] In another aspect, the present invention provides the above-mentioned method, wherein the viscosity of the formulation is reduced by at least 12%.

[0130] In another aspect, the present invention provides the above-mentioned method, wherein the viscosity of the formulation is reduced by at least 50%.

[0131] In another aspect, the present invention provides a liquid pharmaceutical formulation produced by the above-referenced method, which has reduced viscosity compared to the same formulation without the excipient or excipient combination.

[0132] In another aspect, the present invention provides a liquid pharmaceutical formulation as referred to above, comprising a therapeutic protein in a concentration of at least 90 mg / ml up to 250 mg / ml and camphorsulfonic acid as a viscosity-reducing excipient.

[0133] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulation, wherein the concentration of the excipient is less than about 500 mM, in particular less than 200 mM.

[0134] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulation having a pH in the range of between about 4.5 and about 8.0 and comprising a buffer.

[0135] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulation having a pH in the range of between about 4.5 and about 7.5 and comprising a buffer.

[0136] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulation having a pH of about 5 to about 7.2 and comprising a buffer.

[0137] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulations, which comprise a phosphate or acetate buffer.

[0138] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulation, which comprises a stabilizer.

[0139] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulation, which comprises a sugar or a surfactant as a stabilizer.

[0140] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulation, which comprises sucrose as a stabilizer.

[0141] In another aspect, the present invention provides the above-mentioned liquid pharmaceutical formulation, which comprises polysorbate or poloxamer 80 as a stabilizer.

[0142] In another aspect, the present invention provides a method for preparing a lyophilized powder comprising the step of lyophilizing the above-mentioned pharmaceutical composition.

[0143] In another aspect, the invention provides the above-mentioned lyophilized powder comprising a therapeutic protein and camphorsulfonic acid, wherein the camphorsulfonic acid or a combination of camphorsulfonic acid and a cationic excipient is present in an amount sufficient to yield a concentration, upon reconstitution, of less than 500 mM, preferably less than 200 mM.

[0144] In another aspect, the present invention provides a method for reconstituting the above-mentioned lyophilized powder, comprising the step of adding a sterile aqueous diluent.

[0145] In another aspect, the present invention provides the above-mentioned method, wherein the therapeutic protein is selected from the group of antibodies, antibody fragments, minibodies, nanobodies, modified antibodies, antibody-like molecules and fusion proteins.

[0146] In another aspect, the present invention provides a pharmaceutical formulation or composition as mentioned above, wherein the therapeutic protein is selected from the group of antibodies, antibody fragments, minibodies, nanobodies, modified antibodies, antibody-like molecules and fusion proteins.

[0147] In another aspect, the present invention provides the above-mentioned lyophilized powder, wherein the therapeutic protein is selected from the group of antibodies, antibody fragments, minibodies, nanobodies, modified antibodies, antibody-like molecules, and fusion proteins.

[0148] In another aspect, the present invention provides a kit comprising the above-mentioned pharmaceutical formulation or lyophilized powder.

[0149] In another aspect, the present invention provides a kit as referred to above, comprising a freeze-dried or spray-dried preparation of the pharmaceutical composition, obtainable by the method referred to above, which can be made into a solution preparation prior to use.

[0150] In another aspect, the present invention provides the above-mentioned kits comprising ready-to-use lyophilized or spray-dried formulations arranged in 96-well plates.

[0151] In another aspect, the invention provides a kit as referred to above for administration to a patient, including a container, a syringe and / or other administration device (with or without a needle), an infusion pump, a jet injector, a pen device, a transdermal injector, or other needleless injector, and instructions.

[0152] Furthermore, the above-mentioned excipients and excipient combinations have been found to be beneficial in bioprocessing. The excipients and excipient combinations have been found to reduce backpressure on the chromatography column and allow for higher flow rates. This leads to less shear stress pulling on the protein in solution, and therefore aggregation will be reduced. Together, higher yields can be obtained. Beyond this, when the process can be run using higher flow rates, process times will be significantly reduced.

[0153] In the bioprocesses referred to herein, the addition of these excipients, which act as viscosity-reducing additives, leads to improved process economics, in that on the one hand the yield of intact protein is improved and on the other hand the duration of the process can be reduced.

[0154] The foundation of the experiments conducted in this invention is laid by Amicon® centrifugal filtration studies. These experiments resemble any process step in which a solution is forced through a filter membrane by centrifugal force. The resistance to centrifugal force depends on the filter properties (which are assumed to be constant within the context of this experiment) and the viscosity of the solution. The excipients and excipient combinations that provide the most favorable solutions are selected for more complex experiments using stirred cells.

[0155] In experiments using an Amicon® stirred cell, nitrogen gas is used to apply pressure to the solution that has passed through the filter. The resistance to pressure depends on the filter properties (which are assumed to be constant within the context of this experiment) and the viscosity of the solution. The excipient combination that gives the most favorable results is used in experiments using a laboratory-scale tangential flow filtration (TFF) system.

[0156] These two experiments highlight the beneficial effect of viscosity-reducing agents during dead-end filtration approaches. Additionally, in technical approaches (where a solution is forced through a filter or medium (such as a gel bed) by force applied by back-end pressure, e.g., during chromatographic purification), the disclosed viscosity-reducing excipients have been shown to have beneficial effects. While the filtration step is primarily used in downstream processes, viscosity-reducing excipients may also be beneficial in upstream processes. When protein concentrations rise to levels that cause viscosity, with the described negative effects of pressure limitations and shear stresses when a solution is passed through a tube or filter to remove cellular material and debris, the present invention will clearly have beneficial effects.

[0157] A laboratory-scale TFF system was used to measure process efficiency in tangential flow filtration (in contrast to previously used methods, the majority of the flow in the field moves tangentially across the filter surface rather than through the filter). The filtration principle differs from that of previously described methods, but here filtration efficiency depends on the membrane resistance (which also remains constant) and solution viscosity (which is modified by the present invention). Filtration methods are typically used to exchange formulation buffers or to bring the concentration of biomolecules to a desired level. The stirred cell used here represents a dead-end filter, where the feed passes through a filtration material that retains larger molecules on top of the material, releasing filtrate at the other end of the device.

[0158] A common method for buffer exchange and protein concentration is tangential flow filtration, where, in contrast to previously used methods, the majority of the field flow moves tangentially across the surface of the filter, rather than through the filter. As when agitated cells are used in tangential flow filtration, macromolecules are separated from smaller molecules by forcing them through a suitable filter material. In contrast to agitated cells (which represent a form of dead-end filtration), in tangential flow filtration, the feed flow geometry is different to avoid filter cake formation and allow for a continuous process. When agitated cells are used, filter cake formation is also prevented by the use of a stirring device.

[0159] Thus, stirred cell filtration closely resembles tangential flow filtration devices, despite differences in filter geometry. The efficiency of both methods is critically dependent on membrane resistance. High viscosity is also known to reduce the available flux rate and therefore increase processing time, resulting in higher production costs. Therefore, reduced viscosity would enable a more efficient filtration process, while shear stress would remain low, predicted to result in higher protein concentrations in the filtrate. This is highlighted by a study by Hung et al., who stated, "During the production of concentrated monoclonal antibody formulations by tangential flow ultrafiltration (TFF), high viscosity and aggregation often cause extensive membrane blockage, flow spoilage, and low product yields" (Journal of Membrane Science Volume 508, June 15, 2016, Pages 113-126).

[0160] Experiments have shown that camphorsulfonic acid is suitable for improving the bioprocess economics described above. Furthermore, the combination of camphorsulfonic acid with a cationic excipient selected from the group consisting of arginine, meglumine, ornithine, and carnitine as an excipient, and the combination of carnitine and two of the excipients improves the bioprocess economics. In particular, the combination of various ratios depending on the protein solution improves the bioprocess economics described above.

[0161] Therefore, another aspect of the present invention provides a method for reducing the viscosity of a liquid composition in a bioprocess, comprising a protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, the method comprising the step of using a liquid composition having at least camphorsulfonic acid as an excipient in a concentration having a viscosity-reducing effect, preferably in combination with at least one cationic excipient (more preferably selected from the list comprising arginine, meglumine, ornithine and carnitine).

[0162] Another aspect of the present invention is the use of the method for reducing the viscosity of a liquid composition as described above in a bioprocess.

[0163] In accordance with the present invention, all the parameters mentioned above; for example, excipient concentration, protein concentration, excipient ratio, further elements of the composition such as buffers or stabilizers, pH value, viscosity reduction, protein specifications, protein molecular weight, also apply to use in bioprocesses.

[0164] In a preferred embodiment for use in bioprocesses, camphorsulfonic acid is used at a concentration of 75 to 500 mM, more preferably 75 to 150 mM, and most preferably 75 mM or 150 mM. When camphorsulfonic acid is used in combination with a cation selected from the group consisting of arginine, carnitine, meglumine, and ornithine, the concentration of each excipient is 75 to 500 mM, more preferably 75 to 150 mM, and most preferably 75 mM or 150 mM. When camphorsulfonic acid is used in combination with a cation selected from the group consisting of arginine, carnitine, meglumine, and ornithine, the ratio is 1:3 to 3:1, more preferably 1:2 to 2:1, and most preferably 1:1. For example, a combination of 25 mM carnitine and 50 mM camphorsulfonic acid results in an excipient concentration of 75 mM in a solution containing a 1:2 mixture of these excipients.

[0165] Depending on the protein solution and the bioprocess being carried out, different buffer systems can be used as buffers: acetates (such as ammonium acetate or sodium acetate), carbonates (ammonium bicarbonate or sodium bicarbonate), or phosphates (sodium phosphate or triphosphate) can be used here, depending on the conditions during the bioprocess.

[0166] Another aspect of the present invention is to provide a method for reducing the viscosity of a liquid composition in the above-mentioned bioprocess, wherein the permeate flux of the liquid composition in the filtration step is increased compared to the same liquid composition not containing camphorsulfonic acid or compared to the same liquid composition not containing camphorsulfonic acid and at least one cationic excipient (preferably selected from the group consisting of arginine, meglumine, ornithine, and carnitine).

[0167] Another aspect of the present invention is the use of the above-mentioned method in a bioprocess, wherein the permeate flux of the liquid composition in the filtration step is increased compared to the same liquid composition not containing camphorsulfonic acid or compared to the same liquid composition not containing camphorsulfonic acid and at least one cationic excipient (preferably selected from the group consisting of arginine, meglumine, ornithine, and carnitine).

[0168] An increase in permeate flux means a percentage increase of at least 2%, preferably at least 5%, more preferably at least 10%, and most preferably between 10% and 100%.

[0169] Another aspect of the present invention is to provide a method for reducing the viscosity of a liquid composition in the above-mentioned bioprocess, wherein protein recovery and volume reduction in the filter after buffer exchange are increased compared to the same liquid composition without camphorsulfonic acid or compared to the same liquid composition without camphorsulfonic acid and at least one cationic excipient (preferably selected from the group consisting of arginine, meglumine, ornithine, and carnitine).

[0170] Another aspect of the present invention is the use of the above-mentioned method in a bioprocess, wherein protein recovery and volume reduction in the filter after buffer exchange are increased compared to the same liquid composition without camphorsulfonic acid or compared to the same liquid composition without camphorsulfonic acid and at least one cationic excipient (preferably selected from the group consisting of arginine, meglumine, ornithine, and carnitine).

[0171] An increase in protein recovery and volume reduction in the filter after buffer exchange means a percentage increase in protein recovery of at least 1%, preferably at least 2%, more preferably at least 5%, and most preferably 5% to 20%.

[0172] Another aspect of the present invention is to provide a method for reducing the viscosity of a liquid composition in the above-mentioned bioprocess, wherein the processing time of a filtration step (preferably a filtration step in which proteins are concentrated) is reduced compared to the same liquid composition not containing camphorsulfonic acid or not containing camphorsulfonic acid and at least one cationic excipient (preferably selected from the group consisting of arginine, meglumine, ornithine, and carnitine).

[0173] Another aspect of the present invention is the use of the above-mentioned method in a bioprocess, wherein the processing time for the filtration step (preferably the filtration step in which proteins are concentrated) is reduced compared to the same liquid composition not comprising camphorsulfonic acid or not comprising camphorsulfonic acid and at least one cationic excipient (preferably selected from the group consisting of arginine, meglumine, ornithine, and carnitine).

[0174] A reduction in process time for the filtration step means a percentage reduction of at least 5%, preferably at least 10%, more preferably at least 25%, and most preferably between 25% and 100%.

[0175] In a particular embodiment of the invention, the filtration step is tangential flow filtration (TFF).

[0176] Another aspect of the present invention is to provide kits for carrying out the methods described herein. Generally, the kits for carrying out will be adapted to the methods of the present invention, and the form of the parts will depend on their intended function.

[0177] Typically, the kit is packaged and includes a reservoir containing a reagent, the volume of which varies based on the amount of preparation for which the kit is to be evaluated. The reservoir generally contains one or more reagents useful in carrying out the method of the present invention. In some embodiments, the kit is a compartmentalized kit, i.e., the kit contains reagents contained in the same or separate reservoirs. Examples of reservoirs include, but are not limited to, small glass containers, plastic containers, or strips of plastic paper. These or other small reservoirs allow for efficient transfer of reagents from one compartment to another.

[0178] Such solutions may include containers for receiving test samples, containers containing the disclosed proteins or protein solutions, solutions containing materials, containers containing washing reagents, and / or containers containing reagents useful in the application of the kit. The kits may include sources and concentrates of the polypeptides described herein. For larger scale applications, the kits will generally include similar reagents and solutions, but in larger quantities.

[0179] The present invention also provides kits comprising a liquid protein formulation of the present invention, instructions for its administration, optionally together with a container, syringe, and / or other administration device. The present invention further provides kits comprising a lyophilized protein formulation of the present invention, optionally in a container, and instructions for its reconstitution and administration, optionally together with a vial of sterile diluent, and optionally together with a syringe or other administration device. Exemplary containers include vials, tubes, bottles, single- or multi-chamber pre-filled syringes, or cartridges, but also 96-well plates containing ready-to-use lyophilized or spray-dried formulations arranged in the wells. Exemplary administration devices include syringes (with or without needles), infusion pumps, jet injectors, pen devices, transdermal injectors, or other needleless injectors.

[0180] The kit also typically includes instructions for use. The instructions will generally be suitable for enabling the end user to perform the desired preparation or assay. The instructions will generally be in tangible form (e.g., parameters such as reagent concentrations for at least one preparation or assay, relative amounts of reagents and sample to be mixed, maintenance or incubation periods for reagent / sample mixing, temperature requirements or priorities, etc.). The instructions may be printed on the outside or inside of the kit's packaging, present on a booklet, card, or other paper within the kit, and / or on the exterior surface of a container or vessel included in the kit.

[0181] Having described the invention in detail, it will be apparent that modifications and variations can be made thereto without departing from the scope of the invention as defined in the appended claims. Furthermore, it should be understood that all examples in the present disclosure are provided as non-limiting examples.

[0182] The formulation and lyophilization of the solution preparation can be carried out by methods known to those skilled in the art.

[0183] The description of the present invention enables a person skilled in the art to comprehensively implement the present invention. Without further comment, it is assumed that a person skilled in the art can utilize the above description to the widest extent.

[0184] While the present invention has been described in connection with preferred embodiments, it should be understood that various modifications, additions, and variations can be made to the present invention by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims. In particular, the inventive concept is not limited to the particular proteins shown in the examples, but can be transferred to all other proteins as defined above.

[0185] It is understood that if anything is unclear, reference should be made to the publications and patent documents cited and known to those skilled in the art. Consequently, the cited documents should be considered part of the disclosure content of the present description and are incorporated herein by reference.

[0186] For a better understanding and to illustrate the present invention, examples are presented below, which are within the scope of protection of the present invention. These examples also serve to illustrate possible variants.

[0187] Furthermore, in the examples given, and also in the rest of the description, the amounts of constituents present in the compositions always add up only to 100% by weight or mole percent, based on the composition as a whole, and cannot exceed this percentage, even if higher values ​​may result from the percentage range indicated. Unless otherwise stated, % data are therefore % by weight or mole percent, with the exception of proportions expressed in volume data.

[0188] The present invention further provides a method for reducing the viscosity of a liquid composition containing a protein, wherein the protein is infliximab. In the present invention, infliximab is referred to by the abbreviation "mAbC." Preferably, the concentration of infliximab in the pharmaceutical formulation is between 122 mg / ml and 185 mg / ml.

[0189] Infliximab (REMICADE®), developed by Janssen Biotech, Inc., and its biosimilar drug (FUXABI®), developed by Biogen, and (Inflectra), developed by Celltrion, are used for the treatment of rheumatoid arthritis, adult ulcerative colitis, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, and adult and pediatric Crohn's disease (dose / administration: 5 mg / kg). Infliximab is a mAb against tumor necrosis factor alpha (TNF-α) used to treat autoimmune diseases. Infliximab neutralizes the biological activity of TNFα by binding with high affinity to the soluble and transmembrane forms of TNFα and inhibits the binding of TNFα to its receptor.

[0190] It is marketed under the trade name REMICADE® by Janssen Global Services, LLC (“Janssen”) in the United States, Mitubishi Tanabe Pharma in Japan, Xian Janssen in China, and Merck Sharp & Dohme (“MSD”) elsewhere. In some embodiments, the formulation contains a biosimilar of REMICADE® (such as REMSIMA™ or INFLECTRA™). Both REMSIMA™, developed by Celtrion, Inc. (“Celltion”), and INFLECTRA™, developed by Hospira Inc., UK. Infliximab is currently administered via intravenous infusion at doses ranging from about 3 mg / kg to about 10 mg / kg.

[0191] The present invention further provides a method for reducing the viscosity of a liquid composition containing a protein, wherein the protein is evolocumab. In the present invention, evolocumab is referred to by the abbreviation "mAbD." Preferably, the concentration of evolocumab in the pharmaceutical formulation is between 163 mg / ml and 204 mg / ml.

[0192] Evolocumab (REPATHA®), developed by Amgen, is used in the treatment of HeFH, CVD, and the reduction of low-density lipoprotein cholesterol (LDL-C) by targeting PCSK9 (proprotein convertase subtilisin kexin type 9) (monthly dose / dosage: 420 mg).

[0193] The present invention further provides a method for reducing the viscosity of a liquid composition containing a protein, wherein the protein is reslizumab. In the present invention, reslizumab is referred to by the abbreviation "mAbE." Preferably, the concentration of reslizumab in the pharmaceutical formulation is between 178 mg / ml and 224 mg / ml.

[0194] Reslizumab (CINQAIR®) was developed by Teva Pharmaceuticals and is used in severe asthma attacks (exacerbations) (dose / administration: 3 mg / kg).

[0195] example: In the examples, the following abbreviations are used for mAbs: mAbC: Infliximab mAbD: Evolocumab mAbE: reslizumab

[0196] Example 1 Effects of meglumine, L-ornithine, L-carnitine and camphorsulfonic acid on highly concentrated protein solutions: Example 1a) shows that L-arginine, L-carnitine and camphorsulfonic acid (but not L-ornithine and meglumine) reduce the viscosity of mAbC at 98 mg / ml and 148 mg / ml, respectively. Example 1b) shows that meglumine, L-ornithine, L-carnitine and camphorsulfonic acid reduce the viscosity of mAbD at 170 mg / ml and 190 mg / ml. Example 1c) shows that meglumine, L-ornithine, L-carnitine and camphorsulfonic acid reduce the viscosity of mAbE at 179 mg / ml and 223 mg / ml. The control samples in all cases are commercial formulations of the respective mAb without the viscosity-reducing excipients.

[0197] Example 1a The viscosity-reducing effect of L-arginine, L-carnitine and camphorsulfonic acid (but not L-ornithine and meglumine) reduces the viscosity of mAbC formulated in phosphate buffer at pH 7.2.

[0198] Buffer preparation 5 mM phosphate buffer was prepared by mixing sodium dihydrogen phosphate and disodium hydrogen phosphate appropriately to give a pH of 7.2 and dissolving the mixture in ultrapure water. The ratio was determined using the Henderson-Hasselbalch equation. The pH was adjusted, if necessary, using HCl and NaOH. 50 mg / ml sucrose and 0.05 mg / ml polysorbate 80 were added as stabilizers.

[0199] Sample preparation Excipient solutions of 150 mM L-ornithine, L-arginine, L-carnitine, meglumine, and camphorsulfonic acid were each prepared in phosphate buffer pH 7.2. The pH was adjusted, if necessary, using HCl or NaOH. Concentrated mAb solutions containing the desired excipients were prepared using centrifugal filters (Amicon, 30 kDA MWCO) to exchange the original buffer with a buffer containing the relevant excipients and reduce the volume of the solution. The proteins were subsequently diluted to 98 mg / ml and 148 mg / ml, respectively.

[0200] Viscosity measurement The mVROC™ technology (Rheo Sense, San Ramon, California USA) was used for viscosity measurements. Measurements were performed using a 500 μl syringe at a shear rate of 3000 s -1 The assay was performed at 20°C using a 200 μl volume. All samples were measured in triplicate. FIG. 1 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine, arginine and camphorsulfonic acid on mAb C formulated in phosphate buffer pH 7.2 (Example 1a).

[0201] Example 1b Meglumine, L-ornithine, L-carnitine and camphorsulfonic acid reduce the viscosity of mAbD at 170 mg / ml and 190 mg / ml.

[0202] Buffer preparation 20 mM acetate buffer was prepared by mixing 1.2 mg / ml glacial acetic acid with ultrapure water. The pH was adjusted to 5.0 using HCl and NaOH, if necessary. 0.1 mg / ml polysorbate 80 was added as a stabilizer.

[0203] Sample preparation Excipient solutions of 150 mM L-ornithine, L-arginine, L-carnitine, meglumine, and camphorsulfonic acid were each prepared in acetate buffer pH 5.0. The pH was adjusted, if necessary, using HCl or NaOH. Concentrated mAb solutions containing the desired excipients were prepared using centrifugal filters (Amicon, 30 kDA MWCO), exchanging the original buffer with a buffer containing the relevant excipients and reducing the volume of the solution. The proteins were subsequently diluted to 169 mg / ml and 190 mg / ml, respectively.

[0204] Viscosity measurement The mVROC™ technology (Rheo Sense, San Ramon, California USA) was used for viscosity measurements. Measurements were performed for 3000 s with a 500 μl syringe and a 169 mg / ml protein solution. -1 and 2000 s for a 190 mg / ml protein solution. -1 The experiments were carried out at 20°C using a shear rate of 100 rpm. A volume of 200 μl was used. All samples were measured in triplicate. FIG. 2 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine, arginine and camphorsulfonic acid for mAbD formulated in acetate buffer pH 5.0 (Example 1b).

[0205] Example 1c Meglumine, L-ornithine, L-carnitine and camphorsulfonic acid reduce the viscosity of mAbE at 179 mg / ml and 223 mg / ml. Buffer preparation A 20 mM acetate buffer was prepared by mixing sodium acetate and glacial acetic acid with ultrapure water in a ratio that resulted in a buffer of pH 5.5. The ratio of sodium acetate and glacial acetic acid was calculated using the Henderson-Hasselbalch equation. The pH was adjusted, if necessary, using HCl and NaOH. 70 mg / ml sucrose was added as a stabilizer.

[0206] Sample preparation Excipient solutions of 150 mM L-ornithine, L-arginine, L-carnitine, meglumine, and camphorsulfonic acid were each prepared in acetate buffer pH 5.5. The pH was adjusted, if necessary, using HCl or NaOH. Concentrated mAb solutions containing the desired excipients were prepared by exchanging the original buffer with a buffer containing the relevant excipients and using a centrifugal filter (Amicon, 30 kDA MWCO) to reduce the volume of the solution. The proteins were subsequently diluted to 179 mg / ml and 223 mg / ml, respectively.

[0207] Viscosity measurement The mVROC™ technology (Rheo Sense, San Ramon, California, USA) was used for viscosity measurements. Measurements were performed using a 500 μl syringe and 3000 s -1 The experiments were carried out at 20°C using a shear rate of 100 rpm. A volume of 200 μl was used. All samples were measured in triplicate. FIG. 3 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine, arginine, and camphorsulfonic acid on mAbE formulated in acetate buffer pH 5.5 (Example 1c).

[0208] Example 2 Effect of L-arginine, L-carnitine, L-ornithine, and meglumine in combination with camphorsulfonic acid. Example 2a)show that meglumine, L-ornithine, L-carnitine, when combined with camphorsulfonic acid, reduce the viscosity of mAbC to 98 mg / ml and 148 mg / ml, respectively. Example 2b) show that meglumine, L-ornithine, L-carnitine, when combined with camphorsulfonic acid, reduce the viscosity of mAbC at 170 mg / ml and 190 mg / ml. Example 2c) show that meglumine, L-ornithine, L-carnitine, when combined with camphorsulfonic acid, reduce the viscosity of mAbE at 179 mg / ml and 223 mg / ml. Example 2d) shows synergistic viscosity reduction by the combination of L-arginine and camphorsulfonic acid for mAbC formulated in phosphate buffer pH 7.2.

[0209] Example 2a Buffer preparation 5 mM phosphate buffer was prepared by appropriately mixing sodium dihydrogen phosphate and disodium hydrogen phosphate to yield a pH of 7.2 and dissolving the mixture in ultrapure water. The ratio was determined using the Henderson-Hasselbalch equation. The pH was adjusted using HCl and NaOH, if necessary. 50 mg / ml sucrose and 0.05 mg / ml polysorbate 80 were added as stabilizers.

[0210] Sample preparation Excipient solutions of 75 mM L-ornithine hydrochloride, L-arginine, L-carnitine, and meglumine, each supplemented with an additional 75 mM camphorsulfonic acid, were prepared in phosphate buffer pH 7.2. The pH was adjusted using HCl or NaOH, if necessary. Concentrated mAb solutions containing the desired excipients were prepared by exchanging the original buffer with a buffer containing the relevant excipients and using a centrifugal filter (Amicon, 30 kDA MWCO) to reduce the volume of the solution. The proteins were subsequently diluted to 98 mg / ml and 148 mg / ml, respectively.

[0211] Viscosity measurement The mVROC™ technology (Rheo Sense, San Ramon, California USA) was used for viscosity measurements. Measurements were performed using a 500 μl syringe and 3000 s -1 The experiments were carried out at 20°C using a shear rate of 100 rpm. A volume of 200 μl was used. All samples were measured in triplicate. FIG. 4 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine when combined with camphorsulfonic acid for mAbC formulated in phosphate buffer pH 7.2 (Example 2a).

[0212] Example 2b Buffer preparation 20 mM acetate buffer was prepared by mixing 1.2 mg / ml glacial acetic acid with ultrapure water. The pH was adjusted to 5.0 using HCl and NaOH, if necessary. 0.1 mg / ml polysorbate 80 was added as a stabilizer.

[0213] Sample preparation Excipient solutions of 75 mM L-ornithine hydrochloride, L-arginine, L-carnitine, and meglumine, each supplemented with an additional 75 mM camphorsulfonic acid, were prepared in acetate buffer, pH 5.0. The pH was adjusted using HCl or NaOH, if necessary. Concentrated mAb solutions containing the desired excipients were prepared using centrifugal filters (Amicon, 30 kDA MWCO) to exchange the original buffer with a buffer containing the relevant excipients and to reduce the volume of the solution. The proteins were subsequently diluted to 169 mg / ml and 190 mg / ml, respectively.

[0214] Viscosity measurement The mVROC™ technology (Rheo Sense, San Ramon, California USA) was used for viscosity measurements. Measurements were performed for 3000 s with a 500 μl syringe and a 169 mg / ml protein solution. -1 and a shear rate of 190 mg / mls -1 Protein concentration of 2000s -1 The experiments were carried out at 20°C using a shear rate of 100 rpm. A volume of 200 μl was used. All samples were measured in triplicate. FIG. 5 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine when combined with camphorsulfonic acid for mAbD formulated in acetate buffer pH 5.0 (Example 2b).

[0215] Example 2c Buffer preparation 20 mM acetate buffer was prepared by mixing sodium acetate and glacial acetic acid with ultrapure water in a ratio that resulted in a buffer of pH 5.5. The ratio of sodium acetate and glacial acetic acid was calculated using the Henderson-Hasselbalch equation. The pH was adjusted using HCl and NaOH, if necessary. 70 mg / ml sucrose was used as a stabilizer.

[0216] Sample preparation Excipient solutions of 75 mM L-ornithine, L-arginine, L-carnitine, and meglumine, respectively, supplemented with an additional 75 mM camphorsulfonic acid, were prepared in acetate buffer pH 5.5. The pH was adjusted using HCl or NaOH, if necessary. Concentrated mAb solutions containing the desired excipients were prepared by exchanging the original buffer with a buffer containing the relevant excipients and using a centrifugal filter (Amicon, 30 kDA MWCO) to reduce the volume of the solution. The proteins were diluted to 179 mg / ml and 223 mg / ml, respectively.

[0217] Viscosity measurement mVROC™ technology (Rheo Sense, San Ramon, California USA) was used for viscosity measurements. Measurements were performed using a 500 μl syringe and 3000 s -1 The experiments were carried out at 20°C using a shear rate of 100 rpm. A volume of 200 μl was used. All samples were measured in triplicate. FIG. 6 shows the viscosity-reducing effect of meglumine, L-ornithine, L-carnitine when combined with camphorsulfonic acid for mAbE formulated in acetate buffer pH 5.5 (Example 2c).

[0218] Example 2d Buffer preparation, sample preparation and viscosity measurements were performed according to Examples 1a) and 2a). Figure 7 shows the synergistic viscosity-reducing effect of L-arginine when combined with camphorsulfonic acid for mAbC formulated in phosphate buffer pH 7.2 (Example 2d). The "predicted viscosity combination" corresponds to the estimated additive effect of each excipient, L-ornithine and L-arginine, alone at 75 mM. The measured combination exhibited a synergistic lower viscosity at 148 mg / mL for mAbC between these two excipients.

[0219] Example 3 Example 3 shows the effect of camphorsulfonic acid and its combination with L-ornithine or L-arginine on the protein stability of mAbC. Buffer preparation 5 mM phosphate buffer was prepared by appropriately mixing sodium dihydrogen phosphate and disodium hydrogen phosphate to yield a pH of 7.2 and dissolving the mixture in ultrapure water. The ratio was calculated using the Henderson-Hasselbalch equation. The pH was adjusted using HCl and NaOH when necessary. 50 mg / ml sucrose and 0.05 mg / ml polysorbate 80 were used as stabilizers.

[0220] Sample preparation Excipient solutions containing 150 mM L-ornithine, L-arginine, or camphorsulfonic acid were prepared in phosphate buffer pH 7.2. Also, 75 mM L-ornithine hydrochloride or L-arginine, supplemented with an additional 75 mM camphorsulfonic acid, were prepared in phosphate buffer pH 7.2. The pH was adjusted using HCl or NaOH, if necessary. Concentrated mAbC solutions containing the desired excipients were prepared using a centrifugal filter (Amicon, 30 kDA MWCO) to exchange the original buffer with a buffer containing the relevant excipients and to reduce the volume of the solution. The protein was subsequently diluted to approximately 80 mg / ml. The samples were sterilized using a syringe filter (Millex GV, 0.22 μm, PVDF, Art. No.: SLGV013SL) and aliquoted into previously rinsed and sterilized crimped vials. The vials were crimped and stored at 40°C / 75% rH for 28 days.

[0221] Monomer content analysis Monomer content was determined by size exclusion chromatography using an Aquity UPLC Protein BEH SEC column connected to an Agilent 1290 Infinity UHPLC system. Size separation was performed isocratically at 30°C using a potassium salt eluent containing 10% organic solvent. An unstressed mAbC sample (1 mg / mL) was used as a standard (100%). Samples were diluted to 1 mg / mL for analysis. Figure 8 shows the residual monomer content of solutions containing approximately 80 mg / mL mAbC in phosphate buffer pH 7.2, with or without excipients, stored for 28 days at 40°C / 75% rH. Protein stability was less negatively affected when a combination of camphorsulfonic acid and L-arginine or L-ornithine was used instead of when acid was the only excipient.

[0222] Example 4: Benefits for centrifugal filter units Buffer preparation A 10 mM citrate buffer solution was prepared by dissolving citric acid monohydrate in ultrapure water. The pH was adjusted to 5.5 using HCl and NaOH, if necessary. 0.25 mg / mL polysorbate 80 was added as a stabilizer. Excipient solutions of camphorsulfonic acid (CSAcid), ornithine (Orn or OM), arginine (Arg or AG), carnitine (Car), and meglumine (Meg or MG) were prepared at a concentration of 150 mM in citrate buffer, pH 5.5. Combinations containing two of these excipients were prepared at concentrations of 75 mM each or 150 mM each.

[0223] Sample preparation A cetuximab solution containing approximately 14.7 mg / ml was used as the starting material, after which a sufficient volume was calculated to achieve a final concentration of greater than 120 mg / ml in a 500 μL sample, estimating up to 20% sample loss.

[0224] Protein concentration measurement Protein concentrations were determined using absorption spectroscopy, applying the Beer-Lambert law. When the excipient itself had strong absorbance at 280 nm, the Bradford assay was used. Concentrated protein solutions were diluted so that their expected concentrations were measured between 0.3 and 1.0 mg / mL. For absorption spectroscopy, absorbance at 280 nm was measured using a BioSpectrometer® Kinetics (Eppendorf, Hamburg, Germany) with a protein extinction coefficient A of 0.1% at 280 nm = 1.4.

[0225] Protein concentrations were determined using the Bradford assay for excipients that absorbed light at 280 nm. Therefore, a kit from Thermo Scientific™ (Thermo Fisher, Waltham, Massachusetts, USA) was used, as well as cetuximab standards prepared using absorption spectroscopy applying the Beer-Lambert law. Absorbance was measured at 595 nm using a Multiskan™ Wellplate reader (Thermo Fisher, Waltham, Massachusetts, USA). Protein concentrations were determined by appropriate polynomial regression of a standard curve ranging from 125 to 1500 μg / mL.

[0226] Volume measurement The permeate volume was measured using an appropriately sized volumetric flask. For Amicon® centrifugal filter units, the permeate was transferred to a volumetric flask after centrifugation, and for Amicon® stirred cell experiments, the permeate was collected directly into such a flask. The volume of the concentrated protein solution was measured using an appropriately sized Multipette® E3X (Eppendorf, Hamburg, Germany) and Combitips advanced®.

[0227] Buffer exchange and volume reduction Using an Amicon® centrifugal filter with a 30 kDa MWCO, the original buffer was exchanged with a buffer containing the relevant excipients and the volume of the solution was reduced. Five diavolumes were used to exchange the original buffer with a buffer containing the excipient of interest. To measure permeate flux, Amicon® centrifugal filters were centrifuged at 2000×g for 15 minutes and the volume was measured as described above (four replicates were performed and the average was calculated). To achieve the final concentration, Amicon® centrifugal filters were centrifuged in short time steps and the accumulation period was noted upon reaching the 500 μL mark.

[0228] Figures 9-11 highlight the process improvement indicated by the increased permeate flux. 150 mM of each excipient was found to increase flow-through under the experimental conditions. Using combinations containing 75 mM CSAcid and more cationic excipients increases flow-through (especially the combination AG / CSAcid). When the concentration of the individual excipients is increased up to 150 mM, all tested combinations significantly increase the flow-through.

[0229] Another aspect to characterize the benefit of processes using Amicon® centrifugal filters is the time required to reach a certain volume. This aspect is related to, but different from, the average permeate flux, since here the effect of excipients at higher protein concentrations has a strong influence on this parameter.

[0230] Figures 12-14 show the reduction in processing time that can be achieved using 75 mM cationic or anionic excipients, respectively. Reduced processing times can be observed for each of the excipients listed. Figures 13 and 14 highlight the effect of excipient combinations on processing time. Each of the combinations listed reduces processing time and therefore has a beneficial effect on process economics.

[0231] Besides processing time, another aspect that is crucial to process economics is process efficiency. This parameter can be assessed in this experimental setting by protein recovery, which is defined as the protein fraction that can be recovered from the Amicon filter after the volume of the solution has been reduced to 0.5 ml.

[0232] Figures 15 and 16 show the effect of 75 mM cationic and / or anionic excipients, respectively, on protein recovery. Under all test conditions, recovery was improved by the addition of a viscosity reducing agent according to the present invention.

[0233] Example 5: Benefits of stirred cells Excipients and combinations that had a positive effect in processing using Amicon® centrifugal filters were used in Amicon® stirred cell filtration. The Amicon® spin column concentrator is driven by centrifugal force, and the stirred cell is operated by backpressure applied to the solution in the form of nitrogen or airflow. This model system is frequently used to test the processability of solutions and is a closer model system than previously used Amicon® centrifugal filters.

[0234] Buffer preparation See Example 1 Sample preparation The volume of the antibody stock solution was calculated to yield at least 10 mL of solution containing 25 mg / mL cetuximab, assuming up to 20% loss. Protein concentration measurement See Example 1 Volume measurement See Example 1

[0235] Buffer exchange and volume reduction For stirred cell setups, models containing up to 50 mL have a NMWL of 30 kDa and a 13.4 cm 2 The filter was equipped with an ultrafiltration disc filter having an active membrane area of ​​1000 MPa. Each volume of cetuximab stock solution was filled into a stirred cell and the respective buffer was added up to the 50 mL mark. Five diavolumes were used to exchange the original buffer with the relevant excipient or combination.

[0236] To measure the permeation flux, a pressure of 4 bar was applied to an Amicon® stirred cell for 30 minutes (4 times) at a mixing speed of 200 rpm (using a magnetic stir plate). For the final concentration period, the cells were again filled with the respective buffer solution up to the 50 mL mark, and a pressure of 4 bar was applied at an agitation speed of 200 rpm. When the 10 mL mark was reached, the time period was noted, the process was stopped, and the volume and concentration of the resulting antibody solution were measured as described above. As in the previous section, the effect of the formulation on the average permeation flux was first assessed, and the results are depicted in Figure 17 below. Improved permeation flux was observed for each of the excipients and excipient combinations used.

[0237] The next parameter analyzed was process time, where the time it took for the volume of the formulation to be reduced to 10 ml was measured. The results are depicted in Figure 18. For each excipient and excipient combination used herein, a reduction in processing time was observed. Finally, the process yield in the form of protein recovery from stirred cells was determined and is shown in Figure 19. Improved recoveries were observed for all test conditions.

[0238] Example 5: Processing with Amicon: Infliximab Buffer preparation 5 mM phosphate buffer was prepared by appropriately mixing sodium dihydrogen phosphate and disodium hydrogen phosphate to yield a pH of 7.2 and dissolving the mixture in ultrapure water. The ratio was determined using the Henderson-Hasselbalch equation. The pH was adjusted using HCl and NaOH as needed. 50 mg / ml sucrose and 0.05 mg / ml polysorbate 80 were added as stabilizers.

[0239] Sample preparation Excipient solutions with one compound were prepared in phosphate buffer pH 7.2 at a concentration of 150 mM unless otherwise specified. Folic acid was prepared at a concentration of 12 mM. Thiamine pyrophosphate was prepared at a concentration of 75 mM. pH was adjusted using HCl or NaOH, if necessary. The two excipients were combined to prepare each compound at a concentration of 75 mM. Folic acid was used in combination at 12 mM.

[0240] Concentrated infliximab solutions containing the desired excipients were prepared using centrifugal filters (Amicon, 30 kDA MWCO) to exchange the original buffer with a buffer containing the relevant excipients and to reduce the volume of the solution. A solution containing 10 mg / ml infliximab was used as the stock material and the starting volume was calculated such that a concentration of at least 160 mg / ml was achieved in 0.5 ml.

[0241] Protein concentration measurement Protein concentrations were determined using the Bradford assay. Therefore, we used an infliximab standard prepared using a kit from Thermo Scientific™ (Thermo Fisher, Waltham, Massachusetts, USA) and absorption spectroscopy applying the Beer-Lambert law. Absorbance was measured at 595 nm using a Multiskan™ Wellplate reader (Thermo Fisher, Waltham, Massachusetts, USA). Protein concentrations were determined by appropriate polynomial regression of a standard curve ranging from 125 to 1500 μg / mL.

[0242] The processing time to reach a volume of approximately 0.5 ml is depicted in Figures 20 and 21. With each of the excipients used in this study, processing times can be reduced by at least 20 minutes, and in some cases up to 90 minutes. The time reductions that can be achieved with excipient combinations (75 mM each) are depicted in Figure 21. The use of a combination of viscosity-reducing excipients reduces processing time for infliximab when an Amicon filter is used as the model. As previously described, the process demonstrated herein can translate to improved processability even when more realistic models, such as TFF, are used.

[0243] The following was found The viscosity of highly concentrated protein solutions is reduced by L-arginine, L-ornithine, L-carnitine, meglumine, and camphorsulfonic acid. The viscosity of highly concentrated protein solutions is reduced by using camphorsulfonic acid in combination with L-arginine, L-ornithine, L-carnitine, and meglumine. Viscosity is reduced more strongly by the combination of L-arginine and camphorsulfonic acid than the theoretical reduction achieved by the sum of both excipients alone (synergistic combination).

[0244] In relation to viscosity-reducing potential, protein stability was less negatively affected when camphorsulfonic acid was used in combination with L-arginine or L-ornithine compared to when camphorsulfonic acid was the only excipient. Flux through Amicon® centrifugal filters is increased by camphorsulfonic acid and by combinations of camphorsulfonic acid with L-ornithine, L-arginine, L-carnitine, and meglumine. Excipients and combinations that increase flux through Amicon® centrifugal filters can also increase this flux through Amicon® stirred cells.

[0245] The time to achieve the final protein concentration can be reduced by camphorsulfonic acid and its combination with L-ornithine, L-arginine, L-carnitine and meglumine compared to samples without excipients. Antibody recovery after buffer exchange and volume reduction can be increased by camphorsulfonic acid and camphorsulfonic acid in combination with L-ornithine, L-arginine, L-carnitine, and meglumine compared to excipient-free samples. Excipients and combinations that increase protein recovery after buffer exchange and volume reduction in Amicon® centrifugal filters also increase it in Amicon® stirred cells.

Claims

1. 1. A method for reducing the viscosity of a liquid composition comprising a protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, the method comprising combining the liquid composition with at least camphorsulfonic acid as an excipient at a concentration of 70-500 mM and at least one cationic excipient selected from meglumine and ornithine, in concentrations that have a viscosity-reducing effect; wherein the ratio of camphorsulfonic acid to cationic excipient is 1:3 to 3:1, and the protein is a monoclonal antibody; The method.

2. 10. The method of claim 1, wherein the at least one cationic excipient is meglumine.

3. 10. The method of claim 1, wherein the at least one cationic excipient is ornithine.

4. 4. The method of claim 1, 2 or 3, wherein the viscosity is reduced compared to the same liquid composition without camphorsulfonic acid and at least one cationic excipient selected from meglumine and ornithine.

5. The method according to any one of claims 1 to 4, wherein the viscosity of the liquid composition is reduced by at least 12%, preferably by at least 50%.

6. The method of any one of claims 1 to 5, wherein the concentration of the protein is between 90 mg / ml and 250 mg / ml.

7. The method according to any one of claims 1 to 6, wherein the concentration of camphorsulfonic acid is less than about 500 mM, in particular less than 200 mM.

8. The method according to any one of claims 1 to 7, wherein the concentration of camphorsulfonic acid is 75 to 150 mM.

9. the ratio of camphorsulfonic acid to cationic excipient is 1:2 to 2:1; The method according to any one of claims 1 to 8.

10. The method of any one of claims 1 to 9, wherein the pH of the liquid composition ranges from about 3 to about 8 and comprises a buffer.

11. A protein at a concentration ranging from at least 50 mg / ml to 300 mg / ml, and as excipients, at least camphorsulfonic acid in a concentration of 70-500 mM and at least one cationic excipient selected from meglumine and ornithine; A liquid composition comprising: the ratio of camphorsulfonic acid to cationic excipient is 1:3 to 3:1; the protein is a monoclonal antibody, The composition has a reduced viscosity compared to the same liquid composition without the combination of excipients.

12. Use of the method according to any one of claims 1 to 10 in a bioprocess.

13. 13. The use of the method according to claim 12, wherein the permeation flux of the liquid composition in the filtration step is increased compared to the same liquid composition not containing camphorsulfonic acid and at least one cationic excipient selected from meglumine and ornithine.

14. 14. Use of the method according to claim 12 or 13, wherein the protein recovery after buffer exchange and volume reduction in the filtration step is increased compared to the same liquid composition not containing camphorsulfonic acid and at least one cationic excipient selected from meglumine and ornithine.

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