Viscosity reducing excipients and combinations thereof for liquid compositions comprising a protein

Abstract

The present invention relates to a method for reducing the viscosity of a liquid composition comprising a protein and compositions obtainable by the method. Furthermore, the invention relates to combinations of viscosity reducing excipients.

Description

[0001] Viscosity reducing excipients and combinations thereof for liquid compositions comprising a protein

[0002] Technical Field

[0003] The present invention relates to a method for reducing the viscosity of a liquid composition comprising a protein and compositions obtainable by the method. Furthermore, the invention relates to combinations of viscosity reducing excipients.

[0004] Background

[0005] Since the FDA authorized the first biopharmaceutical in 1982, many other biologic drugs have followed suit. Most of these are monoclonal antibodies (mAB) or related formats such as bi-specific antibodies or antibody fragments. While these drugs offer unique opportunities in terms of efficacy, their structure and size pose various challenges.

[0006] Antibodies and other protein therapeutics are usually administered parenterally, for example by intravenous (iv), intramuscular (im) or subcutaneous (sc) route. Subcutaneous injection is particularly popular for the delivery of protein therapeutics due to its potential to simplify patient administration (fast, low-volume injection) and reduce treatment costs (shorter medical assistance). To ensure patient compliance, it is desirable that subcutaneous injection dosage forms be isotonic and can be injected in small volumes (< 2.0 ml per injection site). To reduce the injection volume, proteins are often administered with a concentration of 1 mg / ml to 150 mg / ml.

[0007] At the same time, mAb-based therapies usually require several mg / kg dosing. The combination of high therapeutic dose and low injection volume thus leads to a need for highly concentrated compositions of therapeutic antibodies. However, being large proteins, antibodies possess a multitude of functional groups in addition to a complex three-dimensional structure. This makes their formulation difficult, particularly when a high concentration is required. One of the main problems with high concentration protein solutions is viscosity. At high concentrations, proteins tend to form highly viscous solutions largely due to non-native self-association. Additionally, proteins show an increased rate of aggregation and particle formation at such high concentrations.

[0008] These problems concern both the manufacturing process and the administration to the patient. In the manufacturing process, highly concentrated protein compositions that are highly viscous present particular difficulties for ultrafiltration and sterile filtration. In addition, tangential flow filtration is often used for the buffer exchange and for the increase of protein concentration. However, because viscous solutions show an increased back pressure and shear stress during injection and filtration, the therapeutic protein is potentially destabilized and / or process times are prolonged. Said increased shear stress frequently results in a loss of product. Both aspects adversely affect process economics.

[0009] At the same time, high viscosity is unacceptable when it comes to administration as it significantly limits the injectability of the protein.

[0010] T o solve these problems and / or to improve the stability of the solution, additives and excipients such as sucrose and sodium chloride are usually added in higher concentrations to biopharmaceutical compositions. However, the resulting solutions often cause pain due to high injection forces and resulting tissue damages. Some of these solutions may even be no longer administrable resulting in a lack of therapeutic options for the patient.

[0011] As an alternative, different excipients such as salts, camphor-10-sulfonic acid and specific amino acids, e.g. arginine, histidine, lysine and proline, have been explored as a way of reducing the viscosity of certain high-concentration protein therapeutics. Guo et al. suggest that salts having hydrophobic, bulky, and aliphatic ionic constituents may act as potent viscosity-lowering excipients (Guo Z. et al., Pharmaceutical Research’ 2012, 29(11 ):3102-9). Furthermore, WO 02 / 30463, WO 15 / 196091 , WO 15 / 196187, WO 17 / 070501 and WO 19 / 201904 disclose different viscosity reducing excipients. However, formulating proteins like monoclonal antibodies requires a careful selection of formulation additives and / or excipients to avoid protein denaturation and loss of biological activity. In addition, the excipients need to be pharmaceutically safe and physiologically compatible so to avoid any undesired side effects such as allergic reactions.

[0012] Consequently, the pharmaceutical industry has a strong need for additional pharmaceutically acceptable, viscosity-reducing excipients, especially as an alternative when standard solutions based on NaCI and amino acids such as mentioned above fail.

[0013] The problem to be solved is therefore the provision of excipients that can effectively reduce the viscosity of a protein solution and / or increase stability thereof. Furthermore, the problem to be solved is the provision of excipient combinations that can effectively reduce the viscosity of a protein solution and / or increase stability thereof. Yet another problem to be solved is that many viscosity reducing excipients used at relevant concentrations can adversely affect protein stability.

[0014] During the bioprocess, the solutions have to be pumped through tubing and chromatography columns. At high viscosities, the flow rate through such columns is limited by said viscosity which leads to longer processing times, significant protein losses during chromatography or might lead to complete non-processability of the protein solution. Furthermore, when passing through a connector from the narrow tube into a less narrow column, shear forces may occur. Shear stress is a typical reason for proteins to denature and potentially to aggregate and thereby reducing the yield of the process. Obviously, such shear stress induced aggregation has an adverse effect on process economics. Moreover, the gel bed within the chromatography column may be damaged by the high pressure.

[0015] Additionally, some proteins are formulated into high concentration through tangential flow filtration (TFF). When the viscosity of the solution becomes critical, a gel-like layer may be formed near the membrane. Especially the membrane flux is significantly reduced yielding to an increased processing time and therefore to significant higher manufacturing costs. As discussed before, also during TFF shear stress may occur yielding to insoluble protein aggregates and a reduced yield.

[0016] Generally, it has been observed that highly viscous solutions develop a certain stickiness making it difficult to recover the complete solution from containers, out of tubing or to remove the entire substance from processing systems. This loss of substance leads to a significantly reduced product yield with the obvious adverse effect on process economics.

[0017] Furthermore, a problem to be solved is the provision of excipient combinations that can effectively reduce the viscosity of a protein solution.

[0018] Yet another problem to be solved is that many viscosity reducing excipients used at relevant concentrations can adversely affect protein stability. Hence a further problem to be solved is the provision of excipient combinations that can effectively reduce the viscosity of a protein solution and that show an improved protein stability compared to one viscosity reducing excipient alone used in a higher concentration that results in a similar viscosity reduction compared to the combination.

[0019] High viscosity of protein solutions causes numerous difficulties in bioprocessing. Since known additives which hitherto are used for reducing the viscosity in corresponding protein solutions do not lead to sufficient viscosity-reducing effects in many cases, it is an object of the present invention to find new possibilities whereby corresponding viscosity-lowering effects can be improved and adverse effects on process economics can be reduced.

[0020] Summary of the invention

[0021] The problem is solved by a liquid composition comprising a protein and a viscosityreducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane, N-acetyl- tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or combinations thereof. In a further embodiment, the problem is solved by a liquid composition comprising a protein and a viscosityreducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl- tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

[0022] The use of at least two viscosity reducing excipients allows to overcome the problem that many viscosity reducing excipients used at relevant concentrations can adversely affect protein stability by allowing for the use of a lower amount of each individual excipient and leveraging the stabilizing effect of a second viscosity reducing excipients.

[0023] Likewise, the problem is solved by a method for reducing the viscosity of a liquid composition comprising a protein, comprising the step of combining the liquid composition with a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane, N-acetyl-tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or combinations thereof. In a further embodiment, the problem is solved by a method for reducing the viscosity of a liquid composition comprising a protein, preferably in a concentration in the range of 50 mg / ml to 300 mg / ml, comprising the step of combining the liquid composition with a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

[0024] The problem is also solved by a method for increasing the stability of a protein solution, comprising the steps as described above.

[0025] Furthermore, the problem is solved by a liquid composition comprising a protein and a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane, N-acetyl-tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or combinations thereof. In a further embodiment, the problem is solved by a liquid composition comprising a protein and a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

[0026] Furthermore, the problem is solved by a lyophilized protein composition of the liquid composition as described above.

[0027] Furthermore, the problem is solved by a kit comprising a liquid or lyophilized composition as described above.

[0028] An additional subject of the present invention is the use of the method for reducing the viscosity as described above in a bioprocess.

[0029] Detailed description of the invention

[0030] The invention is directed to a method for reducing the viscosity of a liquid composition comprising a protein, comprising the step of combining the liquid composition with a viscosity-reducing concentration of proline, leucine, alanine, histidine, tryptophane, N-acetyl-tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or combinations thereof.

[0031] In a further embodiment, the invention is directed to a method for reducing the viscosity of a liquid composition comprising a protein, preferably in a concentration in the range of 50 mg / ml to 300 mg / ml, comprising the step of combining the liquid composition with a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin. The term “liquid composition” as used herein, refers to a liquid comprising a protein and at least one viscosity reducing excipient. The liquid composition can refer to a final drug formuation, wherein the protein is a pharmaceutically active protein that is supplied in an acceptable pharmaceutical diluent or one that is reconstituted in an acceptable pharmaceutical diluent prior to administration to the patient. Likewise, the liquid composition can refer to a liquid composition present during manufacturing of a final drug formuation.

[0032] The term “protein” as used herein, refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide. Proteins can be naturally occurring or non-naturally occurring, synthetic, or semisynthetic. The term “protein” is understood to also cover peptides, oligopeptides, polypeptides and any therapeutic protein as defined below. Preferably the protein has a length sufficient to form a detectable tertiary structure.

[0033] Without wanting to be bound by a mechanism, it is believed that the viscosity reducing effect of the liquid compositions according to the invention is based upon an interaction between the excipients and the amino acid residues of the protein. Because all proteins are built from the same pool of amino acids, the effects described herein are thus applicable to all proteins. The liquid compositions according to the invention therefore have an advantageous effect on any protein irrespective of its sequence, size and structure.

[0034] The term “pharmaceutically active protein” as used herein refers to any protein or polypeptide that is administered to a subject with the aim of treating or preventing a disease or medical condition. In particular, the subject may be a mammal or a human. Therapeutic proteins can be administered for different purposes, such as replacing a protein that is deficient or abnormal, augmenting an existing pathway, providing a novel function or activity, interfering with a molecule or organism and delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins encompass antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, antibody drug conjugates (ADCs) and thrombolytics. Therapeutic proteins can be naturally occurring proteins or recombinant proteins. Their sequence can be natural or engineered.

[0035] In a further particularly preferred embodiment, the protein in the liquid compositions according to the invention is a plasma derived protein, in particular IgG or hyperlgG. Some pharmaceutical formulations containing plasma proteins comprise of mixtures of different plasma proteins. The term “plasma derived proteins” herein refers to a protein derived from the blood plasma of a donor by plasma fractionation. Said donor can be human or non-human. One example for plasma proteins are immune globulines. The term “IgG” herein refers to an Immune globbuline type G. The term “IgM” herein refers to an Immune globbuline type M. The term “IgA” herein refers to an Immune globbuline type A. The term “hyper-IgG” herein refers to a formulation of IgGs purified from a donor that has been infected by or vaccinated against a specific disease. Said donor can be human or non-human.

[0036] In a particularly preferred embodiment, the protein in the liquid compositions according to the invention is an antibody, in particular a therapeutic antibody. The term “antibody” herein refers to monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments.

[0037] Antibody fragments comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: Fab fragments, Fab' fragments, Fd fragments, Fd' fragments, Fv fragments, dAb fragments, isolated CDR regions, F(ab')2 fragments as well as single chain antibody molecules, diabodies and linear antibodies.

[0038] In one embodiment, the protein is a biosimilar. A “biosimilar” is herein defined as a biological medicine that is highly similar to another already approved biological medicine. In a preferred embodiment, the biosimilar is a monoclonal antibody.

[0039] In one embodiment, the liquid compositions according to the invention comprise more than one protein species. The invention further provides a liquid composition according to the invention whereas the protein has a molecular weight from 120 kDa to 250 kDa, preferably from 130 kDa to 180 kDa.

[0040] In a preferred embodiment, the protein concentration in the liquid compositions according to the invention is at least 1 mg / ml, at least 50 mg / ml, preferably at least 75 mg / ml and more preferably at least 100 mg / ml. In another preferred embodiment, the protein concentration is between 50 mg / ml and 300 mg / ml, more preferably the protein concentration is between 100 and 250 mg / ml, even more preferable between 120 and 210 mg / ml. The present invention is particularly useful for these high- concentration compositions.

[0041] The term “viscosity reducing excipient”, as used herein, refers to a compound at a suitable concentration which is known to reduce the viscosity of a liquid protein composition, preferably by at least 5% compared to an identical composition not comprising the viscosity reducing excipient.

[0042] According one aspect of the invention, the addition of a viscosity-reducing concentration of proline, leucine, alanine, histidine, tryptophane, N-acetyl- tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or combinations thereof to a liquid composition comprising a protein, preferrably in a concentration in the range of 50 mg / ml to 300 mg / ml, significantly reduces the viscosity of a liquid protein composition and optionally increases the stability of said protein in the liquid composition. The present invention thus provides a method for reducing the viscosity of a liquid composition comprising a protein comprising the step of combining the liquid composition with a viscosity-reducing concentration of the viscosity reducing excipient or the combination.

[0043] The liquid compositions according to the invention show an reduced viscosity and optionally increased stability in comparison to a composition not comprising the first or first and second viscosity reducing excipient. The viscosity reducing excipients proline, leucine, alanine, histidine, tryptophane, N-acetyl-tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin are known to be non-toxic and safe. Thus, their administration is well tolerated.

[0044] According to the invention, the viscosity reducing excipients also include salts or solvates of the excipients. Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. Salts which are not themselves suitable for pharmaceutical uses but can be used, for example, for isolation, purification or storage of the compounds according to the invention are also included.

[0045] Physiologically acceptable salts of the compounds according to the invention include salts of conventional bases, such as, by way of example and preferably, alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 C-atoms, such as, by way of example and preferably, ethylamine, diethylamine, triethylamine, N,N-diisopropylethylamine, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, procaine, dicyclohexylamine, dibenzylamine, N- methylpiperidine, N-methylmorpholine, arginine, lysine and 1 ,2-ethylenediamine.

[0046] Physiologically acceptable salts of the compounds according to the invention include salts of conventional acids, such as, by way of example and preferably, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesul- fonate, bisulfate, butyrate, camphorate, camphorsulfonate, carbonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hy- droxyethansulfonate (isethionate), lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthyl- enesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3- phenylproprionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, and undecanoate.

[0047] Without being limited to specific examples, physiologically acceptable salts can be salts of thiamine monophosphate, e.g. thiamine monophosphate hydrochloride (HCI). Solvates in the context of the invention are designated as those forms of the compounds according to the invention which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of solvates, in which the coordination takes place with water. Hydrates are preferred solvates in the context of the present invention.

[0048] In one aspect of the invention, the compositions according to the invention comprise more than one of the first viscosity reducing excipients. For example, the liquid compositions according to the invention may comprise two, three or four of the first excipients, preferably they contain two of the first excipients.

[0049] In one embodiment, the liquid compositions of the invention comprise proline and leucin, proline and alanine, proline and histidine, proline and tryptophane, proline and N-acetyl-tryptophane, proline and camphorsulfonic acid, proline and benzenesulfonic acid, proline and thiamine monophosphate or proline and pyridoxin.

[0050] In one embodiment, the liquid compositions of the invention comprise leucine and alanine, leucine and histidine, leucine and tryptophane, leucine and N-acetyl- tryptophane, leucine and camphorsulfonic acid, leucine and benzenesulfonic acid, leucine and thiamine monophosphate or leucine and pyridoxin.

[0051] In one embodiment, the liquid compositions of the invention comprise alanine and histidine, alanine and tryptophane, alanine and N-acetyl-tryptophane, alanine and camphorsulfonic acid, alanine and benzenesulfonic acid, alanine and thiamine monophosphate or alanine and pyridoxin.

[0052] In one embodiment, the liquid compositions of the invention comprise histidine and tryptophane, histidine and N-acetyl-tryptophane, histidine and camphorsulfonic acid, histidine and benzenesulfonic acid, histidine and thiamine monophosphate or histidine and pyridoxin.

[0053] In one embodiment, the liquid compositions of the invention comprise tryptophane and N-acetyl-tryptophane, tryptophane and camphorsulfonic acid, tryptophane and benzenesulfonic acid, tryptophane and thiamine monophosphate or tryptophane and pyridoxin.

[0054] In one embodiment, the liquid compositions of the invention comprise N-acetyl- tryptophane and camphorsulfonic acid, N-acetyl-tryptophane and benzenesulfonic acid, N-acetyl-tryptophane and thiamine monophosphate or N-acetyl-tryptophane and pyridoxin.

[0055] In one embodiment, the liquid compositions of the invention comprise camphorsulfonic acid and benzenesulfonic acid, camphorsulfonic acid and thiamine monophosphate or camphorsulfonic acid and pyridoxin.

[0056] In one embodiment, the liquid compositions of the invention comprise benzenesulfonic acid and thiamine monophosphate or benzenesulfonic acid and pyridoxin.

[0057] In one embodiment, the liquid compositions of the invention comprise thiamine monophosphate and pyridoxin.

[0058] According to another aspect of the invention, the addition of a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl- tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin to a liquid composition comprising a protein, preferrably in a concentration in the range of 50 mg / ml to 300 mg / ml, significantly reduces the viscosity of a liquid protein composition and optionally increases the stability of said protein in the liquid composition. The present invention thus provides a method for reducing the viscosity of a liquid composition comprising a protein comprising the step of combining the liquid composition with a viscosity-reducing concentration of the combination. The combination of two or more excipients, preferably two, three or four excipients, can synergistically reduce the viscosity of the liquid compositions comprising a protein and optionally increase the stability of said protein.

[0059] In a preferred embodiment the liquid compositions according to the invention comprise at least one first and at least one second viscosity reducing excipient, wherein the excipients synergistically reduce the viscosity and optionally increase stability in the liquid compositions comprising a protein or in protein solutions.

[0060] According to the invention, a synergistical reduction of the viscosity is given if the viscosity reduction by a combination of two or more excipients is more than the expected sum of the viscosity reduction of each individual excipient. Preferably a synergistical reduction of the viscosity is given if the percentage viscosity reduction by a combination of two or more excipients is more than the expected sum of the percentage viscosity reduction of each individual excipient.

[0061] Additionally, according to the invention, a combination of two or more viscosity reducing excipients is synergistic in case the protein stability reduction by a combination of two or more excipients is less than the expected sum of the stability reduction of each individual excipient.

[0062] In one embodiment, combinations of viscosity-reducing concentrations of two or more viscosity reducing excipients mentioned in the present application result in a synergistic reduction of viscosity and I or synergistic increase of stability of a liquid composition comprising a protein.

[0063] In one embodiment, combinations of a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin are used. In a further embodiment, combinations of a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine and histidine and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin are used.

[0064] In a further embodiment, combinations of at least one first viscosity reducing excipient selected from the group consisting of proline and histidine and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin are used.

[0065] In a further embodiment, combinations of a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin are used.

[0066] In one embodiment, a specific combination of one first viscosity reducing excipient and one second viscosity reducing excipient that synergistically reduce the viscosity of a liquid composition comprising a protein is selected.

[0067] Therefore, a further embodiment is a method for reducing the viscosity of a liquid composition comprising a pharmaceutically active protein, preferably in a concentration in the range of 50 mg / ml to 300 mg / ml, comprising the step of combining the liquid composition with a viscosity-reducing concentration of a combination of one first viscosity reducing excipient and one second viscosity reducing excipient selected from the group comprising proline and camphorsulfonic acid, proline and benzenesulfonic acid, proline and thiamine monophosphate, proline and pyridoxin, leucine and camphorsulfonic acid, leucine and benzenesulfonic acid, leucine and thiamine monophosphate, leucine and pyridoxin, alanine and camphorsulfonic acid, alanine and benzenesulfonic acid, alanine and thiamine monophosphate, alanine and pyridoxin, histidine and camphorsulfonic acid, histidine and benzenesulfonic acid, histidine and thiamine monophosphate, histidine and pyridoxin, tryptophan and camphorsulfonic acid, tryptophane and benzenesulfonic acid, tryptophane and thiamine monophosphate, tryptophan and pyridoxin, N-acetyl-tryptophane and benzenesulfonic acid, N-acetyl-tryptophane and thiamine monophosphate and N-acetyl-tryptophane and pyridoxin.

[0068] More preferred combinations are proline and camphorsulfonic acid, proline and benzenesulfonic acid, proline and thiamine monophosphate, proline and pyridoxin, leucine and camphorsulfonic acid, leucine and benzenesulfonic acid, leucine and thiamine monophosphate, leucine and pyridoxin, alanine and camphorsulfonic acid, alanine and benzenesulfonic acid, alanine and thiamine monophosphate, alanine and pyridoxin, histidine and camphorsulfonic acid, histidine and benzenesulfonic acid, histidine and thiamine monophosphate and histidine and pyridoxin.

[0069] More preferred combinations are proline and camphorsulfonic acid, proline and benzenesulfonic acid, proline and thiamine monophosphate, proline and pyridoxin, histidine and camphorsulfonic acid, histidine and benzenesulfonic acid, histidine and thiamine monophosphate and histidine and pyridoxin.

[0070] In one embodiment, the liquid compositions of the invention comprise proline and camphorsulfonic acid, proline and benzenesulfonic acid, proline and thiamine monophosphate or proline and pyridoxin.

[0071] In one embodiment, the liquid compositions of the invention comprise leucine and camphorsulfonic acid, leucine and benzenesulfonic acid, leucine and thiamine monophosphate or leucine and pyridoxin.

[0072] In one embodiment, the liquid compositions of the invention comprise alanine and camphorsulfonic acid, alanine and benzenesulfonic acid, alanine and thiamine monophosphate or alanine and pyridoxin.

[0073] In one embodiment, the liquid compositions of the invention comprise histidine and camphorsulfonic acid, histidine and benzenesulfonic acid, histidine and thiamine monophosphate or histidine and pyridoxin.

[0074] In one embodiment, the liquid compositions of the invention comprise tryptophane and camphorsulfonic acid, tryptophane and benzenesulfonic acid, tryptophane and thiamine monophosphate or tryptophane and pyridoxin. In one embodiment, the liquid compositions of the invention comprise N-acetyl- tryptophane and camphorsulfonic acid, N-acetyl-tryptophane and benzenesulfonic acid, N-acetyl-tryptophane and thiamine monophosphate or N-acetyl-tryptophane and pyridoxin.

[0075] The term “viscosity-reducing concentration”, as used herein, refers to a concentration or concentration range of the excipient orexcipient concentrations at which the viscosity of the protein formulation is reduced by at least 5%. Both, single and combined excipients can exhibit such a concentration. The viscosity-reducing concentration is variable and not universal for each protein formulation. It is to be adjusted for each protein formulation as described in the literature. For instance, Wang and colleagues (D0l:10.1021 / acs.molpharmaceut.5b00643) performed a detailed study on viscosity lowering effects of amino acids and salts in high concentration mAb formulations and discovered that a) increasing the concentration of an excipient is not necessarily enhancing its viscosity reducing properties and b) distinct amino acids and / or salts act viscosity reducing at a range of concentrations. Another example for variable exceipient concentration in protein formulations comes from protein stability field. Platts et al. (D0l:10.1016 / j.ijpharm.2015.03.051) described the destabilizing effect of arginine at concentrations > 100 mM on proteins, whereas concentrations below 100 mM were used for protein stabiliziation. These examples underline the necessity of using a concentration range for excipients to reduce solution viscosity rather than a fixed concentration. The “viscosity-reducing concentration” can be dependent on the excipient or excipient combination and I or on the protein. For a skilled person in the art, it is a simple and routine process to determine the viscosity-reducing concentration of a certain excipient or excipient combination for a particular protein.

[0076] The liquid compositions according to the invention comprise a viscosity-reducing concentration of the first excipient or a concentration of the first and second excipient sufficient to reduce the viscosity of the composition and / or stabilize the protein. For example, the liquid compositions according to the invention may comprise about 5 mM to about 300 mM, about 5 mM to about 250 mM or about 5 mM to about 150 mM of the first viscosity reducing excipient or of the at least one first and second viscosity reducing excipient respectively and independent of each other. In exemplary embodiments the concentration of the first viscosity reducing excipient or each of the first and second viscosity reducing excipients is 1 , 5, 10, 12, 13, 15, 20, 25, 30, 35, 50, 75, 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 greater. In a preferred embodiment the concentration of the at least one first and second viscosity reducing excipient independent of each other is between 5 mM and 300 mM respectively, more preferably between 10 mM and 150 mM.

[0077] In a preferred embodiment, the compositions according to the invention comprise 5 to 500 mM of proline, more preferably 10 to 300 mM, most preferably 10, 30, 50, 75 or 150 mM when used alone or in combination. In a preferred embodiment, the compositions according to the invention comprise 5 to 500 mM of leucine, more preferably 10 to 300 mM, most preferably 10, 30, 50, 75 or 150 mM when used alone or in combination. In a preferred embodiment, the compositions according to the invention comprise 5 to 500 mM of alanine, more preferably 10 to 300 mM, most preferably 10, 30, 50, 75 or 150 mM when used alone or in combination. In a preferred embodiment, the compositions according to the invention comprise 5 to 500 mM of histidine, more preferably 10 to 300 mM, most preferably 10, 30, 50, 75 or 150 mM when used alone or in combination. In one embodiment, the compositions according to the invention comprise 1 to 50 mM of tryptophane, preferably 5 to 40 mM, more preferably 5, 10, 15, 20, 25, 30 or 40 mM, most preferably 10 mM when used alone or in combination. In one embodiment, the compositions according to the invention comprise 1 to 10 mM of N-acetyl- tryptophane, preferably 5 to 10 mM, more preferably 3, 5, 7, 8 or 10 mM, most preferably 10 mM when used alone or in combination. In a preferred embodiment, the compositions according to the invention comprise 5 to 500 mM of camphorsulfonic acid, more preferably 10 to 300 mM, most preferably 10, 30, 50, 75 or 150 mM when used alone or in combination. In a preferred embodiment, the compositions according to the invention comprise 5 to 500 mM of benzenesulfonic acid, more preferably 10 to 300 mM, most preferably 10, 30, 50, 75 or 150 mM when used alone or in combination. In a preferred embodiment, the compositions according to the invention comprise 5 to 500 mM of thiamine monophosphate, more preferably 10 to 300 mM, most preferably 10, 30, 50, 75 or 150 mM when used alone or in combination. In a preferred embodiment, the compositions according to the invention comprise 5 to 500 mM of pyridoxin, more preferably 10 to 300 mM, most preferably 10, 30, 50, 75 or 150 mM when used alone or in combination.

[0078] In one embodiment the liquid composition according to the invention comprises a combination of two excipients, wherein the molar concentration of the excipients can be identical or different. Preferably the concentrations of each first and second excipient is between 1 mM and 200 mM, more preferably between 25 and 150 mM, most preferably between 50 and 100 mM. The molar ratio of the first and second excipient is between 1 :100 and 100:1 , preferably between 1 :10 and 10:1 , more preferably between 1 :5 and 5:1 , most preferably between 1 :2 and 2:1. In a particular embodiment the molar concentration of the first and second excipient is identical.

[0079] As defined herein, “viscosity” refers to the resistance of a substance (typically a liquid) to flow. Viscosity is related to the concept of shear force; it can be understood as the effect of different layers of the fluid exerting shearing force on each other, or on other surfaces, as they move against each other. There are several ways to express viscosity. The units of viscosity are Ns / m2, known as Pascal-seconds (Pas). Viscosity can be “kinematic” or “absolute”. Kinematic viscosity is a measure of the rate at which momentum is transferred through a fluid. It is measured in Stokes (St). The kinematic viscosity is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equal volume and differing viscosity are placed in identical capillary viscometers and allowed to flow by gravity, the more viscous fluid takes longer than the less viscous fluid to flow through the capillary. If, for example, one fluid takes 200 seconds (s) to complete its flow and another fluid takes 400 s, the second fluid is called twice as viscous as the first on a kinematic viscosity scale. The dimension of kinematic viscosity is Iength2 / time. Commonly, kinematic viscosity is expressed in centiStokes (cSt). The SI unit of kinematic viscosity is mm2 / s, which is equal to 1 cSt. The “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 the milliPascal-second (mPas), where 1 cP=1 mPas. Viscosity may be measured by using, for example, a viscometer at a given shear rate or multiple shear rates. An “extrapolated zero-shear” viscosity can be determined by creating a best fit line of the four highest-shear points on a plot of absolute viscosity versus shear rate, and linearly extrapolating viscosity back to zero-shear. Alternatively, for a Newtonian fluid, 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), wherein absolute viscosity is derived from a change in pressure as a liquid flows through a channel. Viscosity equals shear stress over shear rate. Viscosities measured with microfluidic viscometers can, in some embodiments, be directly compared to extrapolated zero-shear viscosities, for example those extrapolated from viscosities measured at multiple shear rates using a cone and plate viscometer. According to the invention, viscosity of liquid compositions is reduced when at least one of the methods described above show a stabilizing effect. Preferably, viscosity is measured at 20 °C using a microfluidic viscometer. More preferably the viscosity is measured using a RheoSense mVROC microfluidic viscometer at 20 °C. Most preferably the viscosity is measured at 20 °C using a RheoSense mVROC microfluidic viscometer and using a 500 pl syringe, a shear rate of 3000 s-1 or 2000 s-1 and a volume of 200 pl.

[0080] The person ordinary skilled in the art is familiar with the viscosity measurement using a microfluidic viscometer. As microfluidic viscometer the RheoSense mVROC microfluidic viscometer (mVROCTM Technology), especially with the parameters descriped above can be used. Detailed specifications, methods and setting can be found in the 901003.5.1-mVROC_User’s_Manual.

[0081] The limit of convenient injectbility is dependent on many factors, such as the length and inner diameter of the needle, the inner diameter of the syringe barrel. It is known, that drug products with a viscosity of up to 100 mPas can be injected to the patient. However, a viscosity of 60 mPas is preferred. Lower viscosities of below 30 mPas or below 20 mPas are even more preferred due to increased patient convenience.

[0082] “Shear rate” herein 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 plates. This simple case shows the uniform velocity gradient with shear rate (v1-v2) / h in units of (cm / sec) / (cm)=1 / sec. Hence, shear rate units are reciprocal seconds or, in general, reciprocal time. For a microfluidic viscometer, change in pressure and flow rate are related to shear rate. “Shear rate” is to the speed with which a material is deformed. Formulations containing proteins and viscosity-lowering agents are typically measured at shear rates ranging from about 0.5 s-1 to about 200 s-1 when measured using a cone and plate viscometer and a spindle appropriately chosen by one skilled in the art to accurately measure viscosities in the viscosity range of the sample of interest (i.e., a sample of 20 cP is most accurately measured on a CPE40 spindle affixed to a DV2T viscometer (Brookfield)); greater than about 20 s-1 to about 3,000 s-1 when measured using a microfluidic viscometer.

[0083] For classical “Newtonian” fluids, as generally used herein, viscosity is essentially independent of shear rate. For “non-Newtonian fluids,” however, 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., high- concentration) protein solutions, this may manifest as pseudoplastic shear-thinning behavior, i.e., a decrease in viscosity with shear rate.

[0084] In one embodiment, the liquid compositions of the invention show a reduction of viscosity of at least 5%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%, preferably 5%, 10% or 12%, most preferably 5%, compared to an identical composition not comprising the at least one first viscosity reducing excipient or compared to an identical composition not comprising the at least one first and at least on second viscosity reducing excipient.

[0085] In one embodiment, the liquid compositions of the invention show a reduction of viscosity of at least 5%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%, preferably 5%, 10% or 12%, most preferably 5%, compared to an identical composition not comprising the at least one first viscosity reducing excipient or compared to an identical composition not comprising the at least one first and at least on second viscosity reducing excipient. As used herein, the term “stability” encompasses both chemical and physical stability.

[0086] The term “chemical stability” herein refers to the ability of the 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 if less than about 5% of the components are degraded after 24 months at 4 °C. According to the present invention the protein formulation is typically considered chemically stable if less than about 5% of the components are degraded after 24 weeks at 25 °C with a relative humidity of 60%.

[0087] Stability can be assessed in many ways known to the skilled person, including monitoring conformational change over a range of temperatures (thermostability) and / or time periods (shelf-life) and / or after exposure to stressful handling situations (e.g. physical shaking). 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 a specific wavelength, by size exclusion chromatography (in which aggregates of a protein will elute in different fractions compared to the protein in its native active state), HPLC, or other chromatographic methods. Other methods of measuring conformational change can be used, including differential scanning calorimetry (DSC), e.g. to determine the temperature of denaturation, or circular dichroism (CD), which measures the molar ellipticity of the protein. Fluorescence can also be used to analyze the composition. Fluorescence encompasses the emission of light subsequent to absorption of light, which requires a suitable wavelength. Potential readouts are changes in the polar properties of light, light intensity, or emission wavelength. Fluorescence emission can be intrinsic to a protein or can be due to a fluorescence reporter molecule that for example binds to the hydrophobic pockets of partially unfolded proteins. An increase in binding of reporter molecules can be monitored by detection of the fluorescence signal of a protein sample. Other means for measuring stability can be used and are well known to persons skilled in the art. According to the invention, stability of liquid compositions is increased when at least one of the methods described above show a stabilizing effect. In one embodiment, the liquid compositions of the invention show an increase in stability of the protein of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to an identical composition not comprising the at least one first viscosity reducing excipient or compared to an identical composition not comprising the at least one first and at least on second viscosity reducing excipient.

[0088] In one embodiment, the liquid compositions according to the invention comprise a viscosity reducing excipient or excipient combination as mentioned above and show an increased protein stability characterized by an elevated Tm and / or Tagg.

[0089] In one embodiment, the liquid compositions according to the invention have a viscosity between 1 mPas and 60 mPas, preferably between 1 mPas and 50 mPas, more preferably between 1 mPas and 30 mPas, most preferably between 1 mPas and 20 mPas. Preferably the viscosity is measured at 20 °C, using a microfluidic viscometer. More preferably the viscosity is measured using a RheoSense mVROC microfluidic viscometer at 20 °C. Most preferably the viscosity is measured using a RheoSense mVROC microfluidic viscometer at 20 °C, using a 500 pl syringe, a shear rate of 3000 s-1 or 2000 s-1 and a volume of 200 pl.

[0090] In one embodiment, the liquid compositions according to the invention comprise a protein with a molecular weight from 120 kDa to 250 kDa at a concentration between 90 mg / ml to 300 mg / ml, preferably further comprising an acetate buffer or phosphate buffer at a concentration between 10 mM to 50 mM, wherein the composition has a pH between 5 and 7.2, and a viscosity between 1 mPas and 60 mPas when measured at 20 °C, preferably using a microfluidic viscometer, more preferably using a RheoSense mVROC microfluidic viscometer at 20 °C, most preferably using a RheoSense mVROC microfluidic viscometer at 20 °C with a 500 pl syringe, a shear rate of 3000 s-1 or 2000 s-1 and a volume of 200 pl.

[0091] Preferably, liquid composition according to the invention wherein a first and a second viscosity reducing excipient are present, have a protein concentration between 90 mg / ml to 300 mg / ml, more preferably between 100 mg / ml and 200 mg / ml and a concentration of the first and second viscosity reducing excipient between 50 and 200 mM each, more preferably between 50 and 100 mM each, most preferably 75 mM each. Preferably the protein is an antibody and has a molecular weight from 120 kDa to 250 kDa. Preferably the composition has a pH between 4 and 8, more preferably between 5 and 7.2 and the viscosity is between 1 mPas and 60 mPas when measured at 20 °C using a microfluidic viscometer.

[0092] In one embodiment, the liquid composition according to the invention has a pH between 2 and 10, preferably between 4 and 8, more preferably between 5 and 7.2. In one embodiment, the liquid compositions have a pH of 5 or 7.2.

[0093] The liquid composition according to the invention may additionally comprise pharmaceutically acceptable diluents, solvents, carriers, adhesives, binders, preservatives, solubilizers, surfactants, penetration enhancers, emulsifiers or bioavailability enhancers. The skilled person knows how to choose suitable additives for liquid compositions that are safe and well tolerated.

[0094] The liquid composition according the invention may further comprise excipients that are used for purposes other than reducing viscosity, e.g. stabilization, solubilization or preservation.

[0095] In a preferred embodiment, the liquid compositions according to the invention comprise a stabilizer such as a sugar and / or a surfactant. Suitable sugars as stabilizers are known in the literatur, e.g. sucrose or trehalose. In a preferred embodiment, the sugar is sucrose, preferably 50 to 100 mg / ml sucrose. Suitable surfactants are known in the literatur, e.g. polysorbate 20 or polysorbate 80 or poloxamer 188. In another preferred embodiment, the surfactant is polysorbate 80, preferably 0.01 to 0.2 mg / ml, more preferably 0.05 mg / ml of polysorbate 80. The addition of a further stabilizers additionally enhances the stabilizing effect of the compositions according to the inventions.

[0096] The invention further provides a liquid composition according to the invention further comprising a buffer at a concentration of 10 mM to 50 mM. The buffer can be a suitable acetate- or phosphate salt and provide a pH of 5 to 7.2. Furthermore, the invention is directed to a method for increasing the stability and a method of preventing self-association of a protein in a liquid composition comprising the steps as described for the method for reducing the viscosity of a liquid composition above.

[0097] In another aspect, the invention provides liquid compositions obtainable by a method as described above.

[0098] In another aspect, the invention provides lyophilized protein compositions comprising a protein and at least one first excipient as defined above or at least one first viscosity reducing excipient and at least one second viscosity reducing excipient as defined above. Upon reconstitution with a suitable amount of diluent, the compositions exhibit reduced viscosity relative to control compositions with the otherwise same composition but not comprising the excipient. Thus, the at least one excipient is present at an amount effective to reduce viscosity upon reconstitution with diluent.

[0099] In another aspect, the invention provides lyophilized protein compositions comprising a protein and at least one first excipient as defined above or at least one first viscosity reducing excipient and at least one second viscosity reducing excipient as defined above.

[0100] A lyophilized protein composition includes a protein and the at least one excipient according to the invention that has been dried and is present as particles in, for example, powder form. In the present context the expression "powder" refers to a collection of essentially dry particles, i.e. the moisture content being at least below about 10% by weight, 6% by weight, 4% by weight, or lower.

[0101] The invention is also directed to a kit comprising a lyophilized protein composition of the 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-chambered pre-filled syringes, or cartridges, but also a 96-well plate comprising ready-to-use freeze-dried or spray-dried compositions sitting in the wells. Exemplary administration devices include syringes with or without needles, infusion pumps, jet injectors, pen devices, transdermal injectors, or other needle- free injectors.

[0102] All embodiments including any combinations and concentrations of viscosity reducing excipients, concentrations and molecular weight of proteins, pH, buffer and buffer concentrations as mentioned for the method for reducing the viscosity of a liquid composition as described above are also applicable for the liquid composition, lyophilized protein composition, kit, method for increasing the stability and method for preventing self-association.

[0103] In the methods according to the invention, the excipients may be added in any way known to the artisan to the protein solution. When more than one excipient is added, the excipient may be mixed together to form a viscosity-reducing solution which is then added to the protein solution. Likewise, the excipients may be added separately to the protein solution.

[0104] In a preferred embodiment of the invention the liquid composition is a pharmaceutical composition. The invention is also directed to a pharmaceutical composition as described above comprising a therapeutic protein for the treatment of disease.

[0105] In particular pharmaceutical compositions as described above comprising a therapeutic protein are suitable for the treatment of cancer, rheumatoid arthritis, morbus crohn, colitis ulcerose, ankylosing spondylitis, psoriasis-arthritis, psoriasis, hypercholesterolemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, myocardial infaction, peripheral arterial disease or immune deficiency disorders.

[0106] Furthermore, method for reducing the viscosity of a liquid composition comprising a protein of the present invention are beneficial in the bioprocess. Viscosity reducing excipients and viscosity reducing excipient combinations are suitable to reduce the backpressure on chromatography columns and allow for larger flow rates. This leads to less shear forces straining the proteins in the solution and therefore, aggregation will be reduced. Altogether a higher yield can be obtained. Beyond this, when the process can be run using higher flow rates, the process time will be reduced significantly.

[0107] In bioprocesses as meant here, the addition of these excipients, which are found to serve as viscosity-reducing additives, lead to an improved process economy, in that on the one hand the yield of intact protein can be improved, and on the other hand the duration of the process can be reduced.

[0108] The viscosity reducing excipients and combinations of the present invention have beneficial properties during dead-end filtration approaches where a solution is passed through a filter or medium, like a gel bed, by a force applied by back-end pressure, e.g. during chromatographic purifications. While said filtration steps are mainly used in the downstream process, viscosity reducing excipients can also be beneficial in the upstream process. When protein concentrations elevate to levels where they cause viscosity, with the described negative effects of pressure limitations and shear forces when the solution is passed through a tubing or a filter to remove cellular material and debris the presented invention will obviously have beneficial effects. To measure process efficiency in tangential flow filtration, where in contrast to previously used method the majority of the field flow travels tangentially across the surface of the filter, rather than passing through the filter, a laboratory scale TFF system can be used. Filtration methods are typical unit operation used to exchange a composition buffer or to bring the concentration of a biomolecule to the desired level. Also stirred cells can be used as representation of dead-end filters, where a feed is passed through a filtering material that withholds larger molecules on top of the material releasing the filtrate on the other end of the device.

[0109] A frequent method to exchange buffers and to concentrate proteins is tangential flow filtration, where in contrast to previously used methods the majority of the field flow travels tangentially across the surface of the filter, rather than passing through the filter. Like when stirred cells are used in tangential flow filtration the large molecules are separated from smaller molecules by passing said smaller molecules through a suitable filter material. In contrast to stirred cells, which represent one form of dead end filtration, in tangential flow filtration the flow geometry of the feed is different to avoid the formation of a filter cake and allowing for a continuous process. When stirred cells are used the formation of a filter cake is likewise prevented by the use of a stirring device. Therefore, the stirred cells closely resemble a tangential flow filtration device in spite of the differences in filter geometry. The efficiency of both methods is critically depending on the membrane resistance. Also, a high viscosity is known to reduce the flux rate that can be used and therefore increasing processing time resulting in higher production costs. It is therefore expected that a reduced viscosity allows for a more efficient filtration process while shear forces remain low yielding to a higher protein concentration in the filtrate. This is highlighted by the work of Hung et al. who state: “During production of concentrated monoclonal antibody compositions by tangential flow ultrafiltration (TFF), high viscosities and aggregation often cause extensive membrane fouling, flux decay and low product yields” (Journal of Membrane Science Volume 508, 15 June 2016, Pages 113-126)

[0110] Therefore, another aspect of the present invention is to provide a method for reducing the viscosity of a liquid composition comprising a protein, preferably in a concentration in the range of 50 mg / ml up to 300 mg / ml, comprising the step of combining the liquid composition with a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane, N-acetyl-tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or a salt or a solvent thereof, or combinations thereof. In another aspect, a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl- tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin is combined with the liquid composition comprising a protein, preferably in a concentration in the range of 50 mg / ml up to 300 mg / ml. Another aspect of the present invention is the use of the method for reducing the viscosity of a liquid composition comprising a protein as described above in a bioprocess.

[0111] According to the present invention all parameters mentioned above; e.g. combinations of the excipients, concentrations of the excipients, concentrations the protein, ratios of the excipients, further elements of the composition such as buffers or stabilizers, pH values, viscosity reductions, specifications of the protein, molecular weight of the protein also apply to the use in bioprocess.

[0112] Preferably, the at least one first viscosity reducing excipient is selected from the group consisting of proline, leucine, alanine and histidine and the at least one second viscosity reducing excipient is selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

[0113] More preferably, the at least one first viscosity reducing excipient is selected from the group consisting of proline, and histidine and the at least one second viscosity reducing excipient is selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

[0114] In one embodiment a specific combination of two viscosity reducing excipients is used. Preferable viscosity reducing excipient combinations of one first and one second viscosity reducing excipient are selected from the list consisting of proline and camphorsulfonic acid, proline and benzenesulfonic acid, proline and thiamine monophosphate, proline and pyridoxin, leucine and camphorsulfonic acid, leucine and benzenesulfonic acid, leucine and thiamine monophosphate, leucine and pyridoxin, alanine and camphorsulfonic acid, alanine and benzenesulfonic acid, alanine and thiamine monophosphate, alanine and pyridoxin, histidine and camphorsulfonic acid, histidine and benzenesulfonic acid, histidine and thiamine monophosphate, histidine and pyridoxin, tryptophane and camphorsulfonic acid, tryptophane and benzenesulfonic acid, tryptophane and thiamine monophosphate, tryptophane and pyridoxin, N-acetyl-tryptophane and camphorsulfonic acid, N- acetyl-tryptophane and benzenesulfonic acid, N-acetyl-tryptophane and thiamine monophosphate or N-acetyl-tryptophane and pyridoxin.

[0115] Concentration ranges and molar ratios of the first viscosity reducing excipient or the first and second viscosity reducing excipient for the use in the bioprocess are identical to those mentioned above for the liquid compositions comprising a protein.

[0116] Depending on the protein solution and the bioprocess carried out, different buffer systems may be used as buffers. Acetates, like ammonium acetate, or sodium acetate, carbonates like ammonium bicarbonate or sodium bicarbonate, or phosphates, like sodium phosphate or Tris-phosphate may be used here, depending on the conditions during the bioprocess.

[0117] Another aspect of the present invention is to provide a method for reducing the viscosity of a protein solution in a bioprocess as mentioned above, wherein the permeate flux of the protein solution in a filtration step is increased compared to an identical protein solution not comprising at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane, N-acetyl-tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or combinations thereof or compared to an identical protein solution not comprising at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

[0118] Increase of permeate flux means a percentage increase of at least 2%, preferably at least 5%, more preferably at least 10%, most preferred 10% to 100%.

[0119] Another aspect of the present invention is to provide a method for reducing the viscosity of a protein solution in a bioprocess as mentioned above, wherein the protein recovery after buffer exchange and volume reduction in filters is increased compared to an identical protein solution not comprising at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane, N-acetyl-tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or combinations thereof or compared to an identical protein solution not comprising at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

[0120] Increase of protein recovery after buffer exchange and volume reduction in filters means a percentage increase of protein recovery of at least 1%, preferably at least 2%, more preferably at least 5%, most preferred 5% to 20%.

[0121] Another aspect of the present invention is to provide a method for reducing the viscosity of a protein solution in a bioprocess as mentioned above, wherein the process time for a filtration step, preferably a filtration step wherein the protein is concentrated, is reduced compared to an identical protein solution not comprising at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane, N-acetyl-tryptophane, camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate, pyridoxin or combinations thereof or compared to an identical protein solution not comprising at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

[0122] Reduction of process time for a filtration step means a percentage reduction of at least 5%, preferably at least 10%, more preferably at least 25%, most preferred 25% to 100%.

[0123] In a particular embodiment of the invention, the filtration step is a tangential flow filtration (TFF). The term “bioprocess” refers to therapeutic cell manufacturing processes, which can be separated into upstream processes and downstream processes. The upstream process is defined as the entire process prior to separating protein from cellular compounds. The upstream process comprises early cell isolation and cultivation, to cell banking and culture expansion of the cells until final harvest. The downstream part of a bioprocess refers to the part where the target protein is purified from the feed of the upstream and is processed to meet purity and quality requirements. Some type of cells need to be disrupted when entering the downstream process. Yet other cells may secrete the target protein into the media and need to be removed via filtration. Further downstream processing is usually divided into the main sections: a purification section and a polishing section. A bioprocess can be a batch process or a semi-continuous or a continuous process.

[0124] The term “permeate flux” refers to the volume passing through a defined filter within a certain period of time, typically on the order of minutes.

[0125] The term “filtration step” refers to a process step where a liquid is passed through a material with a defined pore size allowing for the separation of materials based on their size. For some filters the pore size is defined in nanometers. Yet for other filters, the pore size is not directly defined, but the weight of a molecule to be withheld is given. Filtering materials can be placed in a way that they block the crosssection of the filtration device (dead-end filtration). Yet filtering materials can be placed in a way that the solution to be filtered is tangentially flowing across the surface of said material, e.g. tangential flow filtration. The filtering material can be a membrane, a glass filter, a metallic filter or a resin. The resin can be held in a chromatography column. The resin can be a cationic or anion exchange resin, an affinity resin, like a Protein A or glutathione resin, or a hydrophobic or hydrophilic resin.

[0126] The term “protein recovery” after buffer exchange and volume reduction refers to the fraction of protein to be retrieved after a process step.

[0127] The term “tangential flow filtration” or “TFF” refers to a method of filtration where a solution passes over a defined filter tangentially. Substances smaller than the filter pores are forced out of the solution through the filter by the pressure resulting from solution flow rate, viscosity, temperature and other factors.

[0128] Examples

[0129] 1. Viscosity reduction of viscosity reducing excipients and combinations thereof on protein formulations

[0130] Infliximab Sample Preparation

[0131] 5 mM phosphate buffer was prepared by appropriately mixing sodium dihydrogenphosphate and di-sodium hydrogenphosphate to yield a pH of 7.2 and dissolving the mixture in ultrapure water. The ratio was determined using the Henderson-Hasselbalch equation. pH was adjusted using HCI and NaOH where necessary. 50 mg / ml sucrose and 0.05 mg / ml polysorbate 80 were added as stabilizers.

[0132] Excipient solutions containing a single excipient at a concentration of 10 mM for (tryptophan (Trp) and N-acteyl-tryptophan (N-Ac Trp) (due to solubility constraints) and 150 mM (proline (Pro), leucine (Leu), alanine (Ala), histidine (His), camphorsulfonic acid (CSacid), benzenesulfonic acid (BSacid), thiamine monophosphate (TMP) and pyridoxin (Pyr)) and excipient solutions containing a combination of two excipients at the same concentration were prepared in phosphate buffer pH 7.2. Excipient combinations and concentrations can be obtained from table 1. The pH was adjusted using HCI or NaOH where necessary.

[0133] A concentrated Infliximab solution containing the desired excipients was prepared using centrifugal filters (Amicon, 30 kDa MWCO) to exchange the original buffer with a buffer containing the respective excipient and to reduce the volume of the solution. The protein was subsequently diluted to 120 mg / ml, respectively.

[0134] Evolocumab Sample Preparation

[0135] 10 mM acetate buffer was prepared by weighing 1.2 g of 100% acetic acid and desolving it in ultrapure water. pH was adjusted using acetic acid and NaOH where necessary. 0.05 mg / ml polysorbate 80 were added as stabilizer. Excipient solutions containing a single excipient at a concentration of 10 mM for (tryptophan (Trp) and N-acteyl-tryptophan (N-Ac Trp) (due to solubility constraints) and 30 mM (proline (Pro), leucine (Leu), alanine (Ala), histidine (His), camphorsulfonic acid (CSacid), benzenesulfonic acid (BSacid), thiamine monophosphate (TMP) and pyridoxin (Pyr)) and excipient solutions containing a combination of two excipients at the same concentration were prepared in phosphate buffer pH 5.0. Excipient combinations and concentrations can be obtained from table 2. The pH was adjusted using HCI or NaOH where necessary.

[0136] A concentrated Evolocumab solution containing the desired excipients was prepared using centrifugal filters (Amicon, 30 kDa MWCO) to exchange the original buffer with a buffer containing the respective excipient and to reduce the volume of the solution. The protein was subsequently diluted to 170 mg / ml, respectively.

[0137] Protein Concentration Measurements

[0138] Protein Concentration was determined using absorption spectroscopy applying Lambert-Beer's law. When excipients themselves had a strong absorbance at 280 nm, a Bradford asssay was used.

[0139] Concentrated protein solutions were diluted so that their expected concentration would lie between 0.3 and 1.0 mg / mL in the measurement.

[0140] For absorption spectroscopy, the absorbance at 280 nm was measured using a BioSpectrometer® kinetic (Eppendorf, Hamburg, Germany) with a protein extinction coefficient of Ao.i%, 280nm=1.428.

[0141] Some excipients have themselves a strong absorption at 280 nm, which makes it necessary to use a Bradford assay for concentration determination.

[0142] For the Bradford assay, a kit as well as Bovine Gamma Globulin Standard from Thermo Scientific™ (Thermo Fisher, Waltham, Massachusetts, USA) were used. Absorption was measured at 595 nm using a Multiskan™ Wellplatereader (Thermo Fisher, Waltham, Massachusetts, USA). Protein concentrations were determined by linear regression of a standard curve from 125 to 1500 pg / ml.

[0143] Viscosity Measurements

[0144] The mVROC™ Technology (Rheo Sense, San Ramon, California, USA) was used for viscosity measurements. Measurements were performed at 20 °C using a 500 l syringe and a shear rate of 3000 s-1 . A volume of 200 l was used. All samples were measured as triplicates.

[0145] Calculation of synergism

[0146] Results of viscosity measurements regarding these experiments can be seen in table 1. The percentual reduction compared to a respective control sample not containing the investigated viscosity reducing excipient was calculated. Using these percentual viscosity reductions an expected viscosity reduction could be calculated if one would combine those.

[0147] Expected values can not be calculated based on absolute values, since two combined excipients which reduce the viscosity by 50% each would result in a viscosity of 0 mPas. From a scientific point this is not feasible, especially in case the excipients reduce the viscosity by more than 50% resulting in negative viscosity values. Hence, the expected viscosity reduction of both excipients was determined based on a consecutive calculation:

[0148] Expected vicosity =

[0149] Viscosity of control * (100% - viscosity reduction [%] 1st excipient) * (100% - viscosity reduction [%] 2nd excipient)

[0150] Example calculation for a combination of two excipients reducing the viscosity by

[0151] (i) 50% each and (ii) 75% each in a solution with a viscosity of 100 mPas:

[0152] (i) Expected viscosity =100 mPas*(100%-50%)*(100%-50%)=25 mPas

[0153] (ii) Expected viscosity =100 mPas*(100%-75%)*(100%-75%)=6,25 mPas

[0154] If for a combination of two excipients a lower viscosity, i.e. higher viscosity reduction is observed compared to the expected one, the combination is found to be synergistic. Relative viscosity is accordingly calculated and can be used as a measure for synergisticy viscosity reduction ( see Table 1 and 2).

[0155] Table 1 is showing the relative viscosity reduction of an Infliximab solution (120 mg / ml) by different excipient (Exc.) combinations as indicated. Table 1

[0156] Table 2 is showing the relative viscosity reduction of an Evolocumab solution (170 mg / ml) by different excipient (Exc.) combinations as indicated.

[0157] Table 2

[0158]

Claims

Claims1. A method for reducing the viscosity of a liquid composition comprising a pharmaceutically active protein in a concentration in the range of 50 mg / ml to 300 mg / ml, comprising the step of combining the liquid composition with a viscosity-reducing concentration of at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

2. The method according to claim 1 , wherein the at least one first viscosity reducing excipient is selected from the group consisting of proline, leucine, alanine and histidine and the at least one second viscosity reducing excipient is selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

3. The method according to claim 1 or 2, wherein the at least one first viscosity reducing excipient is selected from the group consisting of proline and histidine and the at least one second viscosity reducing excipient is selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

4. The method according to claim 1 or 3, wherein the at least one first viscosity reducing excipient is selected from the group consisting of alanine, histidine, tryptophane and N-acetyl-tryptophane and the at least one second viscosity reducing excipient is selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin.

5. The method according to any of claims 1 to 4, wherein the viscosity is reduced by at least 5% compared to an identical composition not comprising the at least one first viscosity reducing excipient and at least one second viscosity reducing excipient.

6. The method according to any of claims 1 to 5, wherein the concentration of the at least one first and second viscosity reducing excipient independent of each other is between 5 mM and 300 mM respectively.

7. The method according to any of claims 1 to 6, wherein the liquid composition has a pH between 4 and 8.

8. The method according to any of claims 1 to 7, wherein the liquid composition further comprises a phosphate buffer or an acetate buffer at a concentration between 5 mM and 50 mM.

9. The method according to any of claims 1 to 8, wherein the protein has a molecular weight from 120 kDa to 250 kDa.

10. The method according to any of claims 1 to 9, wherein the protein is an antibody.

11. A liquid composition obtainable by a method according to any of claims 1 to10.

12. A lyophilized protein composition of the liquid composition according to claim11.

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

14. Use of the method according to any of claims 1 to 10, wherein the permeate flux of the protein solution in a filtration step is increased compared to an identical protein solution not comprising the at least one first viscosity reducing excipient selected from the group consisting of proline, leucine, alanine, histidine, tryptophane and N-acetyl-tryptophane or salts or solvates thereof and at least one second viscosity reducing excipient selected from the group consisting of camphorsulfonic acid, benzenesulfonic acid, thiamine monophosphate and pyridoxin or salts or solvates thereof.

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