Alternative surfactants as stabilizers for therapeutic protein preparations

Abstract

The present invention relates to novel liquid pharmaceutical compositions comprising a protein, preferably an antibody, as defined herein, together with one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG and SL.

Description

[Technical Field]

[0001] Field of Invention This invention relates to the field of aqueous protein formulations, particularly to their stabilization against visible particle formation during storage. [Background technology]

[0002] Background of the Invention The success story of biopharmaceuticals, also known as recombinant therapeutics, began in 1982 with Humulin®, the first genetically modified biopharmaceutical to be approved.[1] Today, biopharmaceuticals are a major field and a rapidly growing market with an increasing number of products under development.[2] Because proteins are amphiphilic polymers, they tend to enter and accumulate at interfaces, where they can aggregate. Therefore, proteins need to be protected from interfaces and interfacial stress to ensure product stability. Interfacial stress can occur not only during many stages of product manufacturing (e.g., freezing, thawing, filtration, pumping, and filling operations) but also during shipment to patients, storage, and administration.[3–5] Protein aggregation can result in increased formation of microscopically visible particles and / or visible particles, or even a potential loss of efficacy or safety (i.e., in the case of immunogenic reactions). Therefore, maintaining formulation stability by preventing protein aggregation is important.[6–8] Various aggregation mechanisms and mitigation strategies to avoid protein aggregation have been identified and disclosed.[9] One common approach is to add excipients such as surfactants to the formulation

[10] . Surfactants are potent stabilizers against interfacial stress, resulting in the suppression or reduction of adsorption of amphiphilic proteins at the interface. Two mechanistic models regarding how surfactants protect proteins have been disclosed: namely (1) the formation of surfactant-protein complexes, and (2) the main mechanisms of preferential competitive adsorption of surfactants at the interface [4,11-14].

[0003] For proven safety and efficacy, three nonionic surfactants are used in the formulation development of commercially available biologics: polysorbate 20, polysorbate 80 (also known as Tween®), or triblock copolymer poloxamer 188 (Kolliphor® P, Pluronic®, or Synperonic®). Polysorbate (PS) is known to be an excellent stabilizer at both the air-water interface present during agitation and agitation stress[15,16] and the silicone oil-water interface mainly found in pre-filled syringes[17-19], but is also present under various other stresses such as freeze / thaw and freeze-drying

[20] . Thus, this type of surfactant is the most widely used in commercial products[21,22]. However, PS is also a chemically heterogeneous mixture consisting mainly of an ethoxylated sorbitan skeleton with up to three different fatty acid side chains, resulting in substantial material variability between suppliers and lots[23-25]. Separately, polysorbates can be degraded by oxidation or hydrolysis, which can lead to problems resulting from (1) protection against interfacial stress or protein reduction, which may be accompanied by protein particle formation, and / or (2) adverse effects of PS degradation products on protein stability [25-27]. Further studies on PS degradation have reported enzymatic cleavage of ester bonds, which may be caused by impurities derived from host cell proteins [28,29]. Both hydrolytic and enzymatic pathways can generate free fatty acid (FFA) degradation products with limited solubility and a tendency to form microscopically visible particles [27,30,31].

[0004] In contrast, poloxamer 188 (Px) has been reported to be more stable and consists of two hydrophilic polyethylene oxide (PEO) units linked by a more hydrophobic polypropylene oxide (PPO) intermediate block [32,33]. The more hydrophilic nature of Px188 (HLB>24) was hypothesized to be due to increased Fc fusion protein adsorption at the silicone oil-water interface in pre-filled syringes compared with PS80 (HLB=15.0)

[19] . Px188 has also been reported to have a higher risk of protein-silicone oil particle formation in vials. Depending on the molecular properties of the protein, the amount of silicone oil, and other characteristics of the formulation composition, challenges may be presented for the use of Px188 as a stabilizer, particularly in pre-filled syringes (PFS), as silicone oil is used as a lubricant, which may account for the majority of particles detected in biopharmaceuticals stored in PFS [8,34,35].

[0005] Recently, several novel molecules have been suggested as alternative surfactants. Maggio reported that alkyl saccharides can stabilize monoclonal antibodies (mAbs) equivalent to interferon and PS [36,37]. Furthermore, alkyl saccharides have been reported to be stable against oxidative degradation

[38] . However, compared to other surfactants, alkyl saccharide surfactants have higher hemolytic activity, particularly n-dodecyl-β-d-maltopyranoside (DDM), making their therapeutic applications more difficult

[39] . Less pronounced hemolytic activity was reported by Schiefelbein et al. for their synthetic trehalose-based surfactants, which also showed promising stabilizing effects on human growth hormone (hGH) during shaking

[40] . Katz et al. synthesized a novel amino acid-based surfactant called FM100 and tested its ability to protect IgG and abatacept from stirring stress compared to polysorbate 20 and 80 and poloxamer 188. FM100 was found to stabilize the interface faster than all three other surfactants while increasing the stabilization of the model protein against agitation-induced aggregation [41,42]. Nevertheless, no clinical or commercial use of any of the above surfactants has yet been reported. Other classes of nonionic surfactants often listed as interface stabilizers are primary alcohol ethoxylates such as Brij® or alkylphenol ethoxylates such as Triton® X [43,44]. However, most of the molecules mentioned above are not approved for parenteral use or there are concerns about the safety of repeated and general parenteral administration.

[0006] Therefore, there is a need for alternative surfactants that do not have problems with inherent stability and adsorption behavior at pharmaceutically relevant interfaces. In particular, there remains a need to investigate novel / alternative surfactants to mitigate existing problems with established surfactants for parenteral administration in order to expand the toolbox for formulation development while ensuring optimal formulation stability. [Overview of the Initiative]

[0007] The present invention solves this problem by proposing a known surfactant for novel use as a stabilizer in therapeutic protein formulations. More specifically, the inventors have performed a comprehensive evaluation of the structural composition of surfactants that have a good protein stabilization effect at the relevant interface and need to be less prone to enzymatic degradation. The inventors have investigated surfactants having a wide range of structural diversity with respect to hydrophobic and hydrophilic molecular moieties (see Figure 1). The inventors have also implemented a screening tool for analyzing surfactants with respect to their stability against enzymatic degradation and their effect on the thermal stability of a model mAb. To eliminate adverse effects on long-term stability, samples were stored at 5°C and 25°C for up to 18 months, as well as at 40°C for 3 months, and analyzed for changes in visible particles and microscope-visible particles, turbidity, color, pH, mAb monomer, and mAb charge. Controls using PS20 and Px188 were run in parallel. [Invention 1001] A liquid pharmaceutical composition comprising a protein and one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL. [Invention 1002] The composition of the present invention 1001, wherein the protein is an antibody; or an immunoconjugate; or an antibody fragment. [Invention 1003] A composition according to the present invention 1001, further comprising a pharmaceutically acceptable excipient or carrier. [Invention 1004] A composition according to any one of the present invention 1001 to 1003, wherein the surfactant (multiple types are permitted) is present in a concentration of 1 mg / mL or less; or in a concentration range of 0.001 mg / mL to 0.01 mg / mL; or 0.01 mg / mL to 0.1 mg / mL; or 0.1 mg / mL to 1.0 mg / mL. [Invention 1005] Use of one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL in the manufacture of liquid pharmaceutical compositions further containing proteins. [Invention 1006] Use of one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL to stabilize the protein and prevent the formation of visible particles in the liquid pharmaceutical composition containing the protein during storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] (Figure 1) Graph representation of all alternative surfactants tested in this study. The structures are classified into four subgroups according to their lipophilic moieties: i) acyl-, ii) alkyl-, iii) sterol-, and iv) other subgroups. Furthermore, the molecules were distinguished by their hydrophilic head groups as i) polyethylene oxide (PEO)-based and ii) sugar-based surfactants. Marked excipients (*) are used in marked parenteral formulations within the FDA and EMA [45,46]. (Figure 2) Representative PS20RP-HPLC chromatograms classified into hydrophilic non-esterified fraction (1) and lipophilic esterified fraction (2) before and after enzymatic digestion (-). The main peak (lipophilic) of the esterified fraction and the free non-esterified (hydrophilic) peak were integrated for evaluation. (Figure 3) Degree of surfactant decomposition. Each value is 0.25 TIFF0007898238000001.tif6128 and 0.5 TIFF0007898238000002.tif6 128 mg / mL PCL / CALB lipase mixture (1:1) or 0.1 mmol sodium hydroxide Before incubation with TIFF0007898238000003.tif6128 The normalized ester main peak area is shown after TIFF0007898238000004.tif6128. **The ester main peak was not observed and was completely decomposed. (Figure 4A) Thermal conformational stability of mAbs in the presence of surfactants. The figure shows T on (A) and T m1 The average of the three individual measurements in (B) is shown. Surfactants with colored values ​​showed significantly reduced thermal stability compared to the control formulation (-) which did not contain a surfactant. (Figure 4B) See the explanation for Figure 4A. (Figure 5A) Control formulation without surfactants TIFF0007898238000005.tif6128 and TIFF0007898238000006.tif51280.1 and Cumulative number of microscopically visible particles ≥ 10 μm / mL for formulations containing 1 mg / mL of surfactant. After horizontal shaking at 5°C (A) and 25°C (B), 5 constitutive freeze-thaw cycles (C) and initial value. SVP count after 12 weeks of storage at 40°C (D) compared with TIFF0007898238000008.tif6128. Formulations where SVP is in the upper segment of the Y axis are classified as USP <787> The number of particles exceeded the standard limit of 6,000 per container, which is ≥ 10 μm in size. (Figure 5B) See the explanation for Figure 5A. (Figure 5C) See the explanation for Figure 5A. (Figure 5D) See the explanation for Figure 5A. (Figure 6A) Different stress conditions and surfactant concentrations Soluble aggregate levels given as increase in HMWS (area %) after TIFF0007898238000009.tif6128: (A) 7 days of horizontal shaking stress at 200 rpm at 5°C or (B) 25°C, (C) 5 constitutive freeze-thaw cycles, and (D) 12 weeks of storage at 40°C. (Figure 6B) See the explanation for Figure 6A. (Figure 6C) See the explanation for Figure 6A. (Figure 6D) See the explanation for Figure 6A. (Figure 7A) mAb formulations at the initial stage TIFF0007898238000010.tif6128 and placebo Cumulative number of microscopically visible particles 2 μm or larger per 1 mL. The sample contained either (A) 0.1 mg / mL or (B) 1 mg / mL of surfactant. (Figure 7B) See the explanation for Figure 7A. (Figure 8A) Typical monomer loss by SE-HPLC of formulations stored at 25°C containing (A) 0.1 mg / mL or (B) 1 mg / mL of surfactant. This graph shows only formulations with significantly altered main peak areas. TIFF0007898238000012.tif6128, standard surfactant PS20 TIFF0007898238000013.tif5128 and Px188 This is compared to TIFF0007898238000014.tif6128. (Figure 8B) See the explanation for Figure 8A. (Figure 9A) Typical decrease in main peak area of ​​mAbs as measured by IE-HPLC. Formulations contained (A) 0.1 mg / mL or (B) 1 mg / mL of surfactant and were stored at 5°C (outlined), 25°C (semi-blacked), and 40°C (blacked). Here, only the CS20 (triangle) formulation, which showed a significant decrease in main peak area, is shown and compared with the standard surfactants PS20 (square) and Px188 (circle). (Figure 9B) See the explanation for Figure 9A. (Figure 10) Physicochemical and structural characteristics of the evaluated surfactants and their current application fields. (Figure 11-1) Formulation attributes of formulations that do not contain surfactants or acyl surfactants for mAb1. a Visible particles are classified into four groups: i) particles with 0 particles, ii) particles with 1 to 5 particles, iii) particles with 6 to 10 particles, and iv) particles with more than 10 particles. b Turbidity is classified into four groups: i) 0-3 NTU, ii) greater than 3-6 NTU, iii) greater than 6-18 NTU, and iv) greater than 18 NTU. c Colors are classified into the following four groups according to the Ph. Eur color scale values: 2.2.2: i) 9-7, ii) 6-5, iii) 4-3, and iv) 2-1. d The value is given as the average of the three individual measurements (standard deviation < 0.4). e The shaking experiment shows the average value of 2-3 analyzed vials. The surface tension of 20 mM His-HCl buffer is 73 mN·m-1. Results for classes II, III, and IV are marked in gray. The darker the color, the higher the class and the worse the stress test result. na=not analyzed. (Figure 11-2) Refer to the description of Figure 11-1. (Figure 11-3) Refer to the description of Figure 11-1. (Figure 11-4) Refer to the description of Figure 11-1. (Figure 12-1) Formulation attributes of a formulation containing an alkyl surfactant for mAb1. a Visible particles are classified into four groups: i) 0 particles, ii) 1 - 5 particles, iii) 6 - 10 particles, and iv) more than 10 particles. b Turbidity is classified into four groups: i) 0 - 3 NTU, ii) more than 3 - 6 NTU, iii) more than 6 - 18 NTU, and iv) more than 18 NTU. c Color is classified into the following four groups according to the color scale values of Ph.Eur. 2.2.2: i) 9 - 7, ii) 6 - 5, iii) 4 - 3, and iv) 2 - 1. d Values are given as the average of three individual measurements (standard deviation < 0.4). e The average value of 2 - 3 analyzed vials is shown by the shaking experiment. The surface tension of 20 mM His-HCl buffer is 73 mN·m-1. The results of classes ii, iii, iv are marked in gray. The darker the color, the higher the class and the worse the stress test results. n.a. = not analyzed. (Figure 12-2) Refer to the description of Figure 12-1. (Figure 12-3) Refer to the description of Figure 12-1. (Figure 12-4) Refer to the description of Figure 12-1. (Figure 13-1) Formulation attributes of a formulation containing a sterol surfactant for mAb1. a Visible particles are classified into four groups: i) 0 particles, ii) 1 - 5 particles, iii) 6 - 10 particles, and iv) more than 10 particles. b Turbidity is classified into four groups: i) 0 - 3 NTU, ii) more than 3 - 6 NTU, iii) more than 6 - 18 NTU, and iv) more than 18 NTU. cThe colors are classified into the following four groups according to the color scale values ​​of Ph.Eur.2.2.2: i) 9-7, ii) 6-5, iii) 4-3, and iv) 2-1. d The value is given as the average of the three individual measurements (standard deviation < 0.4). e The shaking experiment shows the average value of 2-3 analyzed vials. The surface tension of 20 mM His-HCl buffer is 73 mN·m-1. Results for classes II, III, and IV are marked in gray. The darker the color, the higher the class and the worse the stress test result. na=not analyzed. (Figure 13-2) See the explanation for Figure 13-1. (Figure 13-3) See the explanation for Figure 13-1. (Figure 14-1) Formulation attributes of formulations containing a surfactant of class "Other" for mAb1. a Visible particles are classified into four groups: i) 0 particles, ii) 1 to 5 particles, iii) 6 to 10 particles, and iv) more than 10 particles. b Turbidity is classified into four groups: i) 0-3 NTU, ii) greater than 3-6 NTU, iii) greater than 6-18 NTU, and iv) greater than 18 NTU. c Colors are classified into the following four groups according to the Ph. Eur color scale values: 2.2.2: i) 9-7, ii) 6-5, iii) 4-3, and iv) 2-1. d The value is given as the average of the three individual measurements (standard deviation < 0.4). e The shaking experiment shows the average value of 2-3 analyzed vials. The surface tension of 20 mM His-HCl buffer is 73 mN·m-1. Results for classes II, III, and IV are marked in gray. The darker the color, the higher the class and the worse the stress test result. na=not analyzed. (Figure 14-2) See the explanation for Figure 14-1. (Figure 14-3) See the explanation for Figure 14-1. (Figure 14-4) See the explanation for Figure 14-1. (Figure 14-5) See the explanation for Figure 14-1. (Figure 14-6) See the explanation for Figure 14-1. (Figure 15-1) Formulation attributes of formulations that do not contain surfactants and acyl surfactants for mAb2 and mAb3. a Visible particles are classified into four groups: i) particles with 0 particles, ii) particles with 1 to 5 particles, iii) particles with 6 to 10 particles, and iv) particles with more than 10 particles. b Turbidity is classified into four groups: i) 0-3 NTU, ii) greater than 3-6 NTU, iii) greater than 6-18 NTU, and iv) greater than 18 NTU. c The colors are classified into the following four groups according to the Ph. Eur color scale values: 2.2.2: i) 9-7, ii) 6-5, iii) 4-3, and iv) 2-1. Results for classes ii, iii, and iv are marked in gray. The darker the color, the higher the class and the worse the stress test results. (Figure 15-2) See the explanation for Figure 15-1. (Figure 16) Formulation attributes of formulations containing alkyl surfactants for mAb2 and mAb3. a Visible particles are classified into four groups: i) particles with 0 particles, ii) particles with 1 to 5 particles, iii) particles with 6 to 10 particles, and iv) particles with more than 10 particles. b Turbidity is classified into four groups: i) 0-3 NTU, ii) greater than 3-6 NTU, iii) greater than 6-18 NTU, and iv) greater than 18 NTU. c The colors are classified into the following four groups according to the Ph. Eur color scale values: 2.2.2: i) 9-7, ii) 6-5, iii) 4-3, and iv) 2-1. Results for classes ii, iii, and iv are marked in gray. The darker the color, the higher the class and the worse the stress test results. (Figure 17) Formulation attributes of formulations containing sterol surfactants mAb2 and mAb3. a Visible particles are classified into four groups: i) particles with 0 particles, ii) particles with 1 to 5 particles, iii) particles with 6 to 10 particles, and iv) particles with more than 10 particles. b Turbidity is classified into four groups: i) 0-3 NTU, ii) greater than 3-6 NTU, iii) greater than 6-18 NTU, and iv) greater than 18 NTU. c The colors are classified into the following four groups according to the Ph. Eur color scale values: 2.2.2: i) 9-7, ii) 6-5, iii) 4-3, and iv) 2-1. Results for classes ii, iii, and iv are marked in gray. The darker the color, the higher the class and the worse the stress test results. (Figure 18-1) Formulation attributes of formulations containing class "other" surfactants for mAb2 and mAb3. a Visible particles are classified into four groups: i) particles with 0 particles, ii) particles with 1 to 5 particles, iii) particles with 6 to 10 particles, and iv) particles with more than 10 particles. b Turbidity is classified into four groups: i) 0-3 NTU, ii) greater than 3-6 NTU, iii) greater than 6-18 NTU, and iv) greater than 18 NTU. c The colors are classified into the following four groups according to the Ph. Eur color scale values: 2.2.2: i) 9-7, ii) 6-5, iii) 4-3, and iv) 2-1. Results for classes ii, iii, and iv are marked in gray. The darker the color, the higher the class and the worse the stress test results. (Figure 18-2) See the explanation for Figure 18-1. (Figure 18-3) See the explanation for Figure 18-1. [Modes for carrying out the invention]

[0009] Detailed description of the invention Surfactants approved for parenteral use have two structural drawbacks: (1) the intramolecular ester linker makes them susceptible to enzymatic degradation by host cell proteins (HCPs), which can lead to the formation of visible free fatty acid particles; or (2) the charge has been reported to lead to mAb destabilization, possibly through charge-charge interactions

[56] . Therefore, two screening methods were investigated to test these structural components. This allowed for the rapid identification of potential alternative surfactants and facilitated the evaluation of numerous possible candidates.

[0010] The biologics market is growing rapidly, making the development of screening methods to predict the long-term stability of proteins, particularly antibodies, crucial. Several biophysical characterization techniques are known for predicting protein stability [47,48]. Among these, maximizing conformational stability is considered to have a high impact on maintaining long-term drug product quality by preventing unfolding and aggregation of therapeutic proteins. Screening techniques include the measurement of intrinsic protein fluorescence under isothermal chemical denaturation (ICD) or thermal denaturation conditions using DSC (differential scanning calorimetry) or nanoDSF (differential scanning calorimetry)

[49] . Since pH, buffer systems, and excipient compositions can affect the intrinsic conformational stability of proteins, these screenings are often performed in early formulation development

[50] .

[0011] As a second pre-screening method, nanoDSF measurement was investigated. At therapeutic surfactant concentrations of 1 mg / mL or less, most of the tested alternative surfactants were T on and T m It only showed a slight change.

[0012] The inventors have established a simple and rapid method for evaluating the ester stability of various surfactants against enzymatic digestion. The inventors found that surfactants exhibit different degrees of enzymatic degradation depending on the size of their lipophilic skeleton, possibly due to steric interference with the enzyme active site.

[0013] Furthermore, the inventors established the structure-activity relationship of sterol-based surfactants not only under interfacial stress but also during long-term storage. It was found that the small, flexible structure is more effective than bulky surfactants in stabilizing proteins during rapid changes at the interface, for example, during shaking. Similar group behavior was observed under stirring stress for polymer surfactants: poloxamer, tetronic, and polyvinyl alcohol.

[0014] The present invention has surprisingly identified that the surfactants TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL exhibit protein stabilization effects equivalent to or better than those of the established PS20, PS80, and Px188 in the aforementioned protein-containing liquid composition.

[0015] Accordingly, in one embodiment, the present invention provides a liquid pharmaceutical composition comprising a protein and one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL.

[0016] In another embodiment, the present invention provides an aqueous pharmaceutical composition comprising a protein and one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL.

[0017] In another embodiment, the present invention provides an aqueous pharmaceutical composition comprising a protein and one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, and SL.

[0018] In another embodiment, the present invention provides an aqueous pharmaceutical composition comprising a protein and one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, and SL.

[0019] In another embodiment, the present invention provides an aqueous pharmaceutical composition comprising a protein and one or more surfactants selected from TMN-6 and 15-S-15.

[0020] In another embodiment, the present invention provides any of the aforementioned compositions in which a protein is the pharmaceutically active ingredient. In one embodiment, the composition is for use in treating a disease in a patient requiring treatment.

[0021] In another embodiment, the present invention provides any of the aforementioned compositions, wherein the protein is an antibody; or an immunoconjugate; or an antibody fragment.

[0022] In another embodiment, the present invention provides any of the aforementioned compositions, wherein the protein is an antibody contained in any of the antibody products defined herein.

[0023] In another embodiment, the present invention provides any of the aforementioned compositions further comprising a pharmaceutically acceptable excipient or carrier.

[0024] In another embodiment, the present invention provides any of the aforementioned compositions in which the surfactant is present at a concentration of less than 1 mg / mL, or in a concentration range of 0.001 mg / mL to 0.01 mg / mL; or 0.01 mg / mL to 0.1 mg / mL; or 0.1 mg / mL to 1.0 mg / mL. In one embodiment, the surfactant according to the present invention is TPGS and / or PVA at a concentration of 1 mg / mL. In another embodiment, the surfactant according to the present invention is 15-S-15 and / or TMN-6 at a concentration of 1 mg / mL or 0.1 mg / mL.

[0025] In another embodiment, the present invention provides any of the above compositions, wherein the protein is present in any concentration known to those skilled in the art as applicable to aqueous protein or antibody formulations. In one embodiment, particularly if the protein is an antibody approved for use as a human pharmaceutical, the antibody is present in any of their approved concentrations. Information regarding the approved concentrations is readily available to those skilled in the art, for example, in the package insert or product characteristics summary (SmPC) of a given drug. In another embodiment, the protein, particularly the antibody, is present in the composition according to the present invention at concentrations of 5 to 200 mg / ml, or 5 to 100 mg / ml, or 10 to 25 mg / ml.

[0026] In yet another embodiment, the present invention provides the use of one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL in the production of a liquid pharmaceutical composition further comprising a protein.

[0027] In yet another embodiment, the present invention provides the use of one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, and SL in the preparation of a liquid pharmaceutical composition further comprising a protein.

[0028] In yet another embodiment, the present invention provides the use of one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, and SL in the production of a liquid pharmaceutical composition further comprising a protein.

[0029] In yet another embodiment, the present invention provides the use of one or more surfactants selected from TMN-6 and 15-S-15 in the production of a liquid pharmaceutical composition further comprising a protein.

[0030] In another embodiment, the present invention provides any of the aforementioned uses for the preparation of an aqueous pharmaceutical composition comprising an antibody as defined herein. In one embodiment, the composition is an approved pharmaceutical comprising an antibody or a multispecific or bispecific antibody as an active ingredient. In another embodiment, the present invention provides the use of one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL to stabilize a protein and prevent the formation of visible particles in the liquid pharmaceutical composition comprising the protein during storage. In another embodiment, the liquid pharmaceutical composition comprises one or more proteins as active ingredients.

[0031] Furthermore, in another embodiment, the present invention provides one or more surfactants selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL for use in any of the liquid pharmaceutical compositions described herein. In one embodiment, such use means stabilizing the proteins contained in the liquid pharmaceutical composition and preventing the formation of visible particles in the composition during storage.

[0032] In yet another embodiment, the present invention provides one or more surfactants as defined herein, preferably TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15 and / or SL, for substituting PS20, PS80, or poloxamer 188 in commercially available antibody formulations. In another embodiment, 15-S-15 may be used to substitute any of PS20, PS80, or poloxamer 188 in an aqueous pharmaceutical composition containing an antibody as defined herein. In yet another embodiment, TPGS, Px338, Px407, PVA, T1107, TMN-6 and SL may be used to substitute poloxamer 188 in an aqueous pharmaceutical composition containing an antibody as defined herein.

[0033] In yet another embodiment, the present invention provides the use of one or more surfactants as defined herein for the manufacture of a pharmaceutical. In one embodiment, the pharmaceutical is an aqueous pharmaceutical formulation containing any active ingredient that requires stabilization by a surfactant for its authorized use. In another embodiment, one or more surfactants are independently selected from TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, and SL.

[0034] In yet another embodiment, the present invention provides screening methods disclosed herein, either alone or in combination, for identifying surfactants according to the present invention. In one embodiment, the screening method is as disclosed in the appendix examples.

[0035] In this specification, the antibody referred to as "mAb1" is an antibody having INN pertuzumab. Pertuzumab is commercially available, for example, under the trademark name PERJETA®. Pertuzumab is also disclosed, for example, in European Patent No. 2238172. Therefore, in one embodiment, "pertuzumab" (or "Mab 2C4") refers to an antibody containing the variable light and variable heavy amino acid sequences of SEQ ID NOs. 3 and 4, respectively, as disclosed in European Patent No. 2238172. If pertuzumab is an intact antibody, pertuzumab contains the light and heavy chain amino acid sequences of SEQ ID NOs. 15 and 16, respectively, as disclosed in European Patent No. 2238172.

[0036] In this specification, the antibody referred to as "mAb2" is an antibody containing the INN obinutuzumab. Obinutuzumab is commercially available, for example, under the trade names GAZYVA® / GAZYVARO®. Sequence information for obinutuzumab is published, for example, by the WHO in the list of recommended INNs (List 65, WHO Drug Information, Vol. 25, No. 1, 2011). Further information on obinutuzumab is also available, for example, in International Publication No. 2005 / 044859 (B-HH6 is the heavy chain construct and B-KV1 is the light chain construct). See also Tables 2 and 3 of International Publication No. 2005 / 044859 for sequence information.

[0037] In this specification, the antibody referred to as "mAb3" is a bispecific antibody fragment of an investigational drug currently undergoing clinical trials.

[0038] As used herein, the term “surfactant” means TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL.

[0039] TPGS is tocofersolane (D-α-tocopherol polyethylene glycol succinate).

[0040] PVA is poly(vinyl alcohol) 4-88.

[0041] T1107 is Tetronic® 1107 (ethylenediaminetetrakis(propoxylate-block-ethoxylate) tetrol).

[0042] Px338 is Kolliphor® P338 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)).

[0043] Px407 is Kolliphor® P407 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)).

[0044] TMN-6 is Tergitol™ TMN-6 (a branched secondary alcohol ethoxylate having 8EO units).

[0045] 15-S-15 is Tergitol® 15-S-15 (a secondary alcohol ethoxylate containing 15EO units).

[0046] Chol-PEG is mCholesterol-PEG2000.

[0047] SL is REWOFERMSLONE (an aqueous solution of sophorolipid (17-[2-O-(6-O-acetyl-beta-D-glucopyranosyl)-6-O-acetyl-beta-D-glucopyranosyloxy]-9-octadecenoic acid) lactone- and acidic form).

[0048] As used herein, the term “storage” means maintaining a liquid pharmaceutical formulation under conditions commonly applied by those skilled in the art. In one embodiment, such storage includes a period of time up to 6 months, or 12 months, or 18 months, or 24 months, or 30 months. In another embodiment, such storage includes maintaining the liquid pharmaceutical composition under conditions (e.g., temperature) approved by the regulatory authority until its shelf life approved by such regulatory authority. In one embodiment, such shelf life and storage conditions may be found, for example, in the package insert accompanying an approved protein-based drug.

[0049] The term “liquid pharmaceutical composition” preferably means an aqueous composition, formulation, or dosage form for pharmaceutical use. In one embodiment, the liquid pharmaceutical composition is for parenteral administration of a therapeutic protein. In another embodiment, the liquid pharmaceutical composition according to the present invention comprises one or more therapeutic proteins together with pharmaceutically acceptable excipients or carriers. Such excipients are generally known to those skilled in the art. In one embodiment, the term “excipient” refers to a component in the pharmaceutical composition or formulation other than the active ingredient that is nontoxic to the subject. Excipients include, but are not limited to, buffers, stabilizers including antioxidants, or preservatives.

[0050] The term "pharmaceutical composition" refers to a preparation, formulation, or dosage form that is in a form that enables the biological activity of the active ingredient contained herein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0051] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or formulation other than the active ingredient that is non-toxic to the subject. pharmaceutically acceptable carriers include, but are not limited to, the additives defined herein.

[0052] The term "buffer" is well known to those skilled in the art of organic chemistry or pharmacy, for example, in the development of pharmaceutical preparations. As used in this document, buffers refer to acetate, succinate, citrate, arginine, histidine, phosphate, tris, glycine, aspartate, and glutamate buffer systems. Furthermore, in this embodiment, the histidine concentration of the buffer is 5–50 mM.

[0053] The term "stabilizer" is well known to those skilled in the art of organic chemistry or pharmaceuticals, for example, in the development of pharmaceutical preparations. The stabilizer according to the present invention is selected from the group consisting of sugars, sugar alcohols, sugar derivatives, or amino acids. In one embodiment, the stabilizer is (1) sucrose, trehalose, cyclodextrin, sorbitol, mannitol, glycine, or / and (2) methionine, and / or (3) arginine, or lysine. In yet another embodiment, the concentration of the stabilizer is (1) up to 500 mM, or (2) 5 to 25 mM, or / and (3) up to 350 mM, respectively.

[0054] As used in this book, the term "protein" means any therapeutically relevant polypeptide. In one embodiment, the term "protein" means an antibody. In another embodiment, the term "protein" means an immunoconjugate.

[0055] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody classes or structures, including, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit desired antigen-binding activity. In one embodiment, any of these antibodies is a human antibody or is humanized. In another embodiment, the antibody according to the present invention is a human antibody or is humanized, and is a single or bispecific antibody, preferably a monoclonal antibody of the IgG class. The antibody may also comprise a combination of structural elements from various IgG classes or may be conjugated to a pharmacologically active moiety, such as a cell lysate or receptor ligand. In another embodiment, the antibodies include alemtuzumab (LEMTRADA®), atezolizumab (TECENTRIQ®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), pertuzumab (PERJETA®, 2C4, Omnitarg), trastuzumab (HERCEPTIN®), tositumomab (Bexxar®), absiximab (REOPRO®), adalimumab (HUMIRA®), apolizumab, aselizumab, atrizumab, bapineozumab, basiliximab (SIMULECT®), bavituximab, and belimumab (BENLYSTA®). Ankinumab, canakinumab (ILARIS®), sedelizumab, certolizumab pegol (CIMZIA®), cidofcituzumab, cizutuzumab, xixtumumab, crazakizumab, crenezumab, daclizumab (ZENAPAX®), dalotuzumab, denosumab (PROLIA®, XGEVA®), eculizumab ( SOLIRIS (registered trademark), ephalizumab, epratuzumab, erulizumab, emicizumab (HEMLIBRA (registered trademark)), felbizumab, fontrizumab, golimumab (SIMPONI (registered trademark)), ipilimumab, imugatuzumab, infliximab (REMICADE (registered trademark)), rabetuzumab, lebrikizumab, lexatumumab, lintuzumab,Lucatumumab, rulizumab pecol, lumuretuzumab, mapatumumab, matuzumab, mepolizumab, mogamulizumab, motabizumab, motobizumab, muronomab, natalizumab (TYSABRI®), necitumumab (PORTRAZZA®), nimotuzumab (THERACIM®), norobizumab, numabizumab, O Rokizumab, Omalizumab (XOLAIR®), Onartuzumab (also known as MetMAb), Palivizumab (SYNAGIS®), Pascolizumab, Pecufcituzumab, Pectuzumab, Pembrolizumab (KEYTRUDA®), Pexerizumab, Priliximab, Larivizumab, Ranibizumab (LUCEN) TIS (registered trademark), reslivizumab, reslizumab, recibizumab, lobatumumab, lontalizumab, loberizumab, luprizumab, sarilumab, secukinumab, cerivantuzumab, cifalimumab, cibrotuzumab, siltuximab (SYLVANT (registered trademark)), ciprizumab, sontuzumab, tadocizumab, talizumab, tefivazumab, The antibody product is selected from tocilizumab (ACTEMRA®), tralizumab, tuccituzumab, umabizumab, urtoxazumab, ustekinumab (STELARA®), vedolizumab (ENTYVIO®), bicilizumab, zanorimumab, zaltumumab, and obinutuzumab (GAZYVA®). In yet another embodiment, the antibody is pertuzumab or obinutuzumab.

[0056] An "antibody fragment" refers to a molecule other than an intact antibody, containing a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0057] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0058] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to the amino acids of an antibody of non-human origin that utilizes a human antibody repertoire or a sequence encoding a human antibody. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0059] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody substantially contains all of at least one, typically two, variable domains, in which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0060] As used herein, the terms “hypervariable region” or “HVR” refer to each of the regions of the antibody variable domain, such as “complementarity-determining regions” (CDRs), that are hypervariable in the sequence and determine antigen-binding specificity. Generally, an antibody contains six CDRs, three located in the VH (CDR-H1, CDR-H2, CDR-H3) and three located in the VL (CDR-L1, CDR-L2, CDR-L3). Examples of CDRs used herein include: (a) Hypervariable loops arising at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)); (b) CDRs produced at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact occurs at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J.Mol.Biol.262:732-745 (1996)).

[0061] Unless otherwise specified, the CDR will be determined in accordance with Kabat et al. above. Those skilled in the art will understand that the notation of the CDR may be determined in accordance with Chothia above, McCallum above, or any other scientifically recognized nomenclature system.

[0062] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0063] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0064] An "isolated" antibody is an antibody that has been separated from its natural environment. In some embodiments, antibodies are purified to a purity higher than 95% or 99%, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J.Chromatogr.B848:79~87 (2007).

[0065] In one embodiment, as used herein, the term “long-term” in relation to “storage” or “stability” means until the end of the authorized shelf life of any commercially available antibody product as defined herein. In another embodiment, the term “long-term” generally means up to 5 years, or up to 3 years, or up to 24 months, or up to 18 months, or up to 12 months, or up to 6 months, or up to 3 months for antibodies as defined herein. The term “storage” includes conditions, such as temperature and humidity, that are typically required to store antibodies, in particular any of the authorized antibody products as defined herein. Such conditions are well known to those skilled in the art. References to such conditions may be found, for example, in the package insert or summary of product characteristics (SmPC) of a commercially available product among the antibody products as defined herein.

[0066] A. Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody contains a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from those of the parent antibody. A chimeric antibody contains its antigen-binding fragment.

[0067] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies contain one or more variable domains (CDRs or parts thereof) derived from the non-human antibody, and FRs (or parts thereof) derived from the human antibody sequence. Humanized antibodies also optionally contain at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0068] Humanized antibodies and methods for their production are, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods Further information is available in 36:25~34 (2005) (describes specificity-determining region (SDR) glifting); Padlan, Mol.Immunol.28:489~498 (1991) (describes "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describes "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer, 83:252-260 (2000) (describes the "guided selection" approach to FR shuffling).

[0069] Human framework regions that may be used for humanization include, but are not limited to, the following: framework regions selected using the “best fit” method (see, e.g., Sims et al., J.Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al., Proc.Natl.Acad.Sci.USA, 89:4285 (1992); and Presta et al., J.Immunol. 1 See 51:2623 (1993); human maturation (somatic mutation) framework regions or human germline framework regions (e.g., see Almagro and Fransson, Front.Biosci.13:1619-1633 (2008)); and framework regions derived from screening of the FR library (e.g., see Baca et al., J.Biol.Chem.272:10678-10684 (1997) and Rosok et al., J.Biol.Chem.271:22611-22618 (1996)).

[0070] B. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0071] Human antibodies can be prepared by administering an immunogen to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen administration. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, is located extrachromosomally, or is randomly incorporated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing the XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing the HUMAB® technology; U.S. Patent No. 7,041,870 describing the KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing the VELOCIMOUSE® technology. Human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0072] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human xenomyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, those described in U.S. Patent No. 7,189,826 (production of monoclonal human IgM antibody from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0073] Human antibodies can also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0074] C. Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include further non-proteinoid moieties known and readily available in the art. Suitable sites for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody varies, and if multiple polymers are attached, they may be the same molecule or different molecules. In general, the number and / or types of polymers used for derivatization can be determined based on considerations including, but are not limited, the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under defined conditions.

[0075] D. immunoconjugate The present invention also provides immunoconjugates comprising antibodies of this specification conjugated (chemically bound) to one or more therapeutic agents such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant, or animal-derived enzyme-active toxins, or fragments thereof), or radioisotopes.

[0076] In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) to which an antibody is bound to one or more of the aforementioned therapeutic agents. The antibody is typically linked to one or more therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents, drugs, and linkers, is described in Pharmacol Review 68:3~19 (2016).

[0077] In another embodiment, the immunoconjugate includes antibodies that bind to enzyme-active toxins or fragments thereof, as described herein. These include, but are not limited to, diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesin A chain, α-sarcin, tung tree protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crocin, soapwort inhibitor, geronin, mitogenin, restrictosin, phenomycin, enomycin, and trichothecenes.

[0078] In another embodiment, the immunoconjugate comprises an antibody described herein, which is conjugated to a radioactive atom to form a radioactive conjugate. Various radioisotopes are available for the production of radioactive conjugates. Examples include the radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu. When the radioactive material is used for detection, it may include a radioactive atom for scintigraphy studies, e.g., tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0079] Conjugates of antibodies and cytotoxic agents can be prepared, for example, using various bifunctional protein coupling agents: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and diactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See International Publication No. 94 / 11026. The linker may be a "cleavage linker" that facilitates the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photosensitive linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0080] The immunoconjugates or ADCs used herein are, but are not limited to, conjugates prepared using commercially available crosslinking reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0081] E. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, for example, bispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has binding specificity to at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0082] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and the "knob-in-hole" operation (see, e.g., U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Multispecific antibodies can also be used to manipulate the electrostatic steering effect for creating antibody Fc heterodimer molecules (see, e.g., International Publication No. 2009 / 089004); crosslink two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); produce bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992) and International Publication No. 2011 / 034605); and avoid the light chain mispairing problem. These can be produced by using general light chain techniques (see, for example, International Publication No. 98 / 50431); using "diabody" techniques to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv(sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and by preparing triplicate antibodies as described, for example, Tutt et al., J. Immunol. 147:60 (1991).

[0083] For example, this also includes manipulated antibodies having three or more antigen-binding sites, such as "octopus antibodies," or DVD-Ig (see, for example, International Publication Nos. 2001 / 77342 and International Publication Nos. 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in International Publication Nos. 2010 / 115589, 2010 / 112193, 2010 / 136172, 2010 / 145792, and 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include "Dual-Acting FAb" or "DAF" containing antigen-binding sites that bind to two different antigens or two different epitopes of the same antigen (see, for example, U.S. Patent Application Publication 2008 / 0069820 and International Publication 2015 / 095539).

[0084] Multispecific antibodies can also be obtained in an asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, namely by exchanging VH / VL domains (see, e.g., International Publication Nos. 2009 / 080252 and 2015 / 150447), CH1 / CL domains (see International Publication No. 2009 / 080253), or complete Fab arms (see International Publication Nos. 2009 / 080251, 2016 / 016299, Schaefer et al., PNAS, 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20). In one embodiment, a multispecific antibody contains a cross-Fab fragment. The terms “cross-Fab fragment,” “xFab fragment,” or “crossover Fab fragment” refer to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. The cross-Fab fragment includes a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). The asymmetric Fab arm can be manipulated by introducing charged or uncharged amino acid mutations into the domain interface to direct correct Fab pairing. See, for example, International Publication 2016 / 172485.

[0085] Various further molecular formats of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015) 95-106).

[0086] F. Recombination methods and compositions Antibodies can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4816567. For these methods, one or more isolated nucleic acids encoding antibodies are provided.

[0087] For natural antibodies or natural antibody fragments, two nucleic acids are required: one for the light chain or fragment and the other for the heavy chain or fragment. Such nucleic acids encode the amino acid sequence containing the VL and / or VH of the antibody (e.g., one or more of the antibody's light and / or heavy chains). These nucleic acids may be on the same expression vector or on different expression vectors.

[0088] In the case of a bispecific antibody having a heterodimer heavy chain requiring four nucleic acids, one is for the first light chain, one for the first heavy chain containing the first heteromonomer Fc region polypeptide, one for the second light chain, and one for the second heavy chain containing the second heteromonomer Fc region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode amino acid sequences containing the first VL and / or the first VH containing the first heteromonomer Fc region and / or the second VL and / or the second VH containing the second heteromonomer Fc region of the antibody (e.g., the first and / or second light chains and / or the first and / or second heavy chains of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors, and typically these nucleic acids are located on two or three expression vectors, i.e., one vector may contain two or more of these nucleic acids. Examples of these bispecific antibodies are cross-Mabs (see, e.g., Schaefer, W. et al., PNAS, 108(2011)11187-1191). For example, according to EU index numbering, one heteromonomer heavy chain contains a so-called "knob mutation" (T366W and possibly either S354C or Y349C), and the other contains a so-called "hole mutation" (T366S, L368A, and Y407V and possibly Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2(1996)73).

[0089] Regarding the recombinant production of antibodies, for example, the nucleic acids encoding the aforementioned antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using common procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0090] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli). After expression, the antibodies may be isolated from the bacterial cell paste in the soluble fraction and further purified.

[0091] In addition to prokaryotes, useful eukaryotic microorganisms such as filamentous fungi or yeasts, including fungal and yeast strains, whose glycosylation pathways are "humanized," resulting in the production of antibodies with a partially or completely human glycosylation pattern, are also suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, TU, Nat. Biotech. 22(2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24(2006) 210-215.

[0092] Host cells suitable for the expression of (glycosylated) antibodies can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified, and these may be used in combination with insect cells, particularly for the transfection of armyworm (Spodoptera frugiperda) cells.

[0093] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0094] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40(COS-7) transformed monkey kidney CV1 cell line, human embryonic kidney cells (e.g., 293 cells or 293T cells as described by Graham, FL et al., J. Gen Virol. 36 (1977) 59-74, baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described by Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), and TRI cells (e.g., Mather, JP et al., Annals). These include MRC5 cells and FS4 cells (described in NYAcad.Sci.383(1982)44-68). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc.Natl.Acad.Sci.USA 77(1980)4216~4220); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0095] The present invention will now be further explained by the following non-limiting embodiments. [Examples]

[0096] material and method material PCL (lipase derived from Pseudomonas cepacia) and CALB (recombinant lipase B derived from Aspergillus oryzae, Candida antarctica) were purchased from Sigma Aldrich (Steinheim, Germany). The model IgG1 monoclonal antibodies used were supplied by F. Hoffmann-La Roche (Basel, Switzerland) and were formulated as follows: 25 mg / mL (mAb1) in 20 mM His-HCl buffer (Ajinomoto Co., Inc., Tokyo, Japan) containing 240 mM sucrose at pH 7.0 (Pfantiehl Inc., Illinois, USA), 25 mg / mL (mAb2) in 17 mM His-HCl buffer containing 240 mM sucrose at pH 6.0, and 10 mM His-HCl buffer containing 240 mM sucrose at pH 6.0. Each compound was prepared in His-HCl buffer at a concentration of 10 mg / mL (mAb3).

[0097] The screened surfactants were Kolliphor® HS15 (HS15, BASF, Ludwigshafen, Germany), Kolliphor® RH40 (RH40, BASF, Ludwigshafen, Germany), Polysorbate 20 (PS20; Croda International, Snais, UK), Polysorbate 80HX2 (PS 80; NOF Corporation, Tokyo, Japan), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt) (mPEG-DSPE; Avanti Polar Lipids, Alabaster, Alabama), and Rewoferm® SL ONE (SL; Evonik). BASF Industries (Essen, Germany), Kolliphor® CS20 (CS20; BASF (Ludwigshafen, Germany)), Tergitol® 15-S-15 (15-S-15; Sigma Aldrich, Steinheim, Germany), Tergitol® TMN-6 (TMN-6; Sigma Aldrich, Steinheim, Germany), Ecosurf® EH-9 (EH-9; Sigma Aldrich, Steinheim, Germany), Nonanoyl-N-methylglucamide (MEGA-9; Sigma Aldrich, Steinheim, Germany) and Decanoyl-N-hydroxyethylglucamide (HEGA-10; Anatrace, Maumi, Ohio), Sodium deoxycholate (NaDC; Sigma Aldrich, Steinheim, Germany), Sodium glycocholate hydrate (NaGC; Sigma Aldrich) Aldrich (Steinheim, Germany), Chobimalt / cholesterol-β-D-maltopyranosyl-(1→6)-β-D-maltopyranoside (Chobi;Anatrace, Maumi, Ohio), mcholesterol-PEG 2000Supplied by (Chol-PEG; Nanocs Inc, New York), Deoxy-BigCHAP (DBC; Toronto Research Chemicals, North York, Canada), α-Tocopheryl-Polyethylene Glycol-1000-Succinate (TPGS, Toronto Research Chemicals, North York, Canada); Kolliphor® P188 (Px188; BASF, Ludwigshafen, Germany), Kolliphor® P338 (Px338; BASF, Ludwigshafen, Germany), Kolliphor® P407 (Px407; BASF, Ludwigshafen, Germany), Tetronic® 1107 (T1107; BASF, Ludwigshafen, Germany), or Polyvinyl Alcohol 4-88 (PVA; Merck KGaA, Darmstadt, Germany).

[0098] All other reagents, including methanol (MeOH), sodium hydroxide (NaOH), and ammonium acetate, were analytical grade and obtained from Merck KGa KGa in Darmstadt.

[0099] method Measurement of surfactant stability against enzyme ester hydrolysis Each surfactant at a concentration of 0.4 mg / mL was incubated at room temperature for 6 hours in 20 mM ammonium acetate buffer (pH 5.5) with a total of 0.25 or 0.5 mg / mL of a 1:1 lipase mixture of PCL and CALB. Both belong to the carboxyl ester hydrolase group and have been previously reported as important impurities from bioprocesses capable of hydrolyzing PS20

[51] . In cases of no or negligible enzymatic degradation, chemical ester hydrolysis using NaOH was performed as a positive control. For this purpose, the surfactants were incubated with 0.1 mmol of NaOH at room temperature for 6 hours.

[0100] Following the decomposition of the surfactant, reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using a Waters Alliance 2695 instrument equipped with a Waters 2424 evaporative light scattering detector (ELSD) (Waters, Milford, USA) with nitrogen as the carrier gas at a pressure of 25 psi and a drift tube temperature of 95°C. A Phenomenex Luna® C18(2) 100 Å (150 × 4.6 mm, 5 μm) column (Phenomenex, Torrence, USA) was used as the stationary phase. Surfactant analysis was performed using two-component gradient elution with the program shown in Table 1, at a constant flow rate of 0.7 ml / min, an injection volume of 25 μl, a sample temperature of 5°C, and a column temperature of 35°C. Eluent A consisted of a 2% aqueous acetic acid solution, and eluent B consisted of 2% acetic acid in MeOH.

[0101] [Table 1]

[0102] A typical chromatogram is shown in Figure 2. As reported in the literature, the peaks were classified into hydrophilic / non-esterified fractions (1) and lipophilic esterified fractions (2) [52-54]. Data processing was performed using Empower® 3 software. To better compare various surfactants, the degree of surfactant degradation was reported as the normalized ester main peak area, with the initial ester main peak area before degradation set to 100%. The results are given as the average of three individual measurements with a standard deviation of 0.15 or less.

[0103] Evaluation of thermoconformate protein stability in the presence of surfactants The conformational stability of proteins was investigated using the Prometheus NT.Plex (NanoTemper Technologies GmbH, Munich, Germany). This instrument allows for label-free fluorescence analysis of intrinsic protein fluorescence changes from aromatic tryptophan and tyrosine residues using small amounts of solution. Heat-induced protein unfolding was monitored by detecting emission shifts at 330 nm and 350 nm at 5% laser power. Standard nanoDSF-grade capillary tips (NanoTemper Technologies, Munich, Germany) were filled with 10 μL of freshly prepared (t0) formulations containing 25 mg / mL mAb compounded with one of the following specific surfactants at 0.01, 0.1, 1, or 10 mg / mL. Sodium dodecyl sulfate (SDS) was used as a positive control because its charge has been reported to cause protein destabilization, possibly through charge-carrier interactions [55-57]. Samples were heated from 25°C to 95°C with a constant heating gradient of 0.5°C / min. PR.StabilityAnalysis software (NanoTemper Technologies, Munich, Germany) analyzes the melting curve at the start of (T on ) and the first transition point (T m1 The temperature was calculated automatically. The reported data is the average of three individual measurements.

[0104] Evaluation of protein stability after mechanical stress and thermal stability in the presence of surfactants. Surfactant performance screening was performed using 0.1 and 1 mg / mL surfactants in the model mAb formulations described in the Materials section. After formulation, the liquid samples were aseptically filtered through a 0.22 μm Millex Sterivex® GV (Millipore, Bedford, USA) filter unit, filled into 6 mL Type 1 glass vials, and sealed with Φ20 mm Teflon® coated serum stoppers (Daikyo Seiko Co., Ltd., Tokyo, Japan). The sealed vials were crimped using aluminum caps (Infochroma AG, Goldau, Switzerland). Corresponding placebo formulations were prepared and formulated in the same manner and used as controls.

[0105] To evaluate the effect of surfactants on mAb stability, formulations were exposed to various interfacial stress conditions, such as agitation and various freeze-thaw cycles. Vials were placed horizontally and subjected to shaking stress at a constant speed of 200 rpm in a shaker (HS 260 Control Model; IKA Werke GmbH&Co.KG; Stauffen, Germany) for 7 days in the dark at 5°C and 25°C. Freeze-thaw (F / T) stress was performed by exposing vials to five consecutive cycles of freezing at -20°C and thawing at 5°C.

[0106] Thermal stability data was generated by storing liquid protein formulations at 5°C for up to 24 months (mo), at 25°C / 60% relative humidity (rH) for 6 months, and at 40°C / 75% rH for 12 weeks. Samples were analyzed at initial time (t0) and at 1 month, 3 months, 6 months, 12 months, 18 months, and 24 months using the analytical methods described below.

[0107] Visible particles (E / P) Enhanced visual inspection was carried out using a Seidenader V 90-T machine (Seidenader Maschinenbau GmbH, Markt Swaben, Germany) as described above

[58] . The number of particles was classified into the following four classes: Class (I) corresponds to 0 particles, Class (II) corresponds to 1 to 5 particles, Class (III) corresponds to 6 to 10 particles, and Class (IV) corresponds to more than 10 particles.

[0108] Turbidity (milky and transparent) Turbidity was measured using a 2100 AN turbidimeter (Hach Lange GmbH, Düsseldorf, Germany) calibrated with a StablCal® calibration kit (Hach Lange GmbH), according to Ph.Eur.2.2.1, as previously described in the literature. The results are expressed in turbidimetric units (NTU) [58,59].

[0109] colorimetric The colorimetric analysis of the solutions was performed using a LICO 690 colorimeter (Hach Lange GmbH). Classification was carried out according to the color scale described in Ph.Eur2.2.2

[60] . The data presented here indicate the degree of color change according to the color scale values ​​in Ph.Eur2.2.2. Class (I) corresponds to a color scale value of 9, 8, 7, or no color; Class (II) corresponds to a color scale value of 6 or 5; Class (III) corresponds to a color scale value of 4 or 3; Class (IV) corresponds to a color scale value of 2 or 1.

[0110] Obscurity of light Microscopic visible particles (SVP) were counted by light shielding using a HIAC 9703+ liquid particle counting system (Skan AG, Alcheville, Switzerland) and PharmSpec 3 (Hach Lange GmbH) software. The measurement techniques applied were those described in Ph.Eur2.9.19

[61] and USP. <787> The method was adapted from the one described in

[62] . After rinsing the system with the sample solution, it was run four times with a sample volume of 0.2 mL. The final cumulative particle count was obtained by calculating the mean ± SD (standard deviation) from the last three measurements. SVPs of 2, 5, 10 and 25 μm or larger were measured and expressed as the cumulative number per 1 mL of solution.

[0111] Size exclusion rapid chromatography (SE-HPLC) Soluble mAb aggregates were detected and analyzed by SE-HPLC, hereafter referred to as high molecular weight (HMWS), monomers, and low molecular weight (LMWs). The system used was an Alliance 2695 HPLC instrument equipped with a 2487 UV detector (both from Waters Corporation, Milford, Massachusetts). The autosampler temperature was set to 5°C, and a total of 100 μg of mAbs were loaded into the system. Separation was performed using a TSKG 3000 SWXL, 7.8 × 300 mm column (Tosoh Bioscience, Stuttgart, Germany) at an oven temperature of 25°C, with a mobile phase of 200 mM K2HPO4 / KH2PO4 and 250 mM KCl, pH 7.0, and a flow rate of 0.5 mL / min. Signal detection was performed at a wavelength of 280 nm, and the peak area percentage was calculated using Empower 3 Chromatography Data System software (Waters Corporation, Milford, Massachusetts).

[0112] Ion exchange high-performance liquid chromatography (IE-HPLC) The charge heterogeneity of mAbs was evaluated by IE-HPLC using an Alliance e 2695 HPLC instrument equipped with a 2489 UV detector (both Waters Corporation, Milford, Massachusetts). mAbs were digested with carboxypeptidase, and 50 μg was injected into a 4 × 250 mm ProPac® WCX-10 column (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a flow rate of 1.0 mL / min with a column temperature of 34°C. mAb fragment elution was performed using solvents that increased ionic strength (mobile phase A: 20 mM MES, 1 mM Na-EDTA / mobile phase B: 250 mM NaCl, 20 mM MES, 1 mM Na-EDTA, pH 6.0). Signal detection was performed at a wavelength of 280 nm, and Empower 3 Chromatography Data System software (Waters Corporation, Milford, Massachusetts) was used for data processing. The decrease in the main peak was reported as the percentage of total peak area (area %) relative to storage time.

[0113] surface tension measurement Surface tension was measured by a liquid processing station according to the method described by Amrein et al.

[63] . Briefly, the measurement relies on the correlation between droplet mass and the surface tension of the sample. For this study, a fully automated liquid processing station, Freedom 384 EVO 200 (Tecan, Krylsheim, Germany), was equipped with an analytical balance (Mettler Toledo, Columbus, USA). The system was used to process 100 μL·s from a sealed 1 mL round-bottom LoBind® Deepwell Plate (Eppendorf, Hamburg, Germany). -1 Includes a stainless steel fixed tip for aspirating the sample. Add 3 μL·s of sample to a second round-bottom deep-well plate 96 / 500 μL (Eppendorf, Hamburg, Germany) mounted on an analytical balance. -1The sample was dispensed. Weights were continuously recorded using a fully automated routine described in Matlab R2017b (MathWorks, Natick, USA). The surface tension of water (72.6 mN / m) was used as the basis for calculation. The average of three individual measurements (t0) of the sample is reported as the surface tension.

[0114] Counting of microscopically visible particles (SVP) using background membrane imaging (BMI) Microscope-visible particle (SVP) counts were measured by BMI using a Horizon instrument (HaloLabs, Philadelphia, Pennsylvania) for a second set of proteins, mAb2 and mAb3. The system operates on a 96-well polycarbonate membrane filter plate (Halo Labs) with a pore size of 0.4 μm, accepting samples for imaging. Under laminar flow, 50 μL of sterile water for injection was added to each well of the membrane filter plate, the plate was evacuated to 350 mbar, and the wells were measured for background information. Subsequently, 40 μL of sample was transferred to each well of the plate, evacuated to 350 mbar, washed with 50 μL of sterile water for injection, and evacuated again to 350 mbar. The final wells were measured, and image analysis was performed using Horizon Vue software. Particle counts are reported as the average of three measurements and up to 6.4% of the filter plate coverage.

[0115] Example 1: 1.) Measurement of surfactant stability against enzymatic ester hydrolysis Polysorbate hydrolysis with small amounts of co-purified host cell protein poses a significant challenge to the long-term stability of protein formulations. Therefore, assays were developed to test ester stability in the presence of two model lipases, PCL and CALB, at concentrations of 0.25 and 0.5 mg / mL. Figure 2 shows representative chromatograms of PS20 before (solid line) and after (dashed line) enzymatic digestion.

[0116] [Table 2]

[0117] Prior to digestion, the hydrophilic fraction eluted at a lower retention time between 7 and 8 minutes (Table 2), increasing as the size of the average polyethylene oxide subunit increased in the following order: HS15 (15 PEO units) < PS20 (20 PEO units) < TPGS (23 PEO units) < RH40 (40 PEO units) < Chol-PEG (45 PEO units). The broader peak shape of fraction (1) can be explained by the polymeric nature of the PEO moiety and the associated size distribution of the different polymer chains. Due to the more chemically heterogeneous composition of PS20, HS15 and RH40 by the degree of esterification (presence of mono-, di- and partially tri- and tetra-esters), the lipophilic sub-fraction (2) eluted as multiple subsequent peaks, while only one peak was obtained for TPGS and Chol-PEG (data not shown)

[24] . For the latter two surfactants, the previous lipophilicity seems to be more chemically homogeneous.

[0118] After incubation with the lipase mixture, the chromatogram obtained for PS20 showed an increase in the hydrophilic fraction (1) and a complete loss of the lipophilic fraction (2) (dashed line in Figure 2), clearly indicating that all ester bonds had been cleaved. Figure 3 shows the degree of surfactant degradation by enzymatic hydrolysis using lipase mixtures at 0.25 and 0.5 mg / mL. For PS20, HS15 and RH40, strong enzymatic hydrolysis was observed (>95%). No difference in the degree of degradation was observed between the two lipase concentrations tested. In contrast, for TPGS and Chol-PEG, only very slight enzymatic hydrolysis was shown (<0.3%) for both lipase mixtures tested. To rule out the "false positive" results of this test, chemical hydrolysis was performed using 0.1 mmol of NaOH. Under these conditions, the ester bonds of Chol-PEG were completely hydrolyzed (100%), as shown by the disappearance of fraction (2), while TPGS was only partially hydrolyzed (68% of fraction (2) remained).

[0119] The data showed that the HS15 and RH40 surfactants produced a similar degree of degradation to PS20 during the experiment. Since enzymatic ester hydrolysis of polysorbates is a major challenge in protein formulations, surfactants that produced a similar degree of degradation to PS20 were used in subsequent studies.

[0120] Evaluation of thermoconformate protein stability in the presence of surfactants Maximizing conformational stability has been reported to increase long-term formulation quality and / or stability by preventing unfolding and aggregation of therapeutic proteins. High-throughput and low-volume screening techniques involve measuring intrinsic protein fluorescence by DSC (differential scanning calorimetry) or nanoDSF (differential scanning calorimetry) under isothermal chemical denaturation (ICD) or thermal denaturation conditions

[49] . Thermal DSF measurements were performed to eliminate the adverse effects of surfactants on protein conformational stability. Characteristic conformations of multi-domain structures of mAbs containing CH2, CH3, and Fab are important for their binding ability and therapeutic efficacy. In the case of mAbs, independent stepwise unfolding of specific domains is presumed, with CH2 generally being the least stable, followed by Fab and CH3

[48] .

[0121] Unfolding start temperature (T on ) and the first melting transition (T m1 The stability parameter (T) was measured for model mAbs in the presence of surfactants at concentrations ranging from 0.01 mg / mL to 10 mg / mL (see Figure 4). The value of mAbs without surfactant (T) on =61.5±0.3, T m1 Using a baseline of 77.7±0.1, the data was presented as a heatmap: darker colors indicate a stronger temperature decrease due to the presence of the surfactant. In general, most of the conditions tested did not show a significant effect on the conformational stability of mAbs within the surfactant concentrations relevant to the treatment.

[0122] As expected, SDS showed a concentration-dependent destabilizing effect compared to a reference formulation without surfactants. The higher the concentration, the greater the T on and T m1 The decrease is greater in both cases. SDS, known to be a potent destabilizer of protein conformational stability, was used as a positive control [55-57]. Similar to SDS, a significant decrease in conformational stability was observed for negatively charged surfactants such as NaDC, NaGC, and mPEG-DSPE, even at lower concentrations of 0.1 and mg / mL. However, for positively charged molecules such as T1107, the conformational stability parameter remained unaffected. Since the model mAb with an isoelectric point (pI) of 8.7 has a positive net charge overall under selected formulation conditions of pH 7.0, it can be hypothesized that charge-charge interactions between the mAb and the negatively charged surfactant result in the observed different behavior of the charged surfactant. Another interesting finding is that when nonionic sterol-based (Chol-PEG and Chobi) or vitamin E-based (TPGS) surfactants are added at concentrations of 1 mg / mL or higher (Chobi and TPGS) and 10 mg / mL (Chol-PEG), T on The result was a slight increase. Since the surfactant stock solution was measured and did not show a significant fluorescence signal, the possibility of fluorescence signal interference was ruled out. Therefore, it is more likely that these surfactants have a slight stabilizing effect on the native state of the mAb. In contrast, DBC also showed that nonionic sterol surfactants also... on (1 and 10 mg / mL) and T m1(10 mg / mL) decreased slightly. This phenomenon can be explained by different structural characteristics in either of the following sterol group modifications or the presence of glucamide functional groups: (i) DBC contains a free hydroxyl group at the 3-position of the sterol structure (similar to NaDC and NaGC), while Chol-PEG and Chobi retain a sterically larger functional group at this position, and (ii) DBS contains a sterically large hydrophilic gluconamide functionalization at the 20-position of the sterol structure, while for Chobi and Chol-PEG, the original hydrophobic cholesterol structure at this position remained unchanged. Comparing DBC, MEGA-9, and HEGA-10, which commonly have gluconamide functionalization as the hydrophilic part, with T on (1 and 10 mg / mL) and T m1 (10 mg / mL), it seems to be supported by a similar destabilizing effect.

[0123] Comparing the effects of all surfactants, including those with open-ring and closed-ring sugar structures, a destabilizing effect on the conformational stability of mAb at high concentrations was revealed for DBC, MEGA-9, and HEGA-10, as well as for SL. However, no relationship was shown between Chobi and either the open-ring or closed-ring sugar conformations.

[0124] Comparison with alcohol ethoxylates revealed that there was no conformational destabilizing effect of CS20 at all tested concentrations. T on A slight decrease (1 and 10 mg / mL) was observed in the following order: 15-S-15 = EH-9 < TMN-6, while T m1All showed similar decreases at 10 mg / mL. The observed differences can be explained from a structural standpoint as follows: (i) linear (CS20) versus branched alcohol ethoxylates (15-S-15, EH-9, TMN-6), primary versus secondary alcohol ethoxylates, respectively, or (ii) a decrease in the average number of PEO subunits (CS20 (20-24 PEO subunits) > 15-S-15 (15 PEO subunits) > EH-9 (9 PEO subunits) > TMN-6 (6 PEO subunits)). However, CS20 was the only alcohol ethoxylate available in pharmaceutical grade quality and therefore could be assumed to have a higher purity compared to the other three molecules. Residual impurities such as free alkyl residues can also contribute to conformational destabilization effects, a well-known phenomenon for polysorbates.

[0125] A subset of screened surfactants (including some that affect conformational stability) were incorporated into subsequent screenings to evaluate their impact on mAb stability not only during long-term testing but also after mechanical and thermal stress. To clarify the predictive properties of this high-throughput screening (HTS), several potentially high-performing surfactants: mPEG-DSPE, SL, and DBC were further included in the following surfactant performance screenings.

[0126] Example 2: Performance screening of another surfactant 1. Evaluation of protein stability after mechanical stress and thermal stability in the presence of surfactants. The effects of mechanical / interfacial stress on mAb stability were tested using stirring and freeze-thaw studies. Furthermore, the thermal stability of the formulations was tested. Data collected for tested attributes such as visible particles (VP), color, and turbidity were categorized into four categories and presented as heatmaps. Formulations with more undesirable attributes, such as many VPs, strong color changes, or high turbidity, were classified into higher classes and marked with varying gray intensities (darker intensities corresponded to stronger changes in the parameter).

[0127] The tested surfactant levels were kept constant at 0.1 and 1 mg / mL. For readability, surfactants were classified into four subgroups based on their hydrophobic moieties: (i) acyl, (ii) alkyl, (iii) sterol, and (iv) others. Formulations containing no surfactant (see Figure 11), PS20 (see Figure 11), or Px188 (see Figure 14) were used as guidelines for evaluating the performance of alternative surfactants.

[0128] Acyl group In the group containing acyls, mPEG-DSPE showed significantly poor results in many VP and high turbidity values ​​not observed in the placebo formulation. This phenomenon was mainly observed in the 1 mg / mL formulation, and the reason may be the previously described charge-charge interaction that reduces the conformational stability of the mAb (Figure 4). Furthermore, mPEG-DSPE has a relatively high DST, similar to that seen in sterol-based surfactants. mPEG-DSPE has a low critical micelle concentration (CMC: 1 × 10⁻⁶). -6M) These findings were unexpected given its relatively small and flexible structure. While not theoretically bound, the explanation could be charge-charge interaction between mPEG-DSPE and mAb, resulting in a small amount of surfactant molecules at the interface, low diffusivity, and a high apparent molecular weight. Furthermore, the slow degradation of mPEG-DSPE micelles may also be involved, leading to a decrease in interfacial stability. SL, on the other hand, showed better results in most tests, with results for VP, turbidity, and DST (Figure 11) being comparable to PS20. SL showed significantly improved stability at 1 mg / mL, likely with shaking, where changes in HMWS exceeding 10% increased considerably at lower surfactant concentrations (Figure 6). In preliminary screening, both SL and mPEG-DSPE showed conformational destabilization effects at higher concentrations, but no or only slight effects at lower concentrations (Figures 4 and 5). To validate the findings from preliminary screening as a predictive measure, they were compared with the results of thermal stress tests. In the case of mPEG-DSPE, the results appear to support these findings. For this surfactant, only the 1 mg / mL formulation, and not the 0.1 mg / mL formulation, showed a significant increase in SVP and turbidity.

[0129] In the control formulation containing PS20, a small amount of visible particles were observed. Both concentrations, particularly PS20 at 0.1 mg / mL, showed good stabilization during all applied stresses. On the other hand, the Px188 formulation contained many large protein particles during shaking in most stress tests, especially at low surfactant concentrations. A significant difference in dynamic surface tension (DST) was observed at 1 mg / mL of the two compounds, but not at lower surfactant concentrations (Figure 11).

[0130] alkyl group A different group of phenomena was observed, particularly with alkyl surfactants exhibiting higher concentrations and greater VP and turbidity (Figure 12). These particles were often observed even in placebo formulations and were particularly noticeable in EH-9 formulations with large amounts of SVP (larger than 2 μm) (Figure 7). The presence of insoluble impurities may explain the VP in this group, but further investigation was not conducted. The exception was CS20, the only surfactant in this group available in pharmaceutical grade. Here, both concentrations tested showed no visible particles at the initial stage. Similar findings were obtained for 15-S-15. The 1 mg / mL CS20 formulation showed increased degradation under high-temperature conditions, accompanied by increased VP and turbidity (Figure 12). Furthermore, increases in SVP (Figure 7) and HMWS (Figure 6D) were observed. The degradation theory was supported by 1H-NMR measurements (data not shown). TMN-6 performed well and showed no substantial impact on protein quality attributes during stress testing or long-term storage. An increase in turbidity was observed for higher formulations (1 mg / mL). Compared to other surfactant groups, alkyl compounds showed the lowest DST, which can be explained by the flexible structure of these surfactants resulting in high packing density at the interface. Nevertheless, solubility issues made it difficult to interpret the stress test results, and further investigation is needed to identify potential impurities and their effects. Therefore, performance evaluation of these molecules as alternative surfactants would be easier. In summary, 15-S-15 and TMN-6 all showed acceptable quality after applied interfacial and thermal stress (especially for the 0.1 mg / mL formulation), and in some cases were slightly better than PS20.

[0131] sterol group Interestingly, most sterol-based surfactants showed no stabilizing effect or only a negligible one. After shaking stress, these formulations exhibited relatively high VP, high turbidity, and even a strong color change in the case of Chobi. The presence of insoluble impurities may explain the VP in this group, but further investigation was not conducted. The strongest particle formation during shaking was observed with Chobi. This formulation even behaved similarly to formulations without surfactants. Furthermore, the dynamic surface tension of both formulations was similar, at approximately 73 mN / m. DST describes how effectively surfactants can disrupt interfacial cohesive forces and how well they can adapt to changes at the interface, such as those during shaking. The high DST of both the Chobi and control formulations indicates a low protein stabilizing effect at the interface, which again may explain the results of the shaking study. F / T studies for both the surfactant-free Chobi formulation and the control formulation also showed a defect stabilization effect at the interface. In general, most surfactants showed good stabilization with small amounts of VP against freeze-thaw stress. The data suggested some correlation between DST and the results of the shaking test, with increased VP for higher DST values. Further investigation is needed as other factors such as surfactant / impurity solubility or surfactant-protein interactions may also affect VP formation. In addition to Chobi, Chol-PEG and DBC also showed relatively high DST values, with particle formation particularly after agitation. However, Chol-PEG yielded good protein quality attributes (i.e., loss of HMWS and IE-HPLC main peaks) under all test conditions, especially the higher 1 mg / mL formulation condition. Although macroscopically visible particles were observed after the shaking stress test, the test conditions were much harsher than actually observed, and the results were comparable to the Px188 control. Furthermore, the apparatus using 1 mg / mL DBC initially showed many particles (Figure 13), and the placebo formulation showed solubility issues. Despite VP, SVPs larger than 10 μm (Figure 5) and soluble aggregates (Figure 6) increased after shaking, especially when performed at 25°C.Furthermore, Chobi and DBC are USP <787> The number of particles larger than 10 μm per container, which is the standard, exceeded 6,000 at its highest. On the other hand, F / T and thermal stress did not show a significant increase in HMWS or SVP.

[0132] Previous studies on sterol surfactants have reported that the rigid, bulky sterol ring structure is well-suited for long periods to form a favorable conformation at the interface. The orientation of surfactants is more complex in larger surfactants with rigid skeletons, and therefore, the time to reach equilibrium surface tension is longer (more than 2 hours) [64,65]. This data supports the results of our shaking experiments. Sterol surfactants may not react quickly to changes at the interface, for example, during shaking.

[0133] others This group includes polymer surfactants, poloxamers, poloxamines, and polyvinyl alcohols, as well as the tocopherol compound TPGS. All formulations in this group showed lower VP and turbidity values ​​at higher surfactant concentrations (Figure 14). Stress shaking of the 1 mg / mL formulation significantly reduced SVP (Figure 5) and soluble aggregates (Figure 6). Interestingly, the strongest particle formation was observed for Px188. The data suggest a dependence of HLB of poloxamers and poloxamines on their ability to protect mAbs from interfacial stress. Compared to Px188 and Px338, which have relatively high HLBs (over 27), the stress test results for Px407 (HLB: 22) and T1107 (HLB: 18-23) were considerably better, even at concentrations as low as 0.1 mg / mL. Due to the different HLBs and molecular weights of the compounds, slightly different dynamic surface tensions were expected. However, commercially available samples are heterogeneous compositions of molecular mixtures with average molecular weights and HLBs that can explain the results of DST measurements. Generally, the DSTs of surfactants in this group are relatively high, which may explain the low stabilizing effect, especially at low surfactant concentrations. When comparing the molar ratio (mAb:surfactant) with the PS20 reference formulation, it should be noted that the polymer surfactants have a molar ratio of approximately 1:0.004 (mol / mol) at a concentration of 0.1 mg / mL, which is about 10 times lower than 1:0.04 (mol / mol). These findings may also explain the low stabilizing effect during shaking.

[0134] Rather unexpected was the good quality of the formulation containing 1 mg / mL TPGS. The vitamin E-based surfactant TPGS also has a rigid ring structure similar to sterol-based surfactants, but with additional alkyl chains. Furthermore, it showed a fairly high DST, which was nearly constant for both concentrations already observed for sterol-based surfactants. Therefore, similar results after shaking stress were expected, but interestingly, 1 mg / mL TPGS performed very well with respect to VP, SVP, and HMWS. DST measurement does not always provide a reliable prediction of particle formation trends during shaking, and further investigation is needed.

[0135] In summary, formulations containing 1 mg / mL of SL, T1107, Px338, and Px407 were of acceptable quality under all applied stress and storage conditions. Furthermore, at this concentration, formulations containing PVA and TPGS showed very good stabilization effects with excellent product quality. Although the DST of approximately 60 mN / m was relatively high, these compounds are promising alternative surfactant candidates. 15-S-15 and TMN-6 showed acceptable quality under all applied stress and storage conditions at lower surfactant concentrations (0.1 mg / mL).

[0136] 2.) Long-term protein stability Under long-term storage conditions, potential adverse effects on protein stability should be excluded. Therefore, the stability of the formulation was evaluated for storage at 5°C and 25°C for 6 months in terms of the formation of macroscopically and microscopically visible particles, changes in color and turbidity, and monomer content (Figures 11-14).

[0137] A surfactant-free formulation was further used as a reference, and alternative surfactant formulations were compared with the established surfactants PS20 and Px188, which are known not to have adverse effects. The surfactant-free reference formulation showed a large amount of VP after storage for 6 months at both temperatures. Furthermore, the SVP content of ≥10 μm increased slightly compared to the initial state during storage at 25°C. Similar results were observed for PS20. Here, VP formation was observed particularly at high concentrations and high temperatures, but the amount of SVP did not increase (Figure 11). In contrast to the results of the stress-tested formulation with a Px188 concentration of 0.1 mg / mL, long-term storage showed good stability at both temperatures throughout all analyses. Grapentin et al. showed comparable stabilization results for PS20 and Px188 in liquid mAb vial formulations during long-term storage

[66] .

[0138] All three reference formulations showed no changes in color, turbidity, or monomer content (Figures 11 and 12). However, it should be noted that most formulations did not show significant changes during these tests. The number of microscopically visible particles, measured by light shielding, was generally low, and the reported values ​​are in line with the USP Pharmacopoeia. <787> and significantly lower than the maximum acceptable number according to Ph.Eur2.9.19. A slight increase in SVP levels was observed for the CS20 formulation at high temperatures, which can be explained by the aforementioned thermal decomposition. In addition to SVP, higher turbidity and a decrease in monomer content by SE-HPLC were observed (Figure 8). Furthermore, IE-HPLC of the CS20 formulation revealed a decrease in the main peak area compared to the reference formulation with PS20 and Px188 under high stress temperature conditions (Figure 9). This may suggest that the degradation of mAbs was initiated by the CS20 degradation products.

[0139] Increased turbidity was also observed with high concentrations of the alkyl surfactants TMN-6 and EH-9. The previously described phenomenon may be due to potential impurities or solubility issues with the surfactants themselves. Due to their small and flexible structure, good performance against interfacial stress can be expected for these surfactants, but a stronger tendency towards oxidative degradation can also be expected, as has already been described for PS20 [25,26]. Unfortunately, solubility issues, especially at high concentrations, make it difficult to interpret performance from a particle perspective.

[0140] In this study, we were unable to confirm a correlation between long-term stability and conformational stability. While the VP content of both mPEG-DSPE and SL formulations was high at both concentrations, only SL, and not mPEG-DSPE, showed a slight decrease in monomer content at higher concentrations (Figure 8). Evaluation of formulations containing DBC, a third surfactant with slightly reduced structural stability, was also crucial due to solubility issues at high concentrations. In summary, the T observed in our pre-screening (Figure 4) was... on Slight changes in this have so far not provided any predictive properties for protein stability.

[0141] In general, surfactants in the "other" group showed good results during long-term storage at high monomer content, exhibiting low VP and SVP. An exception was the PVA formulation, where VP formation was observed at high temperatures at both concentrations but not at 5°C. Further time-series analysis is needed to describe the performance of PVA as an alternative surfactant at ambient storage temperatures of 2–8°C.

[0142] In contrast to the results of the interfacial stress tests, the stabilization properties of sterol-based surfactants were very good, especially during long-term storage at low concentrations. These surfactants require more time to adsorb to the interface and therefore may not adequately protect proteins during rapid interfacial changes present during shaking, or they may require higher concentrations to achieve sufficient stabilization. However, if the surfactant has sufficient time to adsorb to the interface, it can exert its stabilizing effect. Nevertheless, because the formulation (DP) is subjected to mechanical stress during transport, most of the sterol-based surfactants tested are not suitable as substitutes for the established surfactants PS20 and Px188.

[0143] 3) Evaluation of protein stability of mAb2 and mAb3 in the presence of selected surfactants after mechanical stress and thermal stabilization. The inventors further investigated a selection of surfactants (PS20, PS80, SL, 15-S-15, TMN-6, Chol-PEG, Px188, Px338, Px407, T1107, PVA, and TPGS) in terms of their efficiency in stabilizing protein formulations. Low levels of 0.1 mg / mL surfactants were tested in the presence of two different mAb(2 and 3) at concentrations of 25 mg / mL and 10 mg / mL, respectively. PS80 was added to the PS20 (Figure 15) and Px188 (Figure 18) surfactants to include a supplemental control. This supplemental test was set up to confirm the positive effect of promising surfactant candidates from the first screening (i.e., for mAb1 in this specification) on the stability of two other mAb under mechanical and thermal stress. The conditions were the same as described above: shaking at 200 rpm for 7 days at 5°C and 25°C, followed by 5 freeze / thaw cycles between -20°C and 5°C, and then storage at 5°C, 25°C, and 40°C for 4 weeks.

[0144] While PS20 and PS80 adequately protect the protein under mechanical stress conditions such as shaking and F / T, especially in the case of mAb3, four weeks of high-temperature storage of the PS20 formulation results in numerous microscopic particles in the case of mAb2 (Figure 15).

[0145] Alkyl surfactants, particularly 15-S-15, exhibited good protective effects against mAb2 and mAb3, which are at lower risk of degradation by HCP, under these mechanical stress conditions (Figure 16). 15-S-15 demonstrated comparable or superior protein protection compared to polysorbates PS20 and PS80, as well as Px188, for three different mAb(mAb3) containing bispecific antibody fragments with known lower conformational protein stability. These findings indicate that 15-S-15 is a very promising candidate as an alternative surfactant. Another surfactant tested from the alkyl class, TMN-6, showed good protective effects against mAb2, but many VPs were observed after mechanical and thermal stress on mAb3.

[0146] Compared to PS20 and PS80, surfactants SL, PVA, poloxamer Px338 and Px407, T1107, and TPGS did not perform similarly under shaking conditions, but the protective effect of mAb2 was better than that of Px188, with fewer VPs, especially at 25°C (Figures 15 and 18). For mAb3, no difference in VP counts was observed between these surfactants and Px188, and they all showed fairly high counts. However, the VP numbers of these surfactants and mAb3 when stored for more than 4 weeks were generally lower than that of Px188. For Chol-PEG, the above trend of superior protective effect compared to Px188 was not observed (Figure 17). The trends for SVP were slightly different, with all surfactants and mAb2 formulations being in the same range, while mAb3 formulations showed dramatic differences in SVP numbers among some surfactants, especially under shaking conditions. Furthermore, regarding mAb3 under shaking conditions, the inventors observed that PVA, Px407, T1107, and TPGS showed lower counts than Px188, and TPGS was even in the range close to PS20 and PS80 (Figures 15 and 18).

[0147] Regarding soluble mAb aggregates, most conditions and surfactants showed the same amount of HMWS for mAb2, while only two showed significant differences for mAb3. For mAb3, storage at 40°C resulted in lower HMWS for Chol-PEG, Px338, Px407, T1107, PVA, and TPGS than for polysorbate (Figures 17 and 18), and SL had a surprising effect on soluble aggregates with the lowest overall HMWS levels for mAb3, although high SVP counts still existed (Figure 15).

[0148] As a general result of this selection of surfactant candidates, proteins, and conditions, 15-S-15 can be identified as one of the most efficient surfactants, as it is equivalent to or superior to PS20, PS80, and Px188 across a wide range of formulations and all mAbs tested. TPGS, Px338, Px407, PVA, T1107, TMN-6, and SL exhibited lower protective activity compared to polysorbate, particularly compared to mAb3, during mechanical stress, but their performance was equivalent to or better than Px188. Furthermore, surfactants Px338, Px407, PVA, T1107, and TPGS were tested for mAb2 and mAb3 at surfactant concentrations lower than the ideal range seen in the initial screening for mAb1, yet still showed better protective activity than Px188. For 15-S-15 and TMN-6, the ideal concentration range observed for mAb1 was also used for testing mAb2 and mAb3, where both surfactants retained their overall protective effect. The positive performance of Chol-PEG from the first test (i.e., mAb1) was not confirmed for mAb2 and mAb3. In fact, in this second test, the protective effect of mAb2 and mAb3 was lower compared to Px188 under most conditions (Figure 17).

[0149] In summary, the present invention did not identify any positive effect on the stability of protein formulations based on an entire surfactant class or subgroup, such as those shown in Figure 1. Surprisingly, using TPGS, PVA, T1107, Px338, Px407, TMN-6, 15-S-15, Chol-PEG, and SL, the present invention identified nine surfactants that exhibited protein stabilization effects equivalent to or better than the established PS20, PS80, and Px188. In particular, 1 mg / mL of TPGS and PVA showed very good stabilization properties with small amounts of VP, SVP, and HMWS under both interfacial and thermal stress conditions. However, formulations containing 1 mg / mL of Px338, Px407, T1107, Chol-PEG, and SL also showed superior stabilization effects with smaller amounts of VP and SVP during most applied stresses and stabilizations compared to the conventional surfactant Px188. The SL formulation showed a slight decrease in monomer content after long-term storage at high temperatures, but since the intended storage conditions are 2–8°C, these surfactants are considered potential alternatives to PS20 and Px188. For the 15-S-15 and TMN-6 formulations, even lower surfactant concentrations were sufficient to provide good protein stabilization properties comparable to PS20. Furthermore, even in the presence of mAb2 and mAb3, a lower concentration of 0.1 mg / mL of 15-S-15 provided the best stabilization properties of all surfactants, surpassing PS20 and PS80. TPGS, PVA, Px338, Px407, TMN-6, SL, and T1107 are better than or equivalent to Px188.

[0150] References TIFF0007898238000017.tif236161TIFF0007898238000018.tif245161TIFF0007898238000019.tif249161TIFF0007898238000020.tif23161

Claims

1. The product comprises a protein and one or more surfactants selected from TMN-6 and 15-S-15. A liquid pharmaceutical composition in which the protein is an antibody or an antibody fragment.

2. The composition according to claim 1, further comprising a pharmaceutically acceptable excipient or carrier.

3. The composition according to claim 1 or 2, wherein the one or more surfactants are present in a concentration range of 1 mg / mL or less; or 0.001 mg / mL to 0.01 mg / mL; or 0.01 mg / mL to 0.1 mg / mL; or 0.1 mg / mL to 1.0 mg / mL.

4. A composition according to any one of claims 1 to 3, for stabilizing proteins and preventing the formation of visible particles in a liquid pharmaceutical composition during storage.

5. The use of one or more surfactants selected from TMN-6 and 15-S-15 in the production of a liquid pharmaceutical composition further containing protein, The protein used is an antibody or an antibody fragment.

6. The use of one or more surfactants selected from TMN-6 and 15-S-15 to stabilize the protein and to prevent the formation of visible particles in the liquid pharmaceutical composition containing the protein during storage, The protein used is an antibody or an antibody fragment.

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