EA043419B1 - METHOD FOR OBTAINING LYOPHILIZED PHARMACEUTICAL COMPOSITION BASED ON THERAPEUTIC PROTEIN - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to biopharmaceuticals, in particular to therapeutic proteins, methods of their use, pharmaceutical compositions based on them and methods for producing pharmaceutical compositions. In particular, the present invention relates to methods for preparing lyophilized pharmaceutical compositions.

Prerequisites for creating the invention

Over the past ten years, technological developments have made it possible to obtain a variety of active molecules for pharmaceutical applications. As understanding of the nature of the mechanisms of biological action progresses, these molecules can be developed with specific properties for which small amounts of the product can be effective.

Because these molecules may be larger and/or more complex than traditional organic and inorganic drugs (ie, contain multiple functional groups in addition to complex three-dimensional structures), the formulation of such products poses special challenges. For a product to remain biologically active, the composition must maintain unchanged the conformational integrity of at least the core amino acid sequence of the protein while protecting these several functional groups of the protein from destruction. Pathways for protein degradation may involve chemical instability (i.e., any process that involves modification of a protein through the formation or cleavage of bonds, resulting in the formation of a new chemical entity) or physical instability (i.e., higher order changes in protein structure). Chemical instability may result from deamidation, racemization, hydrolysis, oxidation, beta elimination, or disulfide exchange. Physical instability may result from, for example, denaturation, aggregation, precipitation or adsorption. The three most common pathways of protein degradation are protein aggregation, deamidation, and protein oxidation. Cleland et al. (1993), Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377.

These developed molecules, due to their synthetic nature, are preferably lyophilized (freeze-dried) as this form of release can provide improved storage stability. Freeze drying is a commonly used protein preservation technique that serves to remove water from the protein preparation of interest. Freeze drying or lyophilization is a method by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient may be included in pre-lyophilized formulations to improve stability during the freeze-drying process and/or to improve the storage stability of the lyophilized product. Pikal, M. (1990), Biopharm. 3(9)26-30 and Arakawa et al. (1991), Pharm. Res. 8(3):285-291.

A developed molecule with specific biological targets and resulting product dosage requirements creates new challenges for the manufacturing method. The current state of the art includes a simple method where the product is formulated to a target concentration and then filled into containers of a predetermined volume.

Brief description of the invention

The present invention is directed to a method for producing a lyophilized drug. In particular, it relates to the formulation, dispensing and provision of the required amount of product present after reconstitution with a fixed volume of diluent for the lyophilized product for use.

In accordance with the present invention, there is provided a method for producing a lyophilized pharmaceutical composition based on a therapeutic protein, which includes preparing a composition with a bulk amount of therapeutic protein, measuring the concentration of therapeutic protein in said bulk composition, adjusting the loading weight of the protein in said bulk composition to obtain a fixed dose of protein, and lyophilization formulation with an adjusted protein fill mass to produce a final formulation in a container where the concentration of the product after reconstitution with a fixed volume is within a predetermined range of acceptable values.

In the above method, the protein concentration in the final formulation is preferably about 20 or 25 mg/ml or less, most preferably about 0.5 mg/ml, about 0.05 mg/ml, about 18 mg/ml, about 20 mg/ml and approximately 21 mg/ml. Preferred therapeutic proteins in the methods of the present invention are romiplostim, blinatumomab, infliximab, trastuzumab, AMG 701 and AMG 330. AMG 701 and AMG 330 are bispecific single chain antibody constructs, and other bispecific single chain antibody constructs (e.g., bispecific activators that attract T cells) represent a preferred

- 1043419 new therapeutic proteins in the methods of the present invention. Preferred pharmaceutical excipients present in the formulation include sugars, with trehalose, sucrose and a hydrate of any of these being most preferred. Preferred pharmaceutical excipients also include buffering agents, with histidine, citric acid monohydrate, sodium phosphate, potassium phosphate and glutamic acid being preferred. Preferred excipients further include surfactants, with polysorbate 20 and polysorbate 80 being most preferred. Preferred excipients and therapeutic proteins used in accordance with the methods of the present invention are presented in table. 1 with preferred approximate concentrations of each protein and excipient listed below.

Table 1

Preferred Formulation Components

Protein Sugar Buffer substance Volume-forming agent/solubilizing agent Surface-active substance pH Romiplostim, 0.5 mg/ml Sucrose, 2% w/v Histidine, 10 mM Mannitol, 4% w/v Polysorbate 20, 0.004% w/v 5.0 Blinatumomab, 55 mcg/ml Trehalose, 15% w/v Citric acid monohydrate, 25 mM; L-lysine hydrochloride, 200 mM Polysorbate 80, 0.1% w/v 7.0 Infliximab, 20 ± 1.5 mg/ml Sucrose, 10% w/v Sodium phosphate, 10 mM Polysorbate 80, 0.01% w/v 7.2 Trastuzumab, 21 mg/ml α,αtrehalose dihydrate, 19.1 mg/ml Histidine, 0.303 mg/ml; L-histidine hydrochloride monohydrate, 0.470 mg/ml Polysorbate 20, 0.0840 mg/ml 6.1 AMG 701, 1 mg/ml Sucrose, 9% w/v Lglutamic acid, 10 mM Polysorbate 80, 0.010% w/v 4.2 AMG 330, 0.5 mg/ml Sucrose, 8% w/v Potassium phosphate, 10 mM SBE-CD, 1% w/v Polysorbate 80, 0.010% w/v 6.1

In accordance with the present invention, the composition may additionally contain other excipients described herein below.

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Brief description of graphic materials

Fig. 1 is a response surface map showing an exemplary design field for a low-dose product where the formulation parameters and fill mass would correspond to a typical control strategy. The gray area represents the area where method/recovery variability would have a greater than 50% chance of mismatching the protein concentration. The large square shows the current operating range for formulation development. The smaller box shows the effective operating range.

Fig. 2 is a response surface map showing an exemplary design field for a low-dose product that considers osmolality in addition to bulk product concentration and fill volume. The light gray area shows the drug concentration specification in increments where method/recovery variability would have a greater than 50% chance of mismatching the protein concentration. The dark gray area shows the osmolality specification. The box shows the range of the design field for fill mass (x-axis) and bulk concentration (y-axis) within the operating range for in-process control (IPC) with warning limit (ALOR).

Fig. 3 is a response surface map showing an example of a useful but not error-tolerant strategy for controlling fill mass and formulation parameters. The curve and double-headed arrow indicate the likely error of the target fill parameter. The light gray area represents the drug concentration specification in increments, with the light gray area showing where method/reconstitution variability would have a greater than 50% chance of mismatching the protein concentration. The dark gray area defines the osmolality specification. The rectangle shows IPC/ALOR.

Fig. 4 is a response surface map showing an example of a control strategy for fill mass and formulation parameters with difficult control of range thresholds with an expected lower bulk concentration and lower fill mass. The left curve and double-headed arrow show the likely fill target error when fill volume is low. The right curve and double-headed arrow show the likely fill target error at larger fill volumes. The light gray area represents the drug concentration specification in increments, with the light gray area showing where method/reconstitution variability would have a greater than 50% chance of mismatching the protein concentration. The dark gray zone defines the osmolality specification. The rectangle shows IPC/ALOR.

Fig. 5 is a response surface map illustrating an example of a combined strategy of using the result of concentration selection in a bulk formulation to subsequently adjust the fill mass setpoint based on the total product dose in the target vial, resulting in a suitable, error-tolerant process for producing a lyophilized drug. facilities. The curve and double-headed arrow indicate the likely error of the target fill parameter. The light gray area defines the drug concentration specification in increments as shown in FIG. 1-4. The dark gray area defines the osmolality specification. The rhomboid shows the IPC/ALOR truncated by the osmolality specification at the largest fill volumes within the IPC/ALOR.

In fig. 6 shows the protein product concentration after reconstitution as a function of normalized fill weight values as determined in accordance with Example 1 hereinafter.

In fig. 7 shows osmolality as a function of normalized fill mass values as determined in accordance with Example 1 later herein.

Detailed Description of the Invention

Definition of terms

In the following description, a number of terms are used extensively. To facilitate understanding of the present invention, the following definitions are provided.

Unless otherwise noted, the singular form of nouns and the expression at least one are used interchangeably to mean one or more than one. In addition, unless the context otherwise requires, terms in the singular will include their plural forms, and terms in the plural will include their singular forms.

As used herein, a pharmaceutical composition or composition is a sterile composition of (i) a pharmaceutically active drug, such as a biologically active protein, that is suitable for parenteral administration (including, but not limited to, intravenous, intramuscular, subcutaneous, aerosol, intrapulmonary , intranasal and intrathecal administration) to a patient requiring it, and (ii) one or more pharmaceutically acceptable excipients, diluents and other additives that are considered safe by the Federal Drug Administration or other foreign national authorities.

- 3 043419 by local authorities. Pharmaceutical formulations include liquid (eg, aqueous) solutions that can be directly administered and lyophilized powders that can be reconstituted into solutions by adding a diluent prior to administration. The term pharmaceutical composition, however, specifically excludes compositions for topical administration to patients, compositions for oral administration and compositions for parenteral nutrition.

Shelf life, as used herein, means the period of storage during which the active ingredient (e.g., antibody) in a pharmaceutical composition exhibits minimal degradation (e.g., no more than about 5-10% degradation) upon storage of the pharmaceutical composition under specified storage conditions (eg 2-8°C). Methodologies for assessing degradation vary depending on the nature of the protein in the pharmaceutical formulation. Exemplary techniques include HPLC in size exclusion chromatography (SEC) mode to detect, for example, aggregation; reverse phase (RP) HPLC to detect, for example, protein fragmentation; ion exchange HPLC to detect, for example, changes in protein charge; as well as mass spectrometry, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy to detect protein conformational changes. All of these techniques can be used individually or in combination to evaluate protein degradation in a pharmaceutical formulation and determine the shelf life of that formulation. The pharmaceutical compositions of the present invention preferably exhibit no more than about 5-10% increase in degradation (eg, fragmentation, aggregation or unfolding) over two years when stored at 2-8°C.

As used herein, stable bioactive protein formulations are those that exhibit (i) a reduction in aggregation and/or a reduction in loss of biological activity by at least 20% when stored at 2-8°C for at least 2 years compared to a control sample formulation, or (ii) reduced aggregation and/or reduced loss of biological activity under thermal stress conditions (e.g., 25°C for 1 week to 12 weeks; 40°C for 1 to 12 weeks; 52°C C within 7-8 days, etc.). In one embodiment, a formulation is considered stable if the protein in the formulation retains its physical stability, chemical stability, and/or biological activity.

A protein can be said to retain its physical stability in a formulation if, for example, it does not show signs of aggregation, precipitation and/or denaturation when visually examined for color and/or clarity or when measured by UV scattering or size exclusion chromatography (SEC) ) or electrophoresis, such as for turbidity or aggregate formation.

A protein can be said to retain its chemical stability in its composition if, for example, the chemical stability at a particular point in time is such that modification of the protein by bond formation or cleavage does not result in the formation of a new chemical entity. In a further embodiment, chemical stability can be assessed by identifying and quantifying chemically altered forms of the protein. The chemical change may include, for example, a change in size (eg, truncation), which can be assessed using size exclusion chromatography, SDS-PAGE and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS). Other types of chemical change include, for example, charge change (eg, as a result of deamidation), which can be assessed using ion exchange chromatography. Oxidation is another commonly observed chemical modification.

A protein can be said to retain its biological activity in a pharmaceutical formulation compared to an unmodified protein if, for example, the percentage of biological activity of the formulated protein (e.g., antibody) determined by an assay (e.g., antigen binding assay) compared to a control solution is from about 50% to about 200%, from about 60% to about 170%, from about 70% to about 150%, from about 80% to about 125%, or from about 90% to about 110%. In a further embodiment, a protein may be said to retain its biological activity in a pharmaceutical formulation if, for example, without limitation, the biological activity of the protein at a given time is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

As used herein, the terms "comprising" and "comprising" are intended to indicate that the compositions and methods include the listed elements, but do not exclude other unlisted elements. The terms actually consisting of and actually consisting of, used to define compositions and methods, include listed elements, exclude unlisted elements that change the essential nature of the composition and/or method, but do not exclude other unlisted elements. Thus, a composition actually consisting of the elements defined herein will not exclude trace amounts of other elements, such as contaminants from any isolation and purification processes or pharmaceutically acceptable carriers

- 4 043419 bodies (for example, phosphate-buffered saline), preservatives, etc., but will exclude, for example, additional unspecified amino acids. The terms consisting of and consisting of, when used to define compositions and methods, exclude elements in more than trace amounts from other ingredients and essential steps of the method of administration of the compositions described herein. The embodiments defined by each of these transitional terms are within the scope of the present disclosure and the invention embodied herein.

As used herein, the term isolated refers to a protein (eg, antibody) that has been identified and separated and/or recovered from a component of its natural environment. Contaminants in its natural environment are materials that will interfere with diagnostic or therapeutic applications of the protein, and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the protein will be purified (1) to greater than 95% by weight of the antibody, as determined by the Lowry method, and most preferably to greater than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spin-beaker sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver dye. The isolated protein includes the protein in situ in the recombinant cells, since at least one component of the protein's natural environment will be missing. Typically, however, the isolated protein is obtained through at least one purification step.

The present invention relates to methods for preparing pharmaceutical compositions based on therapeutic proteins such as antibodies. Antibodies (Abs) and their synonym immunoglobulins (Ig) are glycoproteins that share the same structural characteristics. Although antibodies exhibit binding specificity to a particular antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter type, for example, are produced at low levels in the lymphatic system and at increased levels in myelomas. Thus, as used herein, the term antibody or antibody peptide(s) refers to an intact antibody, an antibody derivative, an antibody analogue, a genetically modified antibody, an antibody having a detectable label, an antibody that competes for specific binding with an antibody disclosed herein , or an antigen-binding fragment thereof (eg, Fab, Fab', F(ab') ₂ , Fv, single domain antibody) that competes with the intact antibody for specific binding, and includes chimeric, humanized, fully human and bispecific antibodies. In certain embodiments, antigen binding fragments are produced, for example, using recombinant DNA techniques. In additional embodiments, antigen-binding fragments are produced by enzymatic or chemical digestion of intact antibodies. Antigen binding fragments include, but are not limited to, Fab, Fab', F(ab)2, F(ab')2, Fv and single chain antibodies.

As used herein, the term intact antibodies refers to antibodies containing two heavy chains and two light chains. Thus, the term includes, without limitation, fully human antibodies, genetically engineered antibodies, bispecific antibodies and antibody derivatives, provided that such antibodies contain two heavy chains and two light chains.

As used herein, the term monoclonal antibody is not limited to antibodies produced by hybridoma technology. The term monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic or phage clone, and not to the method of its preparation.

Monoclonal antibodies and antibody constructs formulated in accordance with the present invention specifically include chimeric antibodies (immunoglobulins) in which part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or a subclass of antibodies, while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity ( US Patent No. 4,816,567; Morrison et al (1984), Proc. Natl. Acad. Sci. USA, 81: 6851-6855). Chimeric antibodies of interest herein include primatized antibodies containing antigen binding variable domain sequences derived from a non-human primate (eg, marmosets, apes, etc.) and human constant region sequences. A variety of approaches to the production of chimeric antibodies have been described. See, for example, Morrison et al. (1985), Proc. Natl. Acad. Sci. U.S.A. 81:6851; Takeda et al. (1985), Nature 314:452, Cabilly et al., US Patent No. 4816567; Boss et al., US Patent No. 4816397; Tanaguchi et al., EP 0171496; EP 0173494; and also GB 2177096.

Monoclonal antibodies and antibody constructs formulated in accordance with the present invention specifically include antibodies referred to as human or fully human. Each of the terms human antibody and fully human antibody

- 5 043419 refers to an antibody that has the amino acid sequence of a human immunoglobulin, including antibodies isolated from libraries of human immunoglobulins or from animals transgenic for one or more human immunoglobulins that do not express endogenous immunoglobulins; for example, XenoMouse® antibodies and the antibodies described by Kucherlapati et al. in US Patent No. 5939598.

The term genetically modified antibodies means antibodies whose amino acid sequence differs from the amino acid sequence of the native antibody. Because of the importance of recombinant DNA techniques for the production of antibodies, one should not be limited to the amino acid sequences found in natural antibodies; antibodies can be redesigned to achieve desired characteristics. There are many possible modifications and they range from changes in just one or a few amino acids to complete redesign, for example, of the variable and/or constant region. Changes to the constant region will typically be made to improve or change characteristics such as complement fixation, membrane interaction and other effector functions, as well as processability and viscosity. Variable region changes will be made to improve antigen binding characteristics.

The Fab fragment consists of one light chain, as well as C _H1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The Fab' fragment contains one light chain and one heavy chain, which contains most of the constant region between the _Cw and CH2 domains, so that an interchain disulfide bond can form between the two heavy chains to form the F(ab')2 molecule.

The F(ab') ₂ fragment contains two light chains and two heavy chains containing part of the constant region between the CH and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains.

The terms Fv fragment and single chain antibody refer to polypeptides that contain antibody variable regions from both the heavy and light chains, but lack constant regions. Like whole antibodies, they are capable of selectively binding to a specific antigen. With a molecular weight of only approximately 25 kDa, Fv fragments are much smaller than conventional antibodies (150-160 kDa), which consist of two heavy protein chains and two light chains, and even smaller than Fab fragments (approximately 50 kDa). kDa, one light chain and half a heavy chain).

A single-domain antibody is an antibody fragment consisting of a single-domain Fv unit, for example, VH or _VL . Like whole antibodies, they are capable of selectively binding to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than conventional antibodies (150-160 kDa), which consist of two heavy protein chains and two light chains, and even smaller than Fab fragments (approximately 50 kDa, one light chain and half a heavy chain) and single chain variable fragments (approximately 25 kDa, two variable domains, one light chain and one heavy chain). The first single-domain antibodies were constructed from heavy chain-only antibodies found in camelids. Although most single-domain antibody research is currently conducted using heavy chain variable domains, light chain variable domains and light chain-derived nanobodies have also been shown to specifically bind to target epitopes.

As used herein, the term bispecific means an antibody construct that is at least bispecific, i.e. it comprises at least a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or one target (e.g., CD3) and the second binding domain binds to another antigen or another target (e.g., BCMA; e.g., CD33) . Accordingly, the antibody constructs of the present invention have specificity for at least two different antigens or targets. The term bispecific antibody construct of the present invention also covers multispecific antibody constructs, such as trispecific antibody constructs where the latter contain three binding domains, or constructs with more than three (e.g., four, five, etc.) types of specificity.

Given that the antibody constructs of the present invention are (at least) bispecific, they are not naturally occurring and are markedly different from naturally occurring products. Therefore, a bispecific antibody construct or immunoglobulin is an artificial hybrid antibody or immunoglobulin with at least two different binding sites with different specificities. Bispecific antibody constructs can be generated by a variety of methods, including hybridoma fusion or Fab' binding. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

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The at least two binding domains and variable domains of the antibody construct of the present invention may or may not contain peptide linkers (spacer peptides). The term peptide linker in accordance with the present invention includes an amino acid sequence by which the amino acid sequences of one (variable and/or binding) domain and another (variable and/or binding) domain of the antibody construct of the present invention are linked to each other. An important technical feature of such a peptide linker is that it does not have any polymerization activity. Suitable peptide linkers include those described in US Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344. Peptide linkers can also be used to attach other domains or modules or regions (such as half-life increasing domains) to the antibody construct of the present invention.

If a linker is used, the linker preferably has a length and sequence sufficient to ensure that each of the first and second domains can maintain its differential binding specificity independently of the other. For peptide linkers that connect at least two binding domains (or two variable domains) in an antibody construct of the present invention, preferred are those peptide linkers that contain only a small number of amino acid residues, for example, 12 amino acid residues or less. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are preferred. A peptide linker with less than 5 amino acids is contemplated to contain 4, 3, 2 or one amino acid, with Gly-rich linkers being preferred. A particularly preferred single amino acid in the case of said peptide linker is Gly. Accordingly, said peptide linker may consist of a single amino acid Gly. Another preferred embodiment of the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser (SEQ ID NO: 1), or polymers thereof, i.e. (Gly4Ser)x, where x is an integer equal to 1 or greater (for example, 2 or 3). Preferred linkers are shown under SEQ ID NO: 1-9. The characteristics of said peptide linker, which include not promoting the formation of secondary structures, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol. Immunol. (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Preferred are peptide linkers that, among other things, do not promote the formation of any secondary structures. The connection of these domains with each other can be ensured, for example, using genetic engineering, as described in the examples. Methods for preparing fused and operably linked bispecific single-strand constructs and causing them to be expressed in mammalian or bacterial cells are well known in the art (eg, WO 99/54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

In a particularly preferred embodiment, and as documented in the accompanying examples, the AMG 701 and AMG 330 antibody constructs of the present invention are each a bispecific single chain antibody construct, more preferably a bispecific single chain Fv (scFv). Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined recombinantly using the synthetic linker described above, allowing them to form a single protein chain in which the VL and VH regions are paired with the formation of a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:58795883). These antibody fragments are prepared using conventional techniques known to those skilled in the art, and the fragments are assessed for function in the same manner as whole or full-length antibodies. Therefore, a single chain variable fragment (scFv) is a fusion protein based on the heavy chain (VH) and light chain (VL) variable regions of immunoglobulins, typically linked by a short linker peptide of about ten to about 25 amino acids in length, preferably about 15 to 20 amino acids. The linker is typically rich in glycine for flexibility and serine or threonine for solubility and can connect the N-terminus of VH to the C-terminus of VL or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of constant regions and the introduction of a linker.

Bispecific single chain molecules are known in the art and are described in WO 99/54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loffier, Blood, (2000), 95, 6, 2098-2103, Briihl, Immunol., (2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. The techniques described for the production of single chain antibodies (see, but not limited to, US Pat. No. 4,946,778) can be adapted to produce single chain antibody constructs that specifically recognize the selected target(s).

Bivalent (also called bivalent) or bispecific single chain variable fragments (bi-scFv or di-scFv in (scFv) ₂ format) can be constructed by linking two scFv molecules (eg, with the linkers described above herein). If these two

- 7 043419 scFv molecules have the same binding specificity, the resulting (scFv)2 molecule will preferably be called bivalent (ie it has two valencies for the same target epitope). If two scFv molecules have different binding specificities, the resulting (scFv)2 molecule will preferably be called bispecific. Linking can be accomplished by producing a single peptide chain with two VH regions and two VL regions, resulting in tandem scFvs (see, for example, Kufer P. et al. (2004) Trends in Biotechnology 22(5):238-244) . Another possibility is to create scFv molecules with linker peptides that are too short for the two variable regions to fold together (eg, approximately five amino acids long), thereby forcing the scFv to dimerize. This type is known as diabodies (see, for example, Hollinger, Philipp et al. (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90 (14): 6444-8).

As described herein above, the present invention provides a preferred embodiment wherein the antibody construct is in a format selected from the group consisting of (scFv)2, scFv, single domain mAb, diabodies, and oligomers in any of these formats.

In another preferred embodiment of the antibody construct of the present invention, the heavy chain (VH) and light chain (VL) of the binding domain binding to the target antigen CD3 and CD33 or BCMA are not directly linked by the above-described peptide linker, but the binding domain is formed due to the formation of a bispecific molecule, as described for the diabody. Thus, the VH chain of the CD3 binding domain can be fused to the VL binding domain for CD33 or BCMA using such a peptide linker, while the VH chain of the CD3 binding domain is fused to the VL binding domain for CD33 or BCMA using such a peptide linker. .

The terms amino-terminal and carboxy-terminal and their abbreviated forms N-terminal and C-terminal are used herein to refer to positions in polypeptides. When the context allows, these terms are used with reference to a specific sequence or portion of a polypeptide to indicate proximity or relative position. For example, a particular sequence located carboxyl-terminally relative to a reference sequence in a polypeptide is located at the carboxyl-terminus of the reference sequence, but is not necessarily at the carboxyl-terminus of the full-length polypeptide.

As used herein, the term amino acid refers to natural and/or non-natural or synthetic amino acids, including glycine and both D- and L-optical isomers, amino acid analogues and peptidomimetics, including without limitation N-acetyl-analogs of optical D- or L- isomers (for example, N-acetylarginine). In some aspects, the term amino acid refers to monomeric amino acids.

In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, and techniques related thereto, which are described herein are well known and widely used in this field of technology. The methods and procedures of the present invention are generally carried out in accordance with traditional methods well known in the art and described in various general and more specialized literature references which are cited and discussed throughout this specification unless otherwise indicated. See, for example, Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Ausubel et al. (1992), Current Protocols in Molecular Biology, Greene Publishing Associates, and Harlow and Lane (1990), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Any enzymatic reactions and purification procedures are carried out in accordance with the manufacturer's specifications, as typically performed in the art or as described herein. The terminology used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, and laboratory procedures and techniques related thereto, which are described herein, are well known and widely used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, preparation, formulation and delivery of pharmaceuticals, and treatment of patients.

The use of intermediate concentrations in formulations

The goal of protein formulation is to convert a highly purified solution of recombinant protein (drug substance) into a stable, effective biopharmaceutical dosage form. Kamerzell et al. (2011), 63(13): 1118-59 (incorporated by reference). The first stage, often referred to as preformulation characterization, involves determining the physicochemical properties of the protein and its instability pathways, allowing the development of formulations containing various excipients to ensure protein stability under specific storage conditions. At the same time, it is necessary to develop analytical methods to monitor the physicochemical integrity and biological activity of the protein under formulation conditions (e.g., in the presence of excipients), along with specifications to determine acceptable limits.

- 8 043419 deductions for any changes to these parameters. Specific formulations of varying protein concentrations with target levels of various pharmaceutical excipients are then tested under experimental conditions to ensure stability, solubility and tonicity through shelf life. Additionally, a primary container (eg, a vial, cartridge, or prefilled syringe) is selected to store the protein-excipient mixture and facilitate parenteral administration by the patient or healthcare professional. The entire biopharmaceutical drug or vaccine dosage form (protein, excipients, primary container and delivery device) must be designed to be both scalable to enable commercial production under sterile conditions and to comply with all regulatory guidelines for the preparation and testing of biopharmaceutical dosage units. forms for use in medicine.

Typically, formulation development begins with the identification of a suitable pH and buffer system using a biophysical screening strategy. Buffers are added to a protein solution to stabilize the pH, which in turn stabilizes the protein, since protein stability is typically associated with a characteristic narrow pH range. See Garidel and Bassarab (2009), Impact of formulation design on stability and quality, in: Quality for Biologics: Critical Quality Attributes, Process and Change Control, Production Variation, Characterisation, Impurities and Concerns, Biopharm Knowledge Publishing, London, UK, pp. 94-113 (incorporated by reference). Other formulation auxiliaries such as stabilizers (eg, sugars), bulking agents (eg, mannitol), and surfactants (eg, polysorbate 20) are then added to the buffered protein solution. In the case of a lyophilized formulation, such a mixture is then lyophilized to be formulated in liquid form.

In lyophilization, as in other methods of drug production, the key control parameter is the loading mass of therapeutic protein to deliver the required dose to the patient. For higher concentration products, a simple strategy of controlling the product concentration at the formulation stage, followed by monitoring the fill mass as part of the filling process, is acceptable. However, to deliver a very low dose of product of a few micro- or even milligrams in size, this simple strategy for ensuring dose delivery begins to have problems in ensuring that the patient can receive the required dose. Related concerns are that the product label may not reflect the composition of the product inside the container and that it may be difficult to remove the material for dispensing. Therefore, there is a need for a method that provides greater control over the loading weight of therapeutic protein in a lyophilized pharmaceutical formulation.

As noted above, a simple strategy of controlling product concentration at the formulation stage, followed by controlling the fill mass as part of the fill process, is acceptable for higher concentrations of therapeutic protein, but can lead to problems at lower concentrations. The design margin for fill mass control for a low dose product, when target fill mass and composition parameters are controlled separately, exhibits higher variability with a greater likelihood that the product will not meet product release specifications. Due to the variability of the manufacturing process, precise control of the bulk formulation is not achievable due to the scale of the process and inherent equipment variability during manufacturing. Another problem is that a lyophilized product has increased variability because the product must be reconstituted, which is also a variable process.

A typical control strategy (shown in Figure 1) will have a greater than 50% chance of producing an erroneous protein concentration selection. In fig. 1 The observed overall variability in the lyophilized product homogeneity data is 0.01 mg/mL. To ensure minimum capability, the average operating range should be limited to match the specification range in increments of 3x0.01=0.03 mg/ml. The target white box process is characterized by a quality of less than 0.1% out of specification (OOS) vials per batch. In contrast, the target process in the shaded area has higher OOS scores. As can be seen in FIG. 1, the current operating range (large square) contains the tolerance thresholds for the desired composition. The effective operating range (smaller rectangle in Fig. 1), however, requires tighter fill tolerances than can be achieved through both formulation variability and fill mass control.

Thus, a process that also needs to consider other product quality indicators (e.g., osmolality, pH) as well as protein concentration may not be feasible or suitable using standard methodology. In fig. Figure 2 shows an illustrative design field that considers the osmolality specification. The gray color shows the broad conditions for producing an erroneous result within the operating range for IPC with a warning limit.

A standard methodology may also result in a usable but not error-tolerant system.

- 9 043419 tags for monitoring the filling mass and composition parameters (see Fig. 3). As shown in FIG. 1, IPC/ALOR contains areas that do not meet drug product concentration specifications. Within the drug product concentration out-of-specification zone (the white zone within the IPC/ALOR), there is very little room for target fill parameter error. Thus, as shown in FIG. 3, standard methodology may result in a process with difficult control of range thresholds, suitable but without sufficient robustness to errors.

Increasing the fill volume may not be a sufficient process change to create a suitable formulation with acceptable error tolerance. Fig. 4 is a map of the response surface in which the filled volume is increased. The fill target range can be adjusted to meet IPC/ALOR and product specifications, but error tolerance may still be unacceptable. The range of the filling mass within the permissible concentration range in the bulk formulation has insufficient error tolerance.

If the result of adjusting the concentration in the bulk formulation is used to subsequently adjust the loading mass of the therapeutic protein, the operating range can be changed sufficiently to provide a formulation that is both suitable and reasonably robust. As shown in FIG. 5, the modified operating range (IPC/ALOR) allows the expected range of the target filling parameter to be within the concentration and osmolality specifications. In this way, the range of the target filling parameter can be represented within the specifications for concentration and other parameters with sufficient robustness to errors.

To date, this methodology has been used to define low-dose stock keeping units (SKUs) for SKUs for low-dose romiplostim (Nplate®), blinatumomab (Blincyto®), infliximab, and trastuzumab. Details of this technique are presented in the Demonstrations section later in this document.

Excipients in general

One of the tasks in obtaining formulations is to stabilize the product from types of stress during manufacturing, transportation and storage, which can be achieved with the help of certain auxiliary substances for the formulations. In general, excipients can be classified based on the mechanisms by which they stabilize proteins against various types of chemical and physical stress. Some excipients mitigate the effects of a particular type of stress or regulate the specific susceptibility of a particular protein. Other excipients have a more general effect on the physical and covalent stability of proteins.

Common excipients used in liquid and lyophilized protein formulations are presented in Table. 2 (see Kamerzell et al. (2011), Advanced Drug Delivery Rev. 63(13): 1118-59).

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table 2

Auxiliary components in protein compositions

Auxiliary Component Function Examples Buffering agents Maintaining the pH of the solution Specific interactions of buffer substance ions with protein Citrate Succinate Acetate Glutamate Aspartate G istidine Phosphate Tris Glycine Sugars and carbohydrates Protein stabilization Agents regulating tonicity Carrier for inhaled drugs (lactose) Dextrose solutions used for IV administration Sucrose Trehalose Sorbitol Mannitol Glucose Lactose Cyclodextrin derivatives

- 11 043419

Stabilizers and volume-forming agents Improve product appearance and prevent leakage Ensuring the structural strength of the freeze-dried cake Mannitol Glycine Osmolytes Stabilization from stress caused by environmental influences (temperature, moisture loss) Sucrose Trehalose Sorbitol Glycine Proline Glutamate Glycerol Urea Amino acids Specific interactions with protein Antioxidant (His, Met) Buffering, regulation of tonicity G istidine Arginine Glycine Proline Lysine Methionine Aa mixtures (e.g. Glu/Arg) Proteins and polymers Competitive protein adsorption inhibitors Bulking agents for lyophilization Carriers for drug delivery HSA PVA PVP PLGA P.E.G. Gelatin Dextran Hydroxyethyl starch NES SMS Antioxidants Preventing oxidative protein damage Agents that bind metal ions (if the metal is included in B reducing agents Dissolved oxygen capture agents

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as a cofactor or required for protease activity) Means of capturing free radicals Free radical scavengers Chelating agents (e.g. EDTA, EGTA, DTPA) Ethanol Metal ions Protein cofactors Coordination complexes (suspensions) Magnesium Zinc Specific ligands Stabilizers of native conformation against stress-induced unfolding Providing conformational flexibility Metals Ligands Amino acids Polyanions Surfactants Competitive protein adsorption inhibitor Competitive inhibitor of surface protein denaturation Liposomes as carriers for drug delivery Inhibitor of aggregation during lyophilization Agent that reduces the recovery time of lyophilized products Polysorbate 20 Polysorbate 80 Poloxamer 188 Anionic surfactants (e.g. sulfonates and sulfosuccinates) Cationic surfactants Zwitterionic surfactants Salts Agents that regulate tonicity Stabilizing or destabilizing agents for proteins, in particular anions NaCl KS1 NaSO ₄ Preservatives Protection against the growth of microorganisms in the composition Benzyl alcohol M-cresol Phenol

Other excipients are known in the art and can be found in Powell et al. (1998), Compendium of Excipients for Parenteral Formulations, PDA J. Pharm. Sci. Tech., 52:238-311, which is incorporated herein by reference.

Given the ideas and guidance provided herein, those skilled in the art will know what amount or range of amounts of excipient can be included in any

- 13 043419 specific composition for obtaining the biopharmaceutical composition of the present invention. For example, the amount and type of salt to be included in the biopharmaceutical composition of the present invention can be selected based on the desired osmolality (i.e., isotonicity, hypotonicity, or hypertonicity) of the final solution, as well as the amounts and osmolality of other components that must be included in the composition. Likewise, by way of example, with reference to the type of polyol or sugar included in the formulation, the amount of such excipient will depend on its osmolality.

Those skilled in the art will be able to determine what amount or range of amounts of an excipient that helps maintain the stability of a biopharmaceutical can be included in any particular formulation to formulate the biopharmaceutical composition of the present invention. For example, the amount and type of salt to be included in the biopharmaceutical composition of the present invention can be selected based on the desired osmolality (i.e., isotonicity, hypotonicity, or hypertonicity) of the final solution, as well as the amounts and osmolality of other components that must be included in the composition. Likewise, by way of example, with reference to the type of polyol or sugar included in the formulation, the amount of such excipient will depend on its osmolality.

Approximately 5% w/v. sorbitol, for example, can achieve isotonicity, while the sucrose excipient requires approximately 9% w/v to achieve isotonicity. The choice of the amount or concentration range of one or more excipients that can be included in the biopharmaceutical composition of the present invention has been explained in the example above by reference to salts, polyols and sugars. However, those skilled in the art will appreciate that the considerations described herein and further illustrated by example by reference to specific excipients apply equally to all types and combinations of excipients, including, for example, salts, amino acids, other agents , regulating tonicity, surfactants, stabilizers, bulking agents, cryoprotectors, lyoprotectors, antioxidants, metal ions, chelating agents and/or preservatives.

Additionally, if a particular excipient is indicated in a formulation by, for example, a percentage (%) w/v, those skilled in the art will appreciate that the equivalent molar concentration of that excipient is also considered.

Those of ordinary skill in the art will appreciate that the concentrations of the above excipients are interdependent in a particular formulation. As an example, the concentration of the bulking agent can be reduced if, for example, there is a high concentration of protein/peptide or a high concentration of stabilizing agent. In addition, one of ordinary skill in the art will appreciate that in order to maintain isotonicity of a particular formulation lacking a bulking agent, the concentration of the stabilizing agent may be adjusted accordingly (ie, a tonicity-controlling amount of stabilizer may be used).

Buffering agents Solution pH influences the chemical integrity of protein amino acid residues (eg, Asn deamidation and Met oxidation) and the maintenance of its higher order structure. Thus, those skilled in the art use buffering agents to control the pH of the solution and optimize protein stability. The maximum stability of a protein drug is usually within a narrow pH range. Several approaches (eg, accelerated aging stability studies and calorimetric screening studies) are useful for this purpose (Remmele et al. (1999), Biochemistry, 38(16): 5241-7). Once the formulation is finalized, the drug product should be manufactured and maintained within predetermined specifications throughout its shelf life. Therefore, buffers are almost always used to control the pH of a formulation.

Organic acids, phosphates and Tris are commonly used as buffers in protein formulations (see Table 3). The buffering capacity of buffer compounds is maximum at a pH equal to pKa and decreases as the pH increases or decreases relative to this value. Ninety percent of the buffer capacity is within one pH unit of their pKa. The buffer capacity also increases in proportion to the increase in the concentration of the buffer substance.

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Table 3

Widely used buffers and their _pKa values

Buffer substance pK _a Illustrative drug Acetate 4.8 Neupogen®, Neulasta® Succinate ρΚ _Η ι=4.8, рК _а2 =5.5 Actimmune® Citrate pK _a 1=3.1, pK _a 2=4.8, pK _a3 =6.4 Humira® G istidine (imidazole) 6.0 Xolair® Phosphate _P Ka1=2.15, pKa2=7.2, pK _a z=12.3 Enbrel® (liquid formulation) Tris 8.1 Leukine®

In addition to the above, some therapeutic proteins may be self-buffering at pharmaceutically relevant concentrations. In formulations based on such proteins, the inclusion of a traditional buffer may not be necessary at all. See US Patent Application 2012/0028877, which is incorporated herein by reference.

Sugars and carbohydrates

Sugars are often used to stabilize proteins in both liquid and lyophilized formulations. Disaccharides such as sucrose and trehalose are thought to stabilize proteins through preferential hydration at high concentrations in the liquid state and through specific interactions with proteins and the formation of viscous glassy matrices in the solid state. Sugar molecules can increase the viscosity of monoclonal antibody solutions, apparently through the mechanism of preferential hydration. Sugar alcohols such as sorbitol can stabilize proteins in solution and in the lyophilized state. Mannitol is often used as a bulking agent in lyophilized formulations. Lactose is used as a carrier molecule for protein-based inhalation formulations. Cyclodextrin derivatives can stabilize proteins in liquid formulations based on antibodies, vaccine antigens and smaller proteins such as growth factors, interleukin-2 and insulin. Stabilizers and volume-forming agents.

A bulking agent is typically used in lyophilized formulations to improve the appearance of the product and prevent leakage. Formulation conditions are usually designed such that the bulking agent crystallizes out of the frozen amorphous phase (during freezing or annealing at temperatures above Tg'), imparting structure and volume to the cake. Mannitol and glycine are examples of commonly used bulking agents.

Stabilizers include a class of compounds that can serve as cryoprotectants, lyoprotectants, and glass formers. The action of cryoprotectants is to stabilize proteins during freezing or when frozen at low temperatures (R. Cameron, ed., Good Pharmaceutical Freeze-Drying Practice, Interpharm Press, Inc., Buffalo Grove, IL, (1997)). Lyoprotectants stabilize proteins in freeze-dried solid dosage form by maintaining native-like conformational properties of the protein during the dehydration steps of freeze-drying. Properties in the glassy state have been classified as strength or brittleness depending on the corresponding relaxation properties, which are temperature dependent. It is important that cryoprotectants, lyoprotectants and glass formers remain in the same phase as the protein to impart stability. Sugars, polymers and polyols fall into this category and can sometimes play all three roles.

Polyols cover a class of excipients that includes sugars (eg, mannitol, sucrose, sorbitol) and other polyhydric alcohols (eg, glycerol and propylene glycol). Polyethylene glycol (PEG) polymer is included in this category. Polyols are commonly used as stabilizing excipients and/or isotonic agents in both liquid and lyophilized protein formulations for parenteral administration. In relation to the Hofmeister series, polyols are kosmotropic and are predominantly displaced from the surface of proteins. Polyols can protect proteins from both physical and chemical degradation pathways. Predominantly displaced cosolvents increase the effective surface tension of the solvent at the protein interface, causing most energetically favorable protein conformations to be those with the smallest surface areas.

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Mannitol is a bulking agent often used in lyophilized formulations because it crystallizes from the amorphous protein phase during freeze-drying, imparting structural stability to the lozenge (e.g., Leukine®, Enbrel® Lyo, Betaseron®). It is usually used in combination with a cryo- and/or lyoprotectant such as sucrose. Because of mannitol's tendency to crystallize when frozen, sorbitol and sucrose are preferred tonicity adjusters/stabilizers in liquid formulations to protect the product from the types of freeze-thaw stress encountered during transportation or when freezing the bulk product prior to manufacturing. Sorbitol and sucrose are much more resistant to crystallization and are therefore less likely to separate from the protein as a result of phase separation. It is interesting to note that although mannitol has been used in tonicity-regulating amounts in several commercially available liquid formulations, such as Actimmune®, Forteo®, and Rebif®, the product labels for these formulations contain a warning not to freeze. The use of reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, should be avoided as they can react with the surface lysine and arginine residues of proteins and glycate them through the Maillard reaction between aldehyde groups and primary amino groups (Chevalier F, et al., Nahrung, 46(2): 58-63 (2002); Humeny A, et al., J Agric Food Chem. 50(7): 2153-60 (2002)). Sucrose can be hydrolyzed to fructose and glucose under acidic conditions (Kautz C. F. and Robinson A. L., JACS, 50(4) 1022-30 (1928)) and therefore can cause glycation.

In specific embodiments of the compositions of the present invention, a stabilizer (or combination of stabilizers) is added to the lyophilized formulation to prevent or reduce aggregation and chemical degradation caused by lyophilization or caused by storage. An opalescent or cloudy solution after reconstitution indicates that protein precipitation has occurred. The term stabilizer means an excipient capable of preventing aggregation or other physical degradation as well as chemical degradation (e.g., autolysis, deamidation, oxidation, etc.) in aqueous and solid states. Stabilizers traditionally used in pharmaceutical compositions include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine-HCl, polyhydroxy compounds, including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrins, N-methylpyrrolidone, cellulose and hyaluronic acid, sodium chloride, Carpenter et al. (1991), Develop. Biol. Standard 74:225.

Osmolytes

Osmolytes currently used as excipients in protein formulations are listed in Table. 2. Other osmolytes commonly found in nature that may be useful as excipients include taurine, betaine, trimethylamine-I-oxide (TMAO), choline-O-sulfate and sarcosine.

Proteins and polymers

Protein excipients add complexity to formulation, especially when it comes to developing analytical methods to monitor the stability of a protein drug or vaccine in the presence of a protein excipient. Polymers have been evaluated as excipients (eg, bulking agents) in lyophilized protein formulations. Protein drug and controlled release vaccine formulations are being explored, in which proteins are formulated with polymers such as copolymer of lactic-glycolic acid (PLGA) and polyethylene glycol (PEG). A variety of additional water-soluble polymers (eg, hydroxyethylcellulose (HEC), carboxymethylcellulose (CMC)) have been used to prepare topical protein drug formulations.

PEG can stabilize proteins through two different temperature-dependent mechanisms. At lower temperatures it is preferentially displaced from the protein surface, but has been shown to interact with the unfolded form of the protein at higher temperatures due to its amphipathic nature (Lee and Lee (1987), Biochemistry, 26(24): 7813-9). It may protect proteins by preferential displacement at lower temperatures, but possibly also by reducing the number of productive collisions between unfolded molecules at higher temperatures. PEG is also a cryoprotectant and was used in Recombinate®, a lyophilized formulation based on recombinant antihemophilic factor.

Antioxidants

Many different sources can oxidize residues in proteins. Oxidative damage to proteins can be minimized through careful control of the product's manufacturing and storage processes, including factors such as atmospheric oxygen, temperature, light exposure, and chemical contamination. If such control methods are unacceptable, antioxidant excipients can be included in the composition.

The most widely used pharmaceutical excipients from the group

- 16 043419 antioxidants are reducing agents, dissolved oxygen/free radical scavenging agents or chelating agents. Antioxidants in therapeutic protein formulations must be water soluble and must remain active throughout the shelf life of the product. Reducing agents and dissolved oxygen/free radical scavengers act by removing reactive oxygen species from solution. Chelating agents (eg, EDTA) can be effective by binding trace amounts of contaminants, which are metal ions that contribute to the formation of free radicals. For example, in a liquid formulation based on acidic fibroblast growth factor, EDTA inhibits the oxidation of cysteine residues catalyzed by metal ions. EDTA has been used in commercially available products such as Kineret® and Ontak®.

Metal ions

In general, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical degradation reactions of proteins. However, specific metal ions are included in formulations if they act as cofactors for proteins. Metal ions can also be used in protein-based suspension formulations, where they form coordination complexes (for example, in a zinc suspension of insulin). The use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (WO 2004/039337).

Metal ions have been found to impart stability and/or increased activity in a human deoxyribonuclease based formulation (rhDNase, Pulmozyme®). Ca ⁺² ions (not more than 100 mM) increase the stability of the enzyme through a specific binding site (Chen et al. (1999), J Pharm Sci. 88(4): 477-82). In fact, removal of calcium ions from the EGTA solution caused an increase in deamidation and aggregation. However, this effect was observed only with Ca ⁺² ions; Other divalent cations, Mg ⁺² , Mn ⁺² , and Zn ⁺ , have been observed to destabilize rhDNase.

Similar effects were observed in a formulation based on coagulation factor VIII. Ca ⁺ and Sr ⁺ ions stabilize the protein, while others such as Mg ⁺² , Mn ⁺² and Zn ⁺² , Cu ⁺² and Fe ⁺² destabilize it (Fatouros, et al. (1997), Int. J. Pharm., 155, 121-131). In a separate study with coagulation factor VIII, a significant increase in aggregation rate was observed in the presence of Al ⁺³ ions (Derrick et al. (2004), J. Pharm. Sci., 93(10): 2549-57). The authors note that other excipients, such as buffer salts, are often contaminated with Al ⁺³ ions, and illustrate the need for the use of appropriate quality excipients in formulated products. Vaccines containing live or killed attenuated picornaviruses, such as hepatitis A and polio viruses, are conformationally stabilized with magnesium. Metal ions such as calcium, magnesium and zinc improve the stability of oxytocin in aqueous solution. Insulin can bind to zinc, resulting in the formation of dimers and hexamers in crystalline form, allowing for different formulations with different in vivo release profiles. The chemical and thermal stability of the hexameric insulin formulation varies in the presence of varying levels of zinc and phenol.

Specific ligands

One approach to improve the conformational stability of protein therapeutic drugs is to take advantage of the presence of ligand-binding sites inherent in the protein. For example, Pulmozyme® not only requires the presence of divalent metal ions for its enzymatic activity, but also has improved conformational stability in the presence of calcium ions. Both acidic and basic fibroblast growth factors (aFGF and bFGF) have been evaluated clinically for their ability to promote wound healing, and both proteins naturally bind to highly negatively charged proteoglycans on cell surfaces. A variety of other highly negatively charged compounds also bind to aFGF and substantially stabilize it through interaction with the polyanion binding site of the protein.

Surfactants

Protein molecules have a high propensity to interact with surfaces, making them susceptible to adsorption and denaturation at air-liquid, vial-liquid, and liquid-liquid (silicone oil) interfaces. This degradation pathway is inversely related to protein concentration and results in the formation of soluble or insoluble protein aggregates or loss of protein from solution through adsorption to surfaces. In addition to adsorption to the container surface, surface-induced degradation is enhanced by physical shaking that will occur during transport and handling.

Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphipathic molecules that have the ability to displace proteins from positions at phase boundaries. The hydrophobic portions of surfactant molecules occupy positions at phase boundaries (e.g., air/liquid), while the hydrophilic portions of the molecules remain orientated

- 17 043419 baths to the volume of solvent. At sufficient concentrations (usually around the critical micellar detergent concentration), a surface layer of surfactant molecules serves to prevent the adsorption of protein molecules at the interface. In this way, surface-induced damage is minimized.

The most widely used surfactants are nonionic polyethoxylated sorbitan fatty acid esters, i.e. polysorbate 20 and polysorbate 80 (eg, in Avonex®, Neupogen®, Neulasta®). The two molecules differ only in the length of the aliphatic chain, which gives the hydrophobic character to the molecules, C-12 and C-18, respectively. Polysorbate 80 is more surfactant and has a lower critical micellar concentration than polysorbate 20. Both polysorbate 20 and polysorbate 80 have been shown to provide protection against shake-induced aggregation. Polysorbate 20 and polysorbate 80 also provide protection against stress induced by freezing, lyophilization and reconstitution. Both polysorbate 20 and polysorbate 80 may contain peroxides that can oxidize proteins, and they themselves may be destroyed by oxidation or hydrolysis, having different effects on protein stability. It can also be difficult to control the level of polysorbate 20 or polysorbate 80 in formulations due to their complex behavior during membrane filtration (especially at concentrations at which polysorbates form micelles in solution). Poloxamer 188 surfactant has also been used in several commercially available liquid products such as Gonal-F®, Norditropin® and Ovidrel®. In general, nonionic surfactants are thought to stabilize proteins primarily by pushing protein molecules away from hydrophobic surfaces (e.g., air-water interfaces), thereby preventing proteins from unfolding at these hydrophobic interfaces. Nonionic surfactants can also block the adsorption of protein molecules onto other hydrophobic surfaces present during treatment. In addition, nonionic surfactants can directly interact with the hydrophobic regions of protein molecules. Monoclonal antibodies may affect the critical micellar concentration of polysorbate 20 compared to buffer alone.

Detergents can also affect the thermodynamic conformational stability of proteins. And in this case, the effects of this excipient will be specific to the protein. For example, polysorbates have been shown to reduce the stability of some proteins and increase the stability of other proteins. The destabilization of proteins by detergents can be rationalized in the context of the hydrophobic tails of detergent molecules, which may be involved in specific binding to proteins in a partially or fully unfolded state. These types of interactions can cause a shift in conformational equilibrium toward more unfolded states of the protein (i.e., increasing the accessibility of hydrophobic portions of the protein molecule in addition to polysorbate binding). Alternatively, if the native state protein has some hydrophobic surfaces, detergent binding to the native state protein may stabilize this conformation.

Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. They often contain sufficient peroxides as starting materials to cause oxidation of side chain residues in proteins, particularly methionine. The potential for oxidative damage due to the addition of a stabilizer highlights the fact that the lowest effective concentrations of excipients should be used in formulations. For surfactants, the effective concentration of a given protein will depend on the stabilization mechanism. It has been suggested that if the mechanism of surfactant stabilization is related to the prevention of surface denaturation, then the effective concentration will be around the critical micellar detergent concentration. If the stabilization mechanism is related to specific protein-detergent interactions, then the effective surfactant concentration will be related to the protein concentration and the stoichiometry of the interaction (Randolph et al. (2002), Pharm Biotechnol., 13:159-75).

Surfactants can also be added in appropriate amounts to prevent surface aggregation during freezing and drying (Chang (1996), J. Pharm. Sci. 85:1325). Exemplary surfactants include anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium Nlauroyl sarcosine, lithium dodecyl sulfate, sodium 1-octane sulfonic acid, sodium cholate hydrate, sodium deoxycholate and sodium glycodeoxycholic acid. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate and hexadecyltrimethylammonium bromide. Zwitterionic superficially

- 18 043419 active substances include, without limitation, CHAPS, CHAPSO, SB3-10 and SB3-12. Nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN-20 and TWEEN-80. In another embodiment, the surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene 10, -40, -50 and -60 hydrogenated castor oil, glycerol monostearate, polysorbate 40, 60, 65 and 80, soy lecithin and other phospholipids such as DOPC, DMPG, DMPC and DOPG; sucrose fatty acid ester, methylcellulose and carboxymethylcellulose.

Salts

Salts are often added to increase the ionic strength of the formulation, which can be important for protein solubility, physical stability, and isotonicity. Salts can affect the physical stability of proteins in a variety of ways. Ions can stabilize the native state of proteins by binding to charged residues on the protein surface. Alternatively, they may stabilize the denatured state by binding to peptide groups along the protein backbone (-CONH-). Salts can also stabilize the native conformation of a protein by shielding repulsive electrostatic interactions between residues in the protein molecule. Electrolytes in protein formulations can also shield attractive electrostatic interactions between protein molecules, which can lead to protein aggregation and insolubility.

The effect of salt on protein stability and solubility varies greatly depending on the characteristics of the ion species. The Hoffmeister series originated in the 1880s as a way to rank electrolytes based on their ability to precipitate proteins (Sasase et al. (1997), Quarterly Reviews of Biophysics, 30(3): 241-277). In this report, the Hofmeister series is used to illustrate the scope of the effects of protein stabilization by ionic and nonionic cosolutes. In Table C, cosolutes are ordered by their overall effects on the state of the proteins in solution, from stabilizing (kosmotropic) to destabilizing (chaotropic). In general, the differences in effects among anions are much greater than the differences in effects observed for cations, and for both types, effects are most evident at higher concentrations than those allowed in parenteral formulations. High concentrations of kosmotropes (e.g., >1 mol/L ammonium sulfate) are commonly used to precipitate proteins from solution through a process called salting out, where the kosmotrope is preferentially displaced from the protein surface, reducing the solubility of the protein in its native (folded) conformation. Removing or diluting the salt will return the protein to solution. The term salting-in refers to the use of destabilizing ions (such as guanidine and chloride) that increase the solubility of proteins by solvation of peptide bonds in the protein backbone. Increasing chaotrope concentrations will promote the conformation of the protein in a denatured (unfolded) state, as the solubility of the peptide chain increases. The relative effectiveness of ions in salting-in and salting-out determines their position in the Hofmeister series.

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Table 4

Hofmeister series for salts

Co-solute Stabilization scope Anion Cation Other F (CH ₃ ) ₄ N ⁺ Glycerin/sorbitol Stabilization (salting out) Destabilization (salting in) Cosmotropic Chaotropic RO ₄ (CH ₃ ) ₂ NH ⁺ Sucrose/trehalose SO ₄ " ^nh ₄₊ TMAO snsoo k ⁺ sg Na ⁺ Vg Cs ⁺ G Li ⁺ Mg ²⁺ Guanidine Ca2 ⁺ Arginine Ba ²⁺ Urea

To maintain isotonicity in parenteral formulations, salt concentrations are typically limited to less than 150 mM for monovalent ion combinations. In this concentration range, the mechanism of stabilization by salts is likely due to shielding of electrostatic repulsive intramolecular forces or attractive intermolecular forces (Debye-Hückel shielding). Interestingly, chaotropic salts have been shown to be more effective than kosmotropes at similar concentrations in stabilizing protein structure through this mechanism. It is believed that chaotropic anions bind more tightly than kosmotropic ions. Regarding the breaking of covalent bonds in proteins, differential ionic strength effects are expected with respect to this mechanism according to the Debye-Hückel theory. Accordingly, published reports of protein stabilization with sodium chloride are accompanied by reports in which sodium chloride accelerated the breakdown of covalent bonds. The mechanisms by which salts affect protein stability are protein specific and can vary greatly depending on the pH of the solution. An example where an excipient may be useful to facilitate the delivery of a protein drug is that of some highly concentrated antibody formulations. In the last few years, salts have been shown to be effective in reducing the viscosity of such formulations (Liu et al. (2005, 2006), J. Pharm Sci., 94(9): 1928-40, correction to J Pharm Sci., 95 (1): 234-5).

Preservatives

Preservatives are needed to develop multi-dose parenteral formulations that allow for more than one withdrawal from the same container. Their main function is to inhibit the growth of microorganisms and ensure the sterility of the product throughout the shelf life or period of use of the medicinal product. Commonly used preservatives include benzyl alcohol, phenol and m-cresol. Although preservatives have a long history of use, developing protein formulations that contain preservatives can be challenging. Preservatives almost always have a destabilizing effect on proteins (aggregation), and this has become a major factor limiting their use in multidose protein formulations (Roy et al. (2005), J. Pharm. Sci., 94(2): 382-96) . Benzyl alcohol has also been shown to affect the structure and stability of proteins depending on concentration, temperature and time. Because of these destabilizing effects, many lyophilized protein formulations are reconstituted with a diluent containing benzyl alcohol to minimize contact time with the protein prior to administration.

Most protein drugs are formulated for single use only. However, if multi-dose formulations are possible, they have the added benefit of providing convenience to patients and increasing marketability. A good example is human growth hormone (hGH), in which the development of formulations from up to

- 20 043419 the addition of preservatives led to the appearance on the market of more convenient forms of release in the form of reusable injection pens. At least four such pen devices are currently available, containing hGH-based formulations with added preservatives. Norditropin® (liquid), Nutropin AQ® (liquid) and Genotropin (lyophilized - dual chamber cartridge) contain phenol, while Somatrope® is formulated with m-cresol.

Several aspects must be considered when developing formulations for dosage forms containing added preservatives. The effective concentration of the preservative in the medicinal product must be optimized. To do this, it is necessary to test this preservative in dosage form at concentration ranges that provide antimicrobial effectiveness without compromising protein stability. For example, three preservatives were successfully screened during the development of a liquid formulation for interleukin 1 receptor (type I) using differential scanning calorimetry (DSC). Preservatives were ranked based on their effect on stability at concentrations typically used in marketed products (Remmele et al. (1998), Pharm. Res., 15(2): 200-8).

As expected, developing liquid formulations containing preservatives is more challenging than developing lyophilized formulations. Freeze-dried products can be lyophilized without preservative and reconstituted with a preservative-containing diluent at the time of use. This reduces the time the preservative is in contact with the protein, thereby significantly minimizing the associated stability risks. When liquid formulations are used, the effectiveness and stability of the preservative must be maintained throughout the shelf life of the product (usually approximately 18-24 months). It is important to note that the effectiveness of the preservative must be demonstrated in the final formulation containing the active drug and all excipients.

Some preservatives may cause injection site reactions, which is another factor to consider when choosing a preservative. In a clinical trial evaluating preservatives and buffering agents for Norditropin®, a reduction in pain perception was observed with formulations containing phenol and benzyl alcohol compared to a formulation containing m-cresol (Kappelgaard (2004), Horm. Res. 62 Suppl 3:98-103). Interestingly, among the commonly used preservatives, benzyl alcohol has anesthetic properties (Minogue and Sun (2005), Anesth. Analg. 100(3): 683-6).

Demo examples

All publications, patents and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It should be understood that the disclosed invention is not limited to the specific methodology, protocols and materials described, as these may vary. In addition, it should be understood that the terminology used herein is for purposes of describing specific embodiments only and is not intended to limit the scope of the appended claims.

Those skilled in the art will recognize or be able to determine, through routine experimentation alone, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be covered by the following claims.

Example 1.

This experiment demonstrates that by adjusting target fill parameters, an accurate target protein concentration can be obtained for the corresponding reconstituted drug.

Materials.

Buffer containing: mannitol, sucrose, L-histidine, polysorbate 20 at pH 5.

Container: bottle, volume 3 cubic meters. cm, with internal ring groove on the neck, glass type I, untreated, 13 mm diameter rim with 13 mm diameter stopper, 4432/50 V-50.

Romiplostim is a filtered, purified bulk product.

Way.

1. Dilute the drug to obtain the target product composition (0.5 mg/ml) using the required amount of dilution buffer.

2. Filter the prepared solution using a polyvinylidene difluoride (PVDF) filter with a pore diameter of 0.22 μm.

3. Prepare bottles and stoppers: bottles with a volume of 3 cubic meters. cm are washed and depyrogenated.

4. Fill a sufficient number of vials with the appropriate target filling mass: 0.307, 0.322, 0.342, 0.357, 0.373 g.

5. Partially seal the vials with stoppers and place them in a lyophilizer.

6. Run the required lyophilization cycle with proper freezing, vacuum, primary and secondary drying, followed by capping and discharging the lyophilized

- 21 043419 product in sealed bottles.

7. Reconstitute the product with the specified reconstitution volume of 0.32 ml water for injection.

8. Measure the resulting protein concentration in the vials using ultraviolet (UV) absorption. Absorption coefficient is defined as the amount of light of a certain wavelength that is absorbed when passing through an analyte. The unit of optical density depends on the intrinsic absorption coefficient of the molecule, its concentration and the optical path length of the analyte. The aromatic amino acids phenylalanine, tyrosine and tryptophan in protein molecules absorb light in the UV range of 260-290 nm. UV absorption in this range is usually used to determine the presence of protein in solution.

9. Measure the osmolality of the product using freezing point depression.

10. Analyze to determine the effect of the adjusted target fill parameters on both protein and osmolality.

Table 5

Brief description of filling weight and protein concentration values

Options Conditions experiment A 0.528 IN 0.528 WITH 0.528 D 0.528 E 0.528 Input Concentration in bulk composition (mg/ml) parameter Average value filling mass (g) 0.307 0.322 0.342 0.357 0.373 Day off parameter Average value protein concentration after recovery (mg/ml) 0.459 0.484 0.518 0.546 0.565

A strong linear relationship was observed between reduced protein concentration and fill volume, as evidenced by the R ² value of 0.9964 as shown in FIG. 6. This linear relationship can be explained theoretically using protein mass balance as shown below:

c =(c c restored restored ^v composed loss J filled

[equation 1] _ ^compiled _tterya y restored that filled ^g restored

[Equation 2] where C _recovered means the protein concentration after reconstitution, V _recovered means the volume of product after reconstitution, _Compounded means the protein concentration in the bulk formulation, C _loss means the loss of protein due to binding to the filter and containers, and V _fill means fillable volume in each bottle. Since the bulk concentration, protein loss due to binding, and reconstitution volume are constant in a given run, the protein concentration after reconstitution is proportional to the refill volume as given in Equation 2.

In a pilot scale experiment, the variability in protein concentration of the final reconstituted drug product (DP) was determined using 10 replicates for each loading condition, as shown in Table 1. 6. Observed variability therefore represents both process variability and analytical variability, including variability associated with product recovery.

- 22 043419

Table 6

Variability of (reduced) protein concentration under each condition. filling

A (-10%) IN (-5%) C (Target value) D (+5%) E (+10%) Number of repetitions 10 10 10 10 10 Average value (mg/ml) 0.459 0.484 0.518 0.546 0.565 Standard deviation (SD, mg/ml) 0.0084 0.0133 0.0082 0.0052 0.0069 Relative SD (%) 1.83 2.76 1.58 0.94 1.23

Effect of Fill Mass on Osmolality (Reconstituted DP) The final drug product after reconstitution was tested for osmolality in the filled vials at five target fill mass parameters. The results of osmolality determination before filling and after restoration are summarized in Table. 7. According to the results, at a target filling mass of 0.341 g, the osmolality did not change after filling and reconstitution, indicating that loss of excipients did not occur in appreciable amounts. Osmolality increases slightly at higher fill volumes and decreases slightly at lower fill volumes if the recovery volume is held constant.

Table 7

Brief description of fill mass and osmolality values

Options A IN WITH D E Input parameter Osmolality in bulk composition (mOsm/kg)* 312.4 312.4 312.4 312.4 312.4 Average value filling mass (g) 0.307 0.322 0.342 0.357 0.373 Average fill volume (ml) 0.301 0.316 0.336 0.350 0.366 Normalized volume (%) 89.9% 94.3% 100.2% 104.6% 109.3%

- 23 043419

Output parameter Effective volume after recovery (ml) 0.335 0.335 0.335 0.335 0.335 Average value osmolality after recovery (mOsm/kg) 280 294 313 327 342 Normalized Osmolality (%) 89.6% 94.0% 100.0% 104.6% 109.3%

A linear relationship was found when product osmolality (normalized to target osmolality of 313 mOsm/kg at target fill weight) was plotted against fill weight/volume (normalized to target 0.341 g), as shown in FIG. 7. This linear relationship can be explained by determining osmolality and its dependence on the concentration of buffer components.

Osmolality is a measure of the concentration of a solute, defined as the number of osmoles (Osm) of solute per kilogram of solvent (osmol/kg or Osm/kg). Unlike molarity (mol/L), osmolality measures the number of moles of solute particles (such as a dissociated ion) rather than the number of moles of solute. The osmolality of a solution can be calculated using the following expression:

Osmolality (Osm/kg) = Density

[Equation 3];

where φ denotes the osmotic coefficient, n denotes the number of species (such as ions) into which the molecule dissociates, and C denotes the molar concentration (mol/L) of the solute.

In the case where the filling mass (or volume) is 10% higher than the target volume, the concentration of each type of excipient in the composition is increased by 10% after restoration to a constant volume. Since (φ _i and ni are constant values in a known composition, increasing the concentration of each type of excipient by 10% leads to an increase in osmolality by 10%, as shown in equation 3. Similarly, reducing the filled volume by 10% leads to a decrease in the concentration of excipients substances by 10% and, consequently, to a decrease in osmolality by 10%.

Example 2.

Blinatumomab protein at a concentration of 55 μg/ml was formulated with 200 mM L-lysine-HCl, 25 mM citric acid, 15% w/v. trehalose dihydrate, 0.1% w/v. polysorbate 80 at pH 7.0. The acceptable concentration range of the formulated protein was determined at the filtration stage of the bulk product. Bulk product concentrations ranging from 48.0 μg/mL to 65.0 μg/mL were used. Load target values were calculated from the measured protein concentration to obtain a target reconstituted drug concentration of 12.5 μg/mL after reconstitution with 3 mL water.

C ¹ *V = (C -C ΥΎ restored ^g restored ^composed ^g filled where

WITH .

restored restored

1D SLSVOS SODbelKE restored

The target protein content can then be multiplied by the product density and divided by the measured drug concentration (corrected for loss due to binding if necessary) to determine the target fill weight.

The target fill mass is calculated according to the following formula with a corresponding fill mass value range of 0.634-0.858 g.

The q to be filled is MCG. - g, target dose (Za.5-----) * density (1.07-) vial ml mass (g) concentration DS (x where DS, as is usually understood by specialists in the field, refers to the drug substance. B more generally,

- 24 043419 adjusted fill weight = (target fixed dose of therapeutic protein) x (density) concentration of therapeutic protein in bulk formulation

Example 3.

An infliximab-based drug with 20±1.5 mg/ml infliximab was formulated with 10 mM sodium phosphate, 10% w/v. sucrose, 0.01% w/v. polysorbate 80, at pH 7.2 after reconstitution with 10 ml water for injection.

The target fill weight is calculated according to the following formula with an appropriate fill weight range of 4.85-5.63 g.

Calculation of target filling mass:

_gg (target protein content (100 mg}) x (density (1.042 -))

Target filling _ ml ⁷⁷ mass (g) protein concentration in the released drug / ^mg \ substance (—) ml

Example 4.

Trastuzumab at a concentration of 21 mg/ml was formulated with 0.303 mg/ml L-histidine, 0.470 mg/ml L-histidine hydrochloride monohydrate, 19.1 mg/ml α,α-trehalose dihydrate, 0.0840 mg/ml polysorbate 20, at pH 6.1. The target fill weight is calculated according to the formula below, with varying ranges of fill weight values according to product delivery requirements. The target parameters of the filling mass are in the range of 3.1-21.2 g (depending on the corresponding release form). This product comes in multiple formulations with a total drug concentration of 21 mg/ml.

The target fill weight for each batch of drug product in this Example 4 and subsequent examples is calculated using the following information:

Target fill (target protein content [mg]) x (density [g/ml]) mass [g] total drug concentration [mg/ml]

As an example, the target fill mass for a 150 mg formulation [g] (156 mg) x (1.01 g/ml) total drug concentration [21 mg/ml]

As another example, the target fill mass for a 420 mg [g] formulation (440 mg) x (1.01 g/mL) total drug concentration [21 mg/ppm]

As an additional example, the target fill mass for a 60 mg formulation [g] (65 mg) x (1.01 g/mL) total drug concentration [21 mg/mL]

Example 5.

AMG 701 is a bispecific T cell engagement activator (BiTE®) based on the single chain variable domains of anti-BCMA and anti-CD3 (see NCI Drug Dictionary and other literature). AMG 701 with a protein concentration of 1 mg/ml was formulated with 10 mM L-glutamic acid, 9.0% w/v. sucrose, 0.010% w/v. polysorbate 80, at pH 4.2. The target fill weight is calculated according to the formula below, with varying ranges of fill weight values according to product delivery requirements. The AMG 701 has three release forms with a range of target fill mass parameters for the first release form of 0.47-0.57 g, for the second release form 1.60-1.96 g and for the third release form 3,284.01 g.

For the first release form:

(target protein content (0.50 mg)) x (density (1.032 -)) Target filling mass (g) ₌ ^ml protein concentration of drug released (1.0 -) ml ⁷

For the second release form:

(target protein content (1.71 mg)) * (density (1.032 -))

Target Fill Weight (g) ------------------------------------------- -------- ^ml concentration of oil in the released drug substance (1.0 -)

-

Claims (17)

For the third release form: (target protein content (3.5 mg)) x (density (1.032 -)) Target Fill Weight (g) = ------------------------------------------- ------- ^ml protein concentration in the released drug substance (1.0 Example 6. AMG 330 is a bispecific T-cell engagement activator (BiTE®) based on the single chain variable domains of anti-CD33 and anti-CD3 (see NCI Drug Dictionary and other literature). AMG 300 with a protein concentration of 0.5 mg/ml was formulated with 10 mM potassium phosphate, 8.0% w/v. sucrose, 1.0% w/v. sulfobutyl ether beta-cyclodextrin (SBE-CD), 0.010% w/v. polysorbate 80, at pH 6.1. The target fill weight is calculated according to the formula below, with varying ranges of fill weight values according to product delivery requirements. The target fill mass parameters are in the range of 1.2-1.5 g. The target fill mass is calculated as follows: (target protein content (0.64 mg)) x (density (1.033—)) Target filling mass (g) _ ^ml protein concentration in the drug substance (0.5 -) ml ⁷ In general, the methodology can be used when a target amount of product is required in a container to ensure that the product will be at the desired concentration upon reconstitution. This result is achieved by determining the volume of the recovered product; for example, a volume of 1 ml with a desired therapeutic protein concentration of 1 mg/ml gives a total required protein content of 1 mg. Target protein content [mg]=(protein concentration [mg/ml]xvolume after reconstitution [ml]) The target protein content is then used to calculate the target fill weight using the formula below. In-process or measured drug concentrations are typically measured as part of the formulation process to compensate for any process variability. In some cases, adjustments to the target protein content are made to compensate for loss of product due to binding. Reliability of drug concentration measurements is required to ensure accurate fill target values are obtained. _{tt z} (target protein content [mg]) x (density [g/ml]) Target fill mass (g) = ________________________________________________ measured drug concentration [mg/ml] This is a specific example because in a therapeutic product, both the in-process concentration and the concentration of the reconstituted product can range from a few micrograms (µg or microgram) per milliliter (ml) to several milligrams (mg) per milliliter (ml). Osmolality testing is required as discussed above (based on experimental results). Limits on the permissible measurable drug concentration are established in combination with fill mass targets to achieve filling of the container with the required amount of active drug to ensure that the reconstituted product meets specification limits for both product (active drug or protein) concentration and osmolality. . All publications, patents and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It should be understood that the disclosed invention is not limited to the specific methodology, protocols and materials described, as these may vary. In addition, it should be understood that the terminology used herein is for purposes of describing specific embodiments only and is not intended to limit the scope of the appended claims. Those skilled in the art will recognize or be able to determine, through routine experimentation alone, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be covered by the following claims. CLAIM 1. A method for producing a lyophilized pharmaceutical composition based on a therapeutic protein, which includes a) obtaining a composition with a bulk amount of therapeutic protein, b) measuring the concentration of therapeutic protein in said bulk formulation, - 26 043419 c) determining the fill mass of said bulk formulation in accordance with the formula presented below, resulting in a target fixed dose of therapeutic protein per container: (target fixed dose of therapeutic protein [mg]) x (density [g/ml]) filling mass [g]=------------------------- ----------------------------------concentration of therapeutic protein in bulk [mg/ml] and d) lyophilizing the composition in the container. 2. The method of claim 1, further comprising reconstituting the lyophilized formulation, wherein the concentration of the therapeutic protein in the reconstituted formulation is approximately 20 mg/ml or less. 3. The method of claim 1, wherein the therapeutic protein is selected from romiplostim, blinatumomab, infliximab, trastuzumab, AMG 701 and AMG 330. 4. The method of claim 1, wherein the therapeutic protein is romiplostim and the concentration of romiplostim protein is approximately 0.5 mg/ml. 5. The method of claim 1, wherein the reconstituted formulation contains approximately 0.5 mg/ml romiplostim, approximately 10 mM histidine, approximately 4% w/v. mannitol, approximately 2% w/v. sucrose, approximately 0.004% polysorbate 20 and has a pH of approximately 5.0. 6. The method of claim 2, wherein the therapeutic protein is blinatumomab and the concentration of blinatumomab protein is approximately 55 μg/ml. 7. The method of claim 6, wherein the composition contains approximately 55 μg/ml blinatumomab, approximately 25 mM citric acid monohydrate, approximately 15% w/v. trehalose, approximately 200 mM L-lysine hydrochloride, approximately 0.1% w/v. polysorbate 80 and has a pH of approximately 7.0. 8. The method of claim 2, wherein the therapeutic protein is infliximab and the concentration of infliximab protein is approximately 20 ± 1.5 mg/ml. 9. The method of claim 8, wherein the reconstituted formulation contains approximately 20 mg/ml infliximab, approximately 10 mM sodium phosphate, approximately 10% w/v. sucrose and approximately 0.01% w/v. polysorbate 80 and has a pH of approximately 7.2. 10. The method of claim 2, wherein the therapeutic protein is trastuzumab and the concentration of trastuzumab protein is approximately 21 mg/ml. 11. The method of claim 10, wherein the reconstituted formulation contains approximately 21 mg/ml trastuzumab, approximately 0.303 mg/ml L-histidine, approximately 0.470 mg/ml L-histidine hydrochloride monohydrate, approximately 19.1 mg/ml α dihydrate, α-trehalose and approximately 0.0840 mg/ml polysorbate 20 and has a pH of approximately 6.1. 12. The method of claim 2, wherein the therapeutic protein is AMG 701 and the concentration of AMG 701 protein is approximately 1 mg/ml. 13. The method of claim 12, wherein the reconstituted formulation contains approximately 1 mg/ml AMG 701, approximately 10 mM L-glutamic acid, approximately 9.0% w/v. sucrose and approximately 0.010% w/v. polysorbate 80 and has a pH of approximately 4.2. 14. The method of claim 2, wherein the therapeutic protein is AMG 330 and the concentration of AMG 330 protein is approximately 0.5 mg/ml. 15. The method of claim 14, wherein the reconstituted composition contains approximately 0.5 mg/ml AMG 330, approximately 10 mM potassium phosphate, approximately 8.0% w/v. sucrose, approximately 1.0% w/v. sulfobutyl ether beta-cyclodextrin (SBE-CD) and approximately 0.010% w/v. polysorbate 80 and has a pH of approximately 6.1. 16. The method of claim 1, further comprising reconstituting the lyophilized formulation, wherein the protein concentration in the reconstituted formulation is about 25 mg/ml or less. 17. The method according to claim 1, in which the therapeutic protein is a bispecific construct based on a single chain antibody. -