Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

• Lyophilization or freeze-drying, is a process widely used in the pharmaceutical industry for the preservation of biological and pharmaceutical materials.
• water present in a material is converted to ice during a freezing step and then removed from the material by direct sublimation under low-pressure conditions during a primary drying step.
• a primary drying step During freezing, however, not all of the water is transformed to ice.
• Some portion of the water is trapped in a matrix of solids containing, for example, formulation components and/or the active ingredient. The excess bound water within the matrix can be reduced to a desired level of residual moisture during a secondary drying step. All lyophilization steps, freezing, primary drying and secondary drying, are determinative of the final product properties.
• the present invention encompasses the discovery that freeze-drying may be carried out above the collapse temperature while still retaining product stability, biological activity and other important product attributes.
• the present invention provides, among other things, improved lyophilization methods with significantly shortened primary drying step.
• the present invention provides methods of lyophilizing a liquid formulation including a primary drying step executed at a product temperature at or above the collapse temperature.
• inventive methods include a primary drying step executed without avoiding collapse (e.g., micro-collapse, visually detectable, or macro- collapse) in the lyophilized products.
• the liquid formulation contains a pharmaceutical substance (e.g., protein) at a concentration of at least about 1 mg/ml (e.g., at least about 10 mg/ml, at least about 50 mg/ml, at least about 100 mg/ml, at least about 150 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml, at least about 300 mg/ml, or at least about 400 mg/ml).
• a pharmaceutical substance e.g., protein
• concentration of at least about 1 mg/ml e.g., at least about 10 mg/ml, at least about 50 mg/ml, at least about 100 mg/ml, at least about 150 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml, at least about 300 mg/ml, or at least about 400 mg/ml.
• the liquid formulation is a sucrose-based formulation. [0007] In some embodiments, the liquid formulation is formulated such that the collapse temperature is at least 1 0 C higher than the middle point of glass transition temperature (Tg'). In some embodiments, the liquid formulation is formulated such that the collapse temperature is at least 2 0 C higher than the middle point of glass transition temperature (Tg'). In some embodiments, the liquid formulation is formulated such that the collapse temperature is at least 5 0 C higher than the middle point of glass transition temperature (Tg'). In some embodiments, the liquid formulation is formulated such that the collapse temperature is at least 10 0 C higher than the middle point of glass transition temperature (Tg').
• the primary drying is executed at the collapse temperature or a temperature above collapse but below the eutectic melting temperature (e.g., at least 1 0 C, 2 0 C, 3 0 C, 4 0 C, 5 0 C, 6 0 C, 7 0 C, 8 0 C, 9 0 C, 10 0 C above collapse).
• the eutectic melting temperature e.g., at least 1 0 C, 2 0 C, 3 0 C, 4 0 C, 5 0 C, 6 0 C, 7 0 C, 8 0 C, 9 0 C, 10 0 C above collapse.
• the present invention provides methods of lyophilizing a liquid formulation including a primary drying step at a product temperature at or above the collapse temperature, wherein the liquid formulation includes a pharmaceutical substance (e.g., protein) and a stabilizing agent.
• the ratio of the mass amount of the stabilizing agent and the pharmaceutical substance is no greater than 1000 (e.g., no greater than 500, no greater than 100, no greater than 50, no greater than 10, no greater than 1, no greater than 0.5, no greater than 0.1).
• the pharmaceutical substance is at a concentration of at least about 1 mg/ml (e.g., at least about 10 mg/ml, at least about 50 mg/ml, at least about 100 mg/ml, at least about 150 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml, at least about 300 mg/ml, or at least about 400 mg/ml).
• at least about 1 mg/ml e.g., at least about 10 mg/ml, at least about 50 mg/ml, at least about 100 mg/ml, at least about 150 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml, at least about 300 mg/ml, or at least about 400 mg/ml.
• the stabilizing agent is selected from the group consisting of sucrose, mannose, sorbitol, raffinose, trehalose, glycine, mannitol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran and polyvinylpyrolidone and combinations thereof.
• the present invention provides methods of storing a pharmaceutical substance (e.g., protein) including steps of: (a) lyophilizing the pharmaceutical substance in a liquid formulation comprising a primary drying step executed at a product temperature at or above the collapse temperature; (b) storing the lyophilized pharmaceutical substance for a period longer than 3 months (e.g., longer than 8 months, longer than 12 months, longer than 18 months, longer than 24 months).
• a pharmaceutical substance e.g., protein
• the pharmaceutical substance is at a concentration of at least about 1 mg/ml (e.g., at least about 10 mg/ml, at least about 50 mg/ml, at least about 100 mg/ml, at least about 150 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml, at least about 300 mg/ml, or at least about 400 mg/ml).
• at least about 1 mg/ml e.g., at least about 10 mg/ml, at least about 50 mg/ml, at least about 100 mg/ml, at least about 150 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml, at least about 300 mg/ml, or at least about 400 mg/ml.
• the liquid formulation further contains a stabilizing agent.
• the stabilizing agent is selected from the group consisting of sucrose, mannose, sorbitol, raff ⁇ nose, trehalose, glycine, mannitol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran and polyvinylpyrolidone and combinations thereof.
• the lyophilized product in accordance with the present invention may contain amorphous materials (e.g., fully amorphous materials). In some embodiments, the lyophilized product in accordance with the present invention may contain partly crystalline/partly amorphous materials.
• the present invention provides methods of improving the stability of a lyophilized pharmaceutical substance (e.g., protein) or the efficiency of the lyophilization cycle by lyophilizing the pharmaceutical substance (e.g., protein) in a liquid formulation at a product temperature at or above the collapse temperature.
• a lyophilized pharmaceutical substance e.g., protein
• the efficiency of the lyophilization cycle by lyophilizing the pharmaceutical substance (e.g., protein) in a liquid formulation at a product temperature at or above the collapse temperature.
• the present invention provides methods of evaluating a batch of lyophilized product including steps of (a) evaluating one or more samples from the batch of the lyophilized product, wherein at least one sample is characterized with cake collapse (e.g., micro-collapse, visually-detectable or macro-collapse); and (b) releasing the batch of the lyophilized product based on the evaluation result from step (a).
• cake collapse e.g., micro-collapse, visually-detectable or macro-collapse
• step (a) includes a step of measuring the residual moisture of the one or more samples.
• step (a) includes a step of determining the stability profile of the one or more samples.
• the step of determining the stability profile includes determining a degradation rate.
• the degradation rate is determined by a method selected from the group consisting of SE-HPLC, RP-HPLC, CEX-HPLC, MALS, fluorescence, ultraviolet absorption, nephelometry, CE, and combinations thereof.
• step (a) includes a step of determining an activity of the lyophilized product.
• the activity can be determined by various activity assays (e.g., cell based, ELISA, enzymatic assays).
• the lyophilized product contains a polysaccharide and step (a) includes a step of measuring the conjugation efficiency of the polysaccharide to a carrier protein.
• step (a) includes a step of determining the reconstitution time.
• step (a) does not include a step of evaluating the cake appearance of the one or more samples.
• the present invention provides methods of preparing a pharmaceutical substance (e.g., protein) including steps of: (a) providing a lyophilized pharmaceutical substance (e.g., protein) characterized with cake collapse (e.g., micro- collapse, visually-detectable or macro-collpase); (b) reconstituting the lyophilized pharmaceutical substance, wherein the reconstituted pharmaceutical substance is biologically or pharmaceutically active.
• a lyophilized pharmaceutical substance e.g., protein
• cake collapse e.g., micro- collapse, visually-detectable or macro-collpase
• Inventive methods in accordance with the present invention can be utilized to lyophilize, store, evaluate, and/or prepare pharmaceutical substances, including but not limited to, proteins, nucleic acids (e.g., RNAs, DNAs, or RNA/DNA hybrids, aptamers), chemical compounds, polysaccharides, small molecules, drug substances, natural products, immunogens, vaccines, carbohydrates, and combinations thereof.
• nucleic acids e.g., RNAs, DNAs, or RNA/DNA hybrids, aptamers
• chemical compounds e.g., polysaccharides, small molecules, drug substances, natural products, immunogens, vaccines, carbohydrates, and combinations thereof.
• protein refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds) or combinations of polypeptides.
• Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified.
• a "protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), synthetic polypeptides, or can be a characteristic portion thereof.
• a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
• Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art.
• proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
• Exemplary proteins include, but are not limited to, antibodies (e.g., monoclonal antibodies) or fragments thereof, growth factors, clotting factors, cytokines, fusion proteins, polysaccharide-containing antigens, pharmaceutical drug substances, vaccines (e.g., killed-virus vaccines, attenuated-virus vaccines, toxoid vaccines, subunit vaccines, multi-valent vaccines, conjugate vaccines, live-virus vaccines, and individual components thereof, etc.), enzymes, Small Modular ImmunoPharmaceuticals (SMIPTM).
• SIPTM Small Modular ImmunoPharmaceuticals
• antibodies or antibody fragments include, but are not limited to, intact IgG, F(ab')2, F(ab)2, Fab', Fab, ScFv, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), diabodies, triabodies, tetrabodies.
• the present invention further provides proteins, nucleic acids (e.g., RNAs,
• DNAs, or RNA/DNA hybrids, aptamers chemical compounds, small molecules, drug substances, natural products, polysaccharides, small molecules, drug substances, natural products immunogens, vaccines, carbohydrates, and/or other products lyophilized, stored, and/or prepared using inventive methods in accordance with the present invention.
• any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.
• normal fluctuations of a value of interest may include a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
• Figure 1 illustrates an exemplary freeze-drying cycle graph of 50 mg/ml molecule G at temperature above the collapse temperature: aggressive cycle.
• the formulation also contains 5% sucrose, 10 mM Histidine, 10 mM methionine and 0.01% polysorbate-80.
• Figure 2 illustrates an exemplary cake appearance of molecule G after freeze- drying.
• Left vial represents drying above collapse temperature ( Figure 1), whereas the right vial was freeze-dried well below the Tg'.
• Figure 3 illustrates an exemplary control cycle of molecule G.
• Figure 4 illustrates an exemplary freeze-drying cycle graph of molecule G above collapse temperature but below the eutectic temperature: super aggressive cycle.
• Figure 5 illustrates an exemplary cake appearance of molecule G lyophilized using super aggressive cycle from Figure 3.
• Right vial represent sample from the super aggressive cycle, left vial is the sample form the control cycle.
• Figure 6 illustrates exemplary freeze-drying microscopy images of 10 mg/ml protein J in TMS buffer (10 mM Tris, 4% Mannitol, 1% Sucrose).
• Figure 7 illustrates an exemplary lyophilization cycle for 10 mg/ml protein J in
• TMS buffer performed below the onset of collapse temperature of -18 0 C: working cycle 1.
• Figure 8 illustrates exemplary cake appearance of 10 mg/ml protein J in TMS after working cycle 1. Residual moisture is 0.1%.
• Figure 9 illustrates protein J at 10 mg/ml in TMS: freeze-drying well above collapse temperature but below the melting point of mannitol
• Figure 10 illustrates exemplary cake appearance of 10 mg/ml protein J in TMS lyophilized well above the collapse temperature of amorphous phase. Residual moisture is 0.14%.
• Figure 11 illustrates exemplary formation of the void areas during the isothermal hold at -34 0 C (Top picture) indicating the beginning of collapse at the sublimation front. Total collapse of structure upon lyophilization happened during the isothermal hold at -33° for 30 min (bottom picture).
• Figure 12 illustrates exemplary freeze-drying of Serotype X formulation in 50- ml Schott tubing vials.
• the cake height was approximately 0.5 cm.
• the lyophilization cycle was performed on Benchmark 1000 lyophilizer (SP Industries).
• Figure 13 illustrates exemplary cake appearance of Serotype X lyophilized below the collapse temperature.
• Figure 14 illustrates an exemplary freeze-drying cycle used to lyophilize
• Serotype X polysaccharides which maintained product temperature slightly above the collapse temperature (collapse study 1).
• Figure 15 illustrates exemplary cake appearance of Serotype X polysaccharides freeze-dried slightly above collapse temperature (collapse study 1 , left vial) and well above the collapse temperature (collapse study 2, right vial).
• Figure 16 illustrates an exemplary freeze-drying cycle used to lyophilize
• Serotype X polysaccharides above the collapse temperature (collapse study 2).
• Figure 17 illustrates an exemplary freeze-drying cycle used to lyophilize chicken vaccine below the collapse temperature.
• Figure 18 illustrates exemplary cake appearance of lyophilized chicken vaccine.
• Left vial contains a cake freeze-dried below the collapse temperature.
• Right vial contains a cake freeze-dried above the collapse temperature (the loss of structure could be seen on the bottom of the cake).
• Figure 19 illustrates an exemplary freeze-drying cycle used to lyophilize chicken vaccine above the collapse temperature.
• Figure 20 illustrates exemplary stability analysis results of chicken vaccine during storage at 37 0 C. Filled squares represent materials freeze-dried above the collapse temperature, open triangles represent materials freeze-dried below the collapse temperature. DETAILED DESCRIPTION OF THE INVENTION
• the present invention provides highly efficient and cost-effective lyophilization methods.
• the present invention provides methods of lyophilizing liquid formulations including a primary drying step at a product temperature at or above the collapse temperature.
• inventive methods in accordance with the present invention are particularly useful for freeze-drying liquid formulations containing high concentrations of proteins or other pharmaceutical substances.
• inventive methods in accordance with the present invention improve the stability of lyophilized products.
• Lyophilization also known as freeze-drying
• freeze-drying is often used to store pharmaceutical drug products because chemical and physical degradation rates of the drug products may be significantly reduced in the dried state, allowing for longer product shelf life.
• lyophilization typically adds significantly to the cost of drug manufacturing. This cost can be minimized by developing a cycle that consumes the least amount of time without jeopardizing product quality or stability. For example, increasing product temperature by 1 0 C degree during lyophilization could result in 13% decrease of primary drying time. See, Pikal et al. "The collapse temperature in freeze-drying: dependence of measurement methodology and rate of water removal from the glassy phase," International Journal of Pharmaceutics, 62 (1990), 165-186.
• Lyophilization includes several steps such as freezing, primary drying, and secondary drying. See, Tang X., et al. (2004) "Design of freeze-drying processes for pharmaceuticals: Practical advice," Pharm. Res., 21 :191-200; Nail S. L., et al. "Fundamentals of freeze-drying," In: Development and manufacture of protein pharmaceuticals. Nail S. L., ed. New York: Kluwer Academic/Plenum Publishers, pp 281- 353; Wang, et al. "Lyophilization and development of solid protein pharmaceuticals," Int. J. Pharm., 203:1-60; Williams N. A., et al. "The lyophilization of pharmaceuticals; A literature review.” J.
• the primary drying step which involves sublimation of frozen or unbound water, is the most time-consuming step of the lyophilization cycle. Traditionally, it was considered critical to maintain the product temperature below its collapse temperature during the primary drying in order to keep intact microscopic structure of solid materials present in the frozen solution. It was thought that it is this structure that makes up the freeze-dried cake with a relatively high surface area, allowing low residual moisture and rapid reconstitution after freeze-drying.
• lyophilization in particular, primary drying, may be executed at a product temperature above the collapse temperature while maintaining protein stability and other desirable quality attributes (e.g., residual moisture, reconstitution time, etc.). Even samples with apparent collapse (e.g., visually detectable collapse in vials), which would be normally rejected, exhibited a similar stability profile to the samples lyophilized below the collapse temperature. Moreover, in some cases, the stability of lyophilized products was improved by freeze-drying above the collapse temperature.
• the present invention provides significant economic advantages by providing aggressive and/or fast lyophilization cycles with shorter primary drying time without jeopardizing protein quality and stability. In some cases, the present invention provides improved product stability.
• Another advantage of this invention is an application to the assessment of deviations during the commercial manufacturing. If deviation of process parameters during existing commercial cycle (normally performed below the collapse temperature) results in visually detectable product collapse, the present inventors contemplate that the stability profile of the collapsed product may be comparable to the normal cycle if the residual moisture is within specification. Therefore, this particular batch containing samples with visually detectable cake collapse could be released. Thus, manufacturing of commercial batches with zero or substantially reduced reject rates is possible if the particular product could withstand the collapse. A development robustness study can be performed prior to commercial manufacturing to confirm if the stability of the collapsed materials is comparable to that of the control materials for each particular product.
• Tc cold temperature
• collapse refers to loss of an intact structure or change of the original structure of lyophilized cake.
• collapse includes loss of a microscopic structure (also referred to as micro-collapse).
• micro-collapse is visually undetectable.
• micro-collapse refers to loss of less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01%) of the original intact structure (e.g., a lyophilized cake structure).
• the temperature at or above which the micro-collapse occurs is referred to as the micro-collapse temperature.
• collapse includes loss of gross structures (also referred to as gross collapse or macro-collapse).
• the temperature at or above which the gross collapse occurs is referred to as the gross collapse temperature (or macro-collapse temperature).
• gross collapse or macro-collapse results in visually detectable collapse in the lyophilized product.
• gross collapse “macro-collapse,” and “visually detectable collapse” are used interchangeably.
• gross collapse, macro-collapse or visually detectable collapse refers to loss of at least 0.1% (e.g., at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the original intact structure (e.g., a lyophilized cake structure).
• the temperature at which collapse occurs may not be discrete. Instead, collapse may be a gradual process that takes place over a temperature range with the intact cake structure progressively disappearing over the temperature range.
• the initial change or loss of the intact structure during the lyophilization process is considered the onset of the collapse.
• the temperature at which this initial change was observed is typically referred to as the onset collapse temperature.
• the temperature at which the loss of the structure or the structure change appeared to be complete throughout the cake is referred to as the collapse complete temperature.
• Collapse in the product during lyophilization may be detected by various instruments including, but not limited to, product temperature measurement devices, freeze- drying microscopy or instruments detecting electrical resistance.
• Collapse in lyophilized product e.g., cake
• DSC Differential Scanning Calorimetry
• Collapse phenomenon is sensitive to the nature of the materials involved.
• sucrose dominated formulations are very sensitive to collapse especially if they also contain small molecular species such as salts and buffers (Shalaev et al. "Thermophysical properties of pharmaceutically compatible buffers at sub-zero temperatures: implications for freeze-drying," Pharmaceutical Research (2002), 19(2):195-201).
• collapse usually occurs at temperature close to the mid-point of glass transition.
• the viscosity of amorphous sucrose-salt-buffer systems is very low resulting in massive collapse of structure when product temperature exceeds this critical temperature during primary drying.
• lyophilization is carried out under Tg' whenever possible.
• liquid formulations suitable for the present invention may contain a product (e.g., protein) of interest at a concentration of at least about 1 mg/ml, at least about 10 mg/ml, at least about 20 mg/ml, at least about 30 mg/ml, at least about 40 mg/ml, at least about 50 mg/ml, at least about 75 mg/ml, at least about 100 mg/ml, at least about 150 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml, at least about 300 mg/ml, at least about 400 mg/ml.
• a product e.g., protein
• liquid formulations suitable for the present invention may contain a product (e.g., protein) of interest at a concentration in the range of about 1 mg/ml to 400 mg/ml (e.g., about 1 mg/ml to 50 mg/ml, 1 mg/ml to 60 mg/ml, 1 mg/ml to 70 mg/ml, 1 mg/ml to 80 mg/ml, 1 mg/ml to 90 mg/ml, 1 mg/ml to 100 mg/ml, 100 mg/ml to 150 mg/ml, 100 mg/ml to 200 mg/ml, 100 mg/ml to 250 mg/ml, or 100 mg/ml to 300 mg/ml, or 100 mg/ml to 400 mg/ml).
• a product e.g., protein
• concentration in the range of about 1 mg/ml to 400 mg/ml e.g., about 1 mg/ml to 50 mg/ml, 1 mg/ml to 60 mg/ml, 1 mg/ml to 70 mg
• a suitable formulation contains one or more stabilizing agents (e.g., sucrose, mannose, sorbitol, raff ⁇ nose, trehalose, glycine, mannitol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran or polyvinylpyrolidone).
• the ratio of the mass amount of the stabilizing agent and the pharmaceutical substance is no greater than 1000 (e.g., no greater than 500, no greater than 250, no greater than 100, no greater than 50, no greater than 10, no greater than 1, no greater than 0.5, no greater than 0.1).
• suitable liquid formulations further include one or more bulking agents such as sodium chloride, lactose, mannitol, glycine, sucrose, trehalose and hydroxyethyl starch.
• suitable liquid formulations contain buffering agents such as tris, histidine, citrate, acetate, phosphate and succinate.
• liquid formulations suitable for the present invention contain amorphous materials.
• liquid formulations suitable for the present invention contain a substantial amount of amorphous materials (e.g., sucrose-based formulations).
• liquid formulations suitable for the present invention contain partly crystalline/partly amorphous materials.
• the present invention allows freeze-drying temperatures well above Tg'.
• Tg' glass transition temperature
• the present invention allows freeze-drying at temperatures at least I 0 C, 2 0 C, 3 0 C, 4 0 C, 5 0 C, 6 0 C, 7 0 C, 8 0 C, 9 0 C, or 1O 0 C above the mid-point of Tg'.
• Lyophilized product in accordance with the present invention can be assessed based on product quality analysis, reconstitution time, quality of reconstitution, high molecular weight, moisture, glass transition temperature, and biological or biochemical activity.
• product quality analysis includes product degradation rate analysis using methods including, but not limited to, size exclusion HPLC (SE-HPLC), cation exchange- HPLC (CEX-HPLC), X-ray diffraction (XRD), modulated differential scanning calorimetry (mDSC), reversed phase HPLC (RP-HPLC), multi-angle light scattering detector (MALS), fluorescence, ultraviolet absorption, nephelometry, capillary electrophoresis (CE), SDS- PAGE, and combinations thereof.
• SE-HPLC size exclusion HPLC
• CEX-HPLC cation exchange- HPLC
• XRD X-ray diffraction
• mDSC modulated differential scanning calorimetry
• RP-HPLC reversed phase HPLC
• MALS multi-angle light scattering detector
• Inventive methods in accordance with the present invention can be utilized to lyophilize any materials, in particular, pharmaceutical substances.
• pharmaceutical substances refers to any compounds or entities that alter, inhibit, activate, or otherwise affect biological or chemical events in vivo or in vitro.
• pharmaceutical substances may include, but are not limited to, proteins, peptides, nucleic acids (e.g., RNAs, DNAs, or RNA/DNA hybrids, aptamers), chemical compounds, polysaccharides, small molecules, drug substances, natural products, immunogens, vaccines, carbohydrates, and/or other products.
• the present invention is utilized to lyophilize proteins including, but not limited to, antibodies (e.g., monoclonal antibodies) or fragments thereof, growth factors, clotting factors, cytokines, fusion proteins, polysaccharide antigens, pharmaceutical drug substances, vaccines, enzymes, Small Modular ImmunoPharmaceuticalsTM (SMIPTM).
• antibodies e.g., monoclonal antibodies
• cytokines e.g., cytokines
• fusion proteins e.g., polysaccharide antigens
• pharmaceutical drug substances e.g., vaccines, enzymes, Small Modular ImmunoPharmaceuticalsTM (SMIPTM).
• SMIPTM Small Modular ImmunoPharmaceuticalsTM
• the present invention is utilized to lyophilize antibodies or antibody fragments including, but not limited to, intact IgG, F(ab')2, F(ab)2, Fab', Fab, ScFv, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), diabodies, triabodies, tetrabodies.
• antibodies or antibody fragments including, but not limited to, intact IgG, F(ab')2, F(ab)2, Fab', Fab, ScFv, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), diabodies, triabodies, tetrabodies.
• the present invention is used to lyophilize vaccines or vaccine components.
• Suitable vaccines include, but are not limited to, killed-virus vaccines, attenuated- virus vaccines, toxoid vaccines, subunit vaccines, multi-valent vaccines, conjugate vaccines, live-virus vaccines.
• Suitable vaccine components include, but are not limited to, polysaccharides and carrier proteins. "Polysaccharides,” as used herein, include, without limitation, saccharides comprising a plurality of repeating units, including, but not limited to polysaccharides having 50 or more repeat units, and oligosaccharides having 50 or less repeating units.
• polysaccharides typically have from about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 repeating units to about 2,000 or more repeating units, and preferably from about 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 repeating units to about, 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 repeating units.
• Oligosaccharides typically have about from about 6, 7, 8, 9, or 10 repeating units to about 15, 20, 25, 30, or 35 to about 40 or 45 repeating units.
• Suitable carrier proteins typically include bacterial toxins that are immunologically effective carriers that have been rendered safe by chemical or genetic means for administration to a subject.
• Examples include inactivated bacterial toxins such as diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa.
• Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), or pneumococcal surface proteins BVH-3 and BVH-11 can also be used.
• OMPC outer membrane complex c
• PspA pneumococcal surface protein A
• PsaA pneumococcal adhesion protein
• pneumococcal surface proteins BVH-3 and BVH-11 can also be used.
• carrier proteins such as protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD) can also be used.
• PA protective antigen
• EF Bacillus anthracis
• LF lethal factor
• ovalbumin ovalbumin
• KLH keyhole limpet hemocyanin
• human serum albumin human serum albumin
• BSA bovine serum albumin
• PPD purified protein derivative of tuberculin
• the quality of lyophilized vaccine components can be assessed and determined by their ability to form a conjugate vaccine.
• the quality of lyophilized polysaccharides can be determined by their ability to couple or conjugate to a carrier protein.
• the quality of lyophilized carrier proteins can be determined by their ability to couple or conjugate to a polysaccharide.
• Various methods are known in the art to conjugate a polysaccharide to a carrier protein and the conjugation efficiency can be determined by various analytical methods including, but not limited to, percentage free protein, percentage free polysaccharide, molecular size distribution, saccharide-to-protein ratio ("SPR") and yield rate. Exemplary methods for determining conjugation efficiency are described in the Examples.
• Additional pharmaceutical substances may include, but are not limited to, anti-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-octyl, anti-o-octyl-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-
• AIDS substances anti-cancer substances, antibiotics, immunosuppressants, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti- adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, antihypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, anticoagulants and/or antithrombotic agents, local anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti
• Lyophilization may be performed in a container, such as a tube, a bag, a bottle, a tray, a vial (e.g., a glass vial), syringe or any other suitable containers.
• the containers may be disposable. Controlled freeze and/or thaw may also be performed in a large scale or small scale.
• Inventive methods in accordance with the present invention can be carried out using various lyophilizers, such as, commercial-scale lyophilizers, pilot-scale lyophilizers, or laboratory-scale lyophilizers.
• Lyophilization methods in accordance with the present invention can be applied to any molecules (e.g., proteins) in general.
• the molecules A-J used in the following examples can be any proteins, antibodies, nucleic acids, chemical compounds, vaccines, enzymes, polysaccharides, natural products, small molecules, or any other types of molecules.
• the molecules A-J used in the following examples can be any proteins, antibodies, nucleic acids, chemical compounds, vaccines, enzymes, polysaccharides, natural products, small molecules, or any other types of molecules.
• Example 1 Freeze-drying at or above collapse temperature for completely amorphous materials
• molecule G was freeze-dried at the collapse temperature during primary drying step.
• the formulation contained 50 mg/ml molecule G, 5% sucrose, 10 mM Histidine, 10 mM methionine and 0.01% polysorbate 80 (13).
• An exemplary freeze- drying cycle at collapse temperature is shown in Figure 1.
• freeze-drying at collapse temperature is possible, especially at high protein concentrations such as 50 mg/ml or higher, even for formulations that also contain buffers and sucrose, which normally have low glass transition temperatures.
• all materials examined in this example were amorphous, which was confirmed with the powder X-Ray Diffraction.
• RM is the residual moisture of lyophilized samples.
• Example 2 Freeze-drying at or above collapse temperature for crystalline/amorphous materials
• a pharmaceutical protein J was formulated at a concentration of 10 mg/ml in a
• TMS buffer containing 10 mM Tris, 4% mannitol, 1% sucrose, pH 7.4 (TMS).
• TMS TMS buffer containing 10 mM Tris, 4% mannitol, 1% sucrose, pH 7.4 (TMS).
• Tg' for this formulation was -22.6 0 C.
• Samples were lyophilized at a product temperature well below the glass transition temperature as well as well above Tg'.
• Figure 6 shows exemplary images from freeze-drying microscopy of 10 mg/ml protein J in TMS lyophilized below glass transition temperature (-25 0 C) and well above Tg' (i.e., -18 0 C, -12 0 C and -6 0 C).
• Two lyophilization cycles were performed: one cycle including a primary drying step below the collapse temperature of -18 0 C (onset of collapse) and the other including a primary drying step well above collapse but below the melting point of mannitol.
• the first cycle (working cycle 1 , Figure 7), produces a nice cake with a small degree of shrinkage (Figure 8).
• the second cycle ( Figure 9) was preformed under very aggressive conditions resulting in product temperature being above the collapse temperature of -18 0 C for almost the entire primary drying step. However, the cake appearance ( Figure 10) was acceptable.
• the residual moisture of the samples freeze-dried below the collapse temperature is comparable to that of the samples freeze-dried above the collapse.
• the residual moisture of the samples freeze-dried below the collapse temperature was about 0.1% and the residual moisture of the samples freeze-dried above the collapse temperature was about 0.14%.
• the reconstitution time and even cake appearance of the samples freeze-dried above the collapse temperature were similar to those of the samples freeze-dried below the collapse temperature.
• Example 3 Freeze drying multi-valent vaccine polysaccharide at or above the collapse temperature
• Pneumococcal 13-Valent vaccine (Serotype X) was freeze-dried at three different temperatures during primary drying. The first temperature was below the collapse temperature, the second was slightly above the collapse temperature and the third was approximately 1O 0 C above the collapse temperature.
• the lyophilization formulation contained the polysaccharide, sucrose and Diphtheria CRM 197 protein, with a total dry solids content of 6.5%. Freeze-drying was performed in 50-ml Schott tubing vials filled with 5 ml of solution. Residual moisture, glass transition temperature, reconstitution time, and conjugation efficiency of reconstituted material were the quality attributes used to evaluate lyophilized product freeze-dried under conditions shown below. The target values for these attributes are: a residual moisture of ⁇ 5%, a glass transition temperature of >20°C, and a reconstitution time of ⁇ 1 minute.
• the microscopic collapse temperature for this particular formulation was -33 0 C, less than 2 0 C above the glass transition temperature. Both DSC and FDM showed that the onset of melting endotherm was approximately -2.7 0 C.
• the baseline freeze-drying cycle was performed at a product temperature close to -37°C ( Figure 12), well below the collapse temperature. Freezing was performed in ethanol-dry ice bath; vials with frozen material were loaded on pre-chilled (-5O 0 C) shelves of Benchmark 1000 lyophilizer (SP Industries, Gardiner, NY). Primary drying was completed prior to the secondary drying ramp. The residual moisture of lyophilized material was low, 0.08+0.01%. Two glass transition temperatures were detected: a smaller one at 63 0 C and a larger one at 84 0 C. Despite shrinkage, cake appearance was acceptable ( Figure 13). Reconstitution of lyophilized material was rapid(less than 1 minute, including time for the solution to clear after reconstitution). Conjugation efficiency of lyophilized material was within acceptable range (Table 4). However, the lyophilization cycle was very long (approximately 52 hours) considering the cake height was only 0.5 cm.
• Serotype X solution was freeze-dried in the same freeze-dryer in the same container (with the same fill volume), but at a product temperature almost 1O 0 C higher than the collapse temperature (cycle example is shown in Figure 16).
• Primary drying was completed in 8 hours, much less time when compared to 35 hours for the baseline cycle ( Figure 12) and still less than the 10 hours for collapse study 1 ( Figure 14).
• the refrigeration system was shut down at the end of primary drying while leaving the vacuum pump on ( Figure 16). This combination of stresses resulted in a very high residual moisture of 6.12 +0.15% and poor cake appearance as compared to the other lyophilized materials disclosed herein (Figure 15, right vial).
• the quality of the lyophilized polysaccharides was assessed by the ability of the polysaccharides to conjugate with a carrier protein (e.g., conjugation efficiency) using standard methods known in the art.
• lyophilized polysaccharides are reconstituted and conjugated to a carrier protein CRM 197. Conjugation efficiency was determined using the following criteria:
• Saccharide-to-Protein Ratio an indicator of the reproducibility and efficiency of the conjugation reaction, and is obtained by dividing the saccharide content by the protein content;
• % FS percent free saccharide
• % FP-CE percent free protein - Capillary Electrophoresis
• lyophilized productions from this process have comparable quality when compared to material lyophilized with product temperature below the collapse temperature. Further, the overall lyophilization process takes less time when the product temperature during primary drying is at or above the collapse temperature.
• Example 4 Freeze drying live virus vaccine above collapse temperature
• a live-virus young chicken vaccine was lyophilized both above and below the collapse temperature, and the results compared.
• the quality of vaccine was assessed by cake appearance, residual moisture and titer stability after storage at 37 0 C for 3 months.
• the glass transition temperature was -26.8 0 C measured by modulated DSC.
• the collapse temperature of -18 0 C was measured by freeze-drying microscope (Linkam stage).
• An exemplary lyophilization cycle performed below the collapse temperature is shown in Figure 17.
• the cake appearance of lyophilized material was acceptable ( Figure 18, left vial).
• the residual moisture was 0.5 ⁇ 0.05%.
• the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim.
• any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
• the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
• the invention encompasses compositions made according to any of the methods for preparing compositions disclosed herein.

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