US20050069578A1 - Reconstitution medium for protein and peptide formulations - Google Patents

Reconstitution medium for protein and peptide formulations Download PDF

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US20050069578A1
US20050069578A1 US10/911,865 US91186504A US2005069578A1 US 20050069578 A1 US20050069578 A1 US 20050069578A1 US 91186504 A US91186504 A US 91186504A US 2005069578 A1 US2005069578 A1 US 2005069578A1
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protein
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reconstitution
lipids
ethanol
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Sathyamangalam Balasubramanian
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Research Foundation of State University of New York
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    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers

Definitions

  • This invention relates to compositions for reconstitution of freeze-dried formulations and more particularly provides a composition and method for reconstitution of freeze-dried formulations comprising proteins and lipids.
  • Physical instability is related to protein folding at the molecular level, and denaturation, surface adsorption, aggregation, and precipitation are frequent manifestations of physical instability (1-3). Such instabilities complicate the safety of protein products as the presence of aggregates evokes undesired immune response (4).
  • the loss of protein due to surface adsorption and binding to vial and syringes complicates the therapy.
  • compositions and buffer conditions for preparing the reconstitution solution are disclosed.
  • An example is low concentration of ethanol (less than about 60%, preferably 5-10% vol/vol) in various buffer systems.
  • a solution comprising 0.5 to 10 mM CaCl 2 is another example.
  • FIG. 1 displays the interaction of reconstitution medium with protein.
  • FIG. 2 displays the interaction of reconstitution medium with lipid.
  • DSC Differential Scanning Calorimetry
  • DMPC Dimyristoyl Phosphatidylcholine
  • FIG. 3 displays the effect of reconstitution medium on both lipid and protein.
  • the circular dichroism (CD) melting profile of Factor VIII (5 mM CaCl2 and 100 mM NaCl) in the presence and in the absence of lipid, Phosphatidylserine.
  • the CD spectra were acquired with a heating rate of 15° C./hr.
  • FIGS. 4 a - 4 c displays the effect of reconstitution medium (5 mM CaCl2 and NaCl) on lipidic structures.
  • the LAURDAN profiles of phosphatidylserine containing lipidic particles are shown in the absence of calcium (4a), presence of calcium (4b) and presence of calcium and EDTA (4c).
  • the present invention is directed to compositions for media useful in the reconstitution of dried protein formulations, and the method of use of such compositions.
  • the compositions can be used with dry protein preparations, such as freeze-dried preparations containing protein to form structurally stabilized protein solutions.
  • dry protein preparations such as freeze-dried preparations containing protein to form structurally stabilized protein solutions.
  • lipids when lipids are included, the compositions promote the interaction between the protein hydrophobic domains and the lipid, forming solutions of structurally stabilized, non-aggregating, non-adhering protein/lipid solutions.
  • Alcohols are frequently added as excipients in the formulation of small, conventional organic drug molecules to improve stability and solubility.
  • Intravenous preparations containing up to 50% ethyl alcohol are available for human use (5-7).
  • alcohols are not used in parenteral preparations which include proteins.
  • the present invention demonstrates that alcohols such as ethyl alcohol can be used as excipients for protein formulations and processing containing lipids. It has been discovered that concentrations of alcohol which are below a critical concentration, roughly 60 percent by volume in the case of some common alcohols, such as ethanol, for example, have the effect of structurally stabilizing proteins without greatly impeding their function. However, above the critical concentration, the alcohol induces significant changes in secondary and tertiary structure as demonstrated by the circular dichroism data in Examples 1 and 2, respectively. Such structural changes can be expected to affect the protein's function. Thus, in one embodiment, the present invention provides a stabilized protein solution which is buffered to a physiological pH and which comprises an alcohol at a concentration which is less than 60% by volume and one or more proteins.
  • Stabilization can be determined by methods which probe secondary and tertiary structure such as the disruption of circular dichroism spectra. If circular dichroism bands due to secondary structure are undisrupted, and all of the circular dichroism spectral bands due to tertiary structure are present, it can be inferred that little or no secondary and tertiary structure degeneration has taken place.
  • lipid component when included in the formation of solutions which contain alcohol, the alcohol component promotes the interaction between the protein and the lipid.
  • Protein and lipid can associate in numerous ways to form complexes as simple as the combination of one or a few lipid molecules with hydrophobic protein sites or as complex as phopholipid bilayer, cochleate and other higher order structures.
  • alcohols such as, for example, ethanol, promote the exposure of hydrophobic domains, thereby lowering the free energy of the lipid/hydrophobic association.
  • protein/lipid complexes are stabilized because packing defects are induced and stabilized by alcohol molecules (Example 4), thus decreasing the free energy of the protein embedded in lipid complex.
  • the present invention provides a stabilized protein solution which is buffered to a physiological pH and which comprises one or more lipids, an alcohol at a concentration which is less than 60% by volume, and one or more proteins.
  • the association of proteins with a specific lipid can be a result of electrostatic-interaction
  • This electrostatic component makes some protein/lipid associations particularly stable.
  • FVIII and phosphatidyl serine can associate by electrostatic interaction, and their pairing is thus particularly preferred.
  • the complex may be further stabilized by hydrophobic forces promoted by the formulations of the present invention which lead to the formation of lipid molecular assemblies such as micelles, liposomes and cochleates.
  • the reconstitution of the dry protein preparation may be accomplished in several ways. If it is desired, the dry protein preparation can be combined with a lipid component (0.3 mM to about 4 mM) by combining both protein and lipid, either or both in dry or solvated form before the introduction of alcohol.
  • a lipid component 0.3 mM to about 4 mM
  • the present invention provides a method for reconstituting a dry protein or dry protein preparation to produce a stabilized protein solution which comprises addition of lipidic particles to said protein preparation to form a solution comprised of protein and lipid, and combining said solution with a reconstitution medium, said reconstitution medium comprising alcohol and being buffered to a physiological pH, such that less than about 30% of the dissolved protein aggregates.
  • the present invention provides a method for the reconstitution of a dry protein/lipid preparation to produce a stabilized protein solution comprising combining a dry protein preparation in which protein and lipid which are associated to form protein/lipid complexes with a reconstitution medium, said reconstitution medium comprising alcohol and being buffered to a physiological pH, such that less than about 30% of the dissolved protein forms aggregates.
  • the reconstitution medium with and without ethanol comprises one or more lipids and calcium ions.
  • Suitable concentrations of calcium are 0.5 to 10 mM and preferable concentration is about 5 mM.
  • buffer salts play a prominent role in the stabilizing of protein/lipid interactions.
  • calcium-containing buffers play a role in the stabilization of micellar structures, and are preferred in cases in which micellar structures are preferred over 1) other types of complexes or 2) lower order associations.
  • associative structures other than micellar structures are preferred, such as when a solution is to be administered to a human or animal subject and large structures would trigger an immune response, it is desirable to either lower the calcium ion concentration, or use buffers which contain little or no calcium.
  • the anti-aggregant, anti-adherent effects of the compositions of the present invention will be realized with other buffers as known in the art.
  • physiological pH it is meant that the pH should be in the range which is conducive to the functioning of biological systems, such as, for example, in the range of from about 7.0 to about 7.4.
  • a common physiological pH is about 7.2.
  • Degree of aggregation can be determined by a number of methods. Light scattering methods and size exclusion chromatography are examples of methods which can be used. Due to the range of error inherent in the size exclusion chromatography method, the percent aggregation as determined by size exclusion chromatograph refers to the average value in the range of error.
  • the dried protein preparation may contain amounts of lipid, and may even be a preparation in which the lipid component is already associated with the protein as individuals/small groups or as higher order structures such as phospholipid bilayer, lamellar or cochleate structures.
  • the lipid component can also be introduced either as part of a reconstitution solution containing the alcohol component or after the dry preparation has been reconstituted with the alcohol-containing reconstitution medium. If desired, the alcohol may be added to the solution after 1) a reconstituted protein solution has been formed, and 2) the lipid component has bee added.
  • the dried protein preparation is most conveniently combined with the protein as a freeze dried or lyophilized preparation. It should be noted, however, that a stabilized solution can be formed from protein which has not been subjected to a drying process.
  • a stabilized solution can be formed from protein which has not been subjected to a drying process.
  • the benefits of the inventive medium extend not only to reconstituted solutions which have been prepared from dry protein, but also to solutions which have been formed from proteins introduced as emulsions, suspensions, or other non-desiccated forms.
  • the proteins which can be used in the compositions and methods of the present invention need only have hydrophobic domains which are accessible.
  • accessible it is not meant that the hydrophobic domains must always be at the outer surface of the protein, but the protein must have hydrophobic domains such that when the protein is in the reconstitution medium, the domains can be positioned, through changes in protein conformation, such that they can contact lipids which are part of the medium.
  • the proteins which can be used in the methods and compositions of this invention can range greatly in size, from peptides having fewer than fifty amino acids and weighing several kDa, to much larger proteins, such as a lysozyme or FVIII, with molecular weights in excess of 200 kilodaltons.
  • lipidic molecules which can be used in the compositons and methods of the present invention can vary widely.
  • Phospholipids such as phosphatidyl serine and phosphatidyl choline give excellent results.
  • lipids containing groups having other structures, such as multi-ringed structures including cholesterol can be used for protection against aggregation.
  • the lipid groups need only be long enough to interact with hydrophobic domains and stabilize, through these interactions, protein conformations in which the hydrophobic domains are exposed.
  • the present invention provides compositions comprising alcohols.
  • the alcohols When the compositions are to be used to reconstitute formulations intended for administration to humans or animals, the alcohols should be such that they can be safely administered.
  • Such alcohols include ethanol, glycerol and polyethylene glycol (PEG).
  • Other alcohols, such as sugar alcohols, which can be used include sucrose, glucose, mannitol, and trehalose.
  • Some alcohols, such as benzyl alcohol have been shown to have some stabilizing effect on lipid/protein solutions, but their toxicity to humans makes them of questionable value, except possibly at extremely low concentrations.
  • the alcohol concentration is less than about 60% vol/vol, preferably less than about 20% vol/vol, and even more preferably between about 5 and 10% vol/vol, where vol/vol refers to the ratio of volume of alcohol to total solution volume (including alcohol) at standard temperature and pressure.
  • the protein to lipid ratio can be in the range of from about 1:10 to about 1:1 million mol/mol. Preferred is a ratio in the range of from about 1:10 to about 1:100,000 mol/mol. A ratio in the range of from about 1:30 to about 1:10,000 mol/mol is most preferred.
  • Buffers can be used in concentrations in the range of from about 0.5 to 600 mM, with a preferred range of from about 5 to about 600 mM for NaCl and from about 0.5 to about 10 mM for CaCl 2 .
  • the lipid concentration should be in the range of from about 0.3 mM to about 40 mM and preferably in the range 1 mM to 10 mM.
  • an alcohol can have an anti-aggregant, anti-adherent effect in solutions which are quite high in protein concentration, even as high as about 5 mg/ml per liter.
  • a typical example of composition is ethanol (1-60% vol/vol), protein to lipid (1:30 to 1:10,000 mol/mol) and buffer salts containing 5 mM to 600 mM NaCl and/or 0.5 mM to 10 mM CaCl 2 .
  • Lysozyme was used as a model protein to investigate the effect of ethyl alcohol for following reasons: (i) Lysozyme is a bacteriolytic protein is under investigation as a therapeutic agent for AIDS, and (ii) detailed structural information is available to investigate structure-stability relationships of lysozyme.
  • the unfolding of the protein using thermal stress in the presence and in the absence of ethanol is carried out to investigate the thermal stability of the protein in ethanol-buffer mixtures.
  • the lyophilized lysozyme (660 ug/ml) was mixed with (20%) ethanol containing phosphate buffered saline (pH 7.4) and the protein was subjected to thermal stress.
  • the conformation and aggregation of the protein was followed by CD and turbidity measurements.
  • the lysozyme dissolved in phosphate buffered saline in the absence of ethanol was also carried out ( FIG. 1 ).
  • the data shows that the presence of ethanol prevents the formation of aggregates.
  • the melting profile was distorted especially at temperatures around 65° C. as indicated in the figure.
  • the turbidity measurements and visual inspection of the sample indicated the presence of aggregates and precipitates of the protein.
  • the analysis of unfolding profile of the protein indicated that the melting of the protein follows a two state model and the visual inspection of the sample suggested absence of any significant fraction of aggregates or precipitates.
  • This example describes the effect of ethyl alcohol on tertiary structure.
  • the near-UV CD spectrum is sensitive to the specific orientation of the aromatic groups and tertiary structure.
  • lysozyme displayed three positive bands at 280, 287 and 291 nm; these have been assigned to the transitions of Trp residues.
  • ethanol ⁇ 60% vol/vol
  • enhancement in the CD bands was observed.
  • the ratio of the positive peaks at 280 and 287 nm was sensitive to the presence of ethanol.
  • further increase in ethanol concentrations resulted in the loss of the CD bands, indicating a lack of any appreciable tertiary structure (data not shown).
  • Tm melting temperature
  • ⁇ Hcal calorimetric enthalpy
  • the fluorescence intensity of the probe bound to protein in aqueous environment increased by 10% over that of the unfolded state, whereas the presence of 20% ethanol the fluorescence intensity of the probe increased by 30%.
  • the initial fluorescence intensity of the probe was normalized and the temperature dependent effects were calculated as percent change rather than absolute fluorescence intensity.
  • the decrease in Tm and exposure of hydrophobic domains in solvent containing solutions is possibly because as the protein unfolds, hydrophobic residues come into contact with clusters of solvent molecules, thus thermodynamically favoring the exposure of hydrophobic domains. But such exposure of hydrophobic domains in aqueous environment may lead to aggregation of the protein.
  • the reconstitution medium containing low solvent concentrations may prevent the surface adsorption and aggregation of the protein. Further, denaturation during reconstitution due to agitation, shaking, formation of foam and froth and the contact of the protein with air-aqueous interfaces may be minimized by solvent exposure.
  • the combination of solvent and specific ions may also promote the refolding of the protein to the native conformation (data not shown) as the role of specific ions in the refolding process is well documented.
  • This example describes the effect of reconstitution medium (solvent and buffer salts) on lipid structure.
  • reconstitution medium solvent and buffer salts
  • DSC thermal profiles in the presence and in the absence of ethanol was carried out.
  • the main transition temperature was observed at 23° C. and is consistent with the previous studies reported by others.
  • the transition peak was broadened and a shift in the temperature was observed.
  • Such effects are consistent with ethanol inducing packing defects in the bilayer organization, and thus lend support for the conclusion that ethanol induces such packing defects.
  • the effect of ethanol on protein appears to be to expose the hydrophobic domains and it influences the packing defects in bilayer. This effect can promote the interaction of protein (exposed hydrophobic domains) with lipid bilayer (packing defects) increasing the encapsulation efficiency of proteins in liposomes.
  • This example shows fluorescence spectra acquired on an SLM 8000C or a PTI Quanta Master spectrofluorometer for cochleate and other bilayer structures ( FIG. 4 ).
  • Emission spectra were acquired over the range of 425 to 550 nm, using a slit width of 4 m on the excitation and emission paths. Correction for the inner filter effect was performed by appropriate procedures (9). Samples were maintained at the desired temperature using a water bath (Neslab RTE 110, NESLAB Instruments Inc, Newington, N.H.). Spectra were corrected through the use of an internal reference and further processed using software provided by the manufacturer.
  • the effect of Ca2+ PS interaction was monitored by Laurdan fluorescence.
  • the fluorescence emission spectra of Laurdan was sensitive to the formation of lamellar, cochleate phases ( FIG. 4 ). The presence of Ca2+ ions promote the formation of lipid molecular assemblies such as cochleate phases.
  • Hen egg-white lysozyme was purchased from Sigma (St Louis Mo.) as a crystallized dialyzed and lyophilized powder (Batch No: 57M7045). Recombinant FVIII was expressed in COS-7 cells and purified by chromatography.
  • the lipids were purchased from Avanti Polar lipids (Alabaster Ala.). Spectroscopy grade solvents were purchased from Pharmaco Inc (Brookfield, Conn.) and used without further purification.
  • CD spectra were acquired on a JASCO J715 spectropolarimeter calibrated with d10 camphor sulfonic acid. Temperature scans were acquired using a Peltier 300 RTS unit and the melting profiles were generated using software provided by the manufacturer. The spectra were acquired at heating rates of 60° C./hr and 15° C./hr. The data presented in FIG. 1 is acquired using 60° C./hr heating rate and for FIG. 3 , the heating rate was 15° C./hr. For all the samples, a 10 mm cuvette was used to acquire the data. Samples were scanned in the range of 260 to 200 nm for secondary structural analysis, and the protein concentration used was 20 ⁇ g/ml ( FIG. 3 ).
  • the spectra were acquired in the range of 360 to 270 nm, and the protein concentration used was 0.66 mg/ml ( FIG. 1 ).
  • CD spectra of the protein were corrected by subtracting the spectrum of the solvent alone, and multiple scans were acquired and averaged to improve signal quality.
  • the melting profiles were used to determine the stability of the protein.
  • a two-state unfolding model was applied to analyze the equilibrium unfolding data.
  • Y unf and Y nat are obtained by performing a linear regression analysis of the spectrum plateau region at high and low temperatures, respectively.
  • rFVIII clotting activity was determined by one-stage activated partial thromboplastin time (APTT) assay using micronized silica as activator and FVIII deficient plasma as the substrate.
  • the APTT assay was performed using a COAG-A-MATE coagulation analyzer (Organon Teknika Corporation, Durham, N.C.). Briefly, rFVIII was added to FVIII deficient plasma and the clotting time was monitored. The activity of the rFVIII was then obtained from calibration curve constructed using the clotting times determined from various dilutions of a lyophilized reference concentrate of known activity. The concentration of the protein was determined independently using Bicinchoninic acid (BCA) assay and compared with activity.
  • BCA Bicinchoninic acid
  • ANS (1-anilino-8-naphthalene sulfonate) was dissolved at 1 mg/ml containing 2% ethanol, and a small volume was added to a solution of 10 ⁇ M of lysozyme in water, to give a final probe concentration of 0.3 ⁇ M.
  • the initial fluorescence intensity of the probe was normalized to account for the general solvent effects of ethanol on fluorescence measurements. The data indicated that the presence of ethanol results in the exposure of hydrophobic domains of the protein.
  • the activity of lysozyme was determined by measuring the catalytic activity of the protein as described earlier (10,11).
  • the protein was diluted 20 times into an assay mixture containing a prefiltered cell suspension of 0.16 mg/ml of M.lysodeikticus and the change in absorbance at 450 nm was monitored for the bacteriolytic activity of the protein. Control experiments were performed for the relevant ethanol concentrations and the data indicated that the presence of ethanol did not interfere with the activity of the protein.
  • the particle size of the aggregated protein was determined using NICOMP 315 particle sizer and the turbidity was measured using VARIAN spectrometer. The particle size distribution was analyzed using both Gaussian and NICOMP analysis for unimodal and bimodal distribution. The size of the standard latex beads were measured prior to each measurement. For turbidity measurements, the OD at 350 nm was followed as function of temperature.
  • the relative fraction of aggregated protein was determined using size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • HP-SEC High Performance Size Exclusion Chromatography
  • Biosep SEC S4000 4000 (4.6 mm ⁇ 300 mm).
  • the analytical column was maintained at 20° C. using a Shimadzu CT0-10AC column oven.
  • Chromatograph comprised of a Waters 510 HPLC Pump, Rheodyne injector with a 50 ⁇ l PEEK sample loop and Hitachi F1050 fluorescence detector.
  • Elution of protein was monitored using the intrinsic fluorescence of rFVIII. Excitation and emission were set at 285 nm and 335 nm respectively to monitor the elution of the protein.
  • This example describes the formation of lipid molecular assemblies such as micelle formation in the absence of protein after reconstitution using Ca2+ ions in the reconstitution medium which does not contain alcohol.
  • the formation of lipid molecular assemblies such as micelle formation were monitored using diphenyl-1-3-5-hexatriene (DPH) fluoresecnce.
  • DPH fluorescence was measured using a PTI-Quantamaster fluorescence spectrophotometer (Photon Technology International, Lawrenceville, N.J.) at ⁇ excitation of 360 nm and ⁇ emission of 430 nm. Excitation and emission slit widths were set at 2 nm.
  • a variable path length cuvette was used to minimize the inner filter effect.
  • the aqueous buffer was comprised of 25 mM tris, 300 mM NaCl and 5 mM CaCl 2 (Tris buffer). Calcium chloride was excluded from the buffer for experiments conducted with no Ca 2+ ions.
  • Aqueous solutions of dried DCPS (1 ml) containing various concentrations of lipid (1.0, 2.0, 2.5, 3.0, 3.5, and 4.0 mM) were prepared and mixed with 1 ⁇ l of 0.005 M diphenyl-1-3-5-hexatriene (DPH) solution.
  • DPH diphenyl-1-3-5-hexatriene
  • This example describes the effect of inclusion of protein in the composition of Example 17 on the formation of micelles after reconstitution using Ca2+ ions rFVIII (10 ⁇ g/ml) was reconstituted with tris buffer containing appropriate concentrations much below CMC, 50, 100 and 800 ⁇ M of DCPS. SEC profile of rFVIII shows a single broad peak at 7 minutes. Further, rFVIII can interact with DCPS (both molecular and micellar forms) owing to its interaction with phosphoserine head group of DCPS. Interaction of rFVIII with the molecular form of DCPS causes no significant changes in the molecular volume of the protein.

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US20050123592A1 (en) * 2003-12-08 2005-06-09 Gyurik Robert J. Pharmaceutical compositions and methods for insulin treatment
US20050232867A1 (en) * 2001-06-22 2005-10-20 Gyurik Robert J Pharmaceutical compositions and methods for peptide treatment
US20070116729A1 (en) * 2005-11-18 2007-05-24 Palepu Nageswara R Lyophilization process and products obtained thereby
US20120219538A1 (en) * 2009-11-02 2012-08-30 Therapeomic Ag Stabilized protein formulations and use thereof

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US8110218B2 (en) 2000-11-30 2012-02-07 The Research Foundation Of State University Of New York Compositions and methods for less immunogenic protein-lipid complexes
US7625584B2 (en) 2000-11-30 2009-12-01 The Research Foundation Of State University Of New York Method of complexing a protein by the use of a dispersed system and proteins thereof
GB0615312D0 (en) * 2006-08-02 2006-09-13 Univ Dundee Protein Solubilisation
WO2010148321A1 (fr) * 2009-06-18 2010-12-23 Wyeth Llc Méthode de dissolution lente pour la reconstitution d'un matériau lyophilisé
DE102015107389A1 (de) 2015-05-12 2016-11-17 Valeo Schalter Und Sensoren Gmbh Verfahren zum Betreiben eines Fahrerassistenzsystems eines Kraftfahrzeugs mit Objekterkennung im Nahbereich, Fahrerassistenzsystem sowie Kraftfahrzeug

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CA2534565A1 (fr) 2005-02-24
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CA2534565C (fr) 2012-06-19
EP1668358B1 (fr) 2014-12-10
EP1668358A4 (fr) 2006-10-04
EP1668358A1 (fr) 2006-06-14
WO2005017526A1 (fr) 2005-02-24

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