WO2002035241A2

WO2002035241A2 - High throughput screening for protein formulations

Info

Publication number: WO2002035241A2
Application number: PCT/US2001/050835
Authority: WO
Inventors: Alexey L. Margolin
Original assignee: Altus Biologics Inc.
Priority date: 2000-10-23
Filing date: 2001-10-23
Publication date: 2002-05-02
Also published as: AU2002231339A1; WO2002035241A3

Abstract

This invention relates to methods for rapidly optimizing a protein formulation. This invention provides formulations libraries and methods for high throughput screening of a protein formulations library to obtain protein formulations characterized by protein stability.

Description

HIGH THROUGHPUT SCREENING FOR PROTEIN FORMULATIONS

TECHNICAL FIELD OF THE INVENTION This invention relates to methods for rapidly optimizing a protein formulation. This invention provides formulations libraries and methods for high throughput screening of a protein formulations library to obtain protein formulations characterized by protein stability.

BACKGROUND OF THE INVENTION Combinatorial techniques, which were introduced to the pharmaceutical and other industries in the late 1980s, have, inter alia, revolutionized and dramatically accelerated the drug discovery process. See, e.g., 29 Ace. Chem. Res., 1-170 (1996) ; 97 Chem. Rev. 349-509 (1997) ; S. Borman, Chem. Enσ. News 43-62 (Feb. 24, 1997); A.A. Thayer, Chem. Enq. News 57-64 (Feb. 12, 1996); N. Terret, I. Drug Discovery Today 402 (1996) and WO 00/59627, published on October 12, 2000. In addition, combinatorial methods and sophisticated screening technologies are now being applied to the discovery of inorganic compounds, such as high- temperature superconductors, magnetoresistive materials, luminescent materials, and catalytic materials. (Jandeleit et al . , Angew. Chem. Int. Ed., 38, 2494 (1999) ) .

The ascendance of the postgenomics era is characterized by introduction of the thousands upon thousands of new proteins that have been and will be created for the first time through proteomics. To turn these and other proteins into commercial products, the protein must remain stable in a protein formulation, so that the formulation is efficacious for its intended use and characterized by a suitable shelf life. Such protein formulations are useful for a number of purposes, including pharmaceutical and biomedical applications, such as delivery of therapeutic proteins and vaccines. Additional uses of such protein formulations include protein delivery in human food, agricultural products, such as feeds, veterinary compositions, diagnostics, cosmetics and personal care compositions .

Unlike traditional small molecules, proteins possess higher order structures that are required for their biological activity. While instability of small molecules involves only chemical pathways of degradation, instability of proteins includes both chemical degradation (deamidation, oxidation, β-elimination, etc.) and physical denaturation. The latter relates to the loss of the three-dimensional structure and may lead to aggregation and adsorption on surfaces. Accordingly, early in the development of a protein product for any purpose, it is essential to efficiently and inexpensively design a protein formulation that is reasonably stable during shipping, long-term storage and administration. SUMMARY OF THE INVENTION The above-identified problems are solved by applying high throughput screening (HTS) combinatorial techniques to the development of protein formulations. Accordingly, this invention provides methods for high throughput screening of a protein formulations library to obtain protein formulations that preserve the native structure and efficacy of protein.

According to the present invention, high throughput screening of protein formulations to obtain protein formulations characterized by particular characteristics, such as protein stability, can be carried out by a method comprising the following steps: Step (a) : providing a protein formulations library in suitable containers, such as microtiter plates or batch parallel reactors, wherein each formulation within said library comprises a protein and an excipient or a mixture of excipients in a specific protein to excipient ratio (such as volume, moles or mass) and wherein each formulation within said library differs from another formulation either in the specific ratio of said protein to an excipient or a mixture of excipients or the identity of the excipient or a mixture of excipients but does not differ from another formulation with respect to said protein. Step (b) : providing a sample of said protein without any excipient . Step (c) : assessing the ability of the various formulations within the library to prevent said protein from losing its activity as a result of problems such as denaturation, by measuring the denaturation point (melting point) (Tm) of said protein in each formulation and said protein in the sample of said protein without any excipient using an appropriate calorimetry technique, preferably

Differential Scanning Calorimetry (DSC) , followed by comparing Tm for said protein in each formulation (Tmf) with Tm of said protein in the sample of said protein without any excipient (Tms) and selecting those formulations within said library having said protein characterized by Tmf approximately greater than or equal to 0.75 Tms -- preferably with Tmf greater than or equal to 0.90 Tms, more preferably with Tmf approximately equal to 1.0 Tms . Instead of or in addition to Tm, enthalpy of denaturation (ΔH) or heat capacity (C) can be compared between said protein in each formulation with that of the sample of said protein without any excipient . Alternatively, determining the integrity of the tertiary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient, by fluorescence spectroscopy, circular dichroism ("CD")/ preferably near UV CD (250-350 nm) or UV spectroscop . Step (d) : determining the integrity of the secondary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient, by infrared spectroscopy (IR) , preferably by Fourier Transform IR (FTIR) , followed by obtaining the correlation coefficient (λ) between the IR spectrum of said protein in each formulation with the IR spectrum of said protein in the sample of said protein without any excipient and selecting formulations within said library having said protein characterized by λ approximately greater than or equal to 0.75 -

- preferably with λ greater than or equal to 0.90, more preferably with λ approximately equal to 1.0, indicating that the protein maintained its native conformation. This step can also be carried out by circular dichroism instead of IR. Step (e) : establishing the chemical stability of said protein of the various formulations within the library and assessing the chemical stability of said protein in the sample of said protein without any excipient, by reversed phase HPLC (RP HPLC) or reversed phase capillary electrophoresis (RP CE) , followed by obtaining the correlation coefficient (λ) between the chromatogram of said protein in each formulation with the chromatogram of said protein in the sample of said protein without any excipient and selecting formulations within said library having said protein characterized by λ approximately greater than or equal to 0.75 -

- preferably with λ greater than or equal to 0.90, more preferably with λ approximately equal to 1.0, indicating that the protein maintained its native conformation. Step (f) : assessing the ability of said protein to aggregate in the various formulations within the library and said protein in the sample of said protein without any excipient, by size exclusion, such as HPLC, followed by obtaining the correlation coefficient (λ) between the chromatogram of said protein in each formulation with the chromatogram of said protein in the sample of said protein without any excipient and selecting formulations within said library having said protein characterized by λ approximately greater than or equal to 0.75 -- preferably with λ greater than or equal to 0.90, more preferably with λ approximately equal to 1.0, indicating that the protein maintained its native conformation. Instead of, or in addition to, size exclusion techniques, light scattering may be used to assess protein aggregation.

In a preferred embodiment of this invention, a method is provided in which the above-described steps (c) - (f) can be performed by means of a high throughput robotic handling system, which transports said library to one or more screening stations, wherein said high throughput robotic handling system and said screening stations are controlled by a processor programmed with the operating parameter using a software interface .

In a preferred embodiment, the appropriate calorimetric technique in step (c) is DSC. In another preferred embodiment, the IR in step (d) is FTIR.

In another embodiment of this invention, a method is provided in which the above-described steps (c) - (f) are repeated numerous times (which could be once, for a total of two rounds) .

In another embodiment of this invention, a method is provided in which the above-described steps (c) - (f) are performed in any order. In a preferred embodiment, a method is provided in which the above- described method is performed in the order of steps (a) - (f) . In another preferred embodiment, a method is provided in which the above-described method is performed in the order of steps (a) , (b) , (d) , (c) , (e) and (f) . In another preferred embodiment, a method is provided in which the above-described method is performed in the order of steps (a) , (b) , (d) , (c) , (f) and (e) . In another preferred embodiment, a method is provided in which the above-described method is performed in the order of steps (a) , (b) , (c) , (d) , (f) and (e) .

In another embodiment of the present invention, high throughput screening of protein formulations to obtain protein formulations characterized by particular characteristics, such as protein aggregation or protein stability, can be carried out by a method comprising the following steps: Step (i) : providing a protein formulations library in suitable containers, such as microtiter plates or batch parallel reactors, wherein each formulation within said library comprises a protein and an excipient or a mixture of excipients in a specific protein to excipient ratio (such as volume, moles or mass) and wherein each formulation within said library differs from another formulation either in the specific ratio of said protein to an excipient or a mixture of excipients or the identity of the excipient or a mixture of excipients but does not differ from another formulation with respect to said protein. Step (ii) : providing a sample of said protein without any excipient . Step (iii) : assessing the ability of said protein to aggregate in the various formulations within the library and said protein in the sample of said protein without any excipient by subjecting each formulation to dynamic light scattering analysis ("DLS") . Those formulations in which aggregation is observed are abandoned. Step (iv) : determining the integrity of the tertiary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient, by fluorescence spectroscopy, circular dichroism ("CD") , preferably near UV CD (250-350 nm) or UV spectroscopy. Step (v) : determining the integrity of the secondary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient, by vibration spectroscopy (preferably microscopy/imaging) , preferably FTIR, Raman or FT-Raman, or by CD. Step (vi) : determining the integrity of the primary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient, by capillary zone electrophoresis ("CZE"), mass spectrometry or peptide mapping (RP-HPLC or CZE with online digestion) . In a preferred embodiment of this invention, a method is provided in which the above-described steps (iii) - (vi) can be performed by means of a high throughput robotic handling system, which transports said library to one or more screening stations, wherein said high throughput robotic handling system and said screening stations are controlled by a processor programmed with the operating parameter using a software interface.

In another embodiment of this invention, a method is provided in which the above-described steps (iii) - (vi) are performed in any order. In a preferred embodiment, a method is provided in which the above-described method is performed in the order of steps (i) - (vi) . In another preferred embodiment, a method is provided in which the above-described steps (iii) - (vi) are repeated numerous times (which could be once, for a total of two rounds) .

In certain embodiments of this invention, a method is provided in which the above-steps (iii) - (vi) can be combined with the above-steps (c) - (f) , in any order, and one or more steps, but not all of the steps, may be omitted.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Diagrammatic depiction of an example of a formulations library.

Figure 2. Schematic description of an example of a method for high throughput screening of a formulations library to obtain protein formulations characterized by protein stability. Figure 3. Schematic description of another example of a method for high throughput screening of a formulations library to obtain protein formulations characterized by protein stability. DLS stands for dynamic light scattering. CD stands for circular dichroism.

DETAILED DESCRIPTION OF THE INVENTION In order that the invention herein described may be more fully understood, the following detailed description is set forth.

Protein Constituents

The protein constituents of the formulations may be any protein. The proteins can be naturally or synthetically modified. The proteins may be purified from its natural sources or may be produced by recombinant DNA technology or even synthetically (especially in the case of a short peptide) . The proteins may be glycoproteins, lyoproteins, lipoproteins, phosphoproteins, sulphoproteins, iodoproteins, methylated proteins, unmodified proteins or contain other modifications. Such protein constituents may be any protein, including, for example, therapeutic proteins, prophylactic proteins, including antibodies, cleaning agent proteins, including detergent proteins, personal care proteins, including cosmetic proteins, veterinary proteins, food proteins, feed proteins, diagnostic proteins and decontamination proteins.

The proteins can be fragments (i .e, including peptides) of proteins. The fragments can be produced by any means, including proteolytically, by recombinant DNA technology or naturally.

The proteins can be muteins . The proteins can mutants of naturally occurring proteins, produced, for example, by recombinant DNA technology. The proteins can be conjugated with a small chemical, a toxin, a radioactive isotope or any other compound that can be conjugated to a protein.

The proteins can be fusion proteins. A fusion protein comprises two or more proteins, or fragments thereof.

The proteins may be enzymes, such as, for example, hydrolases, isomerases, lyases, ligases, adenylate cyclases, transferases and oxidoreductases . Examples of hydrolases include elastase, esterase, lipase, nitrilase, amylase, pectinase, hydantoinase, asparaginase, urease, subtilisin, thermolysin and other proteases and lysozyme. Examples of lyases include aldolases and hydroxynitrile lyase . Examples of oxidoreductases include peroxidase, laccase, glucose oxidase, alcohol dehydrogenase and other dehydrogenases . Other enzymes include cellulases and oxidases . Examples of therapeutic or prophylactic proteins include hormones such as insulin, glucogon- like peptide 1 and parathyroid hormone, antibodies, inhibitors, growth factors, postridical hormones, nerve growth hormones, blood clotting factors, adhesion molecules, bone morphogenic proteins and lectins trophic factors, cytokines such as TGF-β, IL-2, IL-4, -IFN, β-IFN, γ-IFN, TNF, IL-6, IL-8, lymphotoxin, IL- 5, Migration inhibition factor, GMCSF, IL-7, IL-3, monocyte-macrophage colony stimulating factors, granulocyte colony stimulating factors, multidrug resistance proteins, other lymphokines, toxoids, erythropoietin, Factor VIII, amylin, TPA, dornase-α, α- 1-antitrypsin, human growth hormones, nerve growth hormones, bone morphogenic proteins, urease, toxoids, fertility hormones, FSH and LSH. Such proteins can be used in pharmaceutical formulations screened according to this invention.

Therapeutic proteins, such as the following, are also included: leukocyte markers, such as CD2 , CD3 , CD4, CD5, CD6, CD7, CD8, CDlla, CDllb, CDllc, CD13, CD14 , CD18, CD19, CE20, CD22, CD23, CD27 and its ligand, CD28 and its ligands B7.1, B7.2 , B7.3 , CD29 and its ligands, CD30 and its ligand, CD40 and its ligand gp39, CD44,

CD45 and isoforms, Cdw52 (Campath antigen) , CD56, CD58, CD69, CD72, CTLA-4, LFA-1 and TCR; histocompatibility antigens, such as MHC class I or II antigens, the Lewis Y antigens, SLex, SLey, SLea and SLeb; integrins, such as VLA-1, VLA-2, VLA-3, VLA- 4, VLA-5, VLA-6 and LFA-1; adhesion molecules, such as Mac-1 and pl50, 95; selectins, such as L-selectin, P-selectin and E-selectin and their counterreceptors VCAM-1, ICAM-1, ICAM-2 and LFA-3; interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, IL- 14 and IL-15; interleukin receptors, such as IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-10R, IL-llR, IL- 12R, IL-13R, IL-14R and IL-15R; chemokines, such as PF4, RANTES, MlPl , MCP1, NAP-2, Gro , Groβ and IL-8; growth factors, such as TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP, EGF family, EGF, PDGF family, endothelin and gastrin releasing peptide (GRP) ; growth factor receptors, such as TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R; GCSF-R and other hematopoietic receptors; interferon receptors, such as IFNαR, IFNβR and IFNγR;

Igs and their receptors, such as IgE, FceRI and FceRII; and blood factors, such as complement C3b, complement C5a, complement C5b-9, Rh factor, fibrinogen, fibrin and myelin associated growth inhibitor.

The protein constituent of the formulations may be any natural, synthetic or recombinant protein antigen including, for example, tetanus toxoid, diptheria toxoid, viral surface proteins, such as CMV glycoproteins B, H and gCIII, HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV envelope glycoproteins, EBV envelope glycoproteins, VZV envelope glycoproteins, HPV envelope glycoproteins, Influenza virus glycoproteins, Hepatitis family surface antigens; viral structural proteins, viral enzymes, parasite proteins, parasite glycoproteins, parasite enzymes and bacterial proteins.

Also included are tumor antigens, such as her2-neu, mucin, CEA and endosialin. Allergens, such as house dust mite antigen, lol pi (grass) antigens and urushiol are included.

Toxins, such as pseudomonas endotoxin and osteopontin/uropontin, snake venom and bee venom are included.' Also included are glycoprotein tumor- associated antigens, for example, carcinoembryonic antigen (CEA) , human mucins, her-2/neu and prostate- specific antigen (PSA) [R.A. Henderson and O.J. Finn, Advances in Immunology, 62, pp. 217-56 (1996)].

Excipients

Examples of the excipient component of the protein formulations are described in the Handbook of Pharmaceutical Excipients, published jointly by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain, the disclosure of which is hereby incorporated by reference.

Preferred excipients include amino acids, surfactants, sugars, bulking agents and antimicrobials. Preferred excipients include: 1) salts of amino acids such as glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline; 2) carbohydrates, e.g. monosaccharides such as glucose, fructose, galactose, mannose, arabinose, xylose, ribose ;

3) disaccharides, such as lactose, trehalose, maltose, sucrose;

4) polysaccharides, such as maltodextrins, dextrans, starch, glycogen;

5) alditols, such as mannitol, xylitol, lactitol, sorbitol ; 6) glucuronic acid, galacturonic acid;

7) cyclodextrins, such as methyl cyclodextrin, hydroxypropyl-β-cyclodextrin and alike;

8) inorganic salts, such as sodium chloride, potassium chloride, magnesium chloride, phosphates of sodium and potassium, boric acid ammonium carbonate and ammonium phosphate;

9) organic salts, such as acetates, citrate, ascorbate, lactate;

10) emulsifying or solubilizing agents like acacia, diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol, poloxamer, polysorbates, sodium lauryl sulfate, stearic acid, sorbitan monolaurate, sorbitan monostearate, and other sorbitan derivatives, polyoxyl derivatives, wax, polyoxyethylene derivatives, sorbitan derivatives; and

11) viscosity increasing reagents like, agar, alginic acid and its salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives propylene carbonate, polyethylene glycol, hexylene glycol, tyloxapol .

A further preferred group of excipients includes sucrose, trehalose, lactose, sorbitol, lactitol, inositol, salts of sodium and potassium such as acetate, phosphates, citrates, borate, glycine, arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol, methoxy polyethylene glycol, gelatin, hydroxypropyl-β- cyclodextrin.

Protein Formulations

A protein formulation comprises a protein and an excipient or a mixture of excipients (i.e. , a protein formulation comprises a protein component and an excipient component, wherein the excipient component may be a single excipient or a mixture of excipients) . Protein formulations in which the protein remains stable are useful for a number of purposes, including as pharmaceutical products and for biomedical applications, such as delivery of therapeutic proteins and vaccines. Additional protein formulations are those used for protein delivery in human food, agricultural products, such as feeds, veterinary compositions, diagnostics, cosmetics and personal care compositions.

Formulations Libraries

In one embodiment of this invention, a library of various protein formulations (a formulations library) is created that is screened for particular characteristics, such as protein stability. First, mixtures of a given protein with excipients (such as amino acids, sugars, surfactants, bulking agents, antimicrobials, preservatives, etc.) are combined in different ratios (volume, moles or mass) . See, e.g. , Figure 1 for an example of a protein formulations library. Hence, each protein formulation of a protein formulations library comprises the same protein and an excipient or a mixture of excipients. The ratio (volume, moles or mass) of a given excipient or a mixture of excipients to the protein, as well as the identity of the excipient or the mixture of excipients, could vary.

Those of skill in the art will appreciate the vast number of possible combinations of different excipients (and ratios of different or the same excipient or mixture of excipients to protein) can be used to form the protein formulations libraries. The preparation of the libraries can be achieved, for example, using known liquid-handling robots to dispense protein and excipients into the microtiter plates or batch parallel reactors, but manual dispensing may also be used. Alternatively, magnetic stirrers may be placed in the vessels and the reactor block may be placed on a heater/stirrer plate to afford agitation and heating. The protein formulations libraries can be prepared in any suitable containers. Examples of suitable containers are microtiter plates and batch parallel reactors. Microtiter plates can be standard 96, 384 or 1,536 microtiter plates. The protein formulations can be in liquid or powder form.

For powder samples, preparation of the protein formulations libraries includes a drying step, such as lyophilization.

High Throughput Screening of Protein Formulations to

Obtain Protein Formulations Characterized by Particular Characteristics, Such as Protein Stability

In one embodiment of this invention, high throughput screening of a protein formulations library to obtain protein formulations characterized by particular characteristics, such as protein stability, is performed according to the following methods. See also Figure 2 for an exemplarary method.

In the first stage, the ability of each protein formulation within a formulations library to prevent a protein from losing its activity as a result of problems such as denaturation is assessed by measuring the denaturation point (Tm) (melting point) of the protein in various formulations within the library and the protein in a sample of that protein without any excipient by Differential Scanning Calorimetry (DSC) or any other appropriate calorimetry techniques. The Tm for that protein in each formulation within the library (Tmf) is compared with Tm of the protein in the sample of the protein without any excipient (Tms) . The formulations having that protein characterized by Tmf approximately greater than or equal to 0.75 Tms -- preferably with Tmf greater than or equal to 0.90 Tms, more preferably with Tmf approximately equal to 1.0 Tms -- are subjected to further screening, while others may be abandoned. For powdered protein formulations, this step can be performed in powder form. Instead of or in addition to Tm, enthalpy of denaturation (ΔH) or heat capacity (C) can be compared between said protein in each formulation with that of the sample of said protein without any excipient. This step can be substituted for a step that determines the integrity of the tertiary structure of the protein in the various formulations within the library and the protein in the sample of that protein without any excipient. This is achieved by applying methods of fluorescence spectroscopy or circular dichroism ("CD"), preferably near-UV circular dichroism (250 - 350 nm) . Those formulations in which significant changes in the fluorescence, or near-UV CD spectrum from that of a protein reference standard may be abandoned. Optionally, other probes of protein tertiary structure such as UV spectroscopy may be used. Perturbations of the near UV absorption bands of aromatic amino acid residues can be used as an indication of changes in the tertiary structure.

The second stage determines the integrity of the secondary structure of the protein in the various formulations within the library and the protein in the sample of that protein without any excipient . This is achieved by applying methods of infrared spectroscopy (IR) , in particular Fourier Transform IR (FTIR) , or by applying methods of circular dichroism (CD) . The FTIR spectrum of that protein in each formulation is compared with that of the protein in the sample without any excipient, and the correlation coefficient (λ) between that protein in each formulation and the protein in the sample without any excipient is determined. See, e.g. , Carpenter et al . , Euro . Journal of Pharmaceutics and Biopharmaceutics , 45, 231 (1998) ; Griebenow and Klibanov, Proc . Natl . Acad. Sci . USA, 92, 10969 (1995) . The formulations having that protein characterized by λ approximately greater than or equal to 0.75 -- preferably with λ greater than or equal to 0.90, more preferably with λ approximately equal to 1.0, indicating that the protein has maintained its native conformation -- are subjected to further screening, while others may be abandoned. For powdered protein formulations, this step can be performed in powder form. This step can also be carried out by circular dichroism instead of IR.

The third stage of screening establishes chemical stability of the protein in the various formulations within the library and assesses the chemical stability of the protein of the sample of the protein without any excipient. To this end, reversed phase HPLC (RP HPLC) or reversed phase capillary electrophoresis (RP CE) is used. The chromatogram of the protein in each formulation is compared to that of the protein in the sample of the protein without any excipient, and the correlation coefficient (λ) is calculated. See, e.g. , Genetic Engineering News, 20, number 17, p. 32 (2000) . The formulations having that protein characterized by λ approximately greater than or equal to 0.75 -- preferably with X greater than or equal to 0.90, more preferably with λ approximately equal to 1.0, indicating that the protein has maintained its native conformation -- are subjected to further screening, while others may be abandoned. For powdered protein formulations, this step is performed with the protein formulation in solution. The sample of the protein without any excipient is also in solution.

In the fourth stage, the ability of the protein in the various formulations within the library and the protein in the sample of the protein without any excipient to aggregate is measured by size exclusion techniques, such as HPLC. Optionally, light scattering can be used in addition to, or instead of, size exclusion techniques, to determine protein aggregation in solution. The chromatogram of the protein in each formulation is compared to the chromatogram of the protein in the sample of the protein without any excipient, and the correlation coefficient (λ) is calculated. The formulations having the protein characterized by λ approximately greater than or equal to 0.75 -- preferably with λ greater than or equal to 0.90, more preferably with λ approximately equal to 1.0, indicating that the protein has maintained its native conformation -- are selected, while the rest may be abandoned. For powdered protein formulations, this step is performed with the protein formulation in solution. The sample of the protein without any excipient is also in solution. Instead of, or in addition to, size exclusion techniques, light scattering may be used to assess protein aggregation. The above sequence can be repeated numerous times in the process of accelerated or long-term stability studies. Optionally, when repeating the entire sequence, one or more steps more be skipped. One or more steps may be repeated several times in a sequence . The order of the steps in each run may be changed.

The order of the steps of the methods for screening a protein formulations library to obtain protein formulations for particular characteristics such as protein stability may be interchanged. The order of the steps may depend on a particular protein. Optionally, one or more, but not all, of the steps of the above-described method for screening a protein formulations library to obtain protein formulations for protein stability may be omitted. In many cases, blank samples (formulation without a protein) may be prepared and screened to serve as control of the screening methods .

In some preferred embodiments of this invention, any one or more of the steps of this embodiment can be combined with any one or more of the steps of embodiment described below. One or more, but not all, of the steps may be skipped. The steps may be performed in any order. One or more of the steps may be repeated. The entire set of the steps may be repeated, with or without changing the order of the steps.

In another embodiment, high throughput screening of a protein formulations library to obtain protein formulations characterized by particular characteristics, such as, for example, protein aggregation or protein stability, is performed according to the following methods. See also Figure 3 for an exemplarary method. In the first stage, the ability of the protein in the various formulations within the library, and the protein in the sample of the protein without any excipient, to aggregate is measured by dynamic light scattering analysis ("DLS"). Those formulations in which significant aggregation is observed may be abandoned. Optionally, size exclusion techniques, such as HPLC, may be used to determine protein aggregation in solution. Optionally, other methods may be used. For powdered protein formulations, this step is performed with the protein formulation in solution.

The second stage determines the integrity of the tertiary structure of the protein in the various formulations within the library and the protein in the sample of that protein without any excipient . This is achieved by applying methods of fluorescence spectroscopy or circular dichroism ("CD"), preferably near-UV circular dichroism (250 - 350 nm) . Those formulations in which significant changes in the fluorescence, or near-UV CD spectrum from that of a protein reference standard may be abandoned. Optionally, other probes of protein tertiary structure such as UV spectroscopy may be used. Perturbations of the near UV absorption bands of aromatic amino acid residues can be used as an indication of changes in the tertiary structure.

When methods of fluorescence spectroscopy are applied, this stage involves comparison of the fluorescence spectrum of the test formulation with that of the reference sample (the protein without any excipient) . These comparisons can be automated to yield a number for each comparison describing the degree of matching or differences between the reference and formulated sample. Such program could be similar to the standard FTIR spectral library search programs commonly found in many FTIR spectrometers (e.g., Nicolet) . When methods of near UV CD are used, the CD spectra of the reference sample and test formulations may be compared the same way. Near UV CD involves electronic transitions of the aromatic groups of the protein. Changes in the tertiary structure (especially native versus unfolded) will be apparent in its comparison to the CD spectrum of the reference material. These spectral changes are due to the immediate chemical environment of the aromatic residues and/or their degrees of rotational freedom. The near UV CD spectrum of the protein can be used as a fingerprint of its tertiary structure.

The third stage determines the integrity of the secondary structure of the protein in the various formulations within the library and the protein in the sample of that protein without any excipient. This is achieved by applying methods of vibration spectroscopy (FTIR, Raman or FT-Raman (FT-Raman is not hampered by protein fluorescence and is readily automated) ) , preferably in microscopy/imaging modes. Formulations exhibiting significant changes in the Amide I band (1600 - 1700 cm-1) from that of the reference standard³ may be abandoned. Optionally, other methods may be used, such as CD or far-ultraviolet CD (185 - 250 nm) , • which is sensitive to the conformations of the peptide backbone (or environment of the amide bonds) .

If the third stage is performed by FTIR, the FTIR spectrum of that protein in each formulation may be compared with that of the protein in the sample without any excipient, and the correlation coefficient (λ) between that protein in each formulation and the protein in the sample without any excipient is determined. See, e.g. , Carpenter et al . , Europ . Journal of Pharmaceutics and Biopharmaceutics, 45, 231 (1998) ; Griebenow and Klibanov, Proc. Natl. Acad. Sci. USA, 92, 10969 (1995) . The formulations having that protein characterized by λ approximately greater than or equal to 0.75 -- preferably with λ greater than or equal to 0.90, more preferably with λ approximately equal to 1.0, indicating that the protein has maintained its native conformation -- are subjected to further screening, while others may be abandoned. For powdered protein formulations, this step can be performed in powder form.

The fourth stage determines the integrity of the primary structure of the protein in the various formulations within the library and the protein in the sample of that protein without any excipient. This is achieved by applying methods of capillary zone electrophoresis ("CZE"), mass spectrometry or peptide mapping (RP-HPLC or CZE with online digestion) . Strictly speaking, CZE does not measure primary structure. CZE, however, is sensitive to changes in stability (deamidation, which can result in changes in protein charge) . Optionally, other methods may be used, such as imaged capillary isoelectric focusing (CIEF) . CIEF is another capillary electrophoresis (CE) technique whereby proteins undergo electrophoresis in a pH gradient. The charge of the protein is dependent on the pH, so that once it is neutral, its electrophoretic mobility is zero and it stops in the capillary. Imaged CIEF is a unique instrument that takes a snapshot of the capillary at 280 nm to display the immobilized protein. From its position relative to known PI standards, the protein's PI can be calculated by interpolation. Changes in the PI of the formulation protein from that of the reference protein are indicative of changes in the primary structure due to degradative processes such as deamidation and oxidation.

One way that peptide mapping (RP-HPLC or CZE with online digestion can be practice is to place a column section containing an immobilized protease before the main analytical portion of the RP-HPLC column or CZE capillary. This enables automated peptide mapping without the need for additional wet chemical steps. See, e.g. , Norberto A. Guzman, "Consecutive protein digestion and peptide derivatization employing an on-line analyte concentrator to map proteins using capillary electrophoresis," in Capillary Electrophoresis In Analytical Biotechnology, P. G. Righetti, Ed., CRC Press, Boca Raton, FL, USA, 1996, Chapter 4.

The above sequence can be repeated numerous times in the process of accelerated or long-term stability studies. Optionally, when repeating the entire sequence, one or more steps more be skipped. One or more steps may be repeated several times in a sequence. The order of the steps in each run may be changed.

The order of the steps of the methods for screening a protein formulations library to obtain protein formulations for particular characteristics, such as protein stability, may be interchanged. The order of the steps may depend on a particular protein. Optionally, one or more, but not all, of the steps of the above-described method for screening a protein formulations library to obtain protein formulations for protein stability may be omitted. In many cases, blank samples (formulation without a protein) may be prepared and screened to serve as control of the screening methods.

For any embodiment of this invention, once the libraries of formulations are created on microtiter plates, a high throughput robotic handling system can then transport the plates to a screening station. This system can be configured to perform multiple screening steps using multiple screening techniques, and there can be more than one screening station. Commercially available robotic systems that preferably include an automated conveyer, robotic arm or other suitable device that is connected to the "control" system can be used to move microtiter plates from each station to the other .

For any embodiment of this invention, the entire high throughput system (high throughput robotic systems and screening stations) can be controlled by a processor, which can be programmed with the operating parameter using a software interface. The software may be commercially available or the person skilled in the art would know how to produce it or modify commercially available software. Typical operating parameters include the coordinates of each of the stations and the initial compositions of the formulations are also be programmed into the system.

All references cited herein are hereby incorporated by reference .

Equivalents

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative of, rather than limiting to, the invention disclosed herein. All changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is claimed is:

1. A method for high throughput screening of a protein formulations library to obtain protein formulations, comprising the steps of:

(a) providing a protein formulations library in suitable containers, wherein each formulation within said library comprises a protein and an excipient or a mixture of excipients in a specific protein to excipient ratio and wherein each formulation within said library differs from another formulation either in the specific ratio of said protein to an excipient or a mixture of excipients or the identity of the excipient or mixture of excipients but does not differ from another formulation with respect to said protein;

(b) providing a sample of said protein without any excipient; (c) assessing the ability of the various formulations within the library to prevent said protein from losing its activity as result of problems such as denaturation, by measuring the denaturation point (melting point) (Tm) of said protein in each formulation and of said protein in the sample of said protein without any excipient using an appropriate calorimetry technique, followed by comparing Tm for said protein in each formulation (Tmf) with Tm of said protein in the sample of said protein without any excipient (Tms) and selecting the formulations within said library having said protein characterized by Tmf approximately greater than or equal to 0.75 Tms; (d) determining the integrity of the secondary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient by infrared spectroscopy (IR) , followed by obtaining the correlation coefficient (λ) between the IR spectrum of said protein in each formulation with the IR spectrum of said protein in the sample of said protein without any excipient and selecting formulations within said library having said protein characterized by λ approximately greater than or equal to 0.75; (e) establishing the chemical stability of said protein in the various formulations within the library and assessing the chemical stability of said protein in the sample of said protein without any excipient, by reversed phase HPLC (RP HPLC) or reversed phase capillary electrophoresis (RP CE) , followed by obtaining the correlation coefficient (λ) between the chromatogram of said protein in each formulation with the chromatogram of said protein in the sample of said protein without any excipient and selecting formulations within said library having said protein characterized by λ approximately greater than or equal to 0.75; (f) assessing the ability of said protein to aggregate in the various formulations within the library and said protein in the sample of said protein without any excipient, by size exclusion, such as HPLC, followed by obtaining the correlation coefficient (λ) between the chromatogram of said protein in each formulation with the chromatogram of said protein in the sample of said protein without any excipient and selecting formulations within said library having said protein characterized by λ approximately greater than or equal to 0.75; wherein steps (c) - (f) can be performed in any order; wherein one or more, but not all, of steps (c) - (f) are omitted.

2. The method according to claim 1, wherein step (c) is changed to: determining the integrity of the tertiary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient by fluorescence spectroscopy or circular dichroism and selecting those formulations within said library whose fluorescence or circular dichroism spectra do not differ substantially from the fluorescence or circular dichroism spectrum of the protein without any excipient .

3. The method according to claim 2 , wherein in step (c) the circular dichroism is near-UV circular dichroism.

4. The method according to any one of claims 1-2, wherein steps (c) - (f) are repeated at least once.

5. The method according any one of claims 1-2, wherein, in step (d) , circular dichroism (CD) is used instead of infrared spectroscopy (IR) .

6. The method according to claim 1, wherein, in step (c) , instead of, or in addition to denaturation point (melting point) (Tm) , enthalpy of denaturation (ΔH) or heat capacity (C) is compared.

7. The method according to any one of claims 1-2, wherein, assessing the ability of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient to aggregate in step (f) is performed or further confirmed by light scattering.

8. The method according to any one of claims 1-2, wherein steps (c) - (f) are performed by means of a high throughput robotic handling system, which transports said library to one or more screening stations, wherein said high throughput robotic handling system and said screening stations are optionally controlled by a processor programmed with operating parameter (s) using a software interface.

9. The method according to any one of claims 1-2, wherein, in step (a) , said containers are selected from the group consisting of microtiter plates and batch parallel reactors.

10. The method according to claim 1, wherein, in step (c) , the appropriate calorimetric technique used is Differential Scanning Calorimetry (DSC) .

11. The method according to any one of claims 1-2, wherein, in step (d) , the infrared spectroscopy (IR) is Fourier Transform IR (FTIR) .

12. The method according to claim 1,> wherein, in step (c) , said appropriate calorimetric technique is Differential Scanning Calorimetry (DSC) ; wherein, in step (d) , the infrared spectroscopy (IR) is Fourier Transform IR (FTIR) and circular dichroism (CD) is not used instead of IR; wherein the steps are performed in the order of (a) -

⁽f⁾; wherein, steps (c) - (f) are performed once; wherein, in step (c) , only denaturation point (melting point) (Tm) and not enthalpy of denaturation (ΔH) or heat capacity (C) is compared between a protein in each formulation and a sample of the protein without any excipient; wherein, none of steps (c) - (f) is omitted; wherein, steps (c) - (f) are performed by means of a high throughput robotic handling system, which transports said library to one or more screening stations, wherein said high throughput robotic handling system and said screening stations are controlled by a processor programmed with operating parameter (s) using a software interface.

13. The method according to claim 1 , wherein the steps are performed in the order of (a) - (f) .

14. The method according to claim 1, wherein the steps are performed in the order of (a) , (b) , (d) ,

(c) , (e) and (f) .

15. The method according to claim 1, wherein the steps are performed in the order of (a) , (b) , (d) , (c) , (f) and (e) .

16. The method according to claim 1, wherein the steps are performed in the order of (a) , (b) , (c) ,

(d) , (f) and (e) .

17. The method according to claim 1, wherein, in step (c) , formulations within said library having said protein characterized by Tmf greater than or equal to 0.90 Tms are selected.

18. The method according to claim 1, wherein, in step (c) , formulations within said library having said protein characterized by Tmf approximately equal to 1.0 Tms are selected.

19. The method according to any one of claims 1-2, wherein, in step (d) , formulations within said library having said protein characterized by X greater than or equal to 0.90 are selected.

20. The method according to any one of claims 1-2, wherein, in step (d) , formulations within said library having said protein characterized by λ approximately equal to 1.0 are selected.

21. The method according to any one of claims 1-2, wherein, in step (e) , formulations within said library having said protein characterized by X greater than or equal to 0.90 are selected.

22. The method according to any one of claims 1-2, wherein, in step (e) , formulations within said library having said protein characterized by λ approximately equal to 1.0 are selected.

23. The method according to any one of claims 1-2, wherein, in step (f) , formulations within said library having said protein characterized by X greater than or equal to 0.90 are selected.

24. The method according to any one of claims 1-2, wherein, in step (f) , formulations within said library having said protein characterized by λ approximately equal to 1.0 are selected.

25. The method according to any one of claims 1-2, wherein steps (c) - (f) are performed by means of a high throughput robotic handling system, which transports said library to one or more screening stations.

26. The method according to claim 25, wherein said high throughput robotic handling system and said screening stations are controlled by a processor programmed with operating parameter (s) using a software interface.

27. The method according to any one of claims 1-2, wherein the formulations library is prepared by using liquid-handling robots to dispense protein and excipients into microtiter plates or batch parallel reactors.

28. The method according to any one of claims 1-2, wherein the formulations library is prepared by manually dispensing protein and excipients into microtiter plates or batch parallel reactors.

29. The method according to any one of claims 1-2, wherein said protein is a therapeutic or a prophylactic protein.

30. The method according to any one of claims 1-2, wherein said protein is an enzyme.

31. The method according to any one of claims 1-2, wherein said protein formulations are pharmaceutical formulations.

32. The method according to any one of claims 1-2, wherein the protein formulations of the protein formulations library are in powder form.

33. The method according to any one of claims 1-2, wherein the protein formulations of the protein formulations library are in solution.

34. A method for high throughput screening of a protein formulations library to obtain protein formulations, comprising the steps of:

(a) providing a protein formulations library in suitable containers, such as microtiter plates or batch parallel reactors, wherein each formulation within said library comprises a protein and an excipient or a mixture of excipients in a specific protein to excipient ratio and wherein each formulation within said library differs from another formulation either in the specific ratio of said protein to an excipient or a mixture of excipients or the identity of the excipient or a mixture of excipients but does not differ from another formulation with respect to said protein;

(b) providing a sample of said protein without any excipient ;

(c) assessing the ability of said protein to aggregate in the various formulations within the library and said protein in the sample of said protein without any excipient by subjecting each formulation to dynamic light scattering analysis;

(d) determining the integrity of the tertiary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient by fluorescence spectroscopy or circular dichroism and selecting those formulations within said library whose fluorescence or circular dichroism spectra do not differ substantially from the fluorescence or circular dichroism spectrum of the protein without any excipient;

(e) determining the integrity of the secondary structure of said protein in the various formulations within the library and said protein in the sample of said protein without any excipient, by vibration spectroscopy, CD or far-ultraviolet CD (185 - 250 nm) ; and

(f) determining the integrity of the primary structure of said protein in the various formulations within the library and said^""' protein in the sample of said protein without any excipient, by capillary zone electrophoresis "CZE" , mass spectrometry, _t capillary isoelctric focusing or peptide mapping (RP-HPLC or CZE with online digestion) ; wherein steps (c) - (f) are performed in any order; wherein one or more, but not all, of steps (c) - (f) may be omitted.

35. The method according to claim 34, wherein in step (d) the circular dichroism is near-UV circular dichroism (250 - 350 nm) .

36. The method according to claim 34, wherein steps (c) - (f) are repeated at least once.

37. The method according to claim 34, wherein steps (c) - (f) are performed by means of a high throughput robotic handling system, which transports said library to one or more screening stations, wherein said high throughput robotic handling system and said screening stations are optionally controlled by a processor programmed with operating parameter (s) using a software interface.

38. The method according to claim 34, wherein in step (a) said containers are selected from the group consisting of microtiter plates and batch parallel reactors.

39. The method according to claim 34, wherein, in step (e) , the vibration spectroscopy is Fourier Transform IR (FTIR) , Raman or FT-Raman.

40. The method according to claim 34, wherein the steps are performed in the order of (a) -

(f) •

41. The method according to claim 34, wherein steps (c) - (f) are performed by means of a high throughput robotic handling system, which transports said library to one or more screening stations .

42. The method according to claim 41, wherein said high throughput robotic handling system and said screening stations are controlled by a processor programmed with the operating parameter using a software interface.

43. The method according to claim 34, wherein the formulations library is prepared by using liquid-handling robots to dispense protein and excipients into microtiter plates or batch parallel reactors.

44. The method according to claim 34, wherein the formulations library is prepared by manually dispensing protein and excipients into microtiter plates or batch parallel reactors.

45. The method according to claim 34, wherein said protein is a therapeutic or a prophylactic protein.

46. The method according to claim 34,^~ - -- wherein said protein is an enzyme.

47. The method according to claim 34, wherein said protein formulations are pharmaceutical formulations .

48. The method according to claim 34, wherein the protein formulations of the protein formulations library are in powder form.

49. The method according to claim 34, wherein the protein formulations of the protein formulations library are in solution.

50. The method according to claim 34, wherein in any one of or more of the steps (c) - (f) are performed in conjunction with any one or more of the steps (c) - (f) according to claim 1; wherein these the steps are performed in any order and wherein these steps can be repeated at least once.