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WO2001002865A1 - Criblage a spectroscopie de masse cyclotronique - Google Patents

Criblage a spectroscopie de masse cyclotronique Download PDF

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Publication number
WO2001002865A1
WO2001002865A1 PCT/US2000/018450 US0018450W WO0102865A1 WO 2001002865 A1 WO2001002865 A1 WO 2001002865A1 US 0018450 W US0018450 W US 0018450W WO 0102865 A1 WO0102865 A1 WO 0102865A1
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Prior art keywords
compositions
library
sample
mass spectrometer
animal
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PCT/US2000/018450
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English (en)
Inventor
Sun Ai Raillard
Willem P. C. Stemmer
Phillip A. Patten
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Maxygen, Inc.
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Priority to AU59152/00A priority Critical patent/AU5915200A/en
Publication of WO2001002865A1 publication Critical patent/WO2001002865A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This invention relates to high throughput methods for mass spectrometry, for example, to monitor half-life and biological transport and permeability, e.g., of protein products of shuffled libraries of genes or other nucleic acids.
  • High throughput screening of biological activity typically involves quantitative detection of one or more component in a biological system.
  • a common detection method for detecting components is mass spectrometry (MS), which allows identification of a particular molecule based on the mass and charge of the component.
  • MS mass spectrometry
  • mass spectrometry is performed in tandem with liquid chromatography to purify and separate the components of interest. This purification typically consists of on-line sequential purification. The sequential nature of the purification limits the ability of mass spectrometry to screen a large number of reaction products in a short amount of time, because the purification must occur in line with and previous to the mass spectrometry.
  • a shuffled library which is constructed, e.g., by homologous exchange of DNA fragments during DNA recombination methods or by synthetic recombination methods, is not easily amenable to expression product detection by traditional mass-spectrometry methods, because of the size of the library and the closely related nature of the library products.
  • encoded activities which are commonly screened in vivo are even more difficult to screen by traditional MS, because of the need for purification of the components of interest from a biological expression system.
  • the need to separate and purify components of interest before injection of the components into a mass spectrometer is time consuming and limits the number of samples that can be analyzed to about 100 samples per day (typical purification runs (e.g., using liquid chromatography) take about 10 minutes/sample. At 6 samples per hour, 144 samples can be run in a 24 hour period).
  • very closely related molecules are not easily resolved by traditional MS, or purified by simple chromatography.
  • Electrospray ionization is a mild method of transferring charged polar organic molecules into the gas phase for mass spectrometry analysis and is applicable for most biologically relevant molecules.
  • the electrospray method eliminates the need for prior derivatization of samples before injection into a mass-spectrometer as in GC/MS and thus shortens the analysis time for mass spectrometry.
  • column separation is still utilized in this technique, limiting throughput as noted above.
  • the present invention relates to the surprising discovery that up to hundreds of compounds can be assessed simultaneously for activity, bioavailablity, transport, half-life and the like, e.g., by cyclotron mass spectrometry.
  • methods of screening a library of compositions are provided.
  • a library comprising a plurality of compositions is provided.
  • the library of compositions includes a plurality of compositions, which may number as few as about 35 compositions, to greater than 150 compositions.
  • This plurality of compositions may be in the form of peptides, proteins, metabolic products, carbohydrates, lipids, small organic molecules, or combinations thereof.
  • the plurality of compositions typically include a plurality of proteins, such as a library of encoded proteins produced by recombination of two or more nucleic acids.
  • the library is administered to a plant or animal, or alternatively, the library is administered to, or tested in, an in vitro system comprising a plant or animal tissue.
  • a sample is obtained from the plant or animal (or the in vitro system) and subjected to cyclotron mass spectrometry.
  • the data generated by mass spectrometry is then used to analyze the library of compositions for a desired property.
  • the sample may be fractionated or purified, for example by off-line parallel purification, prior to performing the mass spectrometry step.
  • the mass spectrometry step may be performed, for example, by injecting the sample into a Fourier transform cyclotron mass spectrometer using electrospray ionization.
  • a method for producing a library of recombinant proteins for use in the method of screening is also described in the present invention.
  • a first nucleic acid that encodes a protein of interest is identified.
  • the first nucleic acid is recombined with at least a second nucleic acid to produce a library of related nucleic acids.
  • the first nucleic acid and the second nucleic acid differ from each other in two or more nucleotides.
  • the library of related nucleic acids are then expressed, thereby creating the library of encoded proteins.
  • a method of screening a library of compositions comprises determining a transfer efficiency for transfer of one or more of the plurality of compositions from a surface or subsurface of an animal to a bloodstream of the animal.
  • this screening method one or more of the plurality of compositions are administered to the surface or subsurface of an animal, including but not limited to a mouth, nose, lung, muscle, rectum, vagina or skin.
  • a blood sample from the bloodstream of the animal is obtained and used to measure the concentration of the one or more of the plurality of compositions.
  • the concentration of the one or more of the plurality of compositions in the blood sample is compared with the concentration of the one or more of the plurality of compositions as administered to the animal, to determine the transfer efficiency of the one or more of the plurality of compositions. In this manner the one or more compositions with the highest transfer efficiency can be identified.
  • the nucleic acids encoding the one or more compositions with the highest transfer efficiency may then be determined, and recombined to form another library of compositions, and subjected to the administering, sample-obtaining, measuring and comparing steps in an iterative fashion, thereby producing one or more compositions with improved transfer efficiency.
  • the composition can be administered to any tissue of a plant by standard methods and any tissue later screened for "transfer" efficiency from one plant tissue to another or from tissue into xylem or phloem.
  • a method of screening a library of compositions is described, wherein the serum half-life of each of the plurality of compositions is determined for use in the screening process.
  • a first serum or other sample is obtained from the plant, animal or in vitro system at a first time ti, and a second serum or other sample is obtained at a second time t 2 .
  • the concentrations of each of the plurality of compositions in the serum samples collected at t] and t are determined.
  • the differences in concentration between the ti and t 2 samples can be used to identify one or more compositions with the longest serum half- life.
  • This method may also be performed in an iterative manner, wherein the nucleic acids encoding the one or more compositions with the longest serum half-life may then be determined and recombined to form an additional library of compositions.
  • the new library of compositions is subjected to the administering, sample-obtaining, measuring and comparing steps in an iterative fashion, thereby producing one or more compositions with improved serum half-life.
  • Yet another embodiment of the present invention describes a method of obtaining a drug with a specified desired property, for example a drug with improved transfer efficiency, or a drug with an increased half-life.
  • a library comprising a plurality of recombinant polynucleotides is created; this library is used to produce a library of polypeptides.
  • the library of polypeptides is administered to a plant, an animal or to an in vitro system comprising a plant or animal tissue or plant or animal tissue derivative.
  • a sample is obtained from the plant, animal or in vitro system and screened for the specified desired property by performing cyclotron mass spectrometry.
  • a method for monitoring serum half-life is described.
  • a library comprising a plurality of protein sequences is provided and administered to a plant or to an animal. A first sample is obtained from the animal at a first time, and a second sample is obtained from the animal at a second time.
  • the amount of each protein sequence of the plurality of protein sequences present in the sample at the first time is determined by injecting the sample into a cyclotron mass spectrometer.
  • the amount of each protein sequence of the plurality of sequences present in the sample at the second time is also determined by injecting the sample into a cyclotron mass spectrometer, and the serum half-life of the plurality of protein sequences is determined.
  • the apparatus comprises a library comprising a plurality of compositions and a cyclotron mass spectrometer injectably coupled to the library.
  • the apparatus may further comprise a column positioned between the library and the mass spectrometer and operably coupled to the mass spectrometer.
  • the column comprises a chromatographic material for purifying or fractionating the library, such that during the operation of the apparatus, the library loads onto the column and flows through the column, resulting in a purified or fractionated library.
  • the apparatus may optionally comprise an automatic sampler that is operably coupled to the mass spectrometer.
  • the apparatus for cyclotron mass spectrometry may also further comprise a computer or computer readable medium and software operably coupled to the apparatus for recording and analyzing data from the mass spectrometer.
  • an apparatus for identifying compositions with a high biological transfer efficiency comprises a library comprised of a plurality of compositions, and a sample obtained from the bloodstream of an animal or a selected portion of a plant at a time t 2 after administration of the plurality of compositions in a predetermined concentration to the plant or animal at a time t ⁇ .
  • the apparatus also includes a cyclotron mass spectrometer injectably coupled to the library, and a computer or computer readable medium operably coupled to the mass spectrometer for recording data obtained from the screening.
  • the computer or computer readable medium, or a second computer or computer readable medium also include software for analyzing the data.
  • the software includes a first instruction set for determining a concentration of each composition in the plurality of compositions at t 2 , a second instruction set for comparing the concentration of each composition in the plurality of compositions at t 2 to the predetermined concentration administered to the plant or animal and determining the transfer efficiency of each composition in the plurality of compositions, and a third instruction set for identifying one or more compositions with high transfer efficiency.
  • a further embodiment of the present invention describes an apparatus for identifying compositions with a long serum half-life.
  • the apparatus is constructed to include a first library comprising a plurality of compositions.
  • the library includes a sample obtained from a plant or animal at a time t] after an injection of a predetermined concentration of the plurality of compositions into the plant or animal.
  • a cyclotron mass spectrometer for screening the first library for presence and concentration of the plurality of compositions is incorporated into the overall system or device.
  • a computer or computer readable medium operably coupled to the mass spectrometer, and comprising software capable of recording and analyzing data obtained from the mass spectrometer is also present in the device.
  • the software includes a first instruction set for detecting the presence of each of the plurality of compositions, a second instruction set for determining a first concentration for each of the plurality of compositions at t], a third instruction set for obtaining the difference between the first concentration and the predetermined concentration, a fourth instruction set for deconvoluting the data to determine the serum half-life of each of the plurality of compositions, and a fifth instruction set for identifying one or more compositions with a long serum half-life.
  • “Screening,” in the present invention refers to a method of examining a number of compositions, e.g., a library of compositions, such as drugs, proteins or the like, for a specified desired property, e.g., serum half-life, transfer efficiency, pharmacokinetic properties and the like.
  • a large number of compositions can be screened at once in the present invention, e.g., 30, 50, 100, 200, 300 compounds or more can be screened at once in a cyclotron mass spectrometer, to determine which of the compounds have, e.g., a high transfer efficiency.
  • the screening is optionally used to determine those compounds that possess a desired property, e.g., a high transfer efficiency or a long serum half-life.
  • a desired property e.g., a high transfer efficiency or a long serum half-life.
  • nucleic acids encoding those components are recombined to create new compounds possessing the desired property in an improved form compared to the original compounds, e.g.. a higher transfer efficiency or a longer serum-half-life.
  • the "specified desired property" of the invention is any desirable property of a composition, e.g., a protein, nucleic acid, pharmaceutical, small organic molecule, and the like, that can be screened or detected by mass spectrometry.
  • a "small organic molecule” is one that has a molecular weight less than about 2000 daltons, more typically less than about 1500 daltons. Examples include, but are not limited to, erythromycin and cholesterol.
  • the specific desired property of the invention is serum half-life or a transfer property, e.g., biological transfer efficiency.
  • transfer efficiency or “biological transfer efficiency” refers to the efficiency or rate of transfer of a composition, e.g., a drug or protein, from the site of administration to the bloodstream of the subject.
  • the transfer efficiency is optionally detected by comparing the amount or concentration of the composition administered to the amount or concentration at a specified time after the administration.
  • the rate is determined by dividing the change in concentration by the change in time.
  • Mass spectrometry is used to detect the presence of the composition in the bloodstream and the concentration of the compositions, thus determining which compositions in a library of compositions have the desired, e.g., the highest, transfer efficiency rate, by detecting the concentration of the compositions in the blood stream, e.g., which are present in the greatest amount in the bloodstream at the specified time. Those compounds are then optionally selected for further testing or recombination.
  • a "high transfer efficiency” refers to a transfer efficiency that is one of the highest in the group of compositions or library being studied.
  • the compositions with a high transfer efficiency will have a transfer efficiency in the top 20% of the library of compositions, more preferably in the top 10% and most preferably in the top 5%.
  • the transfer efficiency is optionally at least 10%, preferably greater than 30%, more preferably greater than 50% and most preferably greater than 60%.
  • serum half-life in the present invention refers to the time in which a given amount of a substance is reduced, e.g., in serum or blood, to 50% of its initial concentration, e.g., by normal turnover such as degradative and metabolic processes of cells and other serum or blood components.
  • the serum half-life is determined by mass spectrometry for a library of compositions, e.g., proteins encoded by a shuffled library, in order to select those with the longest half-lives, e.g., those that will remain in the blood stream the longest amount of time.
  • the half-life can be determined by comparing the concentration of a particular composition at two different times, preferably a time right after administration of the composition to the subject and a later time. Data obtained from a mass spectrometer indicate the presence of the compound at each time measured and is deconvoluted to provide the concentration of the composition. By monitoring at various times the compositions with the longest half-lives are optionally identified as those that are still present at the later times in large concentrations.
  • a "long serum half-life" is a desired property.
  • a “long serum half-life” refers to a half-life that is one of the longest in the group of compositions, e.g., a library of compounds, being studied.
  • the compositions with a long serum-half-life will have a half-life in the top 20% of the library of compositions, more preferably in the top 10% and most preferably in the top 5%.
  • the serum half-life is optionally on the order of minutes, preferably on the order of hours, and more preferably days.
  • the present invention preferably utilizes DNA shuffling methodologies to generate libraries of nucleic acids which are expressed.
  • the expression products are expressed in or administered to a plant, an animal or in vitro system which mimics an aspect of in vivo physiology in a plant or animal.
  • an effect of the expression or administration is observed in the animal or in vitro system, such as the presence or absence of a protein encoded by the library in the animal or in vitro system, or the presence or absence of a molecule affected by the protein in the system.
  • the observation is performed by MS, e.g., by Fourier-transform cyclotron MS on purified or unpurified mixtures of library products, typically by screening tens, or even hundreds of analytes simultaneously.
  • a variety of recombination and recursive recombination (e.g., DNA shuffling) reactions and/or other diversity generating reactions, in addition to or concurrent with standard cloning methods, are optionally used to produce libraries of nucleic acids which are expressed and screened. As adapted to the present invention, these methods are used to make and express libraries, which are then screened by cyclotron MS. A variety of such reactions are known to those of skill in the art, including those developed by the inventors and their co-workers.
  • nucleic acids can be recombined in vitro by any of a va ⁇ ety of techniques discussed m the references above, including e.g., DNAse digestion of nucleic acids to be recombined followed by ligation and/or PCR reassembly of the nucleic acids.
  • nucleic acids can be recursively recombined in vivo, e.g., by allowing recombination to occur between nucleic acids in cells.
  • whole cell genome recombination methods can be used in which whole genomes of cells are recombined, optionally including spiking of the genomic recombination mixtures with desired library components
  • synthetic recombination methods are optionally used, in which oligonucleotides corresponding to different nucleic acids are synthesized and reassembled in PCR or ligation reactions which include oligonucleotides which correspond to more than one parental nucleic acid, thereby generating new recombined nucleic acids.
  • Oligonucleotides can be made by standard nucleotide addition methods, or by t ⁇ -nucleotide synthetic approaches.
  • nucleic acids of the invention are optionally recombined (with each other or with related (or even unrelated) nucleic acids) to produce a diverse set of recombinant nucleic acids, including homologous nucleic acids, thereby providing a very fast way of explo ⁇ ng the manner in which different combinations of sequences can affect a desired result.
  • desired results include, but are not limited to, improved transfer efficiency or serum half-life.
  • DNA shuffling and related techniques provide a robust, widely applicable, means of generating diversity useful for the engineering of proteins, pathways, cells and organisms with improved characteristics.
  • recombination-based methods a variety of diversity generation methods can be practiced and the results (i.e., diverse populations of nucleic acids) evaluated. Additional diversity can be introduced into nucleic acids by methods that result in the alteration of individual nucleotides or groups of contiguous or non-contiguous nucleotides, e.g., mutagenesis methods.
  • Mutagenesis methods include, for example, recombination (PCT/US 98/05223; Publ. No. WO98/42727); oligonucleotide-directed mutagenesis (for review see, Smith, Ann. Rev.Genet. 19: 423-462 (1985)); Botstein and Shortle, Science 229: 1193-1201 (1985); Carter, Biochem. J. 237: 1-7 (1986); Kunkel, "The efficiency of oligonucleotide directed mutagenesis" in Nucleic acids & Molecular Biology, Eckstein and Lilley, eds., Springer Verlag, Berlin (1987)).
  • oligonucleotide- directed mutagenesis Zoller and Smith, Nucl. Acids Res. 10: 6487-6500 (1982), Methods in Enzvmol. 100: 468-500 (1983), and Methods in Enzvmol. 154: 329-350 (1987)) phosphothioate-modified DNA mutagenesis (Taylor et al., Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye and Eckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al., Nucl.
  • making libraries includes the construction of recombinant nucleic acids and the expression of genes in transfected host cells.
  • Molecular cloning techniques to achieve these ends are known in the art.
  • a wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids such as expression vectors are well-known to persons of skill.
  • General texts which describe molecular biological techniques useful herein, including mutagenesis, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol.
  • Methods of transducing cells, including plant and animal cells, with nucleic acids as in library construction are generally available, as are methods of expressing proteins encoded by such nucleic acids.
  • the libraries typically comprise the proteins that are encoded and expressed by such nucleic acids libraries.
  • useful general references for culture of animal cells include Freshney (Culture of Animal Cells, a Manual of Basic Technique, third edition Wiley- Liss, New York (1994)) and the references cited therein, Humason (Animal Tissue Techniques, fourth edition W.H. Freeman and Company (1979)) and Ricciardelli, et al., In Vitro Cell Dev. Biol.25:1016-1024 (1989).
  • the integrated system and methods of the present invention can be used to screen any other library which is advantageously screened by administering pools of library members to a biological system of interest. Because pooling provides a way of increasing screening throughput (i.e., because multiple library members are administered to the biological system simultaneously and are assayed for an activity of interest in an essentially simultaneous fashion), essentially all large libraries of structurally or functionally related compositions benefit from the increase in throughput.
  • a wide variety of libraries are available, including those which can be obtained commercially, e.g., from SIGMA, ALDRICH, or other companies and those which can be generated by standard methods including those taught in Sambrook, Ausubel, and Berger (all supra).
  • One method of purification involves separation of sample components.
  • the samples are optionally separated, e.g., by column chromatography, prior to injection into the mass spectrometer. Separation techniques, such as high performance liquid chromatography or capillary zone electrophoresis, are typically performed in-line with the mass spectrometry analysis. Therefore a column is typically operably coupled to the apparatus of the invention between the library and the mass spectrometer.
  • the sample is typically eluted from the column and directly injected into the mass spectrometer for analysis.
  • the spectrometry provides enough resolution to distinguish the various sample components even when they are not column-separated.
  • the system allows for the off-line parallel purification samples with no time-consuming column separation.
  • Off-line parallel purification is optionally performed as part of sample preparation on a preparation plate. This allows all samples to be sufficiently purified for mass spectrometry analysis without a column separation that is performed sequentially and in-line with the mass spectrometer.
  • the system provides a chemical purification step that is selected based on the type of sample analyzed.
  • this chemical purification step can be performed in the wells of a preparation plate (e.g., a microtiter dish) in an off-line system.
  • off-line chemical purification optionally comprises the use of a different or additional buffer when preparing samples of interest.
  • an off-line parallel purification system comprises the use of an ion exchange resin when preparing the samples of the inventions.
  • off-line parallel purification systems have been developed by the inventors for use with mass-spectrometry systems generally, as in USSN 60/119766 and USSN 09/502,283, which are incorporated herein by reference.
  • no purification is needed prior to ionization.
  • Autosamplers An "automatic sampler” is a robotic handler that transports samples from one location to another.
  • An automatic sampler is used for example, to transport samples from a preparation plate and inject them into a mass spectrometer for analysis.
  • Examples of automatic samplers include the Gilson 8-probe microtiter autosampler and the microtiter autosampler from CTC analytics.
  • Automatic samplers optionally include robotic handlers that are used to pick colonies, such as a Q-bot, and/or add or remove reagents to or from the preparation plate.
  • An autosampler is coupled with the apparatus of the invention to transport samples between the preparation plate, where samples are optionally purified, to the mass spectrometer for injection and analysis.
  • Autosamplers can be purchased from standard laboratory equipment suppliers such as Gilson and CTC Analytics. Such samplers function at rates of about 10 seconds/sample to about 1 min/sample.
  • robotic sampler handlers are optionally used to pick samples into the preparation plate and add reagents in the off-line parallel purification system.
  • robotic handlers include but are not limited to those produced by Beckman instruments and Genetrix (e.g., the Q-bot).
  • Mass spectrometry is a method that allows detection of a large variety of proteins and different small molecule metabolites. Ionspray and electrospray mass spectrometry are used in many different fields for the analysis of organic compounds and for characterization of biomacromolecules. MS is optionally coupled to a separation technique, such as high performance liquid chromatography or capillary zone electrophoresis, which is performed in-line with the mass spectrometry analysis.
  • a separation technique such as high performance liquid chromatography or capillary zone electrophoresis
  • Micromass U.K. produces a variety of instruments such as the Quattro LC (a compact triple stage quadrupole system optimized e.g., for API LC-MS-MS) which utilizes a dual stage orthogonal "Z" spray sampling technique.
  • Quattro LC a compact triple stage quadrupole system optimized e.g., for API LC-MS-MS
  • Other triple stage quadrupole mass spectrometers e.g., the "TSQ" spectrometer
  • the MS in the system herein is preferably a cyclotron MS, e.g., as described in Marshall et al. (1998) "FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPETCTROMETRY: A PRIMER” Mass
  • Electrospray methods are optionally used instead of gas chromatography procedures because no prior derivatization is required to inject the sample.
  • Flow injection analysis methods (FIA) with ionspray-ionization and tandem mass spectrometry further the ability of the present invention to perform high-throughput mass spectrometry analysis.
  • the ionspray method allows the samples to be injected without prior derivatization and the tandem mass spectrometry (MS-MS) allows extremely high efficiency in the analysis.
  • Electrospray ionization is a very mild ionization injection method that allows detection of molecules that are polar and large, which are typically difficult to detect in GC-MS without prior derivatization.
  • Modern electrospray mass spectrometers detect samples in femtomole quantities.
  • sample size is important in case where blood, tissue or serum samples are being collected and tested. Since a couple of microliters are injected, samples are optionally injected in nanomolar concentrations. Quantitation is very reproducible with standard errors ranging from 2% - 5%.
  • Tandem mass spectrometry uses the fragmentation of precursor ions to fragment ions within a triple quadrupole MS.
  • the separation of compounds with different molecular weights occurs in the first quadrupole by the selection of a precursor ion.
  • the identification is performed by the isolation of a fragment ion after collisionally induced dissociation of the precursor ion in the second quadrupole. Reviews of this technique can be found in Kenneth, L. et al. (1988) "Techniques and Applications of Tandem Mass Spectrometry," VCH Publishers, Inc.
  • Triple quadrupole mass spectrometers allow MS/MS analysis of samples.
  • a triple quadropole mass spectrometer with electrospray and atmospheric pressure chemical ionization sources such as a Finnigan TSQ 7000, is optionally used.
  • the machine is optionally set to let one particular parent ion through the first quadrupole which undergoes fragmentation reactions with an inert gas. The most prominent daughter ion can then be singled out in the third quadropole.
  • This method creates two checkpoints for analyte identification.
  • the particle must have the correct molecular mass over charge of both parent and daughter ion. Tandem mass spectrometry thus leads to higher specificity and often also to higher signal to noise ration. It also introduces further separation by distinguishing analyte from impurities with same mass.
  • cyclotrons are particle accelerators that range in size from a few inches up to 236 inches in pole diameter, and in energy from several million electron volts (MeV) up to 700-MeV protons.
  • the larger synchrotrons which have a ring of magnets rather then a solid pole, have diameters up to 800 feet across and produce particle energies of up to 30,000 MeV (30GeV).
  • Machines that accelerate particles in a straight line, such as Van De Graaffs or linear accelerators have lengths from a few feet to 2 miles (e.g., the Stanford linear accelerator). Each of these types of accelerators produces certain types of particles in a particular energy range.
  • a "Fourier transform cyclotron mass spectrometer” is typically used to provide analysis of multiple compounds in a single injection with millidalton resolution, such that proteins that differ in 10 or fewer amino acids or only one amino acid, for example, are distinguished.
  • Fourier transform cyclotron mass spectrometry theory and techniques see, e.g., Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: a Primer, Marshall et al., Mass Spectrometry Reviews, 17, 1-35 (1998) and all references therein. See, also, Ft-Icr/MS : Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Bruce Asamoto, Vch Publisher, Inc. (1991).
  • Control of the other elements of the integrated system and/or the analysis of detected system information are coupled to an appropriately programmed processor or computer, or computer readable medium which functions to instruct the operation of these instrument elements in accordance with preprogrammed or user input instructions, receive data and information from these instruments, and interpret, manipulate and report this information to the user.
  • the computer is typically appropriately coupled to any library storage elements, injection elements, and/or the MS, and/or to any analog to digital or digital to analog converter element as desired.
  • the computer typically includes appropriate software for receiving user instructions, either in the form of user input into set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.
  • the software then converts these instructions to appropriate language for instructing movement of library elements, control of the MS and the like.
  • the computer then receives the data from one or more signal sensor/detectors included within the MS system, and interprets the data, either providing it in a user interpretable format, or uses that data to initiate further instructions, in accordance with the programming, e.g., such as in monitoring and control of injection rates, library selection, temperatures, applied fields, and the like.
  • the computer typically includes software for the monitoring of materials in the MS. Additionally the software is optionally used to control injection or withdrawal of material into or from the MS. The injection or withdrawal is used to select and quantify library members in the system.
  • one or more instruction sets are present in the computer, or on a computer-readable medium such as a computer hard-drive or CD-ROM which include instruction sets for MS operation and signal detection deconvolution.
  • Instruction sets exist in computer memory or on a computer-readable medium such as a computer hard- drive or CD-ROM and are provided by the present invention and accessed by the system for the operation of the instruction sets.
  • a computer commonly used to transform signals from the detection device into reaction rates will be a PC-compatible computer (e.g., having a central processing unit (CPU) compatible with x86 CPUs (e.g., a Pentium I, II or II class machine), and running an operating system such as LINUX, DOSTM, OS/2 WarpTM, WINDOWS/NTTM, WINDOWS/NTTM workstation, or WINDOWS 98TM), or a MacintoshTM (running MacOSTM), or a UNIX workstation (e.g., a SUNTM workstation running a version of the SolarisTM operating system, a PowerPCTM workstation or a mainframe computer), all of which are commercially common, and known to one of skill "in the art.
  • Data analysis software on the computer is then employed to deconvolute signal information.
  • Software for these purposes is available, or can easily be constructed by one of skill using a standard programming language such as Visual Basic, Fortran, Basic, Java, or the like.
  • any, or all, of these components are optionally manufactured in separable modular units, and assembled to form an apparatus or system of the invention.
  • Computers, MS detectors, library manipulation robots, and the like are optionally manufactured in a single unit, but more commonly are constructed as separate modules which are assembled to form an apparatus or system for analyzing a library of components.
  • a computer does not have to be physically associated with the rest of the apparatus to be "operably linked" to the apparatus.
  • a computer is operably linked when data is delivered from other components of the apparatus to the computer.
  • operable linkage can easily be achieved using either conductive cable coupled directly to the computer (e.g., USB, parallel, serial, ethernet, or phone line cables), or using data recorders which store data to computer readable media (typically magnetic or optical storage media such as computer disks and diskettes, CDs, magnetic tapes, but also optionally including physical media such as punch cards, vinyl media or the like) which is then accessed by the computer.
  • computer readable media typically magnetic or optical storage media such as computer disks and diskettes, CDs, magnetic tapes, but also optionally including physical media such as punch cards, vinyl media or the like
  • the library A "library of compositions" in the present invention is optionally composed of proteins, nucleic acids, or pharmacologically active compositions, e.g., drugs, small organic molecules or peptides.
  • the libraries of the present invention comprise mixtures of components that are optionally injected into or administered to subjects, e.g., animals. Samples are then removed from the subject at various times, which samples contain various concentrations of the library components. At this point the concentrations of library components in the sample depend on how much of the component was transferred to the sample material after administration and how much the sample has been degraded in the body of the subject.
  • the libraries are proteins and more typically protein libraries derived from shuffled nucleic acids as described above in the section on making libraries. When using the present invention to obtain a composition with an increased serum half-life or higher transfer efficiency, shuffled libraries are used and the steps of the methods iteratively repeated to produce compositions with improved properties, such as a longer serum half-life.
  • Nucleic acid or “polynucleotide” library components in the present invention refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form which typically encode a protein or protein fragment (e.g., an extein or intein) of interest.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and even peptide -nucleic acids (PNAs).
  • the term "related nucleic acids” is used herein to refer to a group of homologous nucleic acid sequences, for example gene sequences encoding homologous proteins, e.g., pharmacologically active proteins, or protein subunits that have been mutated, e.g., by evolving or shuffling to create new and or related genes that encode proteins with enhanced serum half-lives or transfer properties, either alone or in combination with other genes.
  • recombinant when used with reference to a nucleic acid or protein indicates that the nucleic acid or protein has been modified by the introduction of a heterologous nucleic acid or protein or the alternation of a native nucleic acid or protein.
  • recombinant genes or nucleic acids are those that are not native forms of the genes, nucleic acids, or proteins.
  • polypeptide refers to a polymer of amino acid residues.
  • amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine), N-substitutions, or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
  • encoded proteins refers to proteins that are encoded by nucleic acids.
  • encoded proteins are typically those proteins encoded and/or expressed by a group of related nucleic acids or genes that have been shuffled to create nucleic acids that encode new proteins with enhanced or improved properties, such as higher transfer efficiencies or longer serum half-lives.
  • compositions are those that have a pharmaceutical or biological effect on a living system, e.g., drugs or protein pharmaceuticals.
  • the libraries of the present invention are optionally "unlabeled" libraries.
  • the library members e.g., the components or compositions in the library, do not contain a label moiety or tag. Therefore, the libraries are better for use in screening for transfer properties because no tag is present to interfere with the analysis.
  • the library members can be easily administered to the subject animal because they do not contain a label moiety.
  • the use of cyclotron mass spectrometry can distinguish between the library members, e.g., distinguish between up to 300 or more different peptides, when they are injected into the mass spectrometer in a complex mixture. Therefore, the library is also optionally administered to the subject plant, animal or in vitro system in one complex mixture, instead of requiring injection of each individual library member into a different animal.
  • the libraries in the present invention are optionally purified and/or separated libraries. Purification and/or separation occurs before injection into a mass spectrometer, e.g., to remove components that interfere with the analysis.
  • a library comprising a blood or plant or animal tissue sample is optionally fractionated to remove unwanted components, spun out to remove red blood cells, or purified using monoclonal antibodies to enrich the sample for the components of interest, e.g., protein pharmaceuticals.
  • the crude sample or the semi -purified sample containing the components of interest is optionally injected into the mass spectrometer for analysis.
  • the size of the libraries in the present invention varies.
  • a library typically comprises more than about 20 or more than about 35 compositions.
  • the libraries are between about 50 and about 150 or more compositions and most typically, the libraries comprise more than about 150 compositions.
  • the library optionally comprises about 300 or more peptides or proteins.
  • the library e.g., comprising 300 pharmaceutical peptides or more (e.g., 384 as is conveniently stored and accessed from a 384 well microtiter plate), is optionally injected into an animal in one single dose or administration. Then the sample removed from the animal at a later time comprises those 300 peptides in a concentration indicative of their transfer efficiency and/or serum half-life.
  • a library as described above is administered to an animal, e.g., a mouse, rabbit, human, or the like, and a sample is obtained from the animal at a later time.
  • the delivery of the library of compositions to an animal optionally occurs via a number of routes, such as oral, nasal, pulmonal, mucosal, dermal delivery or the like.
  • the administration is optionally by injection, either intramuscularly, intraperitoneally, or intravenously or the like.
  • administration can be by injection, ballistic delivery, topical delivery, or using a vector for delivery to plant cells which are regenerated to form whole plants.
  • Administration of the library optionally comprises delivery of the entire library in one or a few doses to a single animal or plant, or delivery of one or more components of the library to more than one different plant or animal. In the latter case, resulting samples are optionally pooled into one sample for MS analysis, thereby increasing the throughput of the system.
  • Samples e.g., blood, tissue, serum samples, or the like, are removed from the subject plant or animal, e.g., mouse, rabbit, human, corn plant, soybean plant, or the like, at various times.
  • the sample can be withdrawn from an in vitro analysis system, such as a system comprising multiple chambers separated by biological tissues, or synthetic structures which mimic such tissues, or simply from cell culture.
  • a sample may be taken upon administration of the library of compositions to an animal to determine the initial concentration of compounds in the library and then again at a later time to determine the change in concentration.
  • the sample injected may contain a known concentration of each component delivered, e.g., orally to the subject animal and the sample taken at a later date from the bloodstream of the subject to determine the efficiency of transfer, e.g., from the mouth and/or gastrointestinal tract, to the bloodstream.
  • the samples of the present invention are optionally purified before injection or delivery of the sample to the mass spectrometer. Purification occurs as described above in a chromatographic system or by off-line parallel purification as described in U.S. Application Number 60/119,766, filed February 11, 1999, by Sun Ai Raillard and USSN 09/502,283, filed February 11, 2000. However, the samples are optionally injected as crude serum samples into the mass spectrometers or as semi- purified samples containing non-separated components.
  • samples When purifying the samples, e.g., a blood sample, components that interfere with the analysis are optionally removed. Red blood cells are optionally removed simply by centrifuging the samples. Alternatively, samples may be enriched in the components of interest. For example, for protein drugs, a C-terminal peptide is optionally added to allow purification using monoclonal antibodies. Alternatively conserved epitopes can be used for purification with antibodies. In either case, the mixture of components is optionally injected to the mass spectrometer in one injection for simultaneous analysis. Mass spectrometry
  • the libraries of the present invention are analyzed by mass spectrometry, typically cyclotron mass spectrometry, more typically electrospray ionization Fourier transform cyclotron mass spectrometry.
  • the cyclotron mass spectrometry detects over a range from zero to several hundred thousand daltons and has millidalton resolution.
  • the high resolution capability of the cyclotron mass spectrometer provides separation and independent quantitation of molecules that differ in mass by less than 100 millidaltons.
  • compositions are injected into the mass spectrometer together and detected essentially together, thus allowing a high throughput mass spectrometry screening to occur.
  • Actual analysis of the individual signals can be bifurcated after detection, i.e., signals can be analyzed simultaneously or sequentially.
  • the ability to resolve 100 or more individual components by cyclotron mass spectrometry allows the determination of which components in a library have the highest transfer efficiency. For example, in a blood sample obtained from a rabbit after oral ingestion of a library of protein pharmaceuticals at equal concentrations, or after inhalation of a library of protein pharmaceuticals in equal concentrations, the highest peaks represent the highest concentrations and therefore are typically transferred to the bloodstream more efficiently than the those at lower concentrations in the samples. Of course, to make relative determinations, both the starting concentration of the components in the library and the tested concentrations are typically determined. The components corresponding to the highest transfer efficiency are then subjected to tandem mass spectrometry to characterize the fragments.
  • the cyclotron mass techniques described here and the references included herein are optionally used to perform screening as described below.
  • the present invention provides for screening samples, e.g., blood, tissue, serum samples, or the like, for transfer efficiency.
  • a library of compounds is optionally screened to determine the transfer efficiency of each individual compound in the library. Those compounds with the highest transfer efficiencies are optionally used to develop new compounds with improved transfer efficiencies compared to the original compounds.
  • Mass spectrometry data is used to determine the transfer efficiency of each individual compound by comparing the concentration of a component at the time of administration of the library to the animal with the concentration of the same component at a later time.
  • a sample is optionally administered to an animal, e.g., orally, intransally, intraperintoneally, intramuscularly, or the like.
  • a blood sample comprising a library of compounds, each component at a known concentration, is removed from the animal. By comparing the starting concentration at the time of administration to the concentration in the removed sample, a transfer efficiency is obtained for each component.
  • compositions with the highest transfer efficiency e.g., the top 20%, top 10%, or top 5%, are optionally recombined to produce a new library which is then screened as described above.
  • the selection process can then be iteratively performed to produce new compounds, e.g., pharmaceuticals, with high transfer efficiencies.
  • the present invention is used to determine the serum half-life of a variety of compounds.
  • the invention is used to evolve compounds with an increased serum half-life.
  • cyclotron mass spectrometry is used to screen a library of compounds, e.g., pharmaceuticals, proteins, or the like, for serum half-life.
  • the serum half-life is optionally determined and the compositions with the longest serum half-life are optionally selected for further analysis and manipulation to produce new compounds with a long serum-half-life.
  • the library is co-administered to a subject organism and then samples of serum, or an other tissue or fluid of interest, are removed at a time, ti, and a later time, t .
  • ti is optionally at or shortly after the time of administration and t 2 a few minutes to several hours or days later.
  • the decrease in concentration of each component over time is analyzed to provide the half-life of any analyzed member in serum or any other tissue or biological fluid. Those compounds that have the smallest concentration change over time are those with the longest serum half- life.
  • a known concentration of each component of the library is administered intravenously to the subject.
  • a serum sample is then removed at a later time, e.g., a few hours later.
  • the components with the highest relative concentrations i.e., as compared to the amount administered, as determined by mass spectrometry, at the later time, are those with the longest serum half-lives.
  • This time can be calculated using the computer system described above based on the predetermined beginning concentration injected into the bloodstream and the concentration at the specified later time. Instruction sets for performing this calculation are an optional feature of the integrated system.
  • compositions identified as having the longest serum half-lives in the screening process are optionally selected for recombination, e.g., shuffling as described above.
  • the nucleic acids corresponding to these compositions are recombined to produce new nucleic acids, which are expressed to provide a protein library which is then screened as described above for compounds with a long serum half-life. This process can be repeated iteratively to produce compounds with an improved serum half-life compared to the original and/or to presently available compounds.
  • a library of polypeptides comprising a library of epitope tags is optionally cloned into a vector.
  • the polypeptides of interest are expressed and purified using the epitope tags and a half-life selection assay is performed.
  • the pool of proteins is injected into an animal, e.g., a mouse, a monkey, or the like.
  • the pool of proteins is then isolated from a desired fraction, i.e., a fraction left in the bloodstream of the animal and removed after a desired number of half-lives.
  • the purified protein is then cleaved and the epitope tagged fragments are optionally purified and screened using mass spectrometry, e.g., cyclotron MS, thereby identifying the most abundant peaks, representing those polypeptides with the longest serum half-life.
  • mass spectrometry e.g., cyclotron MS
  • the library is optionally constructed so that there are no tags having a different sequence but the same molecular weight.
  • the sequence of the polypeptide is deduced and the corresponding oligonucleotide is synthesized, screened and subjected to diversity generation as described above to yield polypeptides with improved half-lives.
  • polypeptide selection methods of interest include, but are not limited to, screening uptake by macrophages and degradation in lysozymes, e.g., to get proteins having longer or shorter half lives in lysozymes; oral administration of a protein followed by recovery of intact protein either within the gut, serum, or stool; contacting, e.g., a library of proteins, with a specific protease or with a mixture of proteases and recovering and quantitating, e.g., using the MS screening methods described above, the resistant molecules; and contacting with a ligand, washing, eluting and quantitating by MS.
  • Table I lists proteins that are of particular commercial interest to the pharmaceutical industry and thus optionally used in the libraries of the present invention. The majority of the proteins listed in Table I are either receptors or ligands of pharmaceutical interest. These proteins are all candidates for diversity generation to improve function, e.g., improved pharmaceutical properties, such as serum half-life or transfer efficiency, etc. All are well- suited to manipulation by the techniques of the invention.
  • mutants are screened for activity in a functional assay as described above, e.g., using cyclotron mass spectrometry.
  • the genes from mutants with improved activity relative to wild-type are recovered, and subjected to further diversity generation to improve the phenotype further.
  • Angiostatin antigens e.g., peptide antigens
  • C-X-C chemokines e.g., T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP- 4, SDF-1, PF4, MIG
  • CC chemokines e.g., Monocyte chemoattractant protein- 1, Monocyte chemoattractant protein-2, Monocyte chemoattractant protein-3, Monocyte inflammatory protein- 1 alpha, Monocyte inflammatory protein- 1 beta, RANTES, 1309,
  • CSF Colony stimulating factor
  • GCSF Gonadotropin granulocyte colony stimulating factor
  • Hedgehog proteins e.g., Sonic, Indian, Desert
  • Hemoglobin for blood substitute; for radiosensitization
  • NAF Neutrophil inhibitory factor
  • Soluble interleukin receptors JL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15
  • Superantigens i.e., Staphylococcal enterotoxins (SEA, SEB, SEC1,
  • SEC2, SEC3, SED, SEE Toxic shock syndrome toxin (TSST-1), Exfoliating toxins A and B, Pyrogenic exotoxins A, B, and C, and M. arthritidis mitogen
  • TNF beta Tumor necrosis factor beta
  • TNFR Tumor necrosis factor receptor
  • TNF alpha Tumor necrosis factor-alpha
  • kits optionally include appropriate containers and instructions for using the devices or integrated systems herein as well as necessary reagents, and in cases where reagents are not predisposed in elements of the device, with appropnate instructions for introducing the reagents into the library storage or preparation medium (e.g., a microtiter dish or duplicate dish) or mass spectrometer of the device
  • libraries for performing the preferred function of screening for transfer properties and serum half-life.
  • kits also optionally include appropriate containers and instructions for using the devices or integrated systems herein as well as necessary reagents, and in cases where reagents are not predisposed in elements of the device, with appropnate instructions for introducing the reagents into the library storage or preparation medium (e.g., a microtiter dish or duplicate dish) or mass spectrometer of the device
  • Such kits typically include a preparation plate with necessary reagents, e.g., a shuffled library, predisposed in the wells or separately packaged.
  • reagents are provided in a stabilized form, so as to prevent degradation or other loss du ⁇ ng prolonged storage, e.g., from leakage
  • a number of stabilizing processes are widely used for reagents that are to be stored, such as the inclusion of chemical stabilizers (i.e., enzymatic inhibitors, microcides/bacte ⁇ ostats, anticoagulants), the physical stabilization of the matenal, e.g , through immobilization on a solid support, entrapment m a matnx (i.e., a gel), lyophihzation, or the like.

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Abstract

La présente invention concerne des procédés et des systèmes intégrés permettant de cribler par spectroscopie de masse cyclotronique des banques importantes. Ces procédés, appareil et systèmes intégrés sont conçus pour cribler des banques de composés in vivo et in vitro.
PCT/US2000/018450 1999-07-06 2000-07-05 Criblage a spectroscopie de masse cyclotronique WO2001002865A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US7074334B2 (en) 2001-05-23 2006-07-11 Klaus Wanner Method for determining the binding behavior of ligands which specifically bind to target molecules
US7153655B2 (en) 1998-06-16 2006-12-26 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function involving the use of exonuclease enzyme and two populations of parent polynucleotide sequence
US7262012B2 (en) 2002-05-17 2007-08-28 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function using varied exonuclease digestion in two polynucleotide populations
US7563578B2 (en) 2000-12-12 2009-07-21 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function

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Title
A.S. FANG ET AL.: "Rapid characterization of combinatorial libraries using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry", COMBINATORIAL CHEMISTRY AND HIGH THROUGHPUT SCREENING, vol. 1, no. 1, 1998, Boston MA USA, pages 23 - 33 *
CHEMICAL ABSTRACTS, vol. 126, no. 7, 17 February 1997, Columbus, Ohio, US; abstract no. 89761, XP002150532 *
CHEMICAL ABSTRACTS, vol. 128, no. 23, 8 June 1998, Columbus, Ohio, US; abstract no. 289517, XP002150531 *
J.P. NAWROCKI ET AL.: "Analysis of combinatorial libraries using electrospray Fourier transform ion cyclotron resonance mass spectrometry", RAPID COMMUNICATIONS IN MASS SPECTROMETRY, vol. 10, no. 14, 1996, Gainesville FL USA, pages 1860 - 1864 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7153655B2 (en) 1998-06-16 2006-12-26 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function involving the use of exonuclease enzyme and two populations of parent polynucleotide sequence
US7563578B2 (en) 2000-12-12 2009-07-21 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function
US7074334B2 (en) 2001-05-23 2006-07-11 Klaus Wanner Method for determining the binding behavior of ligands which specifically bind to target molecules
US7262012B2 (en) 2002-05-17 2007-08-28 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function using varied exonuclease digestion in two polynucleotide populations

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