US20030175694A1 - Method for determining biological agents in living target cells - Google Patents

Method for determining biological agents in living target cells Download PDF

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Publication number
US20030175694A1
US20030175694A1 US10/275,253 US27525303A US2003175694A1 US 20030175694 A1 US20030175694 A1 US 20030175694A1 US 27525303 A US27525303 A US 27525303A US 2003175694 A1 US2003175694 A1 US 2003175694A1
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biological agent
target cells
curve
standard
concentration
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Manuel Vega
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Nautilus Biotech SA
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Publication of US20030175694A1 publication Critical patent/US20030175694A1/en
Priority to US11/412,939 priority Critical patent/US7349812B2/en
Abandoned legal-status Critical Current

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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the invention concerns a method for determining the titer (concentration) of biological agents, such as gene transfer viral vectors, in real time, in living target cells, as well as its uses (gene therapy, functional genomics, viral diagnosis, vaccines, recombinant proteins).
  • biological agents such as gene transfer viral vectors
  • vectors initially developed for gene transfer are also used as tools for the screening of gene libraries.
  • the physical methods measure the titer of physical particles (pp) (Mittereder et al., J. Virol., 1996, 70, 11, 7498-7509; Atkinson et al., NAR, 1998, 26, 11, 2821-2823; Nelson et al., Hum. Gene Ther., 1998, 9, 16, 2401-2405), which represents the total number of viral vector particles; usually this titer is estimated either directly by counting the viral particles by electronic microscopy, or indirectly by measuring the nucleic acid content of the vectors (hybridization or optical absorbance (DO 260 ) for AAV and AdV, respectively), or the viral proteins content of the vectors (RT activity and p24 content, for example for MLV and HIV, respectively).
  • the physical particles titer measurement does not reflect the quantity of infectious and biologically-active particles which are present, because of the presence of non-infectious defective particles (defective-interfering particles or DI), without genome or with an incomplete genome.
  • the biological methods allow the determination of an infectious particles titer (ip: infectious units, plaque forming units, transduction units) (Mittereder et al., precited; Salvetti et al., Hum. Gene Ther., 1998, 9, 5, 695-706; Atkinson et al., precited) by the measurement of a biological parameter which reflects the activity of the vector in an infected cells culture: viral replication (AAV), provirus integration (retrovirus, HIV), cellular lysis [formation of plaques or foci of lysis), only in the case of lytic viruses (AdV, HSV)] and transgene expression (all types of vectors).
  • AAV infectious particles titer
  • provirus integration retrovirus, HIV
  • cellular lysis formation of plaques or foci of lysis
  • AdV, HSV lytic viruses
  • the ip measures the number of active particles in the biological process from which the effect is measured.
  • the vector preparations presenting a high titer of infectious particles and a low ratio of physical particles/infectious particles are considered as being of high quality, these two parameters being considered as providing quantitative information concerning the power of a preparation of a gene transfer vector.
  • the methods described are essentially based on: serial dilutions of the vector preparation (about 10 to 20 dilutions in duplicate or triplicate), followed by vector with cells incubation time (1 to 15 days), then by cell treatment (lysis, fixation, coloration, substrate addition, hybridization, PCR), functional parameter measurement, and finally by titer determination; said titer is defined as the end-point dilution, which is the highest dilution at which the value of the biological parameter is below the detection threshold.
  • the titer is generally determined from the curve which represents the values of the biological parameter according to the dilution of the vector:
  • the method includes, in several wells of a micro-titration plate, cell infection with serial dilutions of a viral preparation (10 dilutions in triplicate), replication of the viral genome in the said host cell during 48 to 72 hours, chemical lysis of said cell, hybridization of the nucleic acid, measurement of the relative quantity of viral nucleic acid replicated in each well, and the determination of the titer by linear extrapolation of the curve which represents the measured values according to the dilution of the vector.
  • the result is obtained at a fixed time which, depending on the nature of the measured parameter, varies from a few days (transgene expression) to several weeks (plaques of lysis formation).
  • the delays required to perform these methods are not adapted to rapid determination of gene transfer vectors concentration, screening of vector libraries, control in the course of production or production kinetic monitoring of said vectors.
  • the present invention set itself the objective to supply a method for determining the titer of a biological agent which meets practical needs better than the methods of the prior art in that it allows the analysis of numerous samples in real time.
  • the invention also concerns uses of said method for the screening, the analysis and the production of gene transfer viral vectors, viral vaccines and recombinant proteins, as well as for viral infection diagnosis.
  • the present invention relates to a method for determining the titer of a biological agent interacting with living target cells, characterized in that it comprises at least the following steps:
  • biological agent means a viral or non-viral vector for gene transfer, a virus, an antibody, a vaccine or a recombinant protein.
  • living target cells mean target cells, in vitro or ex vivo, before their modification by a biological agent.
  • titer C of a biological agent means its concentration in particles (virus, gene transfer viral vector, viral vaccine) or in active molecules (recombinant proteins, antibodies), in the reaction biological agent+living target cells (C corresponds to the titer of infectious particles or ip, as defined above for gene transfer viral vectors).
  • reaction biological agent+living target cells means the response of the target cells to the biologic agent or to the biological process, in particular:
  • the product P of the reaction biological agent+living target cells is measurable by a signal; it is determined by the measurement of a parameter which reflects the response of the living target cells to the biological agent.
  • a parameter is: the protein or enzyme quantity which is expressed by a reporter gene or a transgene, the viral vector genome copy number or the cell number.
  • the signal means for example, the fluorescence, the luminescence, the absorbance or the cell numeration.
  • the signal is measured, without limitation, by using optical or fluorescence microscopy, fluorimetry, luminometry and spectrometry.
  • the measurement of the signal intensity means the measurement of the product P of the reaction biological agent+living target cells, without any intervention on the target cells and/or on said reaction from which product P is measured.
  • Standard biological agent means a biological agent identical or similar to the biological agent to analyze; said standard agent presents modifications which do not affect its activity in the reaction from which product P is measured.
  • measure in real time means a measure from which value is obtained instantaneously.
  • the Inventor has shown that the signal intensity i, which reflects the response of the target cells to the biological agent, depends only on two parameters: the concentration C and the time t. Thus, when t increases, the signal intensity i increases, proportionally to the value of C; as a consequence, for a constant value of C, i varies proportionally to t and for a constant value of t, i varies proportionally to C.
  • the standard curve is established simultaneously or before the step (a 1 ), as described above, according to the following steps:
  • the method according to the invention does not require the dilution of the samples; consequently, it is particularly adapted to the analysis of numerous samples such as a gene transfer vectors library.
  • the methods of the prior art require the preparation of 10 to 20 dilutions to determine the titre of 30 vector samples and thus, the manipulation of 300 to 600 cell samples at each step of the method (infection, lysis, fixation, staining, substrate addition, hybridization), which implies 1800 to 2400 manipulations for a method including 3 steps (infection, lysis or fixation, and staining or substrate addition), the method according to the invention does not require sample dilution nor cell manipulation and simply implies 30 signal (i) measurements. Consequently, contrary to the methods of the prior art, the method according to the invention is simple, perfectly standardized and may be automated.
  • the method according to the invention is more accurate than the methods of the prior art, since the measurements are made from the same sample taken at various times t, contrary to the methods of the prior art wherein various sample dilutions are tested separetly, resulting in internal variations between various dilutions.
  • the method according to the invention which uses the time as a variable parameter, presents a number of advantages in that it allows the determination of biological agents concentration in real time, by measuring the signal i, by using techniques such as fluorimetry, luminometry or spectrometry.
  • the experimental values which are available immediately allow a rapid estimation of the vector concentration, in order to monitor the vector production kinetic or to analyze rapidly a gene transfer-vector library.
  • the methods of the prior art do not allow such estimation since no intermediate results are available in the course of the experiment; the final result only is available, once all the data corresponding to the various dilutions have been obtained at the time t and then analyzed, which corresponds to a delay from several days to several weeks, depending on the technique used.
  • said signal is selected from the group consisting of the fluorescence, the luminescence, the absorbance and the cell numeration.
  • the signal is advantageously measured by a technique such as: optical or fluorescent microscopy, fluorimetry, luminometry and spectrometry.
  • said biological agent is selected from the group consisting of viruses, viral and non-viral vectors for gene transfer, vaccines, antibodies and recombinant proteins.
  • the present invention relates also to a kit for quantifying (titration) or detecting a biological agent, characterized in that it comprises:
  • This kit is associated with an appropriate physical mean to measure the signal intensity of the reaction biological agent+living target cells.
  • This kit which allows real time measurements is particularly adapted to the titer determination (titration) of a vector for use in gene therapy, a virus for use as a vaccine, a recombinant protein for use as a biological product (medicament, reagent), or to the quantification and/or to the detection of a virus, for viral infection diagnosis.
  • the invention also comprises other provisions which will become apparent from the following description referring to examples of how to carry out the method forming the subject of the present invention and to the attached drawings, in which:
  • FIG. 1 represents the experimental values of the fluorescence signal intensity i, according to the time t, for each concentration (conc.) of a retroviral vector coding for the EGFP fluorescent protein. The concentrations are expressed for 10 6 infectious particles/ml.
  • FIG. 5 represents the experimental values of the hybridization signal intensity i, according to the time t, for each dilution of an adenovirus associated recombinant vector (rAAV).
  • the biological agent is a retroviral vector called pSI-EGFP (Ropp et al., Cytometry, 1995, 21, 309-317), coding for the fluorescent eukaryotic protein (Eukaryotic Green Fluorescent Protein or EGFP) reporter gene, the target cells are Rat-2 cells (ATC CRL1764) and the reaction biological agent+living target cells is the expression of the EGFP reporter gene.
  • the product P which is measured for determining the titer of said vector concentration in retroviral infectious particles or ip
  • C 0 10 6 infectious particles/ml
  • FIG. 2 shows that the signal intensity is a function of the vector incubation time with the target cells t, and the vector concentration C.
  • the biological agent is a recombinant adeno-associated virus (rAAV), coding for the reporter gene lacZ, the target cells are Hela-repcap32 cell line, the reaction biological agent+living target cells is the viral replication and the product P which is measured for determining the titer of the vector (concentration in active particles or ip) is the rAAV genome copy number. P is measured by Dot-blot hybridization, according to standard techniques known by a skilled person in the art.
  • rAAV recombinant adeno-associated virus
  • the signal intensity which represents the hybridized DNA quantity, is measured by means of a phosphoimager.
  • FIG. 6 shows that the signal intensity is a function of the vector incubation time with cells t and the vector concentration C.
  • the method for determining the titer of biological agents according to the invention allows advantageously the analysis of numerous samples, simultaneously and in real time. Indeed, it is simple, rapid, accurate, standardized, it can be automated and the value of the signal intensity which measures the product of the reaction of the biological agent with the living target cells is obtained immediately, without intervention on the target cells and on the biological reaction from which product is measured.

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US10/275,253 2000-05-09 2001-05-04 Method for determining biological agents in living target cells Abandoned US20030175694A1 (en)

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FR0005852A FR2808804B1 (fr) 2000-05-09 2000-05-09 Procede de determination du titre d'agents biologiques en temps reel dans des cellules cibles vivantes et ses applications
FR00/05852 2000-05-09

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EP (1) EP1281081B1 (de)
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WO (1) WO2001086291A1 (de)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030129203A1 (en) * 2001-08-27 2003-07-10 Nautilus Biotech S.A. Mutant recombinant adeno-associated viruses
US20030134351A1 (en) * 2001-08-27 2003-07-17 Manuel Vega High throughput directed evolution by rational mutagenesis
US20040132977A1 (en) * 2002-09-09 2004-07-08 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20050202438A1 (en) * 2002-09-09 2005-09-15 Rene Gantier Rational directed protein evolution using two-dimensional rational mutagenesis scanning
US20060020396A1 (en) * 2002-09-09 2006-01-26 Rene Gantier Rational directed protein evolution using two-dimensional rational mutagenesis scanning
US20060094655A1 (en) * 2004-11-04 2006-05-04 Thierry Guyon Modified growth hormones
US20060251619A1 (en) * 2005-05-04 2006-11-09 Gilles Borrelly Modified interferon-gamma polypeptides and methods for using modified interferon-gamma polypeptides
US20080003202A1 (en) * 2006-03-28 2008-01-03 Thierry Guyon Modified interferon-beta (IFN-beta) polypeptides
US20080102115A1 (en) * 2006-06-19 2008-05-01 Jorge Oyhenart Modified coagulation factor IX polypeptides and use thereof for treatment
US8252743B2 (en) 2006-11-28 2012-08-28 Hanall Biopharma Co., Ltd. Modified erythropoietin polypeptides and uses thereof for treatment

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EP2461162A1 (de) * 2010-12-03 2012-06-06 Texcell, . Verfahren zur Bestimmung des Titers von Viren durch Verwendung von infektiösen Standardsubstanzen
US11137337B2 (en) 2019-01-21 2021-10-05 Essen Instruments, Inc. Flow cytometry with data analysis for optimized dilution of fluid samples for flow cytometry investigation
US11709116B2 (en) 2020-02-04 2023-07-25 Sartorius Bioanalytical Instruments, Inc. Liquid flourescent dye concentrate for flow cytometry evaluation of virus-size particles and related products and methods

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US5691155A (en) * 1991-01-14 1997-11-25 Centre National De La Recherche Scientifigue Nucleotide sequences encoding the murine β3-adrenergic receptor and their applications
US6444428B1 (en) * 1997-03-21 2002-09-03 Stratagene Polymerase enhancing factor (PEF) extracts, PEF protein complexes, isolated PEF proteins, and methods for purifying and identifying same

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EP2325299A3 (de) * 1997-09-05 2011-10-05 Targeted Genetics Corporation Verfahren zur Erzeugung helferfreier Präparate mit hohem Titer aus rekombinanten AAV-Vektoren
FR2802645B1 (fr) * 1999-12-16 2002-03-08 Meillat Roland Methode d'evaluation de la performance d'un ensemble d'agents biologiques dans des cellules cibles vivantes et ses applications
US7647184B2 (en) * 2001-08-27 2010-01-12 Hanall Pharmaceuticals, Co. Ltd High throughput directed evolution by rational mutagenesis
US20030129203A1 (en) * 2001-08-27 2003-07-10 Nautilus Biotech S.A. Mutant recombinant adeno-associated viruses
US20030224404A1 (en) * 2002-02-25 2003-12-04 Manuel Vega High throughput directed evolution of nucleic acids by rational mutagenesis
US20060020396A1 (en) * 2002-09-09 2006-01-26 Rene Gantier Rational directed protein evolution using two-dimensional rational mutagenesis scanning
US20050202438A1 (en) * 2002-09-09 2005-09-15 Rene Gantier Rational directed protein evolution using two-dimensional rational mutagenesis scanning
AU2003263552A1 (en) * 2002-09-09 2004-03-29 Nautilus Biotech Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US7998930B2 (en) * 2004-11-04 2011-08-16 Hanall Biopharma Co., Ltd. Modified growth hormones

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691155A (en) * 1991-01-14 1997-11-25 Centre National De La Recherche Scientifigue Nucleotide sequences encoding the murine β3-adrenergic receptor and their applications
US6444428B1 (en) * 1997-03-21 2002-09-03 Stratagene Polymerase enhancing factor (PEF) extracts, PEF protein complexes, isolated PEF proteins, and methods for purifying and identifying same

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030129203A1 (en) * 2001-08-27 2003-07-10 Nautilus Biotech S.A. Mutant recombinant adeno-associated viruses
US20030134351A1 (en) * 2001-08-27 2003-07-17 Manuel Vega High throughput directed evolution by rational mutagenesis
US7647184B2 (en) 2001-08-27 2010-01-12 Hanall Pharmaceuticals, Co. Ltd High throughput directed evolution by rational mutagenesis
US8052964B2 (en) 2002-09-09 2011-11-08 Hanall Biopharma Co., Ltd. Interferon-β mutants with increased anti-proliferative activity
US7998469B2 (en) 2002-09-09 2011-08-16 Hanall Biopharma Co., Ltd. Protease resistant interferon beta mutants
US8114839B2 (en) 2002-09-09 2012-02-14 Hanall Biopharma Co., Ltd. Protease resistant modified erythropoietin polypeptides
US8105573B2 (en) 2002-09-09 2012-01-31 Hanall Biopharma Co., Ltd. Protease resistant modified IFN beta polypeptides and their use in treating diseases
US8057787B2 (en) 2002-09-09 2011-11-15 Hanall Biopharma Co., Ltd. Protease resistant modified interferon-beta polypeptides
US20070172459A1 (en) * 2002-09-09 2007-07-26 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20070224665A1 (en) * 2002-09-09 2007-09-27 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US7611700B2 (en) 2002-09-09 2009-11-03 Hanall Pharmaceuticals, Co., Ltd. Protease resistant modified interferon alpha polypeptides
US20070254838A1 (en) * 2002-09-09 2007-11-01 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20060020396A1 (en) * 2002-09-09 2006-01-26 Rene Gantier Rational directed protein evolution using two-dimensional rational mutagenesis scanning
US20050202438A1 (en) * 2002-09-09 2005-09-15 Rene Gantier Rational directed protein evolution using two-dimensional rational mutagenesis scanning
US7650243B2 (en) 2002-09-09 2010-01-19 Hanall Pharmaceutical Co., Ltd. Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20080075672A1 (en) * 2002-09-09 2008-03-27 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20040132977A1 (en) * 2002-09-09 2004-07-08 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20080159977A1 (en) * 2002-09-09 2008-07-03 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20080274081A9 (en) * 2002-09-09 2008-11-06 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20090123974A1 (en) * 2002-09-09 2009-05-14 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US20090131318A1 (en) * 2002-09-09 2009-05-21 Rene Gantier Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
US7884073B2 (en) 2004-11-04 2011-02-08 Hanall Biopharma Co., Ltd. Modified growth hormone
US20080026993A9 (en) * 2004-11-04 2008-01-31 Thierry Guyon Modified growth hormones
US7998930B2 (en) 2004-11-04 2011-08-16 Hanall Biopharma Co., Ltd. Modified growth hormones
US20070249532A9 (en) * 2004-11-04 2007-10-25 Thierry Guyon Modified growth hormones
US20060247170A1 (en) * 2004-11-04 2006-11-02 Thierry Guyon Modified growth hormones
US20060094655A1 (en) * 2004-11-04 2006-05-04 Thierry Guyon Modified growth hormones
US8222209B2 (en) 2004-11-04 2012-07-17 Hanall Biopharma Co., Ltd. Modified growth hormones that exhibit increased protease resistance and pharmaceutical compositions thereof
US20060251619A1 (en) * 2005-05-04 2006-11-09 Gilles Borrelly Modified interferon-gamma polypeptides and methods for using modified interferon-gamma polypeptides
US20080038224A1 (en) * 2006-03-28 2008-02-14 Thierry Guyon Modified interferon-beta (IFN-beta) polypeptides
US20080003202A1 (en) * 2006-03-28 2008-01-03 Thierry Guyon Modified interferon-beta (IFN-beta) polypeptides
US20080102115A1 (en) * 2006-06-19 2008-05-01 Jorge Oyhenart Modified coagulation factor IX polypeptides and use thereof for treatment
US8383388B2 (en) 2006-06-19 2013-02-26 Catalyst Biosciences, Inc. Modified coagulation factor IX polypeptides and use thereof for treatment
US8252743B2 (en) 2006-11-28 2012-08-28 Hanall Biopharma Co., Ltd. Modified erythropoietin polypeptides and uses thereof for treatment

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US20060195268A1 (en) 2006-08-31
ATE384263T1 (de) 2008-02-15
DE60132443D1 (de) 2008-03-06
US7349812B2 (en) 2008-03-25
FR2808804B1 (fr) 2002-08-02
EP1281081B1 (de) 2008-01-16
WO2001086291A1 (fr) 2001-11-15
FR2808804A1 (fr) 2001-11-16
EP1281081A1 (de) 2003-02-05

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