WO2024084463A1 - Procédés et systèmes de détection microbienne à l'aide d'une spectroscopie infrarouge - Google Patents
Procédés et systèmes de détection microbienne à l'aide d'une spectroscopie infrarouge Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Definitions
- the general inventive concepts relate to the field of microbial detection and more particularly to methods and systems for the automated detection of microbes using infrared spectroscopy.
- Bioburden testing is routinely performed as an in-process control (IPC) test for drug substances and for final product release of nonsterile drug product.
- IPC in-process control
- Multiple unit operations in Biologies manufacturing such as the production bioreactor, chromatography purification, and filtration steps are monitored for bioburden as in-process control (IPC) tests to assure microbiological quality and sterility assurance.
- IPC in-process control
- Conventional microbiological testing is based on 100 year-old technology using agar plates for enumerating microbial colony-forming units (CFU).
- Biopharmaceutical manufacturing companies are challenged to increase efficiency in manufacturing to meet the demands of patients for life saving therapies.
- Batch release testing for biopharmaceuticals can take up to 30 days, primarily due to lengthy time it takes to complete safety testing, such as bioburden and endotoxin.
- 3 Efforts in small molecule API pharmaceuticals have successfully incorporated multivariate statistical modeling and process analytical technology (PAT) to enable measurements of both IPCs (i.e. content uniformity) and final DP release testing (i.e. dissolution and assay) enabling release of product within hours of manufacture, effectively achieving real time release.
- PAT statistical modeling and process analytical technology
- RTRT Real-time release testing
- spectroscopy-based PAT sensing for x-line testing of quality attributes required for drug substance and drug product release include protein and excipient concentration, and protein-specific critical quality attributes such as charge, aggregation, glycation, or post-translational modifications PTMs.
- protein and excipient concentration include protein and excipient concentration, and protein-specific critical quality attributes such as charge, aggregation, glycation, or post-translational modifications PTMs.
- 15-18 Recently, there is increased interest in developing new methods to address bioburden and sterility testing for IPC and release. 19 ' 22 To date, these efforts are focused on development of assays that decrease testing time significantly, identify species, and are either intended for the QC laboratory or at-line testing. All of these tests are destructive and require sample preparation (labeling, filtering, or other isolation) prior to testing. A recent effort requires centrifugation of a sample prior to Raman spectroscopy analysis. 25
- a method for detecting a microbe in a sample comprising: measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- the infrared spectrum is measured using an infrared spectrometer.
- the infrared spectrometer comprises a laser, a thermal source or a lamp.
- the laser is a multimode diode laser.
- the laser wavelength is from 100 nm to 250,000 nm, from 200 nm to 200,000 nm, from 300 nm to 150,000 nm, from 500 nm to 150,000 nm, from 600 nm to 100,000 nm, from 700 nm to 100,000 nm, or from 700 nm to 90,000 nm.
- the light source is a narrow bandwidth laser with a wavelength of about 1,600 nm, about 1,500 nm, about 1,400 nm, about 1,300 nm, about 1,200 nm, about 1,000 nm, about 900 nm, about 800 nm, about 785 nm, about 750 nm, about 725 nm, about 700 nm, about 675 nm, about 650 nm, about 625 nm, about 600 nm, about 575 nm, about 550 nm, about 532 nm, about 525 nm, or about 500 nm.
- the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- UF/DF skid a fill finish tank or an incubator.
- the infrared spectrum of the sample is compared to the infrared spectrum of a control sample.
- the spectral range of the sample is from about 400 to about 3600 cm' 1 . In further embodiments, the spectral range of the sample is from about 500 to about 3600 cm' 1 . In further embodiments, the spectral range of the sample is from about 600 to about 3600 cm' 1 . In yet further embodiments, the spectral range of the sample is from about 2800 to about 3100 cm' 1 .
- the spectral range of the control sample is from about 400 to about 3600 cm' 1 . In further embodiments, the spectral range of the control sample is from about 500 to about 3600 cm' 1 . In further embodiments, the spectral range of the control sample is from about 600 to about 3600 cm' 1 . In yet further embodiments, the spectral range of the control sample is from about 2800 to about 3100 cm' 1 .
- the infrared spectrum of the sample is monitored over a period of time.
- the monitoring is automated.
- the monitoring is continuous.
- the monitoring is non-invasive and/or non-destructive.
- the sample has been contacted with an antibody to the microbe.
- the sample is contacted with a fluorescent agent or a bioluminescent agent.
- the method further comprises a means for detecting fluorescence of the sample.
- the means for detecting fluorescence is a fluorimeter.
- the method further comprises a means for detecting bioluminescence of the sample.
- the means for detecting bioluminescence is a luminometer.
- the microbe includes but is not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the measuring of the infrared spectrum is automated.
- the infrared spectrum is subjected to data analysis.
- data analysis of the infrared spectrum comprises modeling performed using principal component analysis -X (PCA-X), orthogonal partial least squares (OPLS), k-nearest neighbors (KNN), orthogonal partial least squares discriminant analysis (OPLS-DA), partial least squares discriminant analysis (PLS-DA), or combinations thereof.
- PCA-X principal component analysis -X
- OPLS orthogonal partial least squares
- KNN k-nearest neighbors
- OPLS-DA orthogonal partial least squares discriminant analysis
- PLS-DA partial least squares discriminant analysis
- the modeling is discriminant modeling.
- the method further comprises incubation with D2O for assessment of microbial viability.
- a sample from a cell culture vessel is diverted to a flow cell chamber for infrared analysis.
- the derivatization is performed prior to measuring the infrared spectrum of the sample.
- the sample comprises a eukaryotic cell.
- the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.
- the eukaryotic cell is a mouse cell, a Chinese hamster ovary (CHO) cell, or a human cell.
- the cell is a T cell, or a B cell.
- the mouse cell is a mouse Sp2/0 cell.
- the cell is HEK293F.
- the cell is PER.C6.
- the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).
- the sample is not centrifuged prior to measuring the infrared spectrum of the sample.
- the microbe is actively growing.
- a method for continuous monitoring for microbial presence in a sample comprising: measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the infrared spectrum of the sample is monitored over a period of time.
- the monitoring is non-invasive and/or non-destructive.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- the infrared spectrum is measured using an infrared spectrometer.
- the infrared spectrometer comprises a laser, a thermal source, or a lamp.
- the laser is a multimode diode laser.
- the laser wavelength is from 100 nm to 250,000 nm, from 200 nm to 200,000 nm, from 300 nm to 150,000 nm, from 500 nm to 150,000 nm, from 600 nm to 100,000 nm, from 700 nm to 100,000 nm, or from 700 nm to 90,000 nm.
- the light source is a narrow bandwidth laser with a wavelength of about 1,600 nm, about 1,500 nm, about 1,400 nm, about 1,300 nm, about 1,200 nm, about 1,000 nm, about 900 nm, about 800 nm, about 785 nm, about 750 nm, about 725 nm, about 700 nm, about 675 nm, about 650 nm, about 625 nm, about 600 nm, about 575 nm, about 550 nm, about 532 nm, about 525 nm, or about 500 nm.
- the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- UF/DF skid a fill finish tank or an incubator.
- the infrared spectrum of the sample is compared to the infrared spectrum of a control sample.
- the spectral range of the sample is from about 400 to about 3600 cm' 1 .
- the spectral range of the control sample is from about 400 to about 3600 cm' 1 .
- the monitoring is automated.
- the monitoring is non-invasive and/or non-destructive.
- the sample has been contacted with an antibody to the microbe.
- the sample has been contacted with a fluorescent agent or a bioluminescent agent.
- the method further comprises detecting fluorescence or bioluminescence of the sample.
- the microbe includes but is not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the measuring of the infrared spectrum is automated.
- data analysis of the infrared spectrum comprises discriminant modeling performed using principal component analysis -X (PCA-X), orthogonal partial least squares (OPLS), k-nearest neighbors (KNN), orthogonal partial least squares discriminant analysis (OPLS-DA), partial least squares discriminant analysis (PLS-DA), or combinations thereof.
- PCA-X principal component analysis -X
- OPLS orthogonal partial least squares
- KNN k-nearest neighbors
- OPLS-DA orthogonal partial least squares discriminant analysis
- PLS-DA partial least squares discriminant analysis
- the method further comprises incubation with D2O for assessment of microbial viability.
- a sample from a cell culture vessel is diverted to a flow cell chamber for infrared analysis.
- the derivatization is performed prior to measuring the infrared spectrum of the sample.
- the sample comprises a eukaryotic cell.
- the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.
- the eukaryotic cell is a mouse cell, a Chinese hamster ovary (CHO) cell, or a human cell.
- the cell is a T cell, or a B cell.
- the mouse cell is a mouse Sp2/0 cell.
- the cell is HEK293F.
- the cell is PER.C6.
- the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).
- CAR-T cell chimeric antigen receptor T cell
- the sample is not centrifuged prior to measuring the infrared spectrum of the sample.
- the microbe is actively growing.
- a method for assaying a test agent comprising: adding a test agent to a sample comprising a microbe; and measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- the infrared spectrum is measured using an infrared spectrometer.
- the infrared spectrometer comprises a laser, a thermal source, or a lamp.
- the laser is a multimode diode laser.
- the laser wavelength is from 100 nm to 250,000 nm, from 200 nm to 200,000 nm, from 300 nm to 150,000 nm, from 500 nm to 150,000 nm, from 600 nm to 100,000 nm, from 700 nm to 100,000 nm, or from 700 nm to 90,000 nm.
- the light source is a narrow bandwidth laser with a wavelength of about 1,600 nm, about 1,500 nm, about 1,400 nm, about 1,300 nm, about 1,200 nm, about 1,000 nm, about 900 nm, about 800 nm, about 785 nm, about 750 nm, about 725 nm, about 700 nm, about 675 nm, about 650 nm, about 625 nm, about 600 nm, about 575 nm, about 550 nm, about 532 nm, about 525 nm, or about 500 nm.
- the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- UF/DF skid a fill finish tank or an incubator.
- the infrared spectrum of the sample is compared to the infrared spectrum of a control sample.
- the spectral range of the sample is from about 400 to about 3600 cm' 1 .
- the spectral range of the control sample is from about 400 to about 3600 cm’ 1 .
- the infrared spectrum of the sample is monitored over a period of time.
- the monitoring is automated.
- the monitoring is continuous.
- the monitoring is non-invasive and/or non-destructive.
- the test agent is a compound, a supplement, an antibiotic, a bacteriophage, or a combination thereof.
- the sample has been contacted with an antibody to the microbe.
- the sample is contacted with a fluorescent agent or a bioluminescent agent.
- the method further comprises detecting fluorescence of the sample.
- the means for detecting fluorescence is a fluorimeter.
- the method further comprises detecting bioluminescence of the sample.
- the means for detecting bioluminescence is a luminometer.
- the microbe includes but is not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus. [0081] In some embodiments, the measuring of the infrared spectrum is automated.
- data analysis of the infrared spectrum comprises discriminant modeling performed using PCA-X, OPLS, KNN, OPLS-DA, PLS-DA, or combinations thereof.
- the method further comprises incubation with D2O for assessment of microbial viability.
- a sample from a cell culture vessel is diverted to a flow cell chamber for infrared analysis.
- the derivatization is performed prior to measuring the infrared spectrum of the sample.
- the sample comprises a eukaryotic cell.
- the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.
- the eukaryotic cell is a mouse cell, a Chinese hamster ovary (CHO) cell, or a human cell.
- the cell is a T cell, or a B cell.
- the mouse cell is a mouse Sp2/0 cell.
- the cell is HEK293F.
- the cell is PER.C6.
- the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).
- the sample is not centrifuged prior to measuring the infrared spectrum of the sample.
- the microbe is actively growing.
- a system for detecting a microbe in a sample comprising: a means for measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the system further comprises a coherent light source.
- the means for measuring an infrared spectrum is an infrared spectrometer.
- the infrared spectrometer comprises a laser, a thermal source or a lamp.
- the laser is a multimode diode laser.
- the laser wavelength is from 100 nm to 250,000 nm, from 200 nm to 200,000 nm, from 300 nm to 150,000 nm, from 500 nm to 150,000 nm, from 600 nm to 100,000 nm, from 700 nm to 100,000 nm, or from 700 nm to 90,000 nm.
- the light source is a narrow bandwidth laser with a wavelength of about 1,600 nm, about 1,500 nm, about 1,400 nm, about 1,300 nm, about 1,200 nm, about 1,000 nm, about 900 nm, about 800 nm, about 785 nm, about 750 nm, about 725 nm, about 700 nm, about 675 nm, about 650 nm, about 625 nm, about 600 nm, about 575 nm, about 550 nm, about 532 nm, about 525 nm, or about 500 nm.
- the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- UF/DF skid a fill finish tank or an incubator.
- the infrared spectrum of the sample is compared to the infrared spectrum of a control sample.
- the spectral range of the sample is from about 400 to about 3600 cm' 1 .
- the spectral range of the control sample is from about 400 to about 3600 cm' 1 .
- the infrared spectrum of the sample is monitored over a period of time. [0101] In some embodiments, the monitoring is automated.
- the monitoring is continuous.
- the monitoring is non-invasive and/or non-destructive.
- the sample has been contacted with an antibody to the microbe.
- the sample is contacted with a fluorescent agent or a bioluminescent agent.
- the system further comprises a means for detecting fluorescence of the sample.
- the means for detecting fluorescence is a fluorimeter.
- the system further comprises a means for detecting bioluminescence of the sample.
- the means for detecting bioluminescence is a luminometer.
- the microbe includes but is not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the measuring of the infrared spectrum is automated.
- the infrared spectrum is subjected to data analysis.
- data analysis of the infrared spectrum comprises modeling performed using principal component analysis -X (PCA-X), orthogonal partial least squares (OPLS), k-nearest neighbors (KNN), orthogonal partial least squares discriminant analysis (OPLS-DA), partial least squares discriminant analysis (PLS-DA), or combinations thereof.
- PCA-X principal component analysis -X
- OPLS orthogonal partial least squares
- KNN k-nearest neighbors
- OPLS-DA orthogonal partial least squares discriminant analysis
- PLS-DA partial least squares discriminant analysis
- the modeling is discriminant modeling.
- the method further comprises incubation with D2O for assessment of microbial viability.
- a sample from a cell culture vessel is diverted to a flow cell chamber for infrared analysis.
- the derivatization is performed prior to measuring the infrared spectrum of the sample.
- the sample comprises a eukaryotic cell.
- the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.
- the eukaryotic cell is a mouse cell, a Chinese hamster ovary (CHO) cell, or a human cell.
- the cell is a T cell, or a B cell.
- the mouse cell is a mouse Sp2/0 cell.
- the cell is HEK293F.
- the cell is PER.C6.
- the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).
- the sample is not centrifuged prior to measuring the infrared spectrum of the sample.
- the microbe is actively growing.
- a system for continuous monitoring for microbial presence in a sample comprising: a means for measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the infrared spectrum of the sample is monitored over a period of time.
- the monitoring is non-invasive and/or non-destructive.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- the infrared spectrum is measured using an infrared spectrometer.
- the infrared spectrometer comprises a laser, a thermal source, or a lamp.
- the laser is a multimode diode laser.
- the laser wavelength is from 100 nm to 250,000 nm, from 200 nm to 200,000 nm, from 300 nm to 150,000 nm, from 500 nm to 150,000 nm, from 600 nm to 100,000 nm, from 700 nm to 100,000 nm, or from 700 nm to 90,000 nm.
- the light source is a narrow bandwidth laser with a wavelength of about 1,600 nm, about 1,500 nm, about 1,400 nm, about 1,300 nm, about 1,200 nm, about 1,000 nm, about 900 nm, about 800 nm, about 785 nm, about 750 nm, about 725 nm, about 700 nm, about 675 nm, about 650 nm, about 625 nm, about 600 nm, about 575 nm, about 550 nm, about 532 nm, about 525 nm, or about 500 nm.
- the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- UF/DF skid a fill finish tank or an incubator.
- the infrared spectrum of the sample is compared to the infrared spectrum of a control sample.
- the spectral range of the sample is from about 400 to about 3600 cm’ 1 .
- the spectral range of the control sample is from about 400 to about 3600 cm’ 1 .
- the monitoring is automated.
- the monitoring is non-invasive and/or non-destructive.
- the system further comprises detecting fluorescence of the sample.
- the sample has been contacted with an antibody to the microbe.
- the sample has been contacted with a fluorescent agent or a bioluminiscent agent.
- the system further comprises a means for detecting fluorescence of the sample.
- the means for detecting fluorescence is a fluorimeter.
- the system further comprises a means for detecting bioluminescence of the sample.
- the means for detecting bioluminescence is a luminometer.
- the microbe includes but is not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the measuring of the infrared spectrum is automated.
- the infrared spectrum is subjected to data analysis.
- data analysis of the infrared spectrum comprises discriminant modeling performed using principal component analysis -X (PCA-X), orthogonal partial least squares (OPLS), k-nearest neighbors (KNN), orthogonal partial least squares discriminant analysis (OPLS-DA), partial least squares discriminant analysis (PLS-DA), or combinations thereof.
- PCA-X principal component analysis -X
- OPLS orthogonal partial least squares
- KNN k-nearest neighbors
- OPLS-DA orthogonal partial least squares discriminant analysis
- PLS-DA partial least squares discriminant analysis
- the method further comprises incubation with D2O for assessment of microbial viability.
- a sample from a cell culture vessel is diverted to a flow cell chamber for infrared analysis.
- the derivatization is performed prior to measuring the infrared spectrum of the sample.
- the sample comprises a eukaryotic cell.
- the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.
- the eukaryotic cell is a mouse cell, a Chinese hamster ovary (CHO) cell, or a human cell.
- the cell is a T cell, or a B cell.
- the mouse cell is a mouse Sp2/0 cell.
- the cell is HEK293F.
- the cell is PER.C6.
- the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).
- the sample is not centrifuged prior to measuring the infrared spectrum of the sample.
- the microbe is actively growing.
- a system for assaying a test agent comprising: a means for measuring an infrared spectrum of a sample to which a test agent has been added.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- the infrared spectrum is measured using an infrared spectrometer.
- the infrared spectrometer comprises a laser, a thermal source, or a lamp.
- the laser is a multimode diode laser.
- the laser wavelength is from 100 nm to 250,000 nm, from 200 nm to 200,000 nm, from 300 nm to 150,000 nm, from 500 nm to 150,000 nm, from 600 nm to 100,000 nm, from 700 nm to 100,000 nm, or from 700 nm to 90,000 nm.
- the light source is a narrow bandwidth laser with a wavelength of about 1,600 nm, about 1,500 nm, about 1,400 nm, about 1,300 nm, about 1,200 nm, about 1,000 nm, about 900 nm, about 800 nm, about 785 nm, about 750 nm, about 725 nm, about 700 nm, about 675 nm, about 650 nm, about 625 nm, about 600 nm, about 575 nm, about 550 nm, about 532 nm, about 525 nm, or about 500 nm.
- the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- UF/DF skid a fill finish tank or an incubator.
- the infrared spectrum of the sample is compared to the infrared spectrum of a control sample.
- the spectral range of the sample is from about 400 to about 3600 cm' 1 .
- the spectral range of the control sample is from about 400 to about 3600 cm' 1 .
- the infrared spectrum of the sample is monitored over a period of time.
- the monitoring is automated.
- the monitoring is continuous.
- the monitoring is non-invasive and/or non-destructive.
- the test agent is a compound, a supplement, an antibiotic, a bacteriophage, or a combination thereof.
- the sample has been contacted with an antibody to the microbe.
- the sample is contacted with a fluorescent agent or a bioluminescent agent.
- the system further comprises detecting fluorescence of the sample.
- the means for detecting fluorescence is a fluorimeter.
- the system further comprises detecting bioluminescence of the sample.
- the means for detecting bioluminescence is a luminometer.
- the microbe includes but is not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the measuring of the infrared spectrum is automated.
- the infrared spectrum is subjected to data analysis.
- data analysis of the infrared spectrum comprises discriminant modeling performed using PCA-X, OPLS, KNN, OPLS-DA, PLS-DA, or combinations thereof.
- the method further comprises incubation with D2O for assessment of microbial viability.
- a sample from a cell culture vessel is diverted to a flow cell chamber for infrared analysis.
- the derivatization is performed prior to measuring the infrared spectrum of the sample.
- the sample comprises a eukaryotic cell.
- the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.
- the eukaryotic cell is a mouse cell, a Chinese hamster ovary (CHO) cell, or a human cell.
- the cell is a T cell, or a B cell.
- the mouse cell is a mouse Sp2/0 cell.
- the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).
- the sample is not centrifuged prior to measuring the infrared spectrum of the sample.
- the microbe is actively growing.
- FIG. 1 shows a visual representation of each spiking study with offline parallel plating.
- FIG. 2 shows a typical spectrum (before and after pre-processing) during a bioreactor media only and cell culture processes with relevant wavenumber regions highlighted.
- FIGs. 3A-3D illustrate principal component analysis (PCA) media-only model overview.
- FIG. 3 A Root mean square error of calibration (RMSEC) and root mean square error of cross- validation (RMSECV) v. principal component (PC).
- FIG. 3B Q residuals v. sample.
- FIG. 3C T2 v. sample.
- FIG. 3D Scores.
- FIGs. 4A-4E illustrate PCA cell culture only model overview.
- FIG. 4A RMSEC and RMSECV v. PC.
- FIG. 4B Q residuals v. sample.
- FIG. 4C T2 v. sample.
- FIG. 4D Scores.
- FIG. 4E Scores.
- FIGs. 5A-5H show an orthogonal partial least squares discriminant analysis (OPLS-DA) cell culture model overview.
- FIG. 5 A RMSEC and RMSECV v PC.
- FIG. 5B T2 v sample.
- FIG. 5C Q residuals v sample.
- FIG. 5D Scores.
- FIG. 5E Calibration error.
- FIG. 5F Y Prediction plot.
- FIG. 5G receiver operating characteristic (ROC) curve.
- FIG. 5H Sensitivity v Specificity.
- FIG. 6A-6B illustrate k-nearest neighbors (KNN) cell culture model overview.
- FIG. 6A Calibration error.
- FIG. 6B Prediction plot.
- the dashed line indicates predicted Y-values in the prediction set (YpredPS) limit of 0.35 to classify spectra as borderline while the solid line indicates YPredPS limit of 0.65 to classify spectra as contaminated.
- the data points for each sample are coded according to contaminated (black), noncontaminated (light gray), and ambiguous (dark gray).
- FIG. 8 shows a table: Study Results Demonstrating the Raman Method Faster Time-to- Detection of Bioreactor Microbial Contamination Compared to Conventional Spread Plating.
- FIGs. 9A-9F show a PLS-DA cell culture model overview for Stelara® (ustekinumab; Janssen) with the following microbial species tested: Staphylococcus epidermidis, Escherichia coli, Candida albicans, Bacillus cereus, Cutibacterium acnes, Bacillus subtilis, and Aspergillus brasiliensis.
- FIG. 9A RMSECV v PC.
- FIG. 9B T2 v sample.
- FIG. 9C Distance to Model (DmodX) v sample.
- FIG. 9D cumulative r-squared (R2Cum) and cumulative Q-square index (Q2Cum) v PC.
- FIG. 9E Scores.
- FIG. 9F Y Prediction plot. The data points for each sample are color coded according to contaminated (black) versus noncontaminated (grey).
- FIGs. 10A-10C show a prediction of OPLS-DA contamination model on CTSS with ambiguous transition spectra for Stelara® (ustekinumab; Janssen) with the following microbial species tested: Staphylococcus epidermidis, Escherichia coli, Bacillus cereus, Bacillus subtilis, and control tests from reactor groupings from the remaining species in the calibration sample set (CSS).
- FIG. 10A predicted values in the workset (Ypred) v sample.
- FIG. 10B Y Prediction plot. The dashed line indicates YPredPS limit of 0.35 to classify spectra as borderline while the solid line indicates YPredPS limit of 0.65 to classify spectra as contaminated.
- FIG. 10C ROC plot.
- the AUC is 0.716.
- the data points for each sample are color coded according to contaminated (black), noncontaminated (light grey), and ambiguous (dark grey).
- FIGs. 11 A-l 1C show prediction of OPLS-DA contamination model on CTSS without ambiguous transition spectra for Stelara® (ustekinumab; Janssen) with the following microbial species tested: Staphylococcus epidermidis, Escherichia coli, Bacillus cereus, Bacillus subtilis, and control tests from reactor groupings from the remaining species in the CSS.
- FIG. 11 A YPred v sample.
- FIG. 1 IB Y Prediction plot. The dashed line indicates YPredPS limit of 0.35 to classify spectra as borderline while the solid line indicates YPredPS limit of 0.65 to classify spectra as contaminated.
- FIG. 11C ROC plot.
- the AUC is 0.997.
- the data points for each sample are color coded according to contaminated (black) and noncontaminated (grey).
- the dashed line indicates YPredPS limit of 0.35 to classify spectra as borderline while the solid line indicates YPredPS limit of 0.65 to classify spectra as contaminated.
- the data points for each sample are color coded according to contaminated (black), noncontaminated (light grey), and ambiguous (dark grey).
- a cell means one cell or more than one cell.
- ‘About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 5%, preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
- coherent light source means a light source that emits light wave(s) having having the same frequency, wavelength and in the same phase, or having a constant phase difference.
- antibody and “antibodies” as used herein are meant in a broad sense and include immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human-adapted, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, and single chain antibodies.
- Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
- IgA and IgG are further sub-classified as the isotypes IgAl , IgA2 , IgGl , IgG2 , IgG3 and IgG4 .
- Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.
- antibody fragments refers to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL).
- HCDR heavy chain complementarity determining regions
- LCDR light chain complementarity determining regions
- VH heavy chain variable region
- VL light chain variable region
- Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a domain antibody (dAb) fragment, which consists of a VH domain.
- Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
- F(ab)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
- a Fd fragment consisting of the VH and CHI domains
- a Fv fragment consisting of the VL and VH domains of a single arm of an antibody
- dAb domain antibody
- VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in PCT Inti. Publ. Nos. WO 1998/44001, WO1988/01649, WO1994/13804, and W01992/01047.
- scFv single chain Fv
- diabody diabody
- isolated antibody refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding CD38 is substantially free of antibodies that specifically bind antigens other than human CD38).
- An isolated antibody that specifically binds CD38 can have cross-reactivity to other antigens, such as orthologs of human CD38, such sMacaca fascicularis (cynomolgus monkey) CD38.
- an isolated antibody may be substantially free of other cellular material and/or chemicals.
- Humanized antibody refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.
- Human antibody refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin.
- a human antibody comprises heavy or light chain variable regions that are "derived from” sequences of human origin wherein the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci as described herein.
- a human antibody may also contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to for example naturally occurring somatic mutations or intentional introduction of substitutions in the framework or antigen binding sites.
- a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.
- Isolated humanized antibodies may be synthetic. Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.
- recombinant antibody includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.
- monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
- a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes.
- epitope means a portion of an antigen to which an antibody specifically binds.
- Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
- An epitope can be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3 -dimensional space through the folding of the protein molecule.
- chimeric antigen receptor or “CAR” as used herein means a synthetic or recombinant receptor comprising an antigen specific domain, a costimulatory domain and an intracellular signaling domain.
- the CAR further comprises an extracellular hinge or spacer region, a transmembrane domain, or combinations thereof.
- the antigen specific domain is an scFv.
- chimeric antigen receptor T cell or “CAR-T” as used herein means a T cell expressing a CAR.
- bioreactor generally refers to a device that supports a biologically active process, such as the culture of cells.
- exemplary bioreactors include stainless steel stirred bioreactors, air-lift reactors, and disposable bioreactors.
- the term “in-line” generally means that the measuring or determining is performed in real time by a probe placed in a container (for example, in a bioreactor) and collection of a sample is not required.
- a range is intended to comprise every integer or fraction or value within the range.
- Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and/or “consisting essentially of’ such features.
- the Raman probe sensed a change in the cell culture through both a change in the metabolic profile (e.g. glucose consumption and lactate generation) as well as a multivariate models that serve as the biological process fingerprint. These models were useful to identify when a process deviated from expected nominal conditions. In this work, infrared multivariate analysis capability was explored to determine whether it also had the specificity to identify different types of microbial contamination (i.e. fast vs. slow, and aerobic vs. anaerobic) and distinguish it from other process faults (under or over feed, pH, or gas control, etc.).
- microbial contamination i.e. fast vs. slow, and aerobic vs. anaerobic
- a method for detecting a microbe in a sample comprising: measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the infrared spectrum of the sample is measured at set intervals. In some embodiments, the infrared spectrum is measured at random intervals. In some embodiments, the infrared spectrum measurement is uninterrupted.
- the infrared spectrum of the sample is monitored over a period of time. In further embodiments, the infrared spectrum of the sample is monitored at set intervals, over a period of time. In yet further embodiments, the infrared spectrum of the sample is monitored at random intervals, over a period of time. In yet further embodiments, the infrared spectrum of the sample is monitored continuously, over a period of time. The period of time may be from about 0.1 to about 1000 hours, from about 0.1 to about 100 hours, from about 0.1 to about 10 hours, from about 0.1 to about 1 hour, or from about 0.1 to about 0.2 hours.
- the monitoring is automated.
- the monitoring is non-invasive and/or non-destructive.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- a method for assaying a test agent comprising: adding a test agent to a sample comprising a microbe; and measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- Infrared Spectrometer Infrared Spectrometer
- the infrared spectrum is measured using an infrared spectrometer.
- the infrared spectrometer may be commercially available, for example a ReactIR from Metier Toledo (Columbus, Ohio, USA).
- the infrared spectrometer comprises a laser, a thermal source or a lamp.
- the laser is a multimode diode laser.
- the laser wavelength is from 100 nm to 250,000 nm, from 200 nm to 200,000 nm, from 300 nm to 150,000 nm, from 500 nm to 150,000 nm, from 600 nm to 100,000 nm, from 700 nm to 100,000 nm, or from 700 nm to 90,000 nm.
- the light source is a narrow bandwidth laser with a wavelength of about 1,600 nm, about 1,500 nm, about 1,400 nm, about 1,300 nm, about 1,200 nm, about 1,000 nm, about 900 nm, about 800 nm, about 785 nm, about 750 nm, about 725 nm, about 700 nm, about 675 nm, about 650 nm, about 625 nm, about 600 nm, about 575 nm, about 550 nm, about 532 nm, about 525 nm, or about 500 nm.
- the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- UF/DF skid a fill finish tank or an incubator.
- the bioreactor can have any suitable volume that allows for the cultivation and propagation of biological cells capable of producing therapeutic proteins (such as Stelara® (ustekinumab; Janssen)).
- the volume of the bioreactor can be about 0.5 liters (L) to about 25,000 L.
- the volume of the bioreactor can be less than or equal to about 250 L.
- the volume of the bioreactor can be about 0.5 liters (L) to about 250 L.
- the volume of the bioreactor can be less than or equal to about 50 L.
- the volume of the bioreactor can be about 1 L to about 50 L.
- the voluem of the bioreactor can be less than or equal to about 25 L. In some embodiments, the volume of the bioreactor can be about 1 L to about 25 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 250 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 100 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 50 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 25 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 10 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 5 L.
- the volume of the bioreactor can be less than or equal to about 1 L. In some embodiments, the volume of the bioreactor can be about 1 L. In some embodiments, the volume of the bioreactor can be about 2 L. In some embodiments, the volume of the bioreactor can be about 5 L. In some embodiments, the volume of the bioreactor can be equal to or about 1,000 L. In some embodiments, the volume of the bioreactor can be about 1,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 10,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 1,000 L. In some embodiments, the volume of the bioreactor can be about 2,000 L.
- the volume of the bioreactor can be about 5,000 L. In some embodiments, the volume of the bioreactor can be about 10,000 L. In some embodiments, the volume of the bioreactor can be about 15,000 L. In some embodiments, the volume of the bioreactor can be about 25,000 L.
- the sample is at a temperature from about 2 °C to about 40 °C. In some embodiments, the sample is at a temperature from about 20 °C to about 40 °C. In some embodiments, the preferred temperature is from about 25 °C to about 37 °C. In further embodiments, the sample is at a temperature from about 2 °C to about 20 °C. In yet further embodiments, the sample is at a temparature from about 2 °C to about 10 °C.
- the sample is grown for about 0.5 to about 14 days prior to measuring the infrared spectrum of the sample. In some embodiments, the sample is grown for about 0.5 to about 7 days prior to measuring the infrared spectrum of the sample. In further embodiments, the sample is grown for about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days prior to measuring the infrared spectrum of the sample.
- the infrared spectrum of the sample is compared to the infrared spectrum of a control sample.
- the spectral range of the sample is from about 400 to about 3600 cm' 1 .
- the spectral range of the control sample is from about 400 to about 3600 cm' 1 .
- the infrared spectrum of the sample is monitored over a period of time. In further embodiments, the infrared spectrum of the sample is monitored at set intervals, over a period of time. In yet further embodiments, the infrared spectrum of the sample is monitored at random intervals, over a period of time. In yet further embodiments, the infrared spectrum of the sample is monitored continuously, over a period of time. The period of time may be from about 0.1 to about 1000 hours, from about 0.1 to about 100 hours, from about 0.1 to about 10 hours, from about 0.1 to about 1 hour, or from about 0.1 to about 0.2 hours.
- the monitoring is automated.
- the monitoring is continuous.
- the monitoring is non-invasive and/or non-destructive.
- the sample has been contacted with an antibody to the microbe.
- the sample is contacted with a fluorescent agent or a bioluminescent agent.
- the method further comprises detecting fluorescence of the sample.
- the means for detecting fluorescence is a fluorimeter.
- the method further comprises detecting bioluminescence of the sample.
- the means for detecting bioluminescence is a luminometer.
- the microbe includes but is not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the measuring of the infrared spectrum is automated.
- the automation is controlled by a central processor, for example a computer.
- the automation is robotic.
- the infrared spectrum is subjected to data analysis.
- data analysis of the infrared spectrum comprises discriminant modeling performed using principal component analysis -X (PCA-X), orthogonal partial least squares (OPLS), k-nearest neighbors (KNN), orthogonal partial least squares discriminant analysis (OPLS-DA), partial least squares discriminant analysis (PLS-DA), or combinations thereof.
- PCA-X principal component analysis -X
- OPLS orthogonal partial least squares
- KNN k-nearest neighbors
- OPLS-DA orthogonal partial least squares discriminant analysis
- PLS-DA partial least squares discriminant analysis
- data analysis comprises a model for a microbe including but not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- a model for a microbe including but not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepaci
- data analysis comprises generating one or more models based on the obtained infrared spectrum data that correlate levels of microbe with the obtained infrared spectrum data.
- the one or more models are regression models.
- data analysis is executed by one or more computing devices.
- the infrared spectrometer is connected to one or more data processors and memory into which instructions can be loaded and executed by the data processors.
- the data processors communicate over a network with one or more computing systems (e.g., servers, personal computers, tablets, loT devices, mobile phones, dedicated control units, etc.) which can execute various algorithms or data analysis as described elsewhere herein.
- the computing systems can also act to change one or more operating parameters associated with the bioreactor.
- the bioreactor can also have network connectivity such that it can communicate with one or more remote computing systems which, in turn, can cause one or more operating parameters of the bioreactor to change.
- the method further comprises incubation with D2O for assessment of microbial viability.
- a sample from a cell culture vessel is diverted to a flow cell chamber for infrared analysis.
- the derivatization is performed prior to measuring the infrared spectrum of the sample.
- the sample comprises a eukaryotic cell.
- the eukaryotic cell produces a therapeutic product.
- the therapeutic product may be released into the cell media, where it may be collected. The methods provided herein allow for detection of contamination.
- the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.
- the eukaryotic cell is a mouse cell, a Chinese hamster ovary (CHO) cell, or a human cell.
- the cell is a T cell, or a B cell.
- the mouse cell is a mouse Sp2/0 cell.
- the cell is HEK293F.
- the cell is PER.C6.
- the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).
- the sample is not centrifuged prior to measuring the infrared spectrum of the sample.
- the sample is not removed from the bioreactor, harvest tank, chromatography skid, chromatography column, ultrafiltration (UF) skid, diafiltration (DF) skid, UF/DF skid, fill finish tank or incubator prior to measuring the infrared spectrum of the sample.
- the microbe is actively growing.
- Advantages of the methods provided herein include but are not limited to risk reduction of encountering false positive process samples due to QC microbiology lab cross contamination, cost reduction, and combinations thereof.
- a system for detecting a microbe in a sample comprising: a means for measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the system further comprises a coherent light source.
- a system for continuous monitoring for microbial presence in a sample comprising: a means for measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the infrared spectrum of the sample is measured at set intervals. In some embodiments, the infrared spectrum is measured at random intervals. In some embodiments, the infrared spectrum measurement is uninterrupted.
- the infrared spectrum of the sample is monitored over a period of time. In further embodiments, the infrared spectrum of the sample is monitored at set intervals, over a period of time. In yet further embodiments, the infrared spectrum of the sample is monitored at random intervals, over a period of time. In yet further embodiments, the infrared spectrum of the sample is monitored continuously, over a period of time. The period of time may be from about 0.1 to about 1000 hours, from about 0.1 to about 100 hours, from about 0.1 to about 10 hours, from about 0.1 to about 1 hour, or from about 0.1 to about 0.2 hours.
- the monitoring is non-invasive and/or non-destructive.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- a method for assaying a test agent comprising: adding a test agent to a sample comprising a microbe; and measuring an infrared spectrum of the sample.
- the infrared spectrum is a near infrared spectrum or a Fourier transform infrared spectrum.
- the sample is exposed to a coherent light source prior to measuring the infrared spectrum of the sample.
- the infrared spectrum is measured using an infrared spectrometer.
- the infrared spectrometer may be commercially available, for example a ReactIR from Metier Toledo (Columbus, Ohio, USA).
- the infrared spectrometer comprises a laser, a thermal source or a lamp.
- the laser is a multimode diode laser.
- the laser wavelength is from 100 nm to 250,000 nm, from 200 nm to 200,000 nm, from 300 nm to 150,000 nm, from 500 nm to 150,000 nm, from 600 nm to 100,000 nm, from 700 nm to 100,000 nm, or from 700 nm to 90,000 nm.
- the light source is a narrow bandwidth laser with a wavelength of about 1,600 nm, about 1,500 nm, about 1,400 nm, about 1,300 nm, about 1,200 nm, about 1,000 nm, about 900 nm, about 800 nm, about 785 nm, about 750 nm, about 725 nm, about 700 nm, about 675 nm, about 650 nm, about 625 nm, about 600 nm, about 575 nm, about 550 nm, about 532 nm, about 525 nm, or about 500 nm.
- the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- UF/DF skid a fill finish tank or an incubator.
- the bioreactor can have any suitable volume that allows for the cultivation and propagation of biological cells capable of producing therapeutic proteins (such as Stelara® (ustekinumab; Janssen)).
- the volume of the bioreactor can be about 0.5 liters (L) to about 25,000 L.
- the volume of the bioreactor can be less than or equal to about 250 L.
- the volume of the bioreactor can be about 0.5 liters (L) to about 250 L.
- the volume of the bioreactor can be less than or equal to about 50 L.
- the volume of the bioreactor can be about 1 L to about 50 L.
- the voluem of the bioreactor can be less than or equal to about 25 L. In some embodiments, the volume of the bioreactor can be about 1 L to about 25 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 250 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 100 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 50 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 25 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 10 L. In some embodiments, the volume of the bioreactor can be less than or equal to about 5 L.
- the volume of the bioreactor can be less than or equal to about 1 L. In some embodiments, the volume of the bioreactor can be about 1 L. In some embodiments, the volume of the bioreactor can be about 2 L. In some embodiments, the volume of the bioreactor can be about 5 L. In some embodiments, the volume of the bioreactor can be equal to or about 1,000 L. In some embodiments, the volume of the bioreactor can be about 1,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 10,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 1,000 L. In some embodiments, the volume of the bioreactor can be about 2,000 L.
- the volume of the bioreactor can be about 5,000 L. In some embodiments, the volume of the bioreactor can be about 10,000 L. In some embodiments, the volume of the bioreactor can be about 15,000 L. In some embodiments, the volume of the bioreactor can be about 25,000 L.
- the sample is at a temperature from about 2 °C to about 40 °C. In some embodiments, the sample is at a temperature from about 20 °C to about 40 °C. In some embodiments, the preferred temperature is from about 25 °C to about 37 °C. In further embodiments, the sample is at a temperature from about 2 °C to about 20 °C. In yet further embodiments, the sample is at a temparature from about 2 °C to about 10 °C.
- the sample is grown for about 0.5 to about 14 days prior to measuring the infrared spectrum of the sample. In some embodiments, the sample is grown for about 0.5 to about 7 days prior to measuring the infrared spectrum of the sample. In further embodiments, the sample is grown for about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days prior to measuring the infrared spectrum of the sample.
- the infrared spectrum of the sample is compared to the infrared spectrum of a control sample.
- the spectral range of the sample is from about 400 to about 3600 cm' 1 .
- the spectral range of the control sample is from about 400 to about 3600 cm' 1 .
- the infrared spectrum of the sample is monitored over a period of time.
- the monitoring is automated.
- the monitoring is continuous.
- the monitoring is non-invasive and/or non-destructive.
- the sample has been contacted with an antibody to the microbe.
- the sample is contacted with a fluorescent agent or a bioluminescent agent.
- the system further comprises a means for detecting fluorescence of the sample.
- the means for detecting fluorescence is a fluorimeter.
- the system further comprises a means for detecting bioluminescence of the sample.
- the means for detecting bioluminescence is a luminometer.
- the microbe includes but is not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- the measuring of the infrared spectrum is automated.
- the automation is controlled by a central processor, for example a computer.
- the automation is robotic.
- the infrared spectrum is subjected to data analysis.
- data analysis of the infrared spectrum comprises discriminant modeling performed using principal component analysis -X (PCA-X), orthogonal partial least squares (OPLS), k-nearest neighbors (KNN), orthogonal partial least squares discriminant analysis (OPLS-DA), partial least squares discriminant analysis (PLS-DA), or combinations thereof.
- PCA-X principal component analysis -X
- OPLS orthogonal partial least squares
- KNN k-nearest neighbors
- OPLS-DA orthogonal partial least squares discriminant analysis
- PLS-DA partial least squares discriminant analysis
- data analysis comprises a model for a microbe including but not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepacia, Salmonella typhimurium, or Bacillus cereus.
- a model for a microbe including but not limited to Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, Burkholderia cepaci
- data analysis comprises generating one or more models based on the obtained infrared spectrum data that correlate levels of microbe with the obtained infrared spectrum data.
- the one or more models are regression models.
- data analysis is executed by one or more computing devices.
- the infrared spectrometer is connected to one or more data processors and memory into which instructions can be loaded and executed by the data processors.
- the data processors communicate over a network with one or more computing systems (e.g., servers, personal computers, tablets, loT devices, mobile phones, dedicated control units, etc.) which can execute various algorithms or data analysis as described elsewhere herein.
- the computing systems can also act to change one or more operating parameters associated with the bioreactor.
- the bioreactor can also have network connectivity such that it can communicate with one or more remote computing systems which, in turn, can cause one or more operating parameters of the bioreactor to change.
- the method further comprises incubation with D2O for assessment of microbial viability.
- a sample from a cell culture vessel is diverted to a flow cell chamber for infrared analysis.
- the derivatization is performed prior to measuring the infrared spectrum of the sample.
- the sample comprises a eukaryotic cell.
- the eukaryotic cell produces a therapeutic product.
- the therapeutic product may be released into the cell media, where it may be collected.
- the systems provided herein allow for detection of contamination.
- the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.
- the eukaryotic cell is a mouse cell, a Chinese hamster ovary (CHO) cell, or a human cell.
- the cell is a T cell, or a B cell.
- the mouse cell is a mouse Sp2/0 cell.
- the cell is HEK293F.
- the cell is PER.C6.
- the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).
- the sample is not centrifuged prior to measuring the infrared spectrum of the sample.
- the sample is not removed from the bioreactor, harvest tank, chromatography skid, chromatography column, ultrafiltration (UF) skid, diafiltration (DF) skid, UF/DF skid, fill finish tank or incubator prior to measuring the infrared spectrum of the sample.
- the microbe is actively growing.
- Advantages of the systems provided herein include but are not limited to risk reduction of encountering false positive process samples due to QC microbiology lab cross contamination, cost reduction, and combinations thereof.
- Example 1 Provided herewith are Examples illustrating methods and systems comprising Raman spectroscopy. See Example 1. Similar or corresponding Examples may be conducted using infrared spectroscopy, as further illustrated in Example 2.
- Test Microorganism Culture Preparation and Plating Method Test microorganisms (Biomerieux, USA, Bioball or equivalent (Remel Quanti-cult plus)) were resuspended according to manufacturers’ instructions. A target suspension of ⁇ 100 CFU per 0.1 mL were prepared to perform the designated inoculation studies.
- CFUs target colony forming units
- Bioreactor samples were collected at least daily unless noted otherwise and spread plating (0.5 mL) performed in triplicate using a 1 pL inoculating loop or equivalent. A PBS only spread plate was used as the negative control. Samples were incubated at 35-37 °C and inspected daily to measure CFUs.
- the reduced scale bioreactors were either 1.5 or 3.0L scale. Each bioreactor was equipped with pH, dissolved oxygen (DO), and Raman probes. The temperature was controlled at 36.5°C. The DO setpoint was 40%. There was no pH control. Proprietary basal media specific to a monoclonal antibody process was used in all studies. A mouse Sp2/0 cell line was used for cell culture reactors.
- Raman data was collected with a Kaiser Optical Systems RXN2 (Endress Hauser) equipped with the RunTime HMI operating system (version 5), 785 nm excitation laser, and a CCD camera maintained at 40°C.
- a fiber optic cable containing excitation and collection fibers were attached to bioreactors. Acquisition parameters were 10s and 75 accumulations for a total collection time of 12.5 min per scan.
- Each Raman spectrum contained 100-3425 cm' 1 spectral region.
- Spectra were preprocessed using derivatives, normalization, and wavelength selection (425-1800, 2800-3100 cm' 1 ). Wavelength selection involved removing peaks due to the optical window material and regions with no Raman information, as documented from instrument vendor. Data were divided between the calibration sample set (CSS) and calibration test set (CTS). Only known non-contaminated and contaminated spectra were used to develop the model with the CSS. Discriminant modeling was performed using PCA-X, OPLS, and KNNs using SIMCA Version 15.0, SIMCA Version 14.1, or Eigenvector PLS Toolbox Version 8.9.2. Data analysis comprised of models for microbes tested.
- a study is defined as the test microorganism and spiking target. See Figure 1.
- a set of four reduced-scale bioreactors were intentionally spiked with different organisms representing slow and fast growing as well as anaerobic and aerobic according to Tables 1-2.
- One reactor served as the control and was not spiked with a microorganism.
- the remaining three bioreactors were spiked.
- Daily triplicate spread plating of bioreactor samples, daily visual inspection for turbidity, and process parameters were documented. The organisms were selected based on compendial methods.
- One set of experiments focused on media-only bioreactors while the second set were cell-culture bioreactors running the process to simulate the first few days of a perfusion based process. For media-only reactors a low and high CFU target was used while the cell culture reactors used low CFU targets to establish the lower limits of detection.
- FIG. 1 A visual representation of each spiking study with offline parallel plating is shown in FIG. 1.
- FIG. 2 A typical spectrum (before and after pre-processing) during a bioreactor media only and cell culture processes with relevant wavenumber regions highlighted is shown in FIG. 2.
- PCA principal component analyis
- FIGs. 4A-4E A PCA cell culture only model overview is shown in FIGs. 4A-4E.
- An orthogonal partial least squares discriminant analysis (OPLS-DA) cell culture model overview is shown in FIGs. 5A-5H.
- KNN k-nearest neighbors
- a contamination trajectory predicted with the OPLS-DA cell culture model for A) uncontaminated manufacturing scale batch and B) contaminated reduced scale batch from CTSS is shown in FIG. 7.
- FIG. 9A-FIG. 12B Study results using Stelara® (ustekinumab; Janssen) are summarized in FIG. 9A-FIG. 12B.
- FIGs. 9A-9F show a PLS-DA cell culture model overview for Stelara® (ustekinumab; Janssen) with several microbial species tested.
- FIGs. 10A-10C A prediction of OPLS-DA contamination model on CTSS with ambiguous transition spectra for Stelara® (ustekinumab; Janssen) with several microbial species tested is shown in FIGs. 10A-10C.
- FIGs. 11A-11C A prediction of OPLS-DA contamination model on CTSS without ambiguous transition spectra for Stelara® (ustekinumab; Janssen) with several microbial species tested is shown in FIGs. 11A-11C.
- FIGs. 12A-12B A batch trajectory prediction for Stelara® (ustekinumab; Janssen) for A) uncontaminated reduced scale batch and B) contaminated reduced scale batch from CTSS containing Bacillus cereus is shown in FIGs. 12A-12B.
- Test microorganisms Biomerieux, USA, Bioball or equivalent (Remel Quanti-cult plus) are resuspended according to manufacturers’ instructions.
- a target suspension of ⁇ 100 CFU per 0.1 mL is prepared to perform the designated inoculation studies.
- CFUs target colony forming units
- Bioreactor samples are collected at least daily unless noted otherwise and spread plating (0.5 mL) performed in triplicate using a l pL inoculating loop or equivalent. A PBS only spread plate was used as the negative control. Samples were incubated at 35-37 °C and inspected daily to measure CFUs.
- the reduced scale bioreactors are either 1.5 or 3.0L scale. Each bioreactor is equipped with pH, dissolved oxygen (DO), and infrared probes. The temperature is controlled at 36.5°C. The DO setpoint is 40%. pH control may or may not be used. Proprietary basal media specific to a monoclonal antibody process is used in all studies. A mouse Sp2/0 cell line is used for cell culture reactors.
- DO dissolved oxygen
- Cell culture bioreactors are inoculated with the mouse cell line at a target 0.3-0.5 xlO 6 cells/mL. The cell culture is allowed to run for at least 24 hrs prior to spiking microorganisms. The bioreactors are then inoculated with the test microorganism suspension and the time of spiking is recorded.
- Infrared data is collected with an infrared spectrometer equipped with an operating system, an excitation laser and a camera.
- a fiber optic cable containing excitation and collection fibers are attached to bioreactors.
- Spectra are preprocessed using derivatives, normalization, and wavelength selection. Wavelength selection involves removing peaks due to the optical window material and regions with no infrared information, as documented from instrument vendor. Data is divided between the calibration sample set (CSS) and calibration test set (CTS). Only known non-contaminated and contaminated spectra are used to develop the model with the CSS. Discriminant modeling is performed using PCA-X, OPLS, and KNNs using SIMCA Version 15.0, SIMCA Version 14.1, or Eigenvector PLS Toolbox Version 8.9.2. Data analysis comprises of models for microbes tested.
- a study is defined as the test microorganism and spiking target.
- a set of four reduced- scale bioreactors are intentionally spiked with different organisms representing slow and fast growing as well as anaerobic and aerobic according to Tables 1-2.
- One reactor serves as the control and is not spiked with a microorganism.
- the remaining three bioreactors are spiked.
- Daily triplicate spread plating of bioreactor samples, daily visual inspection for turbidity, and process parameters are documented. The organisms are selected based on compendial methods.
- One set of experiments focused on media-only bioreactors while the second set are cell-culture bioreactors running the process to simulate the first few days of a perfusion based process. For media-only reactors a low and high CFU target is used while the cell culture reactors use low CFU targets to establish the lower limits of detection.
- IB A method for continuous monitoring for microbial presence in a sample comprising: measuring an infrared spectrum of the sample.
- 2B The method of embodiment IB, wherein the infrared spectrum of the sample is monitored over a period of time.
- a method for assaying a test agent comprising: adding a test agent to a sample comprising a microbe; and measuring an infrared spectrum of the sample.
- 3C The method of claim 2C, wherein the infrared spectrometer comprises a laser, a thermal source, or a lamp.
- 4C The method of claim 3C, wherein the laser is a multimode diode laser.
- test agent is a compound, a supplement, an antibiotic, a bacteriophage, or a combination thereof.
- 15C The method of any one of claims 1C-14C, wherein the microbe is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, or Bacillus cereus.
- 16C The method of any one of claims 1C-15C, wherein the measuring of the infrared spectrum is automated.
- a system for detecting a microbe in a sample comprising: a means for measuring an infrared spectrum of the sample.
- 6D The system of claim 4D or 5D, wherein the laser wavelength is from 100 nm to 250,000 nm.
- 7D The system of any one of claims 1D-6D, wherein the sample is in a bioreactor, a harvest tank, a chromatography skid, a chromatography column, an ultrafiltration (UF) skid, a diafiltration (DF) skid, a UF/DF skid, a fill finish tank or an incubator.
- UF ultrafiltration
- DF diafiltration
- 17D The system of any one of embodiments 1D-16D, wherein the infrared spectrum is subjected to data analysis.
- 18D The system of any one of embodiments 1D-17D, wherein data analysis comprises generating one or more models based on the obtained infrared spectrum data that correlate levels of microbe with the obtained infrared spectrum data.
- 21D The system of any one of embodiments 1D-20D, wherein the microbe is actively growing.
- IE A system for continuous monitoring for microbial presence in a sample comprising: a means for measuring an infrared spectrum of the sample.
- a system for assaying a test agent comprising: a means for measuring an infrared spectrum of a sample to which a test agent has been added.
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Abstract
L'invention concerne un procédé permettant de détecter un microbe dans un échantillon consistant à mesurer un spectre infrarouge de l'échantillon. Dans certains modes de réalisation, la mesure est automatisée. Dans certains modes de réalisation, la surveillance est continue et/ou non invasive. L'invention concerne également un système permettant de détecter un microbe dans un échantillon comprenant un moyen de mesure d'un spectre infrarouge de l'échantillon. Dans certains modes de réalisation, la mesure est automatisée. Dans certains modes de réalisation, la surveillance est continue et/ou non invasive.
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