Endogenous auto-fluorescent biological markers for assessing a biological parameter of a cell

Abstract

The present application related to the use of endogenous fluorescent biological markers to determine a parameter of a cell in a liquid. Because the techniques provided herein provide accurate results in a relatively short amount of time, the methods described herein can be used to monitor and optimize cell culture online as well determine the presence of a cellular contamination in a cell suspension.

What is claimed is: 
     
         1 . A method of determining if the value of a biological parameter of a cell culture in a liquid changes as a function of time, said method comprising:
 (a) irradiating the cell culture or a sample thereof with a light source emitting a specific excitation wavelength to generate a fluorescent signal from an endogenous auto-fluorescent biological marker;   (b) quantifying, at a specific emission wavelength, the fluorescent signal associated with the endogenous auto-fluorescent biological marker to obtain a fluorescent value;   (c) determining if the fluorescent value of step (b) changes as a function of time in the cell culture; and   (d) determining the biological parameter of the cell culture based on the fluorescent value in real time, wherein a modulation in the fluorescent value indicates a modulation in the value of the biological parameter characterizing the value of the biological parameter (i) as changing as a function of time when the fluorescent value is determined, in step (c), to change as a function of time and (ii) as remaining the same as a function of time when the fluorescent value is determined, in step (c) to remain the same;   
       wherein, when the biological parameter is cellular concentration, the endogenous auto-fluorescent biological marker is associated with:
 riboflavin and has the specific excitation wavelength between about 358 and 378 nm and the specific emission wavelength between about 516 and 536 nm; and/or 
 FAD and has the specific excitation wavelength between about 358 and 378 nm and the specific emission wavelength between about 522 and 542 nm. 
 
     
     
         2 . The method of  claim 1 , wherein the sample of the cell culture is filtered prior to step (a) to generate a filtrate and a retentate. 
     
     
         3 . The method of  claim 1 , wherein the cellular concentration is indicative of a cellular contamination in the cell culture. 
     
     
         4 . A method of determining if the value of a biological parameter of a cell culture in a liquid changes as a function of time, said method comprising:
 (a) irradiating the cell culture or a sample thereof with a light source emitting a specific excitation wavelength to generate a fluorescent signal from an endogenous auto-fluorescent biological marker;   (b) quantifying, at a specific emission wavelength, the fluorescent signal associated with the endogenous auto-fluorescent biological marker to obtain a fluorescent value;   (c) determining if the fluorescent value of step (b) changes as a function of time in the cell culture; and   (d) determining the biological parameter of the cell culture based on the fluorescent value in real time, wherein a modulation in the fluorescent value indicates a modulation in the value of the biological parameter characterizing the value of the biological parameter (i) as changing as a function of time when the fluorescent value is determined, in step (c), to change as a function of time and (ii) as remaining the same as a function of time when the fluorescent value is determined, in step (c) to remain the same;   
       wherein, when the biological parameter is biomass concentration (g/L), the endogenous auto-fluorescent biological marker is associated with:
 tryptamine and has the specific excitation wavelength between about 220 and 240 nm and the specific emission wavelength between about 342 and 362 nm; 
 FAD and has the specific excitation wavelength between about 421 and 441 nm and the specific emission wavelength between about 525 and 545 nm; and/or 
 a combination of riboflavin and FAD and has the specific excitation wavelength between about 442 and 462 nm and the specific emission wavelength between about 522 and 542 nm. 
 
     
     
         5 . The method of  claim 4 , wherein the sample of the cell culture is filtered prior to step (a) to generate a filtrate and a retentate. 
     
     
         6 . The method of  claim 4 , wherein the biomass concentration is a dry biomass concentration. 
     
     
         7 . A method of determining if the value of a biological parameter of a cell culture in a liquid changes as a function of time, said method comprising:
 (a) irradiating the cell culture or a sample thereof with a light source emitting a specific excitation wavelength to generate a fluorescent signal from an endogenous auto-fluorescent biological marker;   (b) quantifying, at a specific emission wavelength, the fluorescent signal associated with the endogenous auto-fluorescent biological marker to obtain a fluorescent value;   (c) determining if the fluorescent value of step (b) changes as a function of time in the cell culture; and   (d) determining the biological parameter of the cell culture based on the fluorescent value in real time, wherein a modulation in the fluorescent value indicates a modulation in the value of the biological parameter characterizing the value of the biological parameter (i) as changing as a function of time when the fluorescent value is determined, in step (c), to change as a function of time and (ii) as remaining the same as a function of time when the fluorescent value is determined, in step (c) to remain the same;   
       wherein, when the biological parameter is a rate of biomass accumulation (h −1 ), the endogenous auto-fluorescent biological marker is associated with:
 NAD(P)H and has the specific excitation wavelength between about 265 and 285 nm and the specific emission wavelength between about 438 and 458 nm; and/or 
 pyridoxine and has the specific excitation wavelength between about 313 and 333 nm and the specific emission wavelength between about 384 and 404 nm. 
 
     
     
         8 . The method of  claim 7 , wherein the sample of the cell culture is filtered prior to step (a) to generate a filtrate and a retentate.