DESC:FIELD OF THE INVENTION

The present invention relates to a method for identification and absolute quantification of analytes in a sample using liquid chromatography coupled with mass spectrometry.
BACKGROUND OF THE INVENTION

Targeted metabolomics refers to profiling and/or quantifying, typically a large set of biochemically characterized analytes present in a sample. It allows qualitative and quantitative analysis of samples by comparing with a standard solution and has myriad applications in the analysis of biological samples such as cell suspension, body fluids, tissue or plant extract, or a chemically defined medium, among others.
For example, targeted metabolomics has immense utility in quantifying analytes in a cell culture medium during large scale production of recombinant proteins.
Mammalian cells engineered to produce recombinant proteins are grown in a chemically defined cell culture medium having essential nutrients required for cell growth, viability, and concomitant improved production of the recombinant protein. The quality and composition of the cell culture medium greatly influences the titer and quality of the recombinant product harvested. Such quality and composition can vary over time during the fermentation process. Such variability can arise due to uptake of nutrients by the dividing cells as well as release of metabolites into the medium. Variability can also arise due to degradation of media components because of light, temperature, and other stress factors. As cells grow, such variability can be dynamic for certain components leading to their faster depletion or rapid accumulation (e.g., toxic metabolites). Clearly, in order to maintain the quality and titer of the recombinant protein, it is essential to continuously and rapidly monitor the concentration of the widest array of analytes (comprising nutrients and metabolites) throughout the fermentation process. This is where, targeted metabolomics for such monitoring bears significance.
Owing to the vast array of analytes dynamically varying in their respective concentration ranges, which need to be measured rapidly and simultaneously, existing approaches depend on a combination of orthogonal methods to accommodate detection of a wider range of concentration of analytes. This limits the utility of existing approaches. Thus, although several orthogonal methods such as thin layer chromatography (TLC), nuclear magnetic resonance (NMR) and gas chromatography coupled with mass spectrometry (GC-MS) can be employed, none of these approaches have been reported to allow rapid analysis or measurement of a large range of analytes over a wider range of concentrations with acceptable sensitivity and accuracy.
Multiple reaction monitoring (MRM) is a sensitive mass spectrometry technique that can aid in quantifying several analytes in a complex mixture. Existing MRM-based methods are limited in terms of the number of analytes that can be analyzed with the requirement of multiple sample injections to measure wider concentration ranges in a single injection (eg. nanomolar to millimolar concentration). This poses as a limitation when the number of analytes to be measured is large, and where measurement has to be performed within a short span of time. Thus, it becomes imperative in an industrial setting to identify and quantify as many analytes as possible present at a wide concentration range, rapidly, accurately, and simultaneously, with minimal sample preparation requirement and run time to ensure superior product attributes.
Specifically in the context of the drug manufacturing industry, such rapid and simultaneous monitoring will aid drug development and manufacturing processes in not only monitoring the media composition performance, but also in deciding on the optimal feeding strategy, optimal harvest time and monitoring of product attributes in real-time.

SUMMARY OF THE INVENTION

Accordingly, the present invention discloses a method for precise identification and absolute quantitation of a wide array of analytes present in a sample at nanomolar to millimolar concentration ranges, by mass spectrometry in a single run. Specifically, present method can, in a single run, simultaneously identify and quantify a large number of cell culture media components including amino acids, amino acid derivatives vitamins, organic acids and sugars or a large number of cellular metabolites using liquid chromatography mass spectrometry (LC-MS) coupled with triple quad detector (QqQ) using multiple reaction monitoring (MRM), present at the said concentration range, with substantial accuracy. The method is capable of distinguishing analytes with same or similar molecular mass, is also suitable across production scales and cell culture process types (eg. fed batch or continuous culture) and has immense industrial utility in guiding the cell culture feeding strategy by profiling analytes intermittently during the fermentation process.
Specifically, in the present method, the standard curve for quantifying the identified analytes is prepared by mixing a known concentration of each analyte, followed by serial dilution and generation of area vs concentration plot. This technique of preparation of standard curve imparts an enhanced level of accuracy and precision to the method.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1: Illustrates representative calibration curves arrived at by employing the parameters disclosed in present invention.
Figure 2: Illustrates trends of select analytes over time.
Figure 3: Illustrates observed readings of cell culture medium sample as obtained by employing disclosed method in comparison with corresponding data obtained by NMR analysis.
DETAILED DESCRIPTION OF THE INVENTION

Definitions:
The term “analyte” as used herein refers to a simple organic component present in the sample. Examples of analyte include amino acids, amino acid derivatives, (eg., serine, threonine, hydroxylysine), vitamins (eg., biotin, pyridoxine), organic acids (eg., malic acid, 4-aminobutyric acid), sugars (eg., glucose, sucrose) or cellular metabolites (eg., uric acid, glutathione) which include by-products released from cells during cell culture process etc.
The term “cell culture medium” as used herein refers to a nutrient medium in which a plant or animal cell can grow. The cell culture medium under analysis of present invention can be fresh medium before the inoculation of cells or medium sampled during or at the end of cell culture process.
The term “cell suspension sample” refers to a sample containing cells suspended in a liquid medium that includes a chemically defined or chemically undefined medium.
The term “percentage recovery” or “% recovery” as used herein refers to the difference between the expected and observed concentration values obtained when a solution bearing analytes at known concentrations is analyzed by employing disclosed method.
The term “qualifier” or “qualifier ion” refers to the daughter ion, the corresponding peak of which, on the chromatogram, along with that of the quantifier ion, is used to identify the respective analyte.
The term “quantifier” or “quantifier ion” refers to the daughter ion which helps in quantifying the analyte. The peak corresponding to the quantifier ion is usually the stronger peak on the chromatogram, which, after integration is used to calculate the concentration of the respective analyte.
The term “sample” as used herein refers to a solution comprising organic analytes derived from natural or synthetic origin. For example, the sample can be a cell suspension, extract from living tissues or a chemically defined medium (eg. cell culture medium).
The term “standard mix” as used herein refers to a solution comprising a known concentration of the analytes being identified and measured using disclosed method. The method may employ one or more standard mix solutions which may vary in composition, dilution factor etc.
The term “substantial accuracy” as used herein refers to percentage recovery within 80-120 %.
Abbreviations:
GC-MS: Gas chromatography coupled with mass spectrometry
HCP: Host cell proteins
LC-MS: Liquid chromatography mass spectrometry
MRM: Multiple reaction monitoring
m/z: Mass-to-charge ratio
NMR: Nuclear magnetic resonance
QqQ: Triple quadrupole detector
RPM: Revolutions per minute
RT: Retention time
TLC: Thin layer chromatography

Detailed description of the embodiments
The present invention discloses a method for precisely identifying and quantifying analytes in a sample by liquid chromatography mass spectrometry (LC-MS) coupled with triple quad detector (QqQ) using multiple reaction monitoring (MRM) wherein the method is capable of rapidly and simultaneously quantifying, in a single injection, a large number of analytes comprising amino acids, amino acid derivatives vitamins, organic acids and sugars or a large number of cellular metabolites which range from nanomolar to millimolar concentration range, with substantial accuracy.
Specifically, in the present method, the standard curves for quantifying the identified analytes is prepared by mixing a known concentration of each analyte, followed by serial dilution and generation of area vs concentration plot. This technique of preparation of standard curve imparts an enhanced level of accuracy and precision to the method.
In comparison, none of the existing methods have reported identification and quantification of such a vast array of analytes with wide coverage in detection of concentration range as disclosed in as short as 17 minutes of runtime.
In an embodiment, the present invention discloses a method for identification and absolute quantification of analytes present at nanomolar to millimolar concentration range in a sample with substantial accuracy, wherein the method comprises:
a) optionally treating the sample to remove cellular debris and proteins;
b) optionally serially diluting the treated sample of step a) to obtain multiple dilutions;
c) separating the components in the sample or the diluted samples of step b) by liquid chromatography;
d) ionizing and fragmenting the separated components to obtain an ionized sample comprising daughter ions;
e) identifying one or more analytes in the ionized sample of step d) with a mass spectrometer by detecting the corresponding quantifier daughter ion and at least one qualifier daughter ion of each analyte and;
f) quantifying the thus identified analyte of step e) with a mass spectrometer using area vs. concentration plotted standard curves generated from a standard mix;
wherein the mass spectrometer is equipped with a triple quadrupole analyzer and employs multiple reaction monitoring with polarity switching and settings as specified in Table 2 and Table 4;
wherein the method is capable of identifying and absolutely quantifying analytes comprising amino acids, amino acid derivatives organic acids, vitamins and sugars.
In another embodiment, the present invention discloses a method for identification and absolute quantification of analytes present at nanomolar to millimolar concentration range in a sample with substantial accuracy, wherein the method comprises:
a) optionally treating the sample to remove cellular debris and proteins;
b) optionally serially diluting the treated sample of step a) to obtain multiple dilutions;
c) separating the components in the sample or the diluted samples of step b) by liquid chromatography;
d) ionizing and fragmenting the separated components to obtain an ionized sample comprising daughter ions;
e) identifying one or more analytes in the ionized sample of step d) with a mass spectrometer by detecting the corresponding quantifier daughter ion and at least one qualifier daughter ion of each analyte and;
f) quantifying the thus identified analyte of step e) with a mass spectrometer using area vs. concentration plotted standard curves generated from a standard mix;
wherein the mass spectrometer is equipped with a triple quadrupole analyzer and employs multiple reaction monitoring with polarity switching and settings as specified in Table 3 and Table 5;
wherein the method is capable of identifying and absolutely quantifying analytes comprising cellular metabolites.
In yet another embodiment, the present invention discloses a method for differentiating two or more analytes having same mass present at nanomolar to millimolar concentration range in a sample with substantial accuracy, wherein the method comprises:
a) optionally treating the sample to remove cellular debris and proteins, if present;
b) optionally serially diluting the treated sample of step a) to obtain multiple dilutions;
c) separating the components in the sample or the diluted samples of step b) by liquid chromatography;
d) ionizing and fragmenting the separated components to obtain an ionized sample comprising daughter ions;
e) differentiating the two or more analytes having same mass using mass spectrometer by comparing the corresponding quantifier ions and at least one qualifier daughter ions of the analytes; and
f) quantifying the two or more analytes having same mass with a mass spectrometer by comparing with the area vs. concentration plotted standard curves of the analytes generated from a standard mix;
wherein the mass spectrometer is equipped with a triple quadrupole analyzer and employs multiple reaction monitoring with polarity switching and settings as specified in Table 2 and Table 4 or Table 3 and Table 5.

In an embodiment, the present invention discloses a method for guiding a cell culture feeding strategy during a cell culture process by identification and absolute quantification of analytes present at nanomolar to millimolar concentration range in a sample with substantial accuracy, wherein the method comprises:
a) optionally treating the sample to remove cellular debris and proteins, if present;
b) optionally serially diluting the treated sample of step a) to obtain multiple dilutions;
c) separating the components in the sample or the diluted samples of step b) by liquid chromatography;
d) ionizing and fragmenting the separated components to obtain an ionized sample comprising daughter ions;
e) identifying one or more analytes in the ionized sample of step d) with a mass spectrometer by detecting the corresponding quantifier daughter ion and at least one qualifier daughter ion of each analyte and;
f) quantifying the thus identified analyte of step e) with a mass spectrometer using area vs. concentration plotted standard curves generated from a standard mix;
wherein the mass spectrometer is equipped with a triple quadrupole analyzer and employs multiple reaction monitoring with polarity switching and settings as specified in Table 2 and Table 4;
wherein the method is capable of identifying and absolutely quantifying analytes comprising amino acids, amino acid derivatives organic acids, vitamins and sugars.

In an embodiment, the present invention discloses a method for guiding a cell culture feeding strategy during a cell culture process by identification and absolute quantification of analytes present at nanomolar to millimolar concentration range in a sample with substantial accuracy, wherein the method comprises:
a) optionally treating the sample to remove cellular debris and proteins, if present;
b) optionally serially diluting the treated sample of step a) to obtain multiple dilutions;
c) separating the components in the sample or the diluted samples of step b) by liquid chromatography;
d) ionizing and fragmenting the separated components to obtain an ionized sample comprising daughter ions;
e) identifying one or more analytes in the ionized sample of step d) with a mass spectrometer by detecting the corresponding quantifier daughter ion and at least one qualifier daughter ion of each analyte and;
f) quantifying the thus identified analyte of step e) with a mass spectrometer using area vs. concentration plotted standard curves generated from a standard mix;
wherein the mass spectrometer is equipped with a triple quadrupole analyzer and employs multiple reaction monitoring with polarity switching and settings as specified in Table 3 and Table 5;
wherein the method is capable of identifying and absolutely quantifying analytes comprising cellular metabolites;

In any of the above-mentioned embodiments, the sample comprises a cell, cell suspension, tissue, tissue extract or a chemically defined cell culture medium devoid of cells.

In any of the above-mentioned embodiments step c) to f) is carried out in about 17 minutes.
In any of the above-mentioned embodiments the standard mix used to generate standard curve of step f) is prepared by mixing a known concentration of each analyte based on their relative abundance in the sample and is optionally diluted.
In any of the above mentioned embodiments, the method is capable of identifying and absolutely quantifying upto 70 analytes comprising amino acids, amino acid derivatives organic acids, vitamins and sugars;in a single run.
In any of the above mentioned embodiments, the method is capable of identifying and absolutely quantifying upto 70 analytes comprising cellular metabolites.in a single run.

EXAMPLES

Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of present invention. The invention will now be described in greater detail by reference to the following non-limiting examples.
The following examples illustrate methods using cell culture medium with or without cells in suspension but, of course, should not be construed as in any way limiting its scope to the type of sample that can be analyzed by disclosed method.
Example 1:
An object of present invention is to develop a method to rapidly identify and absolutely quantify a large number of analytes (typically above about 55) present in a sample by LC-MS multiple reaction monitoring (MRM). Existing MRM-based methods are limited in terms of the number of analytes that can be analyzed with the requirement of multiple sample injections to measure wider concentration ranges in a single injection.
Attempts were made to overcome the said challenges. For this, first, a chemically defined cell culture medium suitable for growth of cells was analyzed with MS parameters provided by the supplier. Experiments were conducted with or without serial dilution of the sample as well as using commercially available or in-house standard mix. The extent of the type of analytes that can be tested was also explored. A standard solution comprising the analytes to be tested was prepared with a fixed concentration range as indicated in Table 1. Results of initial experiments are given in Table 1. None of the optimization steps could achieve detection of nanomolar to millimolar concentration range of analytes using a given set of parameters with limited sample dilution, rapidly.
Trial no. No. of analytes Nature of analytes Optimization step % Recovery Outcome & Comments
1 26 Amino acids Fixed conc. of std. mix (0.005-50µM), Sample diluted multiple times Poor • Only 12 out of 26 analytes detected.
• Poor detection range
• Poor % recovery
2 24 Amino acids Fixed conc. of std. mix (0.005-100 µM), Sample diluted multiple times 50-110% • Multiple sample injections required to accommodate detection of all analytes.
• Poor detection range
• Poor % recovery
3 43 Amino acids, Vitamins, Organic acids & sugars Fixed conc. of std. mix (0.005-80 µM), Optimized interface parameters, dwell time, loop time 70-130% • Despite better % sample recovery, multiple sample injections still required to accommodate detection of all analytes.
• Poor detection range

Example 2:
As described in Example 1, previous attempts resulted in better percentage recovery of analytes. However, the method still required multiple sample injections to be carried out so as to allow detection of all the analytes, with respective concentrations varying from nanomolar to millimolar ranges in the sample.
In an attempt to reduce the number of sample injections required to detect maximum number of analytes present at nanomolar to millimolar concentration range, further experiments were performed. Optimization was performed in the following aspects: preparation of standard mix and MS parameters comprising polarity switching, dwell time, collision energy settings etc. For this, a standard mix was prepared by mixing a defined concentration of each analyte based on their relative abundance in the sample and further optionally serially diluted to accommodate the dynamic concentration ranges of all analytes.
For this, stock solution of each individual analyte was initially prepared. Specified volume of each stock solution based on their relative abundance in the sample to be analyzed was then mixed to prepare the said standard mix. The said standard mix was further optionally diluted serially to about 20 times and subject to liquid chromatography based separation and mass spectrometry detection with triple quad detector (LC-MS/MS-QqQ) by multiple reaction monitoring (MRM) method employing polarity switching and as per parameters laid out in Table 2 and Table 4 (to measure amino acids, amino acid derivatives organic acids, vitamins and sugars) or Table 3 and Table 5 (to measure cellular metabolites). During detection, a quantifier ion and at least one qualifier daughter ion(s) were considered to identify a given analyte. A calibration curve of the various analytes was generated by running the standard mix and the respective quantitiation range of each analyte was also recorded. In general, for generating the calibration curve, the acceptable R2 values were that which were greater than or equal to 0.99, and acceptable % accuracy was 80-120% compared to expected concentration values and with at least 8 data points falling exactly within the calibration curve. Representative calibration curves arrived at by employing the criteria mentioned herein are shown in Figure 1.
The standard mix was optionally, serially diluted and run using the MS parameters as tabulated in Table 2 and Table 4 to measure amino acids, amino acid derivatives organic acids, vitamins and sugars and Table 3 and Table 5 to measure cellular metabolites.
Example 3:
Sample preparation is an optional step which is performed depending upon whether the sample contains cells, proteins or cellular debris. For this, the sample was first centrifuged at 14,500 RPM for 10 minutes. The supernatant was collected and filtered through a 0.22 µm filter. The filtered sample was mixed with acetonitrile solution to remove proteins (eg. HCP) and further centrifuged at 14,500 RPM for 10 minutes. Supernatant was collected and was optionally serially diluted to obtain dilutions such as 1:10, 1:100, 1:1000 etc.
Example 4:
The sample with or without sample preparation as described in Example 3 was subject to liquid chromatography based separation and mass spectrometry detection with triple quad detector (LC-MS/MS-QqQ) by multiple reaction monitoring (MRM) method employing polarity switching and as per parameters laid out in Table 2 and Table 4 (to measure amino acids, amino acid derivatives organic acids, vitamins and sugars) or Table 3 and Table 5 (to measure cellular metabolites). In MRM, the instrument scans through multiple mass windows to detect multiple fragmented ions in parallel.

Table 2: MS settings for analysis of amino acids, amino acid derivatives organic acids, vitamins and sugars
Source Parameters Optimal Value
Interface Temperature 400°C
Nebulizing Gas Flow 3 L/min
Heating Gas Flow 15 L/min
De-solvation Temperature 600°C
Drying Gas Flow 3 L/min
Dwell Time 5-15 msec
Collision Energy -45 to +40 V

Table 3: MS settings for analysis of cellular metabolites
Source Parameters Optimal Value
Interface Temperature 400°C
Nebulizing Gas Flow 3 L/min
Heating Gas Flow 15 L/min
De-solvation Temperature 600°C
Drying Gas Flow 3 L/min
Dwell Time 2-97 msec
Collision Energy -47 to +24 V

Table 4: Table showing retention time (RT) range and m/z ratio of precursor and daughter ions for analysis of amino acids, amino acid derivatives organic acids, vitamins and sugars
SlNo Compound Precursor m/z Daughter ions m/z (Quantifier/Qualifier) RT Range (min) Polarity
1 Cystine 241 73.9 0.226 – 2.226 +
241 151.95 +
2 Asparagine 133.1 87.15 0.260 – 2.260 +
133.1 28.05 +
3 Aspartic acid 134 74.05 0.262-2.262 +
134 88.1 +
4 Serine 105.9 60.1 0.263-2.263 +
5 4-Hydroxyproline 132.1 68.05 0.295-2.295 +
132.1 86.05 +
6 Glycine 75.9 30.15 0.303-2.303 +
7 Threonine 120.1 74.15 0.400-2.400 +
120.1 56.05 +
8 Cysteine 122 76.05 0.406-2.406 +
122 59 +
9 Ornithine 133.1 70.1 0.443-2.443 +
133.1 116.05 +
10 Hydroxylysine 163.1 82.2 0.445-2.445 +
163.1 128.2 +
11 Glutamic acid 147.9 84.1 0.475-2.475 +
147.9 56.1 +
12 Alanine 89.9 44.1 0.479-2.479 +
13 Lysine 147.2 130.1 0.499-2.499 +
147.2 56.1 +
14 Citrulline 176.1 70.05 0.522-2.522 +
176.1 159.05 +
15 Histidine 155.9 110.1 0.546-2.546 +
155.9 56.1 +
16 Proline 116.1 70.15 0.580-2.580 +
116.1 28.05 +
17 2-Aminobutyric acid 104 58.1 0.646-2.646 +
104 43.1 +
18 Arginine 175.2 60.1 0.749-2.749 +
116 +
19 4-Aminobutyric acid 104 68.95 1.066-3.066 +
104 45 +
20 Pyridoxalphosphate 248.1 150.15 1.386-3.386 +
248.1 94.15 +
21 Nicotinic acid 124.05 80.05 1.683-3.683 +
124.05 78.05 +
22 Valine 118 57.1 1.734-3.734 +
118 55.1 +
23 Methionine 149.9 56.1 2.098-4.098 +
149.9 104.1 +
24 Niacinamide 123.1 80.05 2.928-4.928 +
123.1 53.1 +
25 Pantothenic acid 220.1 90.15 3.455-5.455 +
220.1 72.05 +
26 Tyrosine 182.1 136.1 3.598-5.598 +
182.1 91.1 +
27 4-Aminobenzoic acid 138.25 60.1 3.707-5.707 +
138.25 77.1 +
28 Cyanocobalamin 608.8 147.1 3.742-5.742 +
608.8 359.05 +
29 Pyridoxal 160.9 150.05 3.799-5.799 +
160.9 94.15 +
30 Folic acid 442 295.15 3.822-5.822 +
442 176.05 +
31 Isoleucine 132.1 69.15 3.854-5.854 +
132.1 86.2 +
32 Riboflavin 377 243.05 3.926-5.926 +
377 172 +
33 Leucine 132.1 30.05 4.026-6.026 +
132.1 86.05 +
34 Biotin 245.1 226.95 4.054-6.054 +
245.1 97.05 +
35 Pyridoxine 169.9 152.05 4.203-6.203 +
169.9 134.05 +
36 Phenylalanine 166.1 103.1 4.405-6.405 +
166.1 120.1 +
37 Pyridoxamine 169 134.1 5.317-7.317 +
169 152.1 +
169 77.1 +
38 Tryptophan 205.1 188.15 5.637-7.637 +
205.1 146.1 +
39 Taurine 124 79.9 0.054-2.054 _
40 Hexose/Glucose 179.2 89.1 0.154-2.154 _
179.2 59.05 _
41 Threonic acid 135.2 75 0.216-2.216 _
42 Sucrose 341.3 89.05 0.316-2.316 _
341.3 179.1 _
43 Glutamine 145.2 127.15 0.351-2.351 _
145.2 109 _
44 Thiamine 263.05 263.05 0.500-2.500 _
45 Malic acid 133.1 114.95 0.582-2.582 _
133.1 71.15 _
46 Isovaleric acid 100.9 100.9 0.583-2.583 _
47 2-Ketoglutaric acid 145 101.15 0.618-2.618 _
145 57.1 _
48 Butyric acid 87.1 87.1 0.625-2.625 _
49 Ascorbic acid 175.2 115 0.697-2.697 _
175.2 87 _
50 Pyruvic acid 86.9 87.05 0.716-2.716 _
86.9 42.95 _
51 Lactic acid 89.3 89.05 0.900-2.900 _
52 Citric acid 191.2 111.1 1.219-3.219 _
191.2 87.05 _
53 Succinic acid 117.3 73 1.701-3.701 _
117.3 99.05 _
54 2-Hydroxy butyric acid 103.2 103.2 2.061-4.061 _
55 Maleic acid 115.2 70.9 2.597-4.597 _
56 4-Hydroxyphenyl pyruvic acid 179 132 3.639-5.639 _
179 116 _

Table 5: Table showing retention time (RT) and m/z ratio of precursor and daughter ions for analysis of cellular metabolites
S.No. Compound Precursor m/z Product m/z ~RT (min) Polarity
1 Indole-3-Lactic acid 205.8 118.05 6.17 +
130.15
2 Serotonin 177.1 160.2 6.47 +
115.1
3 5'-Methylthioadenosine 298.1 136.1 5.9 +
119.1
4 S-Adenosylhomocysteine 385.1 136.15 4.96 +
134.1
5 Kynurenine 209.1 192.05 5.5 +
94.1
6 Kynurenic acid 190.15 144.1 5.8 +
89.1
7 5-Hydroxytryptophan 221.1 204.05 5.02 +
162.15
8 Fumaric Acid 115 71.1 2.69 _
26.95
9 Hydroxykynurenine 225 208.15 4.67 +
208.15
10 Adenine 136 119.05 4.06 +
60
11 Cytidine 244.1 112.05 3.88 +
95
12 4-Pyridoxic acid 184 148.1 4.5 +
166.1
13 Thymidine 243.1 127.1 4.44 +
127.1
14 Xanthosine 284.9 153.05 4.43 +
135.95
15 Thymine 127.1 110.05 3.96 +
54.05
16 Pipecolic acid 130.1 84.05 2.99 +
56.1
17 Uridine 245 113.05 3.05 +
113.05
18 3-Aminoisobutyric acid 104.05 86.1 2.4 +
30.05
19 5-Glutamylcysteine 251.1 84.1 2.59 +
122.1
20 Deoxyadenosine monophosphate 332.1 136.2 3.09 +
81.1
21 Hypoxanthine 137 110 2.97 +
55.05
22 Cytosine 112 95.05 2.160 +
95.05
23 1-Methylhistidine 170 124.05 1.82 +
83.1
24 Homocysteine 136 90.05 2.2 +
56.05
25 N-Acetylaspartic acid 175.9 134.05 2.11 +
88.05
26 Adenosine monophosphate 348 136.05 2.1 +
97.1
27 Uracil 113 70 3.04 +
28 Argininosuccinic acid 291.1 70.05 1.63 +
116.05
29 2-Aminoadipic acid 162.15 55.15 1.7 +
116.2
30 3-Methylhistidine 170 96.1 1.56 +
109.1
31 Glycyl-glutamine 204 84.1 1.59 +
130.1
32 Guanosine monophosphate 364 152.05 1.84 +
135
33 Cytidine monophosphate 324 112.05 1.46 +
95
34 Inosine monophosphate 349.05 137.1 1.6 +
119.2
35 Cystathionine 223 88.05 1.16 +
134
36 Uridine monophosphate 325.05 97 1.24 +
97
37 O-Phosphoethanolamine 142.1 44.2 1.05 +
44.2
38 3-Phenyllactate 160.05 146.95 5.85 _
103
39 Indole-3-carboxylic acid 160.05 116 6.49 _
40 3-Methyl-2-oxovaleric acid 129.2 85.1 5.37 _
41 4-Hydroxyphenyllactic acid 181.2 163 4.81 -_
135.1
42 Xanthine 151 108 2.97 -_
42
43 Uric acid 167.1 123.95 2.25 -_
96.2
44 Alanyl-glutamine 216 154.05 1.8 -_
198.2
45 Orotic acid 155 111 1.79 -_
42.1
46 Methionine sulfoxide 166 74.1 1.25 +
55.95
47 Deoxycytidine monophosphate 308.1 112.05 1.78 +
95.1
48 3-Aminopropanoic acid 90.05 30.15 1.39 +
72.15
49 5-Oxoproline/Pyroglutamic acid 130.05 56.1 2.32 +
41.1
50 Choline 104.1 60.05 2.06 +
45.1
51 Glutathione 308 179.1 2.24 +
52 Deoxyguanosine 268.1 152.15 4.39 +
135.1
53 Urocanic acid 139.05 93.1 4.4 +
66.1
54 3-Hydroxyanthranilic acid 154.05 80.1 2.55 +
136.1
55 Penicillin G 335.1 176 5.57 +
160
56 Formyl Kynurenine 237 146.05 4.82 +
118.15
57 3-Hydroxyisobutyric acid 103.1 73.05 2.69 _-
73.05
58 Guanine 150 133 2.93 _-
66.1
59 Lipoic acid 205.2 171.1 9.12 _-
127.1
60 Thymidine monophosphate 321.05 194.95 2.6 _-
78.9

Representative cell culture samples from day 0 to day 13 of culture were prepared as in Example 3 and were analyzed as described herein. Trends of select analytes over this time period are shown in Figure 2. Thus, present method serves as a suitable tool for rapid determination of concentration trends of media components over days of cell culture and based on this, determine the feeding strategy. Present method is also helpful in monitoring for unexpected changes in analyte trends, thereby helping to closely monitor and maintain product quality.
Example 5:
To assess the veracity and accuracy of the results obtained, two strategies were adopted. First the observed readings of the analytes were compared with that of expected concentrations by running known samples. Second, the readings by present method were compared with data from an orthogonal method (in this case, NMR spectroscopy).
When the observed reading was compared with expected concentration, the percentage (%) recovery of method was found to be 81-105%. Representative % recovery is shown in Table 6.
Table 6: Percentage recovery obtained with disclosed method
Name Expected (µM) Observed (µM) % Recovery Name Expected (µM) Observed (µM) % Recovery
1 Cystine 7 6.45 92.1 19 4-Aminobutyric acid 7 6.35 90.6
2 Asparagine 14 12.33 88.1 20 Nicotinic acid 7 5.69 81.2
3 Aspartic acid 7 6.69 95.6 21 Valine 14 14.32 102.3
4 Sucrose 14 12.56 89.7 22 Methionine 14 13.4 95.7
5 Threonine 14 12.8 91.5 23 Tyrosine 7 6.15 87.9
6 Cysteine 7 6.24 89.2 24 Folic acid 0.7 0.63 90.6
7 Glutamic acid 7 6.25 89.3 25 Riboflavin 0.7 0.61 86.9
8 Alanine 7 6.22 88.8 26 Biotin 0.7 0.63 90.5
9 Malic acid 28 24.42 87.2 27 Isoleucine 7 6.95 99.3
10 2-Ketoglutaric acid 35 36.8 105.2 28 Leucine 14 14.05 100.3
11 Pyruvic acid 28 26.58 94.9 29 Pyridoxine 0.7 0.6 85.8
12 Ornithine 1.4 1.28 91.3 30 Phenylalanine 7 6.33 90.5
13 Proline 7 6.21 88.7 31 Tryptophan 1.4 1.3 93.2
14 Lysine 14 13.52 96.5 32 Maleic acid 28 24.13 86.2
15 2-Aminobutyric acid 14 13.71 97.9 33 4hydroxy pyruvic acid 28 25.43 90.8
16 Histidine 7 6.41 91.6 34 Isovaleric acid 35 36.15 103.3
17 Arginine 7 6.63 94.7 35 B hydroxy butyric acid 28 24.81 88.6
18 Pyridoxalphosphate 7 6.08 86.9 36 Butyric acid 28 27.55 98.4
Next, observed readings of cell culture medium obtained from disclosed method was compared with that of NMR readings. As can be seen from Figure 3, there was no significant difference between the LC-MS QQQ data of present method and NMR data, indicating that the results obtained by present method is accurate and is comparable with that of data obtained from an orthogonal method compared.
Example 6:
Using present method, it is possible to differentiate analytes having same/similar precursor m/z (and consequently, same/similar molecular mass), owing to the fact that the method records a ‘molecular signature’ of a given analyte by way of recording at least two daughter ions (a quantifier ion and a qualifier ion) for that analyte. Thus, the present invention accommodates identification and quantification of a larger set of analytes, even when the apparent molecular mass is same/similar. A few examples of analytes bearing same/similar mass that can be easily differentiated using present method are listed in Table 7.
Table 7: A few examples of analytes bearing same/similar mass
Compound Precursor m/z
Asparagine 133.1
Ornithine 133.1
Malic acid 133.1
Arginine 175.2
Ascorbic acid 175.2
Alanine 89.9
Lactic acid 89.3
Butyric acid 87.1
Pyruvic acid 86.9
Nicotinic acid 124.05
Taurine 124
Isoleucine 132.1
Leucine 132.1
4-Hydroxyproline 132.1
Glutamine 145.2
2-Ketoglutaric acid 145
2-Aminobutyric acid 104
4-Aminobutyric acid 104
3-Aminoisobutyric acid 104.05
Choline 104.1
Pipecolic acid 130.1
5-Oxoproline/Pyroglutamic acid 130.05
Indole-3-Lactic acid 205.8
Lipoic acid 205.2
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments and examples are therefore to be considered in all respects illustrative rather than limiting the invention described herein.

Date: 24th Day of April, 2024 Signature:____________________
V.R. Srinivas, Ph.D., LL.B
(Head–IPM, Biologics)
For, Dr. Reddy’s Laboratories Ltd. ,CLAIMS:CLAIMS
We claim:
1. A method for identification and absolute quantification of analytes, present at nanomolar to millimolar concentration range in a sample, with substantial accuracy, wherein the method comprises:
a) optionally treating the sample to remove cellular debris and proteins;
b) optionally serially diluting the treated sample of step a) to obtain multiple dilutions;
c) separating the components in the sample or the diluted samples of step b) by liquid chromatography;
d) ionizing and fragmenting the separated components to obtain an ionized sample comprising daughter ions;
e) identifying one or more analytes in the ionized sample of step d) with a mass spectrometer by detecting the corresponding quantifier daughter ion and at least one qualifier daughter ion of each analyte and;
f) quantifying the identified analyte of step e) with a mass spectrometer using area vs. concentration plotted standard curves generated from a standard mix;
wherein the mass spectrometer is equipped with a triple quadrupole analyzer and employs multiple reaction monitoring with polarity switching and settings as specified in Table 2 and Table 4;
wherein the method is capable of identifying and absolutely quantifying analytes comprising amino acids, amino acid derivatives organic acids, vitamins and sugars.
2. A method for identification and absolute quantification of analytes, present at nanomolar to millimolar concentration range in a sample, with substantial accuracy, wherein the method comprises:
a) optionally treating the sample to remove cellular debris and proteins;
b) optionally serially diluting the treated sample of step a) to obtain multiple dilutions;
c) separating the components in the sample or the diluted samples of step b) by liquid chromatography;
d) ionizing and fragmenting the separated components to obtain an ionized sample comprising daughter ions;
e) identifying one or more analytes in the ionized sample of step d) with a mass spectrometer by detecting the corresponding quantifier daughter ion and at least one qualifier daughter ion of each analyte and;
f) quantifying the identified analyte of step e) with a mass spectrometer using area vs. concentration plotted standard curves generated from a standard mix;
wherein the mass spectrometer is equipped with a triple quadrupole analyzer and employs multiple reaction monitoring with polarity switching and settings as specified in Table 3 and Table 5;
wherein the method is capable of identifying and absolutely quantifying analytes comprising cellular metabolites.
3. A method for differentiating two or more analytes having same mass, present at nanomolar to millimolar concentration range in a sample, with substantial accuracy, wherein the method comprises:
a) optionally treating the sample to remove cellular debris and proteins;
b) optionally serially diluting the treated sample of step a) to obtain multiple dilutions;
c) separating the components in the sample or the diluted samples of step b) by liquid chromatography;
d) ionizing and fragmenting the separated components to obtain an ionized sample comprising daughter ions;
e) differentiating the two or more analytes having same mass using mass spectrometer by comparing the corresponding quantifier ions and at least one qualifier daughter ions of the analytes; and
f) quantifying the two or more analytes having same mass with a mass spectrometer by comparing with the area vs. concentration plotted standard curves of the analytes generated from a standard mix;
wherein the mass spectrometer is equipped with a triple quadrupole analyzer and employs multiple reaction monitoring with polarity switching and settings as specified in Table 2 and Table 4 or Table 3 and Table 5.
4. The method as claimed in claim 1, claim 2 or claim 3, wherein step c) to f) is carried out in about 17 minutes.
5. The method as claimed in claim 1, claim 2 or claim 3, wherein the standard mix used to generate standard curve of step f) is prepared by mixing a known concentration of each analyte in the sample and is serially diluted.
6. The method as claimed in claim 1, claim 2 or claim 3 wherein the method is capable of guiding a cell culture feeding strategy during a cell culture process.

Date: 24th Day of April, 2024 Signature:____________________
V.R. Srinivas, Ph.D., LL.B
(Head–IPM, Biologics)
For, Dr. Reddy’s Laboratories Ltd.