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Introduction
Experimental
Pharmaceutical manufacturing is increasingly using biotechnologybased processes for formation of product molecules. Monitoring and
controlling these processes can be extremely challenging due to the
complexity of the cellular mechanisms and pathways that govern cell
growth and product formation. Individual amino acids, carbohydrates,
vitamins, trace metals, and metabolites can have a significant effect
on product quality and yield even when present in low levels.1 Quantification of these individual components can lead to a much greater
understanding of the cell culture process and the underlying cellular
mechanism that is leading to changes in the product. Identification of
these critical individual components and utilization of on-line analytical
methods with sufficient specificity and sensitivity to quantitate them can
lead to feedback control mechanisms that can continuously drive the
cell culture to produce the desired product quality and maximize yield.
This increased level of detailed understanding can be extremely valuable
in the process development stage of a pharmaceutical product when the
optimum process conditions are being identified and fine tuned, as well
as in production when troubleshooting of an unexpected event may
be required.
The complexity of a typical bioreactor, where thousands of molecules
are present2 during normal operation, creates a situation where measurement specificity and low-level detection limits are extremely important. On-line liquid chromatography (LC) is an analytical technique that
is uniquely suited to the challenges of bioreactor monitoring. LC with
integrated pulsed amperometric detection has the capability of directly
analyzing these complex mixtures for many of these important components, such as amino acids and carbohydrates in cell culture media.3
400
12
13
17
11
nC
1
5
23
70
Peaks:
10
10
9
15
14 16
20
25
22
30
35
Minutes
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Isoleucine
Leucine
Methionine
Histidine
Phenylalanine
System
Glutamate
Aspartate
Cystine
Tyrosine
Tryptophan
23
20 21
19
6
15
18
1. Arginine
2. Ornithine
3. Methionine
sulfoxide
4. Glucose
5. Glutamine
6. Asparagine
7. Alanine
8. Threonine
9. Glycine
10. Valine
11. Hydroxyproline
12. Serine
13. Proline
24
40
45
50
55
60
65
22981-01
11
Typical cell culture
10
9
8
7
6
5
4
3
2
1
0
24
48
72
96
120
144
168
192
216
250
200
150
24
48
72
96
120
144
168
192
288
312
50
264
26718
100
240
350
300
Monitoring Product Attributes, Media Components, and Metabolites with On-Line Liquid Chromatography
Acknowledgement
1800
The author gratefully acknowledges the data provided for this presentation by Ms. Tina Larson of the Genentech Process Development group
in South San Francisco, CA, USA.
1500
1200
References
900
600
300
0
24
48
72
96
120
144
168
192
Conclusions
Integrated pulsed amperometric detection provides an excellent detection method for quantification of amino acids and carbohydrates in
bioreactors without pre- or post-column derivatization. This detection
method was used in an on-line LC to provide automated analysis of
19 amino acids and glucose in a mammalian cell culture bioreactor
approximately once per hour. The on-line amino acid values compared favorably with off-line analysis methods except for high levels
of glutamine, which occurred at the beginning of a bioreactor run, and
alanine, which was not well resolved from glucose at high glucose
concentrations. The additional data generated by the on-line LC
provided increased understanding of metabolism differences across cell
lines with far less labor than would have been required using off-line
analysis. The increased amount of data allowed derived values, such
as amino acid uptake rate, to be predicted with increased accuracy. The
higher data frequency also made transient events that occurred in the
bioreactor much more readily apparent.
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