Application Note

December 16, 2022
Keywords or phrases
Spent Media Analytics, Media Optimization, MediaDevelopment, Media Benchmarking, amino acid analysis,vitamin analysis, trace element analysis

How Spent Media Analytics Can Support
Process Optimization
Dr. Tim Steffens
Sartorius Xell GmbH, Alte Verler Str. 1, 33689 Bielefeld, Germany
Correspondence
Email: tim.steffens@sartorius.com

Abstract
Cell culture involves growth of cells in an artificial environment. Cell culture medium is a liquid designed to support thegrowth of cells in this environment. Usually, it contains a mixture of an energy source like carbohydrates in the form of sugars;

 amino acids since they are essential building blocks of proteins; fatty acids and lipids are particularly important in serum-free media;
 inorganic salts to maintain osmotic balance;

 vitamins and trace elements essential for growth and proliferation of cells;
 buffering system to maintain the pH in the physiological range.

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Introduction

Concentration [%]Spent media is the media that remains after cell cultivation.
Nutrient depletion and metabolite accumulation occur in themedium during cell growth (Figure 1). Additionally, the target
product also accumulates which, in certain circumstances,
can also become toxic if concentrations become too high.

At Sartorius Xell, we offer spent media analysis in four
service tiers, ranging from basic insights into full process
understanding (Table 1):
1. Analysis of only primary parameters like pH value,osmolality, glucose, lactate, glutamine and ammonium.
2. Analysis of essential parameters like amino acids,dipeptides, and vitamins.
3. Measurement and analysis of trace elements and ions for acomprehensive understanding of the bioprocess.
4. Measurement of additional features such as organic acids,
polyamines, for in-depth understanding of the bioprocess.
Table 1: Spent Media Analysis for Various Components.

Nutrients
Metabolites

Ions and Elements

Equipment

Trace Elements

ICP-MS

Phospate

Multimodereader

Ammonium

Multimodereader

Amino Acids

Time [d]Free Amino Acids

UHPLC-DAD

Figure 1: Exemplary Change in Nutrient andMetabolite Levels During Cell Cultivation.

Total Amino Acids

UHPLC-DAD

Spent media analysis is the study of the used media over thecourse of the production process. The information gatheredfacilitates the selection of the right cell culture medium andfeed combination, as well as the development of suitable
feeding strategies. It is a useful tool that enables us to gain
insight into the metabolic processes as well as to track andunderstand changes in medium composition.

Cell Culture Process Formats
A batch process is inoculated with the medium of choice andno further nutrients are added during the cultivation.
In a fed-batch process, additional substrates or supplements
are added to increase process duration, cell density andproductivity. The feed supplement can be added in bolus orcontinuous mode. The feeding strategy should be adapted
to the cell’s requirements which can be initially determinedusing spent media analysis.
Continuous processes can be performed with (perfusionmode) and without cell retention (chemostat mode). Freshnutrients are continuously added to the culture while spentmedium (and cells) are harvested at the same time. With thisprocess strategy, toxic metabolites can be removed and theprocess is especially suited for unstable products.
2Amines and Peptides
Dipeptides

UHPLC-MS/MS

GlutathioneUHPLC-MS/MS

Polyamines (Putrescine,Spermidine, Spermine)

UHPLC-MS/MS

Organic Acids
Tartaric acid, Malate, Malonic acid,Citrate, Pyruvate, Succinate,
KetoGlutaric acid, Fumarate,
Maleic acid

UHPLC-MS

Vitamins
Ascorbate

UHPLC-MS/MS

Water-soluble Vitamins

UHPLC-MS/MS

Vitamin E (Tocopherol)

UHPLC-DAD

Pyridoxal Phosphate

UHPLC-DAD

SugarsGlucoseChip-Sensor

Lactate

Chip-Sensor

SucroseChip-Sensor

Methods
Since different mammalian cells have different features, theculture media should be designed for optimal growth of eachunique cell type or clone. Challenges occur when the composition of the cell culture medium does not match the cells’
requirements. An ill-suited medium could underfeed or overfeed the cells. The depletion of nutrients and the accumulation of growth inhibitory metabolites can cause cessation ofcell growth. Oversupply of substrates can lead to unwanted
overflow metabolism and inefficient use of provided nutrients. In addition, accumulating waste metabolites negatively
impact process parameters and performance of cultures.

CHO GS cells were cultured in TCX10D medium in one batchprocess and four fed-batch conditions. Glucose, lactate,asparagine, and serine levels were measured in spent media
across the different production modes.

Therefore, regular monitoring of changes in sugars, metabolites, vitamins, and amino acids is a valuable way to select theappropriate medium, optimize feed strategies, and determine harvest times. Together, these modifications promotehigh cell viability, maximize production titer, and ensure highproduct quality.
In this application note, we demonstrate how monitoring
just four media components (glucose, lactate, serine, andasparagine) can inform an optimization strategy that leads
to significant improvements in the overall productivity of abioprocess.

3

Results
Analysis of Glucose and Lactate Metabolism
As glycolysis proceeds, lactate levels increase in both batch
and fed-batch processes, peaking after around four days inculture (Figure 2A and B). This is due to a metabolic switch,
called the lactate shift, where the cells transition from a
lactate producing to a lactate consuming culture, resulting
in the subsequent decrease in lactate levels at day six. The
lactate shift is a desirable characteristic, as the accumulationof lactate would limit cell growth, but switching to lactate
consumption would prevent acidification of the medium andnot slow cell growth.

AB

Batch

Fed-Batch

6655Glucose [g/L]

Glucose [g/L]

Typically, mammalian cells are glycolytic and will produce
lactate from glucose in the medium. Figure 2A demonstrates
that glucose levels gradually decrease and are eventuallydepleted in the batch processes. Identifying this limitationof glucose by spent media analysis facilitates the design ofan optimized feeding strategy for a fed-batch process where
feed supplements are added periodically, limiting the depletion of glucose in the media (Figure 2B).

43210432102468101214016024Time (Day)
Batch

D
2.5

2.0

2.0

1.5
1.0
0.5
0

24

68

10Time (Day)10121416121416Fed-Batch

2.508Time (Day)

Lactate [g/L]

Lactate [g/L]C

6121416

1.5
1.0
0.5
002468

10Time (Day)

Figure 2: Spent Media Analysis of Glucose (A, B) and Lactate (C, D) in a Batch and Several Fed-Batch Processes.

4Analysis of Amino Acid Levels
Analysis of select amino acids from spent media from thebatch process showed that both asparagine (Figure 3A)
and serine (Figure 3C) were depleted by day six and eight,
respectively. Fed-batch supplementation strategies wereable to recover serine levels better than asparagine (Figure3B and 3D). The fed-batch strategy (dark blue) that reduced
the asparagine and serine depletion also showed higher
productivity (Figure 4B), highlighting the value that spent
media analytics can bring to process optimization.

BBatchFed-Batch

7766Asparagine [mM]

Asparagine [mM]A

5432100246810121454321016024Time (Day)Batch

D65543210246810Time (Day)101214161416

Fed-Batch

608Time (Day)Serine [mM]

Serine [mM]C

612141643210024681012Time (Day)

Figure 3: Spent Media Analysis of Amino Acids Asparagine (A, B) and Serine (C, D) in a Batch and Several Fed-Batch Processes.

5Overall Improvements in Productivity
Analysis of the spent media for productivity (measured bybiomass, Figure 4B) and antibody titer (Figure 4C) show thatthe highest cellular productivity was achieved in fed-batchprocess 1 (Figure 4B, dark pink). However, antibody titer wasmaximized with fed-batch process 4 (Figure 4C, dark blue).
A100
90
2080

BatchFed-Batch 1Fed-Batch 2Fed-Batch 3Fed-Batch 4

157060501040Viability [%]Viable Cell Density [10E6 Cells/mL]

2530
520
1000123456789101112131415160Time (Day)
CBBatchFed-Batch 1
Fed-Batch 2

121086420

Process Strategies

BatchFed-Batch 1
Fed-Batch 2

1800

Productivity [pg/(cell × day)]

Productivity [pg/(cell × day)]14Fed-Batch 3
Fed-Batch 4

1600
1400
1200
1000
800
600
4002000

Process Strategies

Figure 4: Cell Density as (A) Well as Titer and Productivity (B, C) Measures for Various Feed Strategies.

6Fed-Batch 3
Fed-Batch 4

Discussion
The data shown here demonstrates that modification of thefeeding strategy by monitoring glucose, lactate, asparagineand serine levels lead to an increase in overall process yield.
Both cell density and titer were increased following modifications, and cell-specific productivity in the modified process
was maintained at 20% increment compared to a standard
batch process. This shows how spent media analysis can aid
in delivering a significant titer increase.
Only glucose, lactate, asparagine, and serine levels weremeasured for the purpose of this study. A full panel of spent
media analysis for samples taken all throughout the production run provides deeper insights to optimize the feed andmaximize bioprocess yield.
Using our long-standing experience in cell culture media
development we have established fast and robust methods
for measuring the concentrations of a variety of components
in cell culture media with our state-of-the-art equipment(Table 1).

Germany
Sartorius Stedim Biotech GmbH
August-Spindler-Strasse 1137079 Goettingen
Phone +49 551 308 0Sartorius Xell GmbH
Waldweg 2133758 Schloss Holte-StukenbrockPhone +49 521 96989 200	For more information, visit
www.sartorius.com

Specifications subject to change without notice.
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