Christian Priesnitz

Elmar Heinzle

J Niklas

Jens Niklas

JO Krömer

Thomas Rose

V Blanchard

Volker Sandig

BMC Proceedings

English

PubMed

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MEETING ABSTRACT Open AccessProducer vs. parental cell – metabolic changesand burden upon a1-antitrypsin production inAGE1.HN®Jens Niklas1*, Christian Priesnitz1, Volker Sandig2, Thomas Rose2, Elmar Heinzle1From 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based TechnologiesVienna, Austria. 15-18 May 2011BackgroundThe human designer cell line AGE1.HN® represents apromising production system for biopharmaceuticals,particularly for those needing human-type post-transla-tional modifications [1,2]. For further rational improve-ment of the cell line and the cultivation process adetailed understanding of the metabolism and metabolicchanges during glycoprotein production is desirable.Metabolism and metabolic burden upon production ofa1-antitrypsin (A1AT) were analyzed by comparing par-ental cells and a derived clone, AGE1.HN.AAT. Thequestions addressed were (i), which changes occur incell growth and metabolism upon A1AT production and(ii), what are specific cellular properties distinguishingthe producer clone AGE1.HN.AAT from the parentalcell population.Materials and methodsCultivation and analysisCultivations of the human neuronal cell line AGE1.HN®(ProBioGen AG, Berlin, Germany) were carried out inshake flasks (Corning, NY, USA). Extracellular metabo-lites were quantified using different HPLC methods[3,4]. A1AT was quantified using an activity assay. Iden-tification of extracellular proteins was done usingMALDI-ToF/ToF MS (Applied Biosystems). Biomasscomponents were quantified using classical biochemicalmethods.Model for A1AT productionModel for A1AT production was set up using the KyotoEncyclopedia of Genes and Genomes (http://www.kegg.com) pathway database for Homo sapiens.Metabolic flux analysisFluxes were estimated using standard methods [1,5] byincorporating extracellular and anabolic rates usingMatlab R2007b (The Mathworks, Natick, MA, USA).ResultsCell growth was similar in the first 5 days. After 5 daysAGE1.HN.AAT cells were growing less than the paren-tal cell line and the viability dropped faster. After 2 daysdry weight was slightly higher in the parental cell line.A1AT production was low in the first 2 days and thenincreasing up to a final concentration of ~0.4 g/l in theend of the cultivation. The total cell mass was increasingsimilarly in the first 4 days. After 4 days AGE1.HN.AATcell mass remained constant whereas the extracellularprotein concentration was increasing. This was differentin the AGE1.HN parental cells where the cell mass wasstill increasing after 4 days and the increase in totalextracellular protein mass was less. Intracellular proteinconcentration was decreasing in AGE1.HN.AAT after 4days. Specific cellular proteins might be degraded uponnutrient deprivation to maintain the production of therecombinant glycoprotein A1AT. This was clearly differ-ent in the parental cell line. Fractions of the biomassconstituents RNA, lipid and phosphatidylcholine wereincreased in AGE1.HN.AAT. The total mass of the ana-lyzed components in the end of the cultivation wassimilar in the cultivations of both cell lines being around2.5 g/l. The final A1AT concentration which was 0.4 g/lwas ~30 % of the total protein in the culture.* Correspondence: j.niklas@mx.uni-saarland.de1Biochemical Engineering Institute, Saarland University, 66123 Saarbrücken,GermanyFull list of author information is available at the end of the articleNiklas et al. BMC Proceedings 2011, 5(Suppl 8):P85http://www.biomedcentral.com/1753-6561/5/S8/P85© 2011 Niklas et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.Similar time courses for most extracellular metaboliteswere observed. Glycine and glutamate production washigher in AGE1.HN.AAT whereas uptake of arginineand aspartate as well as alanine production were lower.Glutamine was consumed in the cultivations of both celllines at ~4 days which resulted also in reduced growth.Especially the increase in glycine and glutamate produc-tion points to differences in C1 metabolism and cellularnucleotide demand.The differences that can be seen in the metabolismupon production of the glycoprotein were explainedby using an appropriate model of the anabolicdemand needed to produce active A1AT. The wholecellular production process of a glycoprotein includesits expression (transcription), synthesis of the aminoacid chain (translation), posttranslational modificationin endoplasmic reticulum (ER) and Golgi apparatus(Golgi) and secretion of the mature protein (Figure 1).Metabolite demand for A1AT production was finallysimulated. These results indicate that one couldexpect an increase in the production of glutamate andglycine with increasing intracellular nucleotidedemand which was observed for AGE1.HN.AAT; thisis caused by differences in cellular composition of theproducer, partly originating from recombinant A1ATproduction.The differences that were found in the biomass com-position between both cell lines were included in ametabolic network model and intracellular metabolicfluxes were calculated for both cell lines. Fluxes in cen-tral energy metabolism (glyolysis, TCA cycle) were simi-lar. Differences were observed in C1 and glutamatemetabolism including changes in activities of severaltransaminases. The observed changes in metabolic fluxreflect the anabolic differences between producer andparental cells.Figure 1 Schematic presentation of the glycoprotein production process in mammalian cells.Niklas et al. BMC Proceedings 2011, 5(Suppl 8):P85http://www.biomedcentral.com/1753-6561/5/S8/P85Page 2 of 3ConclusionsThis improved understanding of the metabolism andcellular changes during glycoprotein production sup-ports the identification of targets for further improve-ment of (i) cell line, e.g., by genetic modifications and(ii) cultivation process, e.g., by improved feeding strate-gies. The presented data indicate that nucleotide andlipid metabolism might be interesting targets for furtherengineering of the AGE1.HN® cell line.Author details1Biochemical Engineering Institute, Saarland University, 66123 Saarbrücken,Germany. 2ProBioGen AG, 13086 Berlin, Germany.Published: 22 November 2011References1. Niklas J, Schräder E, Sandig V, Noll T, Heinzle E: Quantitativecharacterization of metabolism and metabolic shifts during growth ofthe new human cell line AGE1.HN using time resolved metabolic fluxanalysis. Bioprocess Biosyst Eng 2011, 34(5):533-545.2. Blanchard V, Liu X, Eigel S, Kaup M, Rieck S, Janciauskiene S, Sandig V,Marx U, Walden P, Tauber R, Berger M: N-glycosylation and biologicalactivity of recombinant human alpha1-antitrypsin expressed in a novelhuman neuronal cell line. Biotechnol Bioeng 2011, 108:2118-2128.3. Niklas J, Noor F, Heinzle E: Effects of drugs in subtoxic concentrations onthe metabolic fluxes in human hepatoma cell line Hep G2. Toxicol ApplPharmacol 2009, 240(3):327-336.4. Krömer JO, Fritz M, Heinzle E, Wittmann C: In vivo quantification ofintracellular amino acids and intermediates of the methionine pathwayin Corynebacterium glutamicum. Anal Biochem 2005, 340(1):171-173.5. Niklas J, Heinzle E: Metabolic Flux Analysis in Systems Biology ofMammalian Cells. Adv Biochem Eng Biotechnol 2011, DOI: 10.1007/10_2011_99. [Epub ahead of print]..doi:10.1186/1753-6561-5-S8-P85Cite this article as: Niklas et al.: Producer vs. parental cell – metabolicchanges and burden upon a1-antitrypsin production in AGE1.HN®®.BMC Proceedings 2011 5(Suppl 8):P85.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitNiklas et al. BMC Proceedings 2011, 5(Suppl 8):P85http://www.biomedcentral.com/1753-6561/5/S8/P85Page 3 of 3

Producer vs. parental cell – metabolic changes and burden upon α1-antitrypsin production in AGE1.HN®

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MEETING ABSTRACT Open Access
Producer vs. parental cell – metabolic changes
and burden upon a1-antitrypsin production in
AGE1.HN®
Jens Niklas1*, Christian Priesnitz1, Volker Sandig2, Thomas Rose2, Elmar Heinzle1
From 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies
Vienna, Austria. 15-18 May 2011
Background
The human designer cell line AGE1.HN® represents a
promising production system for biopharmaceuticals,
particularly for those needing human-type post-transla-
tional modifications [1,2]. For further rational improve-
ment of the cell line and the cultivation process a
detailed understanding of the metabolism and metabolic
changes during glycoprotein production is desirable.
Metabolism and metabolic burden upon production of
a1-antitrypsin (A1AT) were analyzed by comparing par-
ental cells and a derived clone, AGE1.HN.AAT. The
questions addressed were (i), which changes occur in
cell growth and metabolism upon A1AT production and
(ii), what are specific cellular properties distinguishing
the producer clone AGE1.HN.AAT from the parental
cell population.
Materials and methods
Cultivation and analysis
Cultivations of the human neuronal cell line AGE1.HN®
(ProBioGen AG, Berlin, Germany) were carried out in
shake flasks (Corning, NY, USA). Extracellular metabo-
lites were quantified using different HPLC methods
[3,4]. A1AT was quantified using an activity assay. Iden-
tification of extracellular proteins was done using
MALDI-ToF/ToF MS (Applied Biosystems). Biomass
components were quantified using classical biochemical
methods.
Model for A1AT production
Model for A1AT production was set up using the Kyoto
Encyclopedia of Genes and Genomes (http://www.kegg.
com) pathway database for Homo sapiens.
Metabolic flux analysis
Fluxes were estimated using standard methods [1,5] by
incorporating extracellular and anabolic rates using
Matlab R2007b (The Mathworks, Natick, MA, USA).
Results
Cell growth was similar in the first 5 days. After 5 days
AGE1.HN.AAT cells were growing less than the paren-
tal cell line and the viability dropped faster. After 2 days
dry weight was slightly higher in the parental cell line.
A1AT production was low in the first 2 days and then
increasing up to a final concentration of ~0.4 g/l in the
end of the cultivation. The total cell mass was increasing
similarly in the first 4 days. After 4 days AGE1.HN.AAT
cell mass remained constant whereas the extracellular
protein concentration was increasing. This was different
in the AGE1.HN parental cells where the cell mass was
still increasing after 4 days and the increase in total
extracellular protein mass was less. Intracellular protein
concentration was decreasing in AGE1.HN.AAT after 4
days. Specific cellular proteins might be degraded upon
nutrient deprivation to maintain the production of the
recombinant glycoprotein A1AT. This was clearly differ-
ent in the parental cell line. Fractions of the biomass
constituents RNA, lipid and phosphatidylcholine were
increased in AGE1.HN.AAT. The total mass of the ana-
lyzed components in the end of the cultivation was
similar in the cultivations of both cell lines being around
2.5 g/l. The final A1AT concentration which was 0.4 g/l
was ~30 % of the total protein in the culture.
* Correspondence: j.niklas@mx.uni-saarland.de
1Biochemical Engineering Institute, Saarland University, 66123 Saarbrücken,
Germany
Full list of author information is available at the end of the article
Niklas et al. BMC Proceedings 2011, 5(Suppl 8):P85
http://www.biomedcentral.com/1753-6561/5/S8/P85
© 2011 Niklas et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Similar time courses for most extracellular metabolites
were observed. Glycine and glutamate production was
higher in AGE1.HN.AAT whereas uptake of arginine
and aspartate as well as alanine production were lower.
Glutamine was consumed in the cultivations of both cell
lines at ~4 days which resulted also in reduced growth.
Especially the increase in glycine and glutamate produc-
tion points to differences in C1 metabolism and cellular
nucleotide demand.
The differences that can be seen in the metabolism
upon production of the glycoprotein were explained
by using an appropriate model of the anabolic
demand needed to produce active A1AT. The whole
cellular production process of a glycoprotein includes
its expression (transcription), synthesis of the amino
acid chain (translation), posttranslational modification
in endoplasmic reticulum (ER) and Golgi apparatus
(Golgi) and secretion of the mature protein (Figure 1).
Metabolite demand for A1AT production was finally
simulated. These results indicate that one could
expect an increase in the production of glutamate and
glycine with increasing intracellular nucleotide
demand which was observed for AGE1.HN.AAT; this
is caused by differences in cellular composition of the
producer, partly originating from recombinant A1AT
production.
The differences that were found in the biomass com-
position between both cell lines were included in a
metabolic network model and intracellular metabolic
fluxes were calculated for both cell lines. Fluxes in cen-
tral energy metabolism (glyolysis, TCA cycle) were simi-
lar. Differences were observed in C1 and glutamate
metabolism including changes in activities of several
transaminases. The observed changes in metabolic flux
reflect the anabolic differences between producer and
parental cells.
Figure 1 Schematic presentation of the glycoprotein production process in mammalian cells.
Niklas et al. BMC Proceedings 2011, 5(Suppl 8):P85
http://www.biomedcentral.com/1753-6561/5/S8/P85
Page 2 of 3
Conclusions
This improved understanding of the metabolism and
cellular changes during glycoprotein production sup-
ports the identification of targets for further improve-
ment of (i) cell line, e.g., by genetic modifications and
(ii) cultivation process, e.g., by improved feeding strate-
gies. The presented data indicate that nucleotide and
lipid metabolism might be interesting targets for further
engineering of the AGE1.HN® cell line.
Author details
1Biochemical Engineering Institute, Saarland University, 66123 Saarbrücken,
Germany. 2ProBioGen AG, 13086 Berlin, Germany.
Published: 22 November 2011
References
1. Niklas J, Schräder E, Sandig V, Noll T, Heinzle E: Quantitative
characterization of metabolism and metabolic shifts during growth of
the new human cell line AGE1.HN using time resolved metabolic flux
analysis. Bioprocess Biosyst Eng 2011, 34(5):533-545.
2. Blanchard V, Liu X, Eigel S, Kaup M, Rieck S, Janciauskiene S, Sandig V,
Marx U, Walden P, Tauber R, Berger M: N-glycosylation and biological
activity of recombinant human alpha1-antitrypsin expressed in a novel
human neuronal cell line. Biotechnol Bioeng 2011, 108:2118-2128.
3. Niklas J, Noor F, Heinzle E: Effects of drugs in subtoxic concentrations on
the metabolic fluxes in human hepatoma cell line Hep G2. Toxicol Appl
Pharmacol 2009, 240(3):327-336.
4. Krömer JO, Fritz M, Heinzle E, Wittmann C: In vivo quantification of
intracellular amino acids and intermediates of the methionine pathway
in Corynebacterium glutamicum. Anal Biochem 2005, 340(1):171-173.
5. Niklas J, Heinzle E: Metabolic Flux Analysis in Systems Biology of
Mammalian Cells. Adv Biochem Eng Biotechnol 2011, DOI: 10.1007/
10_2011_99. [Epub ahead of print]..
doi:10.1186/1753-6561-5-S8-P85
Cite this article as: Niklas et al.: Producer vs. parental cell – metabolic
changes and burden upon a1-antitrypsin production in AGE1.HN®®.
BMC Proceedings 2011 5(Suppl 8):P85.
Submit your next manuscript to BioMed Central
and take full advantage of: 
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at 
www.biomedcentral.com/submit
Niklas et al. BMC Proceedings 2011, 5(Suppl 8):P85
http://www.biomedcentral.com/1753-6561/5/S8/P85
Page 3 of 3


C: In vivo quantification of intracellular amino acids and intermediates of the methionine pathway in Corynebacterium glutamicum. Anal Biochem

Heinzle E: Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2. Toxicol Appl Pharmacol

Heinzle E: Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis. Bioprocess Biosyst Eng

Metabolic Flux Analysis in Systems Biology of Mammalian Cells. Adv Biochem Eng Biotechnol

N-glycosylation and biological activity of recombinant human alpha1-antitrypsin expressed in a novel human neuronal cell line. Biotechnol Bioeng

Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3285021

Producer vs. parental cell – metabolic changes and burden upon α1-antitrypsin production in AGE1.HN®

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