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Development of a protein-free fed-batch process for NS0 cells: studies on regulation of proliferation
KTH, School of Biotechnology (BIO).
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The overall objective of this study was to investigate how NS0 cell proliferation is regulated in protein-free media. The hypothesis was that during the adaptation to growth factor-free media, animal cell lines start to produce their own autocrine growth factors to support proliferation, and after some time in a culture the effects of these factors are lost which results in cessation of proliferation. A chemically defined, protein-free and animal component-free medium was developed for the NS0 cells. This medium was comprised of a basal hybridoma medium to which phosphatidyl¬choline, cholesterol, β-cyclodextrin, ferric citrate and amino acids were added. A fed-batch process was then developed in this medium. The feed profile was optimised in a step-wise manner with a final feed solution containing glucose, glutamine, lipids, amino acids, vitamins, sodium selenite and ethanolamine. Specifically, supplementation with lipids (cholesterol) had a drastic effect on cell growth. Calcium, magnesium and potassium were not depleted during culture and a feed containing also iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield further. More than 8 x 106 viable cells mL-1 and 600 mg antibody L-1 was obtained in the final fed-batch. This corresponded to a 4.3-fold increase in viable cell yield and an 11.4-fold increase in product yield compared to bioreactor batch culture when the dilution of the fed-batch culture was also accounted for. The presence of autocrine growth factors in NS0 cell cultures was initially investigated by studying the effects of conditioned medium (CM). Concentrated CM had a significant positive effect on cell growth and part of this effect could be attributed to factor(s) eluting from a gel-filtration column at 20-25 kDa. In the search for cell-derived factors affecting cell growth the following proteins were identified as released/secreted by the NS0 cells; cyclophilin A, cyclophilin B, cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerise, macrophage migration inhibitory factor (MIF), β2-microglobulin, niemann pick type C2, secretory leukocyte protease inhibitor (SLPI), thioredoxin-1, TNF-α, tumour protein translationally controlled-1 and ubiquitin. Zymogram electrophoresis further identified aspartic acid, papain-like cysteine (including cathepsin L) and serine protease activity in the CM. Pro/cathepsin L, CypB, EGF, IFN-α/β/γ, IGF-I/II, leukaemia inhibitory factor, IL-6, IL-11, IL-25, MIF, oncostatin M, TGF-β and TNF-α were excluded as involved in autocrine regulation of NS0 cell proliferation. The serine protease activity was suggested to affect the cells negatively and since the serine protease inhibitor SLPI is also present in NS0 CM, a balance in serine protease activity may be crucial for optimal cell growth. Further, the receptor gp130, known to be associated with myeloma cell growth, was shown to be essential for NS0 cell proliferation as demonstrated by siRNA gene silencing. The results suggested that autocrine regulation of proliferation in NS0 cell cultures involves the receptor subunit gp130.

Place, publisher, year, edition, pages
Stockholm: KTH , 2006. , 44 p.
Keyword [en]
NS0 myeloma cells, protein-free medium, fed-batch, conditioned medium, autocrine growth factors, abortive proliferation, protease activity, aprotinin, gp130
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4162ISBN: 91-7178-466-7 (print)OAI: oai:DiVA.org:kth-4162DiVA: diva2:10996
Public defence
2006-11-17, FB54, AlbaNova, Roslagstullsbacken 21, Stockholm, 09:00
Opponent
Supervisors
Note
QC 20100920Available from: 2006-11-01 Created: 2006-11-01 Last updated: 2010-09-20Bibliographically approved
List of papers
1. Defined protein-free NS0 myeloma cell cultures: stimulation of proloferation by conditioned medium factors
Open this publication in new window or tab >>Defined protein-free NS0 myeloma cell cultures: stimulation of proloferation by conditioned medium factors
2005 (English)In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 21, no 1, 87-95 p.Article in journal (Refereed) Published
Abstract [en]

A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, β-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 × 106 cells mL -1 in this medium. The antibody yield was 195 mg L-1 in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 × 10 6 cells mL-1, had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20-25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes.

Keyword
Antibodies, Cell culture, Growth kinetics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6312 (URN)10.1021/bp049822g (DOI)000226934800012 ()2-s2.0-13544262478 (Scopus ID)
Note
QC 20100907Available from: 2006-11-01 Created: 2006-11-01 Last updated: 2017-12-14Bibliographically approved
2. Protease activity in protein-free NSO myeloma cell cultures
Open this publication in new window or tab >>Protease activity in protein-free NSO myeloma cell cultures
2005 (English)In: In vitro Cellular & Developmental Biology-Animal, ISSN 1071-2690, E-ISSN 1543-706X, Vol. 41, no 10, 330-336 p.Article in journal (Refereed) Published
Abstract [en]

Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that thiscell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identifiedas aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular massesof 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using anenzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid andcysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product oraffecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, butaddition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibodyproduct was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acidproteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM,one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotininsignificantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminalamino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitorand cystatin C.

Keyword
conditioned medium, zymography, aprotinin, cathepsin L, SLPI, cystain C
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6313 (URN)10.1007/s11626-005-0004-4 (DOI)000236070000004 ()16448222 (PubMedID)2-s2.0-33645019941 (Scopus ID)
Note

QC 20100920

Available from: 2006-11-01 Created: 2006-11-01 Last updated: 2017-12-14Bibliographically approved
3. Defined protein and animal component-free NS0 fed-batch culture
Open this publication in new window or tab >>Defined protein and animal component-free NS0 fed-batch culture
2007 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 98, no 6, 1183-1194 p.Article in journal (Refereed) Published
Abstract [en]

A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a stepwise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for nonmeasurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L-1 was obtained as compared to 2.3 X 10(6) cells mL(-1) and 70 mg antibody L-1 in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6-fold increase in total cell yield, a 10.5-fold increase 6.2 in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not i significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.

Keyword
NSO cells, protein-free medium, fed-batch culture, cholesterol, vitamins
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6314 (URN)10.1002/bit.21509 (DOI)000250813200007 ()2-s2.0-36749087591 (Scopus ID)
Note
QC 20100907. Uppdaterad från Submitted till Published (20100907)Available from: 2006-11-01 Created: 2006-11-01 Last updated: 2017-12-14Bibliographically approved
4. Cell-derived proteins and their role in proliferatin of protein-free NS0 cell cultures
Open this publication in new window or tab >>Cell-derived proteins and their role in proliferatin of protein-free NS0 cell cultures
(English)Article in journal (Refereed) Submitted
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6315 (URN)
Note
QC 20100920Available from: 2006-11-01 Created: 2006-11-01 Last updated: 2010-09-20Bibliographically approved

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