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Defined protein and animal component-free NS0 fed-batch culture
KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
2007 (Engelska)Ingår i: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 98, nr 6, s. 1183-1194Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2007. Vol. 98, nr 6, s. 1183-1194
Nyckelord [en]
NSO cells, protein-free medium, fed-batch culture, cholesterol, vitamins
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-6314DOI: 10.1002/bit.21509ISI: 000250813200007PubMedID: 17516495Scopus ID: 2-s2.0-36749087591OAI: oai:DiVA.org:kth-6314DiVA, id: diva2:10994
Anmärkning
QC 20100907. Uppdaterad från Submitted till Published (20100907)Tillgänglig från: 2006-11-01 Skapad: 2006-11-01 Senast uppdaterad: 2020-03-09Bibliografiskt granskad
Ingår i avhandling
1. Development of a protein-free fed-batch process for NS0 cells: studies on regulation of proliferation
Öppna denna publikation i ny flik eller fönster >>Development of a protein-free fed-batch process for NS0 cells: studies on regulation of proliferation
2006 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2006. s. 44
Nyckelord
NS0 myeloma cells, protein-free medium, fed-batch, conditioned medium, autocrine growth factors, abortive proliferation, protease activity, aprotinin, gp130
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-4162 (URN)91-7178-466-7 (ISBN)
Disputation
2006-11-17, FB54, AlbaNova, Roslagstullsbacken 21, Stockholm, 09:00
Opponent
Handledare
Anmärkning
QC 20100920Tillgänglig från: 2006-11-01 Skapad: 2006-11-01 Senast uppdaterad: 2010-09-20Bibliografiskt granskad

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