Rekombinant insanlaştırılmış anti tumör nekrozis faktör-α monoklonal antikor üretim prosesinin optimizasyonu /
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Tarih
2016
Yazarlar
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Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Tümör nekrozis faktör alfa (TNF-α) bağışıklık sisteminin önemli hücre sinyal bileşenidir. Anti-TNF-α monoklonal antikorlar (mAbs) kronik enflamatuvar hastalıkların tedavisinde sıklıkla kullanılmaktadır. Bu çalışmanın amacı, farklı üretim sistemleri ve farklı besleme stratejilerinin CHO hücre büyümesi, mAb verim/verimlilik ve kalitesine olan etkisinin araştırılarak ticari anti-TNF-α mAb üretimi için uygun bir proses tasarlamaktır. Bu tez çalışmasında, stabil olarak insanlaştırılmış anti TNF-α mAb salgılayan glutamin sentetaz (GS) vektör sistemine sahip Çin hamster yumurtalık (CHO) hücreleri kullanıldı. Hücreler öncelikle kesikli olarak faklı ticari kimyasal olarak tanımlanmış (CD) besi ortamlarında ve düşük kültür sıcaklıklarda (33, 35 ve 37°C) kültüre edildi. Ardından farklı kesikli-beslemeli (FB) kültür stratejilerinin hücrelerin üreme parametreleri, ürün verim/verimlilikleri ve ürün kalitesi üzerine etkileri belirlendi. 1L karıştırmalı tank biyoreaktörde pH, çözünmüş oksijen (DO), havalandırma hızı ve karıştırma hızı proses parametreleri optimize edildi. Hücrelerin farklı üretim sistemlerindeki büyüme potansiyellerinin belirlenmesi için, hücrelerin glikoz tüketim ve laktik asit üretimleri HPLC yöntemiyle, mAb üretimleri ise indirekt ELISA yöntemiyle belirlendi. Üretilen mAb'lar protein A affinite kromotografisi ile kısmi olarak saflaştırıldı. 4L üretimden elde edilen süpernatant çapraz akış filtrasyon sistemi ile konsantre edildi. Ürünün saflığı ve üretilen mAb'ın indirgenmiş kütlesi SDS-PAGE ile belirlendi. Ürün ayrıca konformasyonel olarak da karakterize edildi. Karıştırmalı tank biyoreaktörde, CD2 ortamında (CD-Forti-CHO, Gibco, Invitrogen, ABD)), FB5 stratejisinde (CHO CD EfficientFeed KitTM, Invitrogen, ABD)) ve bifazik 35°C kültür sıcaklığında yaklaşık olarak 2g/L mAb elde edildi. Biyoreaktör %40±10 DO ve pH 7,2±0,5'de kontrol edildi. Bu koşullarda üretilen mAb'ın, orjinatör molekülü (HUMIRA®) ile yüksek konförmasyonel benzerlik gösterdiği bulundu.
Tumor necrosis factor alpha (TNF-α) is an important cell-signaling factor of the immune system. Anti-TNF-α monoclonal antibodies (mAbs) are frequently used as therapeutic agents in chronic immune-mediated inflammatory diseases. The aim of this study is to investigate the effects of the different production systems and different feeding strategies on the growth parameters of this cell line, product yield/productivity and quality of the product and accordingly to design a feasible process for anti-TNF-α mAb manufacturing. In this thesis, glutamine synthetase (GS) - Chinese hamster ovary (CHO) cells that constantly produce humanized anti-TNF-α mAb, were cultivated in different culture conditions including different commercially available chemically defined (CD) media in batch culture. To evaluate the effect of low culture temperature on mAb production, the cells were cultivated at 33, 35, and 37°C. Thereafter, different fed-batch cultivation approaches were used to cultivate the cells. pH, dissolved oxygen (DO), aeration rate and stirring speed were optimized in 1L stirred tank bioreactor. mAb production process was successfully scaled up to 4L. In order to assess the growth parameters of the cells in different production systems glucose consumption, lactate production were determined by HPLC and mAb productivities of the systems were determined by indirect ELISA. mAbs were partially purified by protein A affinity chromatography. The supernatants from the 4L bioreactor were concentrated by cross flow filtration system. SDS- PAGE was applied to prove the both purity of the product and mAbs' reduced size. Products were also characterized according to their conformational structure. Approximately 2 g/L mAb was observed in CD2 medium with FB5 cultivation mode by using biphasic 35°C culturing temperature in 1L bioreactor. The bioreactor was controlled at 40±10% DO and the culture pH was kept at 7,2±0,5. The produced mAb under those conditions gave high conformational similarity with its originator molecule (HUMIRA®).
Tumor necrosis factor alpha (TNF-α) is an important cell-signaling factor of the immune system. Anti-TNF-α monoclonal antibodies (mAbs) are frequently used as therapeutic agents in chronic immune-mediated inflammatory diseases. The aim of this study is to investigate the effects of the different production systems and different feeding strategies on the growth parameters of this cell line, product yield/productivity and quality of the product and accordingly to design a feasible process for anti-TNF-α mAb manufacturing. In this thesis, glutamine synthetase (GS) - Chinese hamster ovary (CHO) cells that constantly produce humanized anti-TNF-α mAb, were cultivated in different culture conditions including different commercially available chemically defined (CD) media in batch culture. To evaluate the effect of low culture temperature on mAb production, the cells were cultivated at 33, 35, and 37°C. Thereafter, different fed-batch cultivation approaches were used to cultivate the cells. pH, dissolved oxygen (DO), aeration rate and stirring speed were optimized in 1L stirred tank bioreactor. mAb production process was successfully scaled up to 4L. In order to assess the growth parameters of the cells in different production systems glucose consumption, lactate production were determined by HPLC and mAb productivities of the systems were determined by indirect ELISA. mAbs were partially purified by protein A affinity chromatography. The supernatants from the 4L bioreactor were concentrated by cross flow filtration system. SDS- PAGE was applied to prove the both purity of the product and mAbs' reduced size. Products were also characterized according to their conformational structure. Approximately 2 g/L mAb was observed in CD2 medium with FB5 cultivation mode by using biphasic 35°C culturing temperature in 1L bioreactor. The bioreactor was controlled at 40±10% DO and the culture pH was kept at 7,2±0,5. The produced mAb under those conditions gave high conformational similarity with its originator molecule (HUMIRA®).
Açıklama
Anahtar Kelimeler
GS-CHO, anti-TNF-alfa mAb, Adalimumab, Kesikli Beslemeli., Sıcaklık Değişimi, Karıştırmalı Tank Biyoreaktör, Optimizasyon, CHO, Fed-Batch, Temperature Shifting, Stirring Tank Bioreactor, Optimization
