Monoklonal antikor üreten rekombinant çin hamster yumurtalık (rCHO) hücreleri için serumsuz ortam (SFM) tasarımı
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Dosyalar
Tarih
2016
Yazarlar
Dergi Başlığı
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Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Serum, hücrelerin büyümesi için doğal ortamı sağlarken, hayvansal kaynaklı olduğundan ve içeriği tam olarak bilinemediğinden uluslararası düzenleyici kuruluşlarca biyofarmasötiklerin üretimde serumun kullanımına izin verilmemektedir. Bu çalışmanın amacı, özellikle biyobenzer monoklonal antikor (mAb) üretiminde sıklıkla kullanılan rCHO hücreleri için serumsuz ortam geliştirmektir. Bu çalışmada, serumun yerini alabilecek ortam bileşiklerinin kültürün üremesi ve mAb üretimine etkisi sırasıyla Plackett-Burman ve faktöriyel tasarım istatistiksel optimizasyon programları kullanılarak belirlendi. Bu amaçla, Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12) bazal ortamına rekombinant insan insülini, bitki peptonu, linoleik asit-albümini, dekstran sülfat, Pluronik F68, maya ekstraktı, L-glutamin, esansiyel olmayan aminoasit karışımı (NEAA), transferrin, sığır serum albümini (BSA) ve hipoksantin-timidin (HT) eklenerek bu maddelerin hücrelerin spesifik büyüme hızı ve mAb üretimi üzerine etkileri belirlendi. En uygun kompozisyonun belirlenmesinin ardından başlangıç hücre konsantrasyonunun ürün verimine etkisi incelendi. Farklı üretim modlarında erlenlerde ve son olarak da 1L çalışma hacmine sahip karıştırmalı tank biyoreaktörde ölçek büyütme yapıldı. Sonuçlar ürün verimi/verimliliği ve maliyet analizi açısından referans ticari ortamlar ile karşılaştırılarak değerlendirildi. Ayrıca üretilen mAb'ın orjinatör olan HUMIRA® ile yapısal benzerliği karakterize edildi. Tasarlanan serumsuz ortamın bazal ortamına eklenen %1 HT supplement, %0,05 MSX, 0,5 mg/L insan insülini, 5 mg/ml bitki peptonu, 0,1 mg/mL dekstran sülfat, 1 mg/mL linoleik-asit albümini, 10 mM NEAA karışımı ve 0,05 mg/mL transferrinden oluştuğu belirlendi. Bu ortamın maliyeti ticari CD-Opti CHO ortamınının maliyetinin 1,42 katı iken ticari CD-Forti CHO ortamının da 1,28 katı olduğu hesaplandı. İkilenme süresini kısaltıp mAb verimliliğini artırmak için başlangıç hücre konsantrasyonunun 3x105 hücre/mL olması gerektiği ve hücrelerin erlene aktarılmasından önce statik kültürde 2-3 pasaj geliştirilen ortama adapte edilmesi gerektiği saptandı. Tez çalışması kapsamında geliştirilen serumsuz ortam ve optimize üretim şartları kullanılarak üretilen mAb'ın, orijinatör HUMIRA® ile yüksek yapısal benzerlik gösterdiği belirlendi.
Serum is used to support growth of animal cell culture. However, it is not allowed to use in production of biopharmaceuticals because of unknown contents and produced from fetal bovines. The aim of this study is to make an attempt to design serum-free medium for rCHO cells which are especially used for the commercial production of biosimilar monoclonal antibodies. In this study, medium components that can replace with serum was determined with statistical optimization approaches based on Plackett–Burman design and factorial designs, respectively. For that purpose, a basal medium was arranged by adding Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12) with recombinant human insulin, plant peptone, linoleic acid-albumin, dextran sulfate, pluronic F68, yeast extract, glutamine, non-essential aminoacids (NEAA) mix, transferrin, bovine serum albumin (BSA) and hypoxanthine and thymidine (HT) supplement which are generally contained in SF medium formulations. All those components were evaluated due to their effect on specific growth rate and mAb production. After the optimization of medium components, effects of different initial cell concentrations on mAb production yields were determined. The production of mAb was carried out in different culture operating modes in Erlenmeyer and process was scaled up 1L stirred tank bioreactor. All results were compared with commercially available CD-Opti CHO and CD-Forti CHO media in terms of mAb yield, mAb productivity and cost. Also, conformational similarity between the produced mAb and its originator HUMIRA® has been observed. Consequently, the developed SF medium was included basal medium with %1 HT supplement, %0,05 MSX, 0,5 mg/L human insulin, 5 mg/mL plant peptone, 0,1 mg/mL dextran sulphate, 1 mg/mL linoleic acid-albumin, 10 mM NEAA mix and 0,05 mg/mL transferrin. The cost of this SF medium was 1,42 and 1,28 times higher than commercial CD-Opti CHO and CD-Forti CHO medium, respectively. For reducing doubling time and increasing mAb yield, 3x105 cells/mL initial cell concentration was determined and before transfering cells to Erlenmeyer, they should be adapted to the developed SF medium. In addition, the produced mAb under those conditions gave high conformational similarity with HUMIRA®.
Serum is used to support growth of animal cell culture. However, it is not allowed to use in production of biopharmaceuticals because of unknown contents and produced from fetal bovines. The aim of this study is to make an attempt to design serum-free medium for rCHO cells which are especially used for the commercial production of biosimilar monoclonal antibodies. In this study, medium components that can replace with serum was determined with statistical optimization approaches based on Plackett–Burman design and factorial designs, respectively. For that purpose, a basal medium was arranged by adding Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12) with recombinant human insulin, plant peptone, linoleic acid-albumin, dextran sulfate, pluronic F68, yeast extract, glutamine, non-essential aminoacids (NEAA) mix, transferrin, bovine serum albumin (BSA) and hypoxanthine and thymidine (HT) supplement which are generally contained in SF medium formulations. All those components were evaluated due to their effect on specific growth rate and mAb production. After the optimization of medium components, effects of different initial cell concentrations on mAb production yields were determined. The production of mAb was carried out in different culture operating modes in Erlenmeyer and process was scaled up 1L stirred tank bioreactor. All results were compared with commercially available CD-Opti CHO and CD-Forti CHO media in terms of mAb yield, mAb productivity and cost. Also, conformational similarity between the produced mAb and its originator HUMIRA® has been observed. Consequently, the developed SF medium was included basal medium with %1 HT supplement, %0,05 MSX, 0,5 mg/L human insulin, 5 mg/mL plant peptone, 0,1 mg/mL dextran sulphate, 1 mg/mL linoleic acid-albumin, 10 mM NEAA mix and 0,05 mg/mL transferrin. The cost of this SF medium was 1,42 and 1,28 times higher than commercial CD-Opti CHO and CD-Forti CHO medium, respectively. For reducing doubling time and increasing mAb yield, 3x105 cells/mL initial cell concentration was determined and before transfering cells to Erlenmeyer, they should be adapted to the developed SF medium. In addition, the produced mAb under those conditions gave high conformational similarity with HUMIRA®.
Açıklama
Anahtar Kelimeler
Serumsuz Ortam Tasarımı, rCHO, Biyobenzer Üretimi, Optimizasyon, Serum-Free Medium Design, Biosimilar Production, Optimization
