Production of therapeutic human etanercept and characterization of DM-etanercept, a biosimilar of etanercept
- 발행기관 서강대학교 일반대학원
- 지도교수 주봉건
- 발행년도 2026
- 학위수여년월 2026. 2
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/sogang/000000082378
- UCI I804:11029-000000082378
- 본문언어 영어
- 저작권 논문은 저작권에 의해 보호받습니다.
초록(요약문)
바이오의약품 시장은 지난 수년간 빠르게 성장해 오면서, 주요 바이오의품의 특허만 료에 따라 바이오시밀러의 개발 필요성이 전세계적으로 크게 증가하였다. 엔브렐은 (Enbrel®) 종양괴사인자 알파 (TNF-α) 억제제로서 1998년 류마티스 관절염 치료제로 첫 승인을 받은 이 후 류마티스 관절염을 포함하여 주로 건선성 관절염, 강직성 척추염, 소아 특발성 관절염, 판상건선 등의 자가 면역질환 치료에 사용되고 있으며, 2024년 전세계 매 출 52-75억 달러를 기록하고 있는 의약품이다. 현재 전세계적으로 에타너셉트 (성분명: Etanercept, 상품명: Enbrel®) 바이오시밀러에 대한 필여성도 증가함에 따라, 대조약인 에 타너셉트와 동등한 주요 품질 특성을 확보하면서도 비용 효율적이고 견고한 제조 플랫폼 의 필요성이 커지고 있다. 한편, 에타너셉트의 구조는 인간 TNFR2의 세포외 도메인과 IgG1 Fc 영역이 융합된 단백질 구조이며, 29개의 디설파이드 결합과 다수의 N-글리칸과 O-글리칸 부위가 특이적으로 배열된 고도로 정교한 분자구조를 특징으로 한다. 본 연구에서는 이러한 엔브렐의 바이오시밀러 후보물질인 DM-etanercept의 개발 과 정을 다루며, 세포주 개발 및 공정개발, 그리고 광범위한 분석 및 기능적 비교평가를 수행 하였다. 우선 CHO-DHFR⁻ 세포주를 호스트로 사용하여 재조합 발현 시스템을 구축하였 고, pAD11-Etanercept-pro 발현벡터를 형질전환한 후 메토트렉세이트(MTX) 농도 단계 적 증가와 한계희석클로닝을 통해 고생산성 단일세포 클론을 선별하였다. 생산성, 세포 안 정성, 전하 이질성 패턴, 글리코실화를 기준으로 1차 및 2차 선별 단계를 통해 84-1-21 클론을 구축하였고, 100일동안 장기 계대안정성 시험을 통해 우수한 생산성과 증식 특성 이 유지됨을 확인하였다. 제조공정으로서 배양공정인 업스트림 공정은 초기 10리터에서 200리터 규모의 생물 반응기를 이용하여 단계적으로 스케일업하였으며, 고밀도 접종 세포에서 30℃ 저온 배양, xviii ManNAc 등의 전구물질 및 영양분 등을 보충하는 유가배양 조건에서 단백질 역가 1.03- 1.12 g/L를 안정적으로 달성하였다. 정제공정 단계인 다운스트림 공정에서는 Protein A 친화크로마토그래피로 타깃 단백질을 우선 포획한 뒤 양이온 교환(CEX)과 소수성 상호작 용(HIC) 크로마토그래피를 연속적용하여 공정 및 제품 관련 불순물을 효과적으로 제거하 면서 60% 이상의 전체 회수율을 확보하였다. 구조적 동등성 검증을 위해 펩타이드 맵핑과 MALDI-TOF MS, ESI-QTOF MS를 이용한 분자량 분석, N-말단 및 C-말단 서열 분석을 수행한 결과, DM-etanercept는 Enbrel® 과 동일한 아미노산 서열 및 약 124.5 kDa 분자량을 보였다. 원편광이색성(CD) 분광법 에서는 2차·3차 구조의 동등성이 관찰되었다. 번역후변형 분석에서는 글리칸에 대한 포괄적 프로파일링을 통해 G0F, G1F, G2F 등 주요 N-글리칸 패턴과 O-글리칸 패턴이 엔브렐과 유사함을 확인하였고, 약 50%에 이르는 높은 시알산화 수준의 글리코실화 특 성을 학인하였다. 물리화학적 특성 평가에서는 DM-etanercept의 고분자종(응집체) 비율은 0.85–1.09% 로 엔브렐의 3.72–4.31%에 비해 매우 낮아 응집체 제거능이 우수함을 보였으며, 등전 점전기영동(IEF) 분석에서는 유사한 전하 이성질체의 분포와 패턴을 보여 주었다. 생 물학적 활성 평가로서 L929 세포를 기반으로 평가된 TNF-α 중화능은 상대 역가 92.7– 105.7%를 보여 주어 엔브렐과 동등함을 확인하였으며, FcRn 및 C1q 결합 활성도 유사 한 결과를 보여 혈청 반감기와 보체 결합 기능이 엔브렐과 동등하게 유지될 수 있음을 확인하였다. FcγRIIIA 결합 친화도는 엔브렐대비 51-56%로 감소된 결과를 보였으나, 에타너셉트의 치료 메커니즘이 ADCC가 아닌 TNF-알파 중화임을 고려할 때 임상적으로 두 물질간의 유의미한 차이는 없을 것으로 판단된다. 공정관련 불순물 평가에서 DM-etanercept의 용출 protein A의 수준과 숙주세포 DNA 수준은 엔브렐 대비 높은 수준이었지만 규재허용기준 이하였다. 엔도톡신은 엔브 렐과 유사한 수준이었고. 숙주세포 유래 단백질 함량은 엔브렐보다 약 3배 낮은 수치 를 보여주었다. 결론적으로, 본 연구에서 확립된 세포주 및 공정 플랫폼과 분석법 개발은 바이오 시밀러로서 DM-etanercept의 동등성을 입증할 수 기반을 만들었으며, 특히 응집체 등 불순물 제거 측면에서 우수함을 보여 잠재적인 면역원성의 위험성을 조절하면서 동등 한 효능을 제공할 수 있음을 보여주었다. 더욱이, 본 연구에서 사용된 QbD 접근에 의 한 체계적인 개발방법은 향후 복잡한 Fc-융합단백질 바이오시밀러를 포함한 생물의약 품의 성공적인 개발 효율성을 높이는데 기여할 수 있을 것이다.
more초록(요약문)
The biopharmaceutical market has grown rapidly over the past decade, and with the patent expiry of major originator biologics, the need for biosimilar development has increased globally. Enbrel® (etanercept), a tumor necrosis factor-alpha (TNF-α) inhibitor, was first approved in 1998 for rheumatoid arthritis and is now widely used for autoimmune diseases including psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and plaque psoriasis. It remains a major therapeutic with global sales of 5.2-7.5 billion USD in 2024. As global demand for etanercept biosimilars rises, there is growing need for a cost effective and robust manufacturing platform that can achieve critical quality attributes equivalent to the reference product. Etanercept has a complex structure consisting of the extracellular domain of human TNFR2 fused to the IgG1 Fc region, featuring 29 disulfide bonds and multiple N and O glycosylation sites arranged in a highly sophisticated molecular architecture. This study describes the development of DM-etanercept, a biosimilar candidate to Enbrel®, including cell line development, process development, and comprehensive analytical and functional comparability assessment. A recombinant expression system was established using CHO DHFR⁻ cells as the host, followed by transfection with the pAD11 Etanercept pro expression vector. High productivity single cell clones were selected through stepwise methotrexate (MTX) amplification and limiting dilution cloning. Based on productivity, cell stability, charge heterogeneity pattern, and glycosylation, clone 84 1 21 was selected after primary and secondary screening, and its excellent productivity and growth characteristics were confirmed through long term serial passage stability testing over 100 days. For manufacturing, the upstream process was scaled up stepwise from 10 L to 200 L bioreactors. Using high cell density seeding, low temperature culture at 30 ℃, and fed batch conditions supplemented with precursors such as ManNAc and nutrients, a stable protein titer of 1.03-1.12 g/L was achieved. In the downstream purification process, the target protein was first captured by Protein A affinity chromatography, followed by sequential cation exchange (CEX) and hydrophobic interaction (HIC) chromatography, effectively removing process- and product-related impurities while maintaining an overall recovery of over 60%. To verify structural similarity, peptide mapping, molecular weight analysis by MALDI TOF MS and ESI QTOF MS, and N terminal and C terminal sequencing were performed. DM etanercept showed the same amino acid sequence and a molecular weight of approximately 124.5 kDa as Enbrel®. Circular dichroism (CD) spectroscopy demonstrated similarity in secondary and tertiary structure. Comprehensive glycan profiling confirmed that major N glycans (G0F, G1F, G2F) and O glycan patterns were similar to Enbrel®, with a high sialylation level of about 50%. Physicochemical characterization showed that the high molecular weight species (aggregates) in DM etanercept, as measured by size exclusion HPLC (SE HPLC), were 0.85-1.09%, significantly lower than Enbrel® (3.72-4.31%), demonstrating superior aggregate control. Isoelectric focusing (IEF) analysis revealed a similar distribution and pattern of charge variants similar to Enbrel®. Biological activity was assessed in an L929 cell based TNF α neutralization assay, where DM etanercept achieved a relative potency of 92.7-105.7%, confirming biological similarity to Enbrel®. FcRn and C1q binding activities also showed comparable results, indicating that serum half-life and complement-binding function are maintained at levels equivalent to Enbrel®. FcγRIIIA binding affinity was reduced to 51-56% of Enbrel®, but given that etanercept's primary therapeutic mechanism is TNF α neutralization rather than ADCC, there is no clinically meaningful difference between the two products. In the assessment of process-related impurities, the level of leached Protein A and host cell DNA in DM-etanercept was higher than that in Enbrel, but still below the regulatory acceptance limits. The endotoxin levels were comparable between the two products, while the host cell protein content of DM-etanercept was approximately threefold lower than that of Enbrel. In conclusion, the established cell line, process platform, and analytical method development in this study provided a solid foundation for demonstrating the biosimilarity of DM-etanercept to Enbrel. In particular, the process showed superior performance in removing impurities such as aggregates, thereby controlling the potential risk of immunogenicity while maintaining equivalent efficacy. Furthermore, the systematic development strategy based on the QbD approach applied in this study is expected to enhance the efficiency of future biopharmaceutical development, including complex Fc-fusion protein biosimilars.
more목차
CHAPTER 1 INTRODUCTION 1
1.1 Biological medicines 1
1.2 Biosimilars 3
1.3 Development of biosimilars 4
1.3.1 Timeline and cost efficiency 5
1.3.2 Development phases. 6
1.3.3 Manufacturing processes 6
1.3.4 Analytical characterization.18
1.3.5 Regulatory framework and guideline for approval pathways..29
1.4 Etanercept30
1.4.1 Etanercept originator (Enbrel®).30
1.4.2 Structural complexity of etanercept 31
1.4.3 Etanercept biosimilars35
CHAPTER 2 MATERIALS & METHODS 39
2.1 Reference product39
2.2 Construction of the expression vector39
2.3 Establishment of stable recombinant (CHO) cell lines expressing
etanercept (DM-etanercept) 39
2.3.1 Cell transfection and selection 39
2.3.2 Preparation of research cell bank (RCB) and master cell bank
(MCB)42
2.4 Production of etanercept43
2.4.1 Development of upstream (cell culture) processes43
2.4.2 Development of downstream (protein purification) processes ..
52
2.5 Structural characterization59
2.5.1 Amino acid composition analysis59
2.5.2 Amino acid sequence analysis.61
2.5.3 N-terminal sequence identification62
2.5.4 C-terminal sequence identification63
2.5.5 N-terminal heterogeneity63
2.5.6 C-terminal heterogeneity64
2.5.7 Peptide mapping 65
2.5.8 Carbohydrate content 66
2.5.9 N-glycan profiling 67
2.5.10 O-glycan profiling (normal phase-HPLC)69
2.5.11 Sialic acid determination 70
2.6 Physicochemical properties 71
2.6.1 Molecular weight determination71
2.6.2 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) analysis72
2.6.3 Isoelectric focusing (IEF) analysis73
2.6.4 Size-exclusion high-performance liquid chromatography (SE–
HPLC) analysis74
2.6.5 Ion-exchange high-performance liquid chromatography (IE–
HPLC) analysis75
2.6.6 Hydrophobic interaction high-performance liquid
chromatography (HI–HPLC) analysis76
2.6.7 Extinction coefficient determination77
2.6.8 UV-visible spectral analysis78
2.6.9 Circular dichroism (CD) spectroscopy78
2.7 Biological characterization79
2.7.1 TNFα neutralization activity assay.79
2.7.2 TNFα binding affinity assay (ELISA-based)80
2.7.3 FcRn binding affinity assay (ELISA-based).82
2.7.4 C1q binding affinity assay (ELISA-based)84
2.7.5 FcγRIIIA binding affinity assay (SPR-based)85
2.7.6 FcRn binding affinity assay (SPR-based) 86
2.8 Impurity analysis 87
2.8.1 Leached protein A quantification (ELISA-based).87
2.8.2 Endotoxin determination89
2.8.3 Host cell protein (HCP) quantification 89
2.8.4 Host cell DNA (HCD) quantification90
CHAPTER 3 RESULTS 92
3.1 Construction and verification of the DM- etanercept expression
vector92
3.2 Establishment and selection of stable recombinant CHO cell lines
expressing DM-etanercept 92
3.3 Production of DM-etanercept 105
3.3.1 Development of upstream (cell culture) processes105
3.3.2 Development of downstream (protein purification) processes
111
3.4 Structural characterization 117
3.4.1 Amino acid composition analysis.117
3.4.2 Amino acid sequence analysis119
3.4.3 N-terminal sequence identification 121
3.4.4 C-terminal sequence identification 127
3.4.5 N-terminal heterogeneity130
3.4.6 C-terminal heterogeneity134
3.4.7 Peptide mapping137
3.4.8 Carbohydrate content141
3.4.9 N-glycan profiling 143
3.4.10 O-glycan profiling (normal-phase HPLC)154
3.4.11 Sialic acid determination 158
3.5 Physicochemical properties 161
3.5.1 Molecular weight determination by MALDI-TOF MS161
3.5.2 SDS-PAGE analysis165
3.5.3 IEF analysis 167
3.5.4 SE–HPLC analysis.169
3.5.5 IE–HPLC analysis173
3.5.6 HI–HPLC analysis175
3.5.7 Extinction coefficient measurement179
3.5.8 UV-visible spectral analysis.181
3.5.9 CD spectroscopy analysis184
3.6 Biological characterization190
3.6.1 TNFα neutralization activity assay190
3.6.2 TNFα binding affinity assay (ELISA-based) 192
3.6.3 FcRn binding affinity assay (ELISA-based)194
3.6.4 C1q binding affinity assay (ELISA-based).196
3.6.5 FcγRIIIA binding affinity assay (SPR-based).198
3.6.6 FcRn binding affinity assay (SPR-based) 202
3.7 Impurity analysis 205
3.7.1 Leached protein A quantification (ELISA-based)205
3.7.2 Endotoxin determination 205
3.7.3 Host cell protein (HCP) quantification 208
3.7.4 Host cell DNA (HCD) quantification.208
CHAPTER 4 DISCUSSION 211
4.1 Manufacturing process development.211
4.2 Analytical similarity assessment214
4.2.1 Glycosylation profile comparability 214
4.2.2 Product-related impurities and product advantages.215
4.2.3 Charge heterogeneity differences215
4.3 Functional characterization216
4.4 Process-related impurities and safety profile 216
4.5 Potential original contributions in this study217
4.6 Limitations and future considerations218
CHAPTER 5 CONCLUSION 220
REFERENCES 221
ABSTRACT (ENGLISH)
