Protein engineering and process optimization of 3D8 scFv for therapeutic application

다국어 초록 (Multilingual Abstract)

The development process of biological products has made significant progress, showing potential in treating viral infections, but it often faces serious challenges during industrial development. High viral mutation rates, complex production processes, and difficulties in scaling up frequently hinder the transition from laboratory research to mass production.
In this research focuses on the development of 3D8 single-chain variable fragment (scFv), a broad-spectrum antiviral candidate with nucleic acid hydrolyzing activity, as a therapeutic option. This research begins by addressing a critical issue concerning the stability of 3D8 scFv, which is essential for industrial applications. Based on in silico analysis, mutations were introduced to enhance stability and reduce aggregation of 3D8, resulting in stable and effective mutant candidates. The methods of inducing these mutations and evaluating both their stability and efficacy are detailed. Next, to optimize production for commercial applications, specific recombinant protein
expression systems were used, significantly increasing the production yield of both 3D8 wild type and mutants. Advanced fermentation and purification processes were also developed to ensure high purity and scalability, which are essential for preclinical and clinical applications. Lastly, the antiviral efficacy of the selected 3D8 scFv candidates, particularly Y101P, against respiratory viruses, including hCoV OC43 and SARS-CoV-2, was compared. Both in vitro and in vivo tests demonstrated strong antiviral activity, safety, and minimal toxicity, positioning 3D8 as a promising antiviral therapeutic candidate.
This research presents a comprehensive approach to addressing challenges in stability, productivity, and efficacy in the development of biological antiviral agents, paving the way for the future therapeutic application of 3D8 scFv.

국문 초록 (Abstract)

생물학적 제제의 개발 공정은 많은 발전을 거치며 바이러스 감염 등의 치료에 가능성을 보여주지만 산업적 개발 과정 중 심각한 어려움에 직면할 수 있습니다. 높은 바이러스 돌연변이율, 복잡한 생산 공정, 규모 확장의 어려움으로 인해 실험실 연구에서 대량 생산으로 전환하는 데 방해가 되는 경우가 많습니다.
이 연구는 치료 옵션으로서 핵산 가수분해 활성을 갖는 광범위한 항바이러스제 후보인 3D8 단일 사슬 가변 단편(scFv)의 개발에 중점을 두고 있습니다. 이 연구에서는 산업 응용에 필수적인 3D8 scFv의 안정성에 대한 중요한 문제를 해결하는 것으로 시작됩니다. 먼저, in silico 분석을 기반으로 안정성을 향상시키고 3D8의 응집을 감소시켜 안정적이고 효과적인 돌연변이 후보를 도출하기 위한 및 돌연변이 유발 방법 및 안정성과 효능 평가에 대해 자세히 설명합니다. 다음으로 상업적 적용을 목적으로 대량 생산을 하기 위한 특정 재조합 단백질 발현 시스템을 사용하여 이러한 3D8 야생형과 돌연변이의 생산성을 최적화하여 생산 수율을 크게 높였습니다. 또한 전임상 및 임상 적용에 필수적인 높은 순도와 확장성을 보장하기 위해 고급 발효 및 정제 공정이 개발되었습니다. 마지막으로 앞에서 선택된 3D8 scFv 후보, 특히 Y101P의 hCoV OC43 및 SARS-CoV-2를 포함한 호흡기 바이러스에 대한 항바이러스 효능을 비교합니다. 시험관 내 및 생체 내 테스트 모두 강력한 항바이러스 활성과 안전성 및 최소 독성을 입증하여 3D8을 유망한 항바이러스 치료 후보로 자리매김했습니다.
이 연구는 생물학적 항바이러스제 개발에서 안정성, 생산성 및 효능 문제를 해결하기 위한 포괄적인 접근 방식을 제시하여 3D8 scFv의 향후 치료 적용을 위한 길을 제안합니다.

목차 (Table of Contents)

• Literature Review 1
• 1. Research and Industrialization of Biological Products 2
• 2. Industrialization of Single Chain Variable Fragment (scFv) Therapies 4
• 3. Antibody-Based Antiviral Therapies 7
• 4. 3D8 scFv 9
• 5. Improving Stability and Solubility for the Industrialization of Protein Therapeutics 13
• 6. Discussion 15
• References 18
• Chapter 1. Stability Optimization of Recombinant 3D8 scFv Using Protein Engineering Approaches 28
• 1. Introduction 29
• 2. Materials & methods 33
• A. Cell lines and Viruses 33
• B. In silico Analysis of 3D8 scFv Sequence for Increased Stability of 3D8 scFv 33
• C. Construction of 3D8 scFv mutants 34
• D. 3D8 scFv protein production 36
• E. Verification of Expression Levels of Produced 3D8 scFv Before Purification 38
• F. Verification of Purity Post-Purification Using SE-HPLC 38
• G. Abzyme Test to Confirm Nuclease Activity 39
• H. Cell Toxicity Test Before Cell-Based Experiments 40
• I. Post-Treatment Method for Confirming Antiviral Effect of 3D8 scFv 40
• J. Analysis of Viral RNA Expression Using qRT-PCR 41
• 3. Results 41
• A. In silico analysis of protein sequence for the stability enhancement of 3D8 scFv 43
• B. Construction of 3D8 scFv Mutants 44
• C. Verification of Expression of Constructed 3D8 scFv Mutants 47
• D. Stability Verification of Expressed 3D8 scFv Mutants (SDS-PAGE & SE-HPLC) 52
• E. Verification of Activity of Expressed 3D8 scFv Mutants (Nuclease Activity) 56
• F. Verification of Cytotoxicity of Expressed 3D8 scFv Mutants 59
• G. G. Verification of Antiviral Activity of Expressed 3D8 scFv Mutants 61
• 4. Discussion 64
• References 68
• Chapter 2. Process Optimization for High-Yield Production of Therapeutic scFv 3D8 72
• 1. Introduction 73
• 2. Materials & methods 77
• A. Lonza expression system 77
• B. Fermentation of 3D8 scFv 77
• C. Purification of 3D8 scFv 78
• D. Verification of 3D8 scFv Titer During Cultivation and Purification Yield Using Sandwich ELISA 81
• E. SDS-PAGE Analysis for 3D8 scFv Purity Verification 83
• F. Buffer Exchange and Concentration 83
• G. SE-HPLC Analysis for 3D8 scFv Purity Verification 84
• H. Endotoxin Analysis of 3D8 scFv 84
• I. Verification of Nuclease Activity of 3D8 scFv 85
• J. Analysis of Viral RNA Expression Using qRT-PCR 85
• 3. Results 87
• A. Verification of ELISA Applicability for 3D8 Mutants 87
• B. Application of 3D8 scFv to Lonza XS Expression Platform 89
• C. Establishing Cultivation Process for High-Quality 3D8 scFv Production 92
• D. Establishment of a purification process for high-quality 3D8 scFv production 98
• E. Endotoxin Removal for Industrial Application 101
• F. Verification of Purity After Improved Purification Methods (SDS-PAGE and SE-HPLC Analysis) 105
• G. Verification of 3D8 scFv Activity After Process Improvement: Antiviral Effect 107
• 4. Discussion 109
• References 112
• Chapter 3. Evaluation of Antiviral Efficacy of Optimized 3D8 scFv Against Respiratory Viruses 115
• 1. Introduction 116
• 2. Materials & methods 119
• A. Production of 3D8 scFv (Culture and Purification) 119
• B. Purification of 3D8 scFv 119
• C. Purity Analysis of 3D8 scFv 121
• D. Endotoxin Analysis of 3D8 scFv 122
• E. Host Cell Protein Analysis for Impurity Confirmation 123
• F. Host Cell DNA Analysis for Impurity Confirmation 123
• G. Nuclease Activity test of 3D8 scFv 123
• H. Viral RNA Quantification by qRT-PCR 123
• I. Antiviral Efficacy Against COVID-19: Immuno Fluorescence Assay 124
• J. In Vivo Toxicity Evaluation 125
• K. In Vivo Efficacy Evaluation 126
• 3. Results 127
• A. Production and Purity Confirmation of 3D8 scFv through Established Process 127
• B. Activity Confirmation of Expressed 3D8 scFv (WT, Y101P) (Nuclease Activity & Antiviral Effect) 129
• C. Verification of Antiviral Efficacy of 3D8 scFv: Confirmation of Antiviral Efficacy Against SARS-CoV-2 132
• D. In Vivo Toxicity Evaluation of 3D8 scFv Produced by the New Process 134
• E. In Vivo Efficacy Evaluation of 3D8 scFv Produced by the New Process (hCoV OC43) 141
• 4. Discussion 145
• References 149
• 논문 요약 153