Validation of Recombinant Growth Hormone Manufacturing Processes
- 주제(키워드) Validation , Growth Hormone
- 발행기관 고려대학교 대학원
- 지도교수 김찬화
- 발행년도 2010
- 학위수여년월 2010. 2
- 학위구분 박사
- 학과 일반대학원 생명공학과
- 세부전공 생물법제학 전공
- 원문페이지 251 p
- 실제URI http://www.dcollection.net/handler/korea/000000021080
- 본문언어 영어
- 제출원본 000045592513
초록/요약
Validation of Recombinant Human Growth Hormone (r-hGH) Manufacturing Processes by Byeong Jo Min Process validation defined by “Guidance of Process Validation in FDA” is to assure absolutely that some process consistently produce the suitable product at the already fixed standard and specification (titer, purity, stability and safety etc), and to document with these results. Also, it must be confirmed the eligibility test on the facilities and equipments for the production of interest before process validation. It has been taken process validation for r-hGH manufacturing, and its purpose is to confirm that the fermentation and purification processes for the production of the r-hGH solutions that fulfill the pre-established criteria at each step, and get the reproducibility. For the production of r-hGH, cultivation was carried out in 150 L fermentor operated in the fitted conditions such as agitation speed, temperature, pressure, dissolved oxygen, pH, and 3-IAA as inducer for leading cell expression during For the production of r-hGH, cultivation was carried out in 150 L fermentor operated in the fitted conditions such as agitation speed, temperature, pressure, dissolved oxygen, pH, and 3-IAA as inducer for leading cell expression during cultivation was added into the culture broth. After fermentation, when the cell density of culture broth and SDS-PAGE to confirm the r-hGH expression were measured, O.D600 was kept in standard values between 80 and 120, and the band obtained from SDS-PAGE was detected at the same position comparing with standard band. The cell recovery ratio and concentration volume were measured after the cells produced were washed and concentrated by using micro-filtration and then, the cell disruption ratio and the humidity weight of precipitate were also measured after the cell was disrupted and centrifuged to isolate the insoluble inclusion body. The results obtained from on each step were satisfied with the acceptance criteria pre-established as follows; cell recovery ratio: over 90%, cell disruption ratio: over 90%, humidity weight of precipitate: 1,500~2,300 g. Inclusion body was dissolved to activate the insoluble protein by controlling stirring time and pH. After the dissolution of inclusion body, stirring time (3~5 hr), pH (10.3~10.7) and the purity of Met-hGH protein (over 50%) fit in the standard. When the activated r-hGH solution was treated with phosphoric acid and precipitated and centrifuged to separate between Met-hGH protein and impurity protein, the yield Met-hGH (20~45%) of supernatant was also satisfied with the acceptance criteria pre-established. The activated supernatant was loaded into the first anion exchange chromatography and eluted with buffer. When the eluate was assessed in accordance with the pre-established sampling plan and quality control tests, the results satisfied the acceptance criteria pre-established for the following purification process as follows; endotoxin: ≤ 350 EU/mg, ECP: ≤ 42 ppm, IEF: same removal distance, r-hGH content in Native-PAGE: > 73%, purity by HPLC: ≥ 86%, purification yield by UV scanning: 32~36%. Therefore, the first anion exchange chromatography process was well validated and consistently yields crude r-hGH solutions that fulfill the pre-established criteria for the next purification process. For producing natural hGH, the methyl group of N-terminal was removed in conversion process with immobilized enzyme. Buffer pH and temperature in conversion reaction were 7.0, 44~47℃, respectively, in standard value. When the conversion reaction was completed, endotoxin (≤ 800 EU/mg Met-hGH), ECP (≤ 50 ppm), IEF (fitness in main band), Native PAGE (identified, over 70%) and purity (≥80%) tests were exactly in standard value and the yield was also in 95~100%. Also, HPLC was performed to identify the removal of methyl group of N-terminal. We checked that there was no signal after RT 34 min and also confirmed that conversion reaction was completed perfectly. The converted solution was used in the second anion exchange chromatography. The results of endotoxin (≤ 20 EU/mg hGH), ECP (≤ 30 ppm), IEF (fitness in main band, not to be strong than main band), Native PAGE (Identified, over 90%) and purity (≥ 90%) were in approval standard, and the yield was in 60~80%. Secondary completed solution was taken to Reverse-Phase chromatography and then took some sample. The results of IEF (fitness in main band, not to be stronger than main band), Native PAGE (Identified, over 92%) and purity (≥ 92%) were in standard, and the yield was in 80~95%. I took it to go via the dialysis. After test, it was taken to the third anion exchange chromatography and then took some sample. The results of endotoxin (≤ 5 EU/mg hGH), ECP (≤ 10 ppm), IEF (fitness in main band, not to be strong than main band), Native PAGE (Identified, over 98%) and purity (≥ 94%) were in approval standard, and the yield was in 80~95%. Finally in manufacturing bulk process, raw solution which the last purification was completed showed that it was in approval standard and was absolutely taken to final product process. Therefore, the consequence of process validation is that this process has reproducibility and is approval for high quality product as protocol. I think that this study would help being the industrialization of protein medical supplies and developing the best product process as r-hGH taken to industrial validation. Furthermore, this study could be good instance to confirm bulk production of protein medical supplies.
more초록/요약
공정밸리데이션이란 “FDA 공정 밸리데이션 가이드라인”에 규정하고 있는 바와 같이 ‘어떤 공정이 미리 설정한 규격과 품질특성 (예를 들면, 역가, 순도, 안정성, 안전성 등)에 적합한 제품을 일관되게 생산할 수 있다는 것을 확실하게 보증할 수 있고 그 결과를 문서화하는 것’을 말한다. 그리고 공정 밸리데이션을 실시하기 전에 제조설비나 기기에 대해서 적격성 확인을 하지 않으면 아니 된다. 인 성장 호르몬을 제조하기 위해서 공정 밸리데이션을 실시하였는데 이는 hGH 발효 및 정제 공정이 각 단계마다 미리 정한 품질에 적합한 원액이 생산되고 또한 재현성을 가지고 있다는 것을 확인하는 것에 그 목적이 있다. 재조합된 인성장호르몬을 생산하기 위하여 교반속도, 온도, 압력, 용존 산소 그리고 pH 조건이 맞춰진 150 L 배양조에서 발효하고, 배양 중 세포의 발현 유도를 위해 Inducer인 3-IAA를 첨가시켰다. 배양이 완료된 액의 세포밀도와 인 성장 호르몬의 발현 유무를 확인하기 위해 O.D 측정 및 SDS-PAGE 전기 영동을 실시하였다. 그 결과 O.D600 80~120에서 세포밀도가 일정하게 유지되어 기준에 적합하였고 표준품과 동일한 위치에 있는 밴드가 확인되었다. 발효가 완료된 액을 세척하고 농축 후 세포 회수율과 농축 볼륨을 측정하였다. 또한 재조합된 세포가 배양조에서 대량생산될 경우 세포내에 불용성의 봉입체가 형성하는데 이를 분리하기 위해서는 세포 파쇄 및 봉입체 분리 과정이 필요하다. 세포 파쇄 후 파쇄율을 측정하고 원심분리 후 침전된 봉입체의 습윤중량을 확인하였다. 그 결과 세포 회수율 및 파쇄율은 90% 이상이고, 습윤중량은 1,500~2,300g으로 기준에 적합하였다. 불용성의 단백질을 활성화시키기 위해서 봉입체를 용해시키기 위하여 교반 시간 및 pH를 조절하여 실시한 결과 pH 10.3~10.7와 교반 시간은 3~5 hr으로 기준 내에 적합하였고, Met-hGH 단백질의 순도 또한 ≥ 50%로 적합함을 확인하였다. 활성화가 완료된 액에 Met-hGH와 다른 불순 단백질과의 분리를 위해 pH를 기준 내에 적합하도록 조절하여 침전이 형성되도록 한 후 수율을 확인한 결과 Met-hGH 단백질 량이 20~45%로 기준에 적합하였다. 활성화 상등액을 1차 음이온교환 크로마토그래피 정제 공정을 실시하고 정제완료액을 채취하여 엔도톡신 (≤ 1000 EU/mg Met-hGH), ECP (≤ 50 ppm), IEF (표준품 주밴드와 이동거리 일치), Native PAGE (Identified, 70% 이상), 순도 (≥ 80%)를 확인한 결과 기준 내에 적합하였고, 수율 또한 20~45%로 기준에 적합하였다. N말단의 met기를 제거하여 천연형 hGH를 생산하기 위해서 전환반응 공정을 실시하였다. 전환반응에서 완충액의 pH는 7.0이며 온도는 44~47℃로 유지되어 기준 내에 적합하게 공정이 진행되었음을 확인하였다. 전환 반응이 완료된 액을 채취하여 엔도톡신 (≤ 800 EU/mg Met-hGH), ECP (≤ 50 ppm), IEF (표준품 주밴드와 이동거리 일치), Native PAGE (Identified, 70% 이상), 순도 (≥ 80%)를 확인한 결과 기준 내에 적합하였고 수율 또한 95~100%로 기준에 적합하였다. N말단의 met기의 제거를 확인하기 위하여 HPLC를 통해 RT (34분) 이후에 피크가 존재하지 않음을 확인하고 전환 반응이 100% 이루어 졌음을 검증하였다. 전환 완료액을 2차 음이온교환 크로마토그래피 정제 공정을 실시하고 정제완료액을 채취하여 엔도톡신 (≤ 20 EU/mg hGH), ECP (≤ 30 ppm), IEF (표준품 주밴드와 이동거리 일치, 주밴드 보다 진해서는 안됨), Native PAGE (Identified, 90% 이상), 순도 (≥ 90 %)를 확인한 결과 기준 내에 적합하였고 수율 또한 60~80%로 기준에 적합하였다. 2차 정제완료액을 역상액체크로마토그래피 정제 공정을 실시하고 정제완료액을 채취하여 IEF (표준품 주밴드와 이동거리 일치, 주밴드 보다 진해서는 안됨), Native PAGE (Identified, 92% 이상), 순도 (≥ 92%)를 확인한 결과 기준 내에 적합하였고 수율 또한 80~95%로 기준에 적합하였다. 투석을 실시하고 완료된 액을 3차 음이온교환 크로마토그래피 정제 공정을 실시하고 정제완료액을 채취하여 엔도톡신 (≤ 5 EU/mg hGH), ECP (≤ 10 ppm), IEF (표준품 주밴드와 이동거리 일치, 주밴드 보다 진해서는 안됨), Native PAGE (Identified, 98% 이상), 순도 (≥ 94%)를 확인한 결과 기준 내에 적합하였고 수율 또한 80~95%로 기준에 적합하였다. 마지막으로 Manufacturing Bulk 공정에서는 정제가 완료된 원액이 정해진 허용기준에 적합하게 최종의 제형화 공정을 거쳐 완료되었음을 나타내었다. 공정 밸리데이션 결과 본 생산공정은 재현성 있게 정해진 품질에 적합한 제조 공정이라 할 수 있겠다. 본 연구는 인 성장 호르몬의 산업화된 생산 공정을 밸리데이션하므로서 단백질 의약품의 산업화와 최적생산공정을 개발하는데 도움을 줄 것이며 더 나아가 본 연구는 단백질 의약품의 대량생산체계를 검증하는데 좋은 예가 될 수 있다
more목차
ABSTRACT i
ABSTRACT in Korean v
CONTENTS ix
LIST OF FIGURES xv
LIST OF TABLES xvi
LIST OF APPENDICES xx
I. INTRODUCTION 1
1.1 r-hGH 1
1.2 Process validation 3
1.2.1 A sort of process validation 4
1.2.1 Prospective validation 5
1.2.2 Concurrent validation 6
1.2.3 Retrospective validation 6
1.2.4 Revalidation 7
1.3 Chromatography 8
1.4 Overall scheme of r-hGH production 10
1.5 Validation Protocol of Manufacturing Process for r-hGH 13
1.5.1 Seed culture 13
1.5.2 Main culture 13
1.5.3 Cell concentration by microfiltration 14
1.5.4 Cell disruption 15
1.5.5 Isolation of inclusion body 15
1.5.6 Solubilization of inclusion body 16
1.5.7 Refolding 16
1.5.8 Acid precipitation 16
1.5.9 1st Anion exchange chromatography (IEC-1) 17
1.5.10 Concentration of IEC-1 pooling solution 17
1.5.11 Met-hGH conversion to natural hGH 18
1.5.12 Concentration of natural r-hGH solution 18
1.5.13 2nd Anion exchange chromatography (IEC-2) 19
1.5.14 Reverse phase chromatography (RPC) 19
1.5.15 Dialysis of RPC pooling solution 20
1.5.16 3rd Anion exchange chromatography (IEC-3) 20
1.5.17 Formulation and filtration 20
1.5.18 Bulk solution of final product 21
1.6 Objectives of the study 21
II. MATERIALS AND METHODS 23
2.1 Production of r-hGH by fermentation 23
2.1.1 Microorganism 23
2.1.2 1st Seed culture 23
2.1.3 2nd Seed culture 24
2.1.4 Main fermentation 24
2.2 Cell disruption and solubilization of inclusion body 28
2.2.1 Stock solution preparation 28
2.2.2 Micro-filtration 28
2.2.3 Cell disruption 28
2.2.4 Inclusion Body (IB) isolation 32
2.2.5 Inclusion Body solubilization 32
2.2.6 Refolding 37
2.2.7 Acid precipitation 37
2.3 Purification of hGH 37
2.3.1 1st Anion Exchange Chromatography (IEC-1) 37
2.3.2 Ultra-filtration 41
2.3.3. Met-hGH conversion to natural hGH 41
2.3.4 Ultra-filtration 46
2.3.5 2nd Anion Exchange Chromatography (IEC-2) 46
2.3.6 Reverse Phase Chromatography (RPC) 51
2.3.7 Dialysis 51
2.3.8 3rd Anion Exchange Chromatography (IEC-3) 51
2.3.9 Formulation and filtration 56
2.4 Specification of r-hGH drug substance 60
2.5 Analysis 60
2.5.1 Filter integrity test 60
2.5.2 Media sterility test 60
2.5.3 Contamination test 62
2.5.4 Cell density (OD) 62
2.5.5 Endotoxin test 62
2.5.6 Bioburden test 63
2.5.7 E. coli host cell protein (ECP) test 64
2.5.8 Iso-Electric Focusing 65
2.5.9 Native PAGE 66
2.5.10 Quantitation of hGH 67
2.5.11 Protein assay 67
III. RESULTS AND DISCUSSION 68
3.1 Cell Growth and Fermentation 68
3.1.1 1st Seed culture 68
3.1.1.1 Cell thawing 68
3.1.1.2 1st Seed culture process 70
3.1.2 2nd Seed culture 70
3.1.3 Main fermentation 73
3.2 Cell recovery and disruption 73
3.2.1 Stock solution preparation 73
3.2.2 Buffer A solution preparation 75
3.2.3 Micro-filtration 77
3.2.4 Cell disruption 77
3.3 Production of active Met-hGH 77
3.3.1 Inclusion Body (IB) isolation 77
3.3.2 Inclusion Body solubilization 81
3.3.3 Refolding 81
3.3.4 Acid precipitation 84
3.4 Purification of natural hGH 84
3.4.1 1st Anion Exchange Chromatography (IEC-1) 84
3.4.1.1 Quality control of buffer solution for IEC-1 84
3.4.1.2 Validation of the elution process of IEC-1 87
3.4.1.2.1 Endotoxin test 87
3.4.1.2.2 E. coli host cell protein (ECP) test 91
3.4.1.2.3 Iso-Electric Focusing (IEF) test 91
3.4.1.2.4 Native-PAGE test 93
3.4.1.2.5 Protein purity and yield 95
3.4.1.3 Ultra-filtration 99
3.4.2 Met-hGH conversion to natural hGH 102
3.4.2.1 Ultra-filtration 105
3.4.3 2nd Anion Exchange Chromatography (IEC-2) 108
3.4.4 Reverse Phase Chromatography (RPC) 111
3.4.4.1 Dialysis 112
3.4.5 3rd Anion Exchange Chromatography (IEC-3) 115
3.4.5.1 Formulation dialysis and filtration 118
3.4.6 Specification of r-hGH drug substance 120
IV. CONCLUSION 122
V. REFERENCES 125
