恩博 凍晶注射劑 - pfizer€¦ · 性關節炎、幼年型慢性關節炎、乾癬性關節炎、僵直性 脊椎炎、乾癬或幼 年乾癬之診斷及治療。 於皮下注射前,每瓶Enbrel
生技藥品產業發展 現況及趨勢 - imi.ym.edu.tw · 乾癬性關節炎、...
Transcript of 生技藥品產業發展 現況及趨勢 - imi.ym.edu.tw · 乾癬性關節炎、...
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生技藥品產業發展現況及趨勢
紀威光 博士
財團法人生物技術開發中心
June 1, 2017
Outline
•生技藥品簡介
•生技藥品產業
•新藥開發-DCB
•生技藥品生產技術 – case studies
• Q&A
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Outline
•生技藥品簡介
•生技藥品產業
•新藥開發-DCB
•生技藥品生產技術 – case studies
• Q&A
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Source: BioExecutive International; ITIS Program, DCB
Major Classes of Biopharmaceutical Products
Non-rDNA Monoclonal Antibodies Radio-immune Conjugates Vaccines Toxins Enzymes Cultured Cells and Tissues Blood Products, Human Immune Globulins Blood Products, Animal Immune Globulins
Biopharmaceuticals
rDNA Proteins rDNA Monoclonal Antibodies
Gene Therapy
Genetically Engineered Products
Biopharmaceuticals
Pharmaceutical Products Manufactured by Biotech Methods
(involving live organisms, bioprocessing)
Non-recombinant DNA Products
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The Emergence of Biotech Industry
Source: DCB ITIS Program
1940 → 1950 → 1960 → 1970 → → →1980 → → → → →→ → 1990 → → → → 2000 → → →2010
1953 DNA Double-helix
1973 Recombinant DNA
Technology
1975 Hybridoma Technology
2008 Revenues of Publicly
Traded Biotechs Grew 12% to US$89.7 Billion
1983 PCR
Technology
Genentech Acquired by Roche
for US$46.8 Billion
1982 Insulin
launched
1986 Interferon launched
1989 EPO
launched
1980 Amgen
founded
1976 Genentech
founded
1980 Genentech went public
1983 Amgen
went public
1992 Listed in
Fortune 500
Herbert Boyer & Stanley Cohen
Originators of Genetic Engineering
Robert Swanson & Herbert Boyer The father of the Biotechnology Industry
Amgen Revenue US$ 15 Billion
Ranked 11th Among Pharmas
Outline
•生技藥品簡介
•生技藥品產業
•新藥開發-DCB
•生技藥品生產技術 – case studies
• Q&A
6
生技藥品成長潛力大
7
生技藥品優點 滿足unmet medical need 安全性、臨床效果佳 抗藥性較少
CAGR 2007~2011 2011~2016
全球藥品市場 6.1% 4.75%
全球生技藥品市場 10.0% 5.48%
資料來源:IMS;生物技術開發中心產業資訊組整理
生技藥品成長潛力大
8
生技藥品優點 滿足unmet medical need 安全性、臨床效果佳 抗藥性較少
165
187
278
4.2
0
2
4
6
8
10
12
0
50
100
150
200
250
300
2011 2012 2013 2014 2015e 2016f 2017f 2018f 2019f 2020f
成長率(
%)
銷售額(十億美元)
年
資料來源:EvaluatePharma, Medtrack(2016.07);財團法人生物技術開發中心(DCB) ITIS研究團隊整理推估
抗體藥品於生技藥品市場扮重要角色
• 抗體藥品優點多
– 標的明確、開發時程短、成功率高
– 臨床表現佳,能逐步擴充不同適應
症
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2015銷售額排名 產品名
2015年 銷售額
(百萬美元) 主要適應症
生技藥品 整體藥品
1 2 Humira 14,950 類風濕性關節炎
2 3 Lantus 11,458 糖尿病
3 4 Enbrel 9,471
類風濕性關節炎、乾癬性關節炎、斑塊型乾癬、僵直性脊椎炎
4 6 Remicade 8,195
牛皮癬、關節炎、克隆氏症、潰瘍性大腸炎、僵直性脊椎炎
5 9 Rituxan/ MabThera
6,298 非何杰金氏淋巴瘤
6 10 Avastin 6,183
轉移性結腸直腸癌、多形惡性神經膠質瘤二線療法、轉移性腎細胞癌、非小細胞肺癌
7 13 NovoRapid 5,612 糖尿病
8 14 Herceptin 5,596 乳癌
9 19 Copaxone 5,050 多發性硬化症
mAb銷售額占53%
PhRMA: 907項
資料來源: IMS Health, PhRAM (2016.07);財團法人生物技術開發中心(DCB) ITIS研究團隊整理
2015年美國FDA核准上市之生技藥品
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產品名 主成分 廠商名 適應症 核准日期
(月.日) 產品類別
CDER核准之生技藥品 Cosentyx secukinumab Novartis 斑塊型乾癬 01.21 單株抗體
Natpara parathyroid hormone
NPS Pharma 副甲狀腺功能低下患者之低血鈣症 01.23 重組蛋白質
Unituxin dinutuximab United Therapeutics 神經母細胞瘤 03.10 單株抗體 Praluent alirocumab Sanofi 降低低密度膽固醇 07.24 單株抗體
Repatha evolocumab Amgen 降低低密度膽固醇 08.27 單株抗體
Praxbind idarucizumab Boehringer Ingelheim 逆轉dabigatran引起之抗凝血作用 10.16 單株抗體
Strensiq asfotase alfa Alexion 低磷酸酯酶症 10.23 重組蛋白質 Nucala mepolizumab GlaxoSmithKline 重度哮喘 11.04 單株抗體
Darzalex daratumumab Johnson & Johnson 多發性骨髓瘤 11.16 單株抗體
Portrazza necitumumab Eli Lilly 非小細胞肺癌 11.24 單株抗體
Empliciti elotuzumab Bristol-Myers Squibb 多發性骨髓瘤 11.30 單株抗體
Kanuma sebelipase alfa Alexion 發病初期溶酶體酸脂肪酶缺乏症 12.08 重組蛋白質
CBER核准之生物藥品
BEXSERO meningococcal group B vaccine Novartis 腦膜炎球菌B型引起的侵襲性腦膜炎 01.23 疫苗
Anthrasil anthrax immune globulin Cangene 吸入性炭疽 03.24 重組蛋白質
Quadracel Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed and Inactivated Poliovirus Vaccine
Sanofi 預防百日咳、白喉、破傷風、脊髓灰質炎
03.24 疫苗
Ixinity coagulation Factor IX Cangene B型血友病 04.30 重組蛋白質 Anavip crotalidae immune F(ab')2 Instituto Bioclon 抗蛇毒血清 05.06 血液製劑 Nuwiq Antihemophilic Factor Octapharma A型血友病 09.04 重組蛋白質
Source Plasma - Hemarus 血漿 09.11 血液製劑
Coagadex coagulation Factor X Bio Products Laboratory 遺傳性X 因子缺陷 10.20 血液製劑
Imlygic lalimogene laherparepvec Amgen 黑色素瘤 10.27 核酸
Adynovate antihemophilic Factor Baxalta A型血友病 11.13 重組蛋白質 Fluad influenza vaccine, adjuvanted Novartis 季節性流感 11.24 疫苗 Vonendi von Willebrand factor Baxalta 類血友病 12.08 重組蛋白質
資料來源:UISFDA(2016.07);財團法人生物技術開發中心(DCB) ITIS研究團隊整理
Nature Biotech. 35: 108-112
2016 Biologics Approvals
12 biologics
3 biosimilars
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18% 18% 19% 19% 21% 23% 24% 25% 26% 27% 28% 29% 29% 30% 30%
650
778
1121$Bn
'08yr
'16(e)
'22(f)
生技藥物占比
生技藥品占巿場份量趨重 2000~2016年美國FDA上巿新藥 2008~2022年全球百大生技製藥公司藥品銷售額
生技藥品占比
資料來源:USFDA,EvaluatePharma(2017.01);財團法人生物技術開發中心(DCB) ITIS研究團隊整理
專利到期高峰
2724
1721
31
1818 16
21 1915
24
33
2530
33
15
25
7
6
5
2 4
2
3 6
6
6
6
2
11 12
7
31.8
0
5
10
15
20
25
30
35
40
45
50
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
占
比
(%
)
藥
品
數
量
年
NCE BLA 生技藥品占比
生物藥品占所有新藥研發比例持續增長
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小分子藥品
61.1%
生物藥品
34.9%
天然物
2.4%
其他
1.7%
1,597
729
469 437 432 417 412 386 349 345
25.6
13.2
21.3
41.8
10.3 13.9
-5
0
5
10
15
20
25
30
35
40
45
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
成長率(
%)
件數
2015年1月 2016年1月 成長率
全球新藥研發件數之藥物類別以小分子藥品為居多,占61.1%,生物藥品居次,占34.9%,值得注意的是生物藥品成長較小分子藥品快,占比由2015年的34.2%
提升至2016年的34.9%。
前10大治療技術的研發新藥皆為生物藥品,抗體藥物及生物相似性藥品研發數量持續成長
資料來源:Pharmaproject(2016.07);財團法人生物技術開發中心(DCB) ITIS研究團隊整理
2008
111bn
USD
Biologics33%
SM Drugs
Biologics44%
SM Drugs
2014
131bn
USD
2020(f)
135bn
USDBiologics
64%
SM Drugs
73%
62.2bn
專利到期高峰Biosimilar潛力看漲 全球Top 20 sales藥品之生技藥品銷售額占比
註:SM Drugs(Small Molecule Drugs ,小分子藥品) 資料來源:EvaluatePharma, IMS Health(2017.01);DCB產資組ITIS 研究團隊整理
2016~2020年間專利到期之全球暢銷生技藥品 14,950
9,471
8,195
6,298
4,737
910
Humira
adalimumab
Enbrel
etanercept
Remicade
infliximab
Rituxan/
MabThera
rituximab
Neulasta
peg-figrastim
Gonal-f
follitroplin alfa
2015年銷售額(百萬美元)
暢銷藥物專利到期吸引Biosimilars投入
15
Drug INN EU expiry
date
US expiry
date
Humira Adalimumab 2018 2016
Enbrel Etanercept 2015 2028 (extended)
Remicade Infliximab 2014 2018
Lantus Insulin Glargine 2014 2014
Mabthera Rituximab 2013 2016
Avastin Bevacizumab 2019 2017
Avonex, Rebif Interferon β-1A 2012 Expired
Herceptin Trastuzumab 2015 2015
Novomix,
Novorapid
Insulin Aspart 2014 2019
Copaxone Glatiramer Acetate 2017 2015
Neulasta Pegfilgrastim 2015 2014
Lucentis Ranibizumab 2016 2016
銷售額~670億美元
生物相似性藥品的研發pipeline
資料來源:IMS Health, MedTrack(2017.03);DCB產資組ITIS 研究團隊整理
M A PA PIII P II P I PC R
Rituxan,
MabThera 14 1 1 8 4 11 7
Avastin 5 2 7 4 14 10
Herceptin 6 1 4 2 6 11 9
Humira 3 1 2 9 1 3 12 8
Remicade 5 5 6 4
Erbitux 3 1 5 6
Xolair 1 4 3
Lucentis 2 1 3 1
Stelara 5 2
Synagis 3 3
Actemra 4 2
ReoPro 2 1 1
Simponi 2 2
Prolia 2 2
Cimzia 2 1
Campath,
MabCampath 1 2
Perjeta 1 2
註:M(Marketed); A(Approved); PA(Pending Approval);
PIII(Phase III); PC(PreClinical); R(Research)
單株抗體Biosimilars核准上市
16
Product name
INN Therapeutic Area Authorization
Date Co. name (Country)
Remsima infliximab 僵直性脊椎炎、克羅恩病、乾癬性關節炎、牛皮癬、類風濕關節炎、潰瘍性大腸炎
2013.09.10 Celltrion (Korea)
Inflectra infliximab 僵直性脊椎炎、克羅恩病、乾癬性關節炎、牛皮癬、類風濕關節炎、潰瘍性大腸炎
2013.09.10 Hospira (USA)
2009年起進行
合作
韓國於2012年7月核准第一個生物相似性單株抗體上市
Celltrion的Remsima (infliximab)
其他亞洲國家、南美approval in progress (end-2013)
EMA已核准18項Biosimilars上市
目前審核中有3項產品
資料來源:EMA, GaBi;生物技術開發中心ITIS計畫整理
2013桃竹苗CEO策略餐會_131120_Copyright 2013 DCB
預估3~4年後,
40% EU Market,↓40% price
台灣在生技藥品產業有發展機會
2015年全球生技藥品的銷售額為1,870億美元,生技藥品佔全球臨床開發中藥品之40%
至2020年,將有12項以上蛋白質藥品專利過期,相似或改良藥品將搶食670億美元之龐大商機
蛋白質藥品附加價值高,技術、臨床及品管門檻也高,適合台灣發展
至少有50家廠商積極投入蛋白質藥物發展
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台灣生物製藥產業現況
項目 2011年 2012年 2013年 2014年 2015年
生物藥品產值 12.3 13.6 11.8 14.0 10.6
生物藥品產值成長率 -1.9 10.5 -13.4 19.3 -24.2
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產品分類 進口值 出口值
2013年 2014年 2015年 2013年 2014年 2015年
血液代用品與血漿代用品及基因重組
製劑 1,930.7 2,088.4 1,868.6 0 0 0
血液製劑 6,468.1 6,616.5 8,168.1 349.4 312.4 234.7
人類醫藥用疫苗 2,515.2 2,585.7 3,170.9 0 1.2 0
合計 10,914.0 11,290.6 13,207.7 349.4 313.6 234.7
2015年我國生物藥品產業總產值為新台幣10.6億元,主要產品為血液製劑、抗蛇毒血清、人用疫苗等,基因工程蛋白質則仍未有產品上巿,目前主要來自外銷長效型干擾素產品及藥物或相關技術授權金的貢獻。
我國生物藥品一向是貿易逆差,多仰賴進口,2015年進口值達新台幣132.1億元,較2014年成長17%,主要進口之生物藥品為血液製劑,占進口值的61.8%。而出口值則為新台幣2.3億元,較前一年衰退25.2%,使貿易逆差額擴大到129.7億元
資料來源:經濟部統計處工業產銷存動態調查,ITIS智網臺灣進出口資料庫(2016.07);財團法人生物技術開發中心(DCB) ITIS研究團隊整理
台灣產業界積極投入生技藥品領域
我國從事生技藥品開發的公司已有50-60家,以開發基因重組蛋白質藥物及單株抗體為主。
業者亦投入風險較低、成功率較高之生物相似性藥品(biosimilar)及第二代產品(superbiosimilar或biobetter)研發,如:永昕、賽德、金樺、及天福等。
19 資料來源:財團法人生物技術開發中心(DCB) ITIS研究團隊整理
我國廠商之生技藥品研發進入後期臨床階段
• 台灣廠商所開發之生技藥品,現階段在國內外已進行臨床階段的產品共計有47項,獲美國FDA許可進入臨床試驗階段的有25項
• 以疾病領域分,以癌症藥品最多,共21項;其次是抗感染的19項
20
註1:資料統計至2016.06.15,本統計不包括細胞治療產品 註2:新藥臨床試驗進程以全球最新臨床階段進行分類 資料來源:財團法人生物技術開發中心(DCB) ITIS研究團隊調查整理
16
20
11
0
5
10
15
20
25
Phase I Phase II Phase III
件數
中樞神經
自體免疫
血液
感染
癌症
在國內外進行臨床階段之我國生物藥品
1
3
10
11
0
2
4
6
8
10
12
Phase I Phase II Phase II/III Phase III
件數
自體免疫
感染
癌症
獲美國FDA許可進入臨床階段之我國生物藥品
Outline
•生技藥品簡介
•生技藥品產業
•新藥開發-DCB
•生技藥品生產技術 – case studies
• Q&A
21
Nonprofit Organization Founded in 1984
Funded Mainly by Ministry of Economic Affairs
(MOEA), National Science Council and the
Industry
380 Employees (20% Ph.D., 60% M.S.)
DCB
22
1984 Founded on Mar. 14th
1984 Spun off Lifeguard Co.
1987 Chang-Hsin Headquarters Inaugurated in Jan.
1994 Xizhi R&D Area Building Constructions Finished
2000 Spun off Taiwan Advance Bio-Pharm for diagnostic products on May 25th
2004
2011
Headquarters moved from Chang-Hsin to Xizhi & Nankang
GLP Toxicology Lab acquired by QPS Taiwan on
Jan. 1st
Brief History
23
24
Hepatitis B vaccine in-licensing (1984), spun off Lifeguard
Biological pesticide (Bio-Bac) production permit (1999), transferred to BionTech
AAALAC accreditation of animal facility in toxicology laboratory (2001)
GMP certification of biopharmaceuticals pilot plant facility (2005)
First-in-Class anticancer lead optimization (2011, with Taivex)
“Taiwan’s Firsts” by DCB
25
Uniqueness
Protein Drug Antibody Application Manufacturing Service (alliance with 台康生技, 啟弘生技)
Preclinical Development Integrated Capability (alliance with 昌達生技on GLP Toxicology)
Business Promotion Technology Commercialization Coordination & Incubation Academia-Industry Linkage
26
Biopharmaceutical Development:
Biologic Drugs,
Small Molecule & Botanical Drugs
From Lead Optimization to IND
Lead
Discovery Preclinical
ADME Tox
IND
NDA
Validation
Optimization
Clinical Trials
Phase I, II, III
Market
Core Business
建立「新藥產業化」之關鍵設施及能力
27
● GLP Toxicology Lab (毒理試驗)
● DMPK Lab (藥品代謝及藥動)
● BioSafety Lab (生技藥品檢驗)
● cGMP Pilot Plant (製程試驗量產)
● IND Filing (臨床試驗申請)
產業化 Commercialization
生技中心建立之平台
人體臨床
試驗
Clinical Trial
新藥探索
研發
Discovery
藥品毒理及前臨床試驗中心 (2011/1起由QPS經營)
GLP Lab Facilities (OECD GLP、DOH GLP) 符合國際規範
Accreditations: AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care), TAF ISO17025, TAF OECD GLP 通過國際認證
81 ISO 17025 Test Services
28
認證: TFDA GLP
服務項目:體外藥物動力試驗 (In vitro ADME)、體內藥物代謝、藥物動力及藥效試驗(In vivo DMPK)及實驗室生物檢體分析研發服務(Bioanalytical Method Development) 。
藥品代謝及藥動(DMPK)實驗室
29
• 動物設施取得DOH GLP及TAF OECD GLP
• 癌症藥理實驗室,取得肺腺癌皮下動物試驗
TAF ISO17025 之認證。
動物藥理實驗室
30
31
A mammalian cell & microbial based biologics production facility
cGMP certificate issued by TFDA, Taiwan
US FDA Drug Master File (Type V, No. 19164)
PIC/S international cGMP compliance
生技藥品製程量產試驗工廠(2013/4起由台康生技經營)
(BioPharma Asia 2011 Best CMO Award)
32
Cell Bank Characterization
Virus Clearance Validation
Bulk & Lot Release Testing
Bioassay Development
Clinical Sample Analysis
Certifications
In vitro / In vivo Lab
Alliance Partners
TFDA GLP TAF OECD GLP ISO
P2+ Lab P2 In vitro Lab P2 Animal Facility Bacterial Lab R&D Lab
ATIT Vitrology (Acquired by SGS) University of Queensland
32
Protein Drug / Safety Testing(2016/10起由啟宏經營)
Recent Case : 510(K) Accreditation for Biogenic Technology, Inc.
GLP Testing Facility for Biological Safety
Target & Lead Discovery
Preclinical Evaluation
IND
NDA
Lead Optimization
Clinical Trials Phase I, II, III
Market Launch
33
• Drug Design & Synthesis
• Animal Pharmacology
• Formulation
• DM/PK (ADME)
• Toxicity Testing
• GMP Production
Preclinical Development / Integrated Capability
IND core-team integrates chemistry, biology, pharmacology, toxicology etc. Receive drug leads from academia & industry and optimize into drug-like candidates for IND filing and entry into clinical stage
Calcitonin Injection,1993 Granisetron Long-Acting Injection, 2008
Calcitonin Nasal Spray, 1996, 2005
Misoprostol Long-Acting,1994 Misoprostol Fast-Acting,1997
Bio-Bac Biological Pesticides (百泰 1995;Japan Market 2004)
(R)-HPBA ACE Inhibitor Intermediate (駿瀚 1998)
Fluconazole Synthetic Process (Matrix Lab.2002; USA Market)
Anti-HSV mAb (聯亞生技, 2012)
Transferred Technologies/Products
Anti-HiB Vaccine 2012
34
mTOR Anticancer Injection, 2011
DCB-WH1 Botanical Drug, 2007 LT platform 2014
Raf 2015
Anti-ENO-1 2015
Antibody Engineering Platform
•High yield cell line screening (CHO, E. coli, Pichia)
•Medium optimization •Micro-bioreactor system
• Biosafety test -Cell line identity characterization -Process validation of impurity -Bulk & lot release tests -Clinical sample analysis -Cell-based bioassays
•Tissue cross reactivity, TCR • Extinction coefficient
•Protein expression/ purification- - Scale-down bioreactor process development (250 ml)
- Scale-up to 2 – 5 liter - Purification process development
•Antibody engineering -Humanization -Affinity maturation -Glyco-emgineered Ab
• Phage display • B cell/plasma cell • Automated library screening system
• Ab types- IgG, BsAb, scFv, Fab, ADC
Preclinical development
Lead antibody
Candidate antibody
Lead optimization
Antibody screening
IND filing
• Antibody characterization -physical, chemical, biological, formulation and stability studies
35
Antibodies from Single B Cell/Plasma Cell
36
Medical Infectious disease and cancer patient
Single B cell
Antibodies screening of single B-cell
Automation screening
system
Antibodies expression and characterization
Cell sorter
Antibody characterization : • Affinity • Epitope • Potency
Animal studies ADCC/CDC
Library Constructed Mouse immunize Library Size > 10-9
Time: 3 month
Panning and Screening >10,000 clones assay Time: 1month
Full length antibodies >100 antibodies Time: 1month
Antibody Screening is Facilitated by Automation
38
Cell line – Bioreactor – Purification – Scale-up – Tox material production
39
Feed supplement evaluation
Cell line development Basal medium adaptation
Process development
Process validation
A B C
D E
Batch & Simple fed-batch
F1 F2 F3
F4 F5 F6
F7 F8 F9 F10
Fed-batch
Basal medium &
Feed medium
DO pH
Temp Agitation Aeration
Feeding strategies
Cell Line Development and Process Development
WL FITC Before After
Fluorescence intensity
Enhanced Single Cell Screening by Automation
40
CHO scale-down process development
41
250ml mini-bioreactor (DASGIP) 5L Bioreactor
Titer optimization from shake flask, 250 ml mini-bioreactors
and 2 – 5 liter bioreactors
With ATF
CHO scale-down process development with shake flask
3764
4877
0
1000
2000
3000
4000
5000
6000
6 7 8 9 10 11 12 13 14
Tite
r m
g/L
Day
cell line A cell line B cell line C
7.4
35.0
13.5
0
5
10
15
20
25
30
35
40
6 7 8 9 10 11 12 13 14
Qp
Day
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
0 3 4 5 6 7 8 9 10 11 12 13 14
Via
bili
ty %
VC
D 1
0X
6 c
ells
/ml
Day
cell line A cell line B cell line C
cell line A cell line B cell line C
2568
3070
2478
0
500
1000
1500
2000
2500
3000
3500
6 7 8 9 10 11 12 13 14
Tite
r m
g/L
Day
Flask 250ml-mini bioreactor 50L single-use bioreactor
Case study
2294
6187
1942
6491
0
1000
2000
3000
4000
5000
6000
7000
7 8 9 10 11 12 13 14
Tite
r m
g/L
Day
FB cell lineA CFB cell linA
FB cell lineB CFB cell lineB
2.7 fold
3.3 fold
Antibody titer enhancement through Bioreactor with ATF
2-5L Bioreactor AFT2
Concentrated fed-batch
489.24
556.39
651.03
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
5 6 7 8 9 10 11 12 13
Tite
r (
mg/
L)
Day
cell line A
Continuous perfusion culture
Case study
Continuous purification
Stirred tank reactor
Continuous purification of scFv, BsAb and Fab
Batch purification
Advantages-
・ Constant product quality
・ Higher efficiency
・ High product titer
・ Cost down (about 1/10 – 1/20
resin)
・ Saves processing times
Continuous bioprocess
Batch
bioprocess
IgG: Purification yield is > 78 % 5-50L
Operation of three-column PCC Purification
Column: Dgal column 1.6*2.5 cm Linear Flow rate: 120 cm/hr Sample: LT-BI01- UH3-20161123 Buffer A: 50mM Tris, 0.2M NaCl pH 7.5 , cond. 23 ms/cm Buffer B : 50mM Tris, 0.2M NaCl, 0.2M D-galatose pH 7.5 , cond. 21.9 ms/cm Buffer C : 50mM Tris, 0.2M NaCl, 1M D-galatose pH 7.5 , cond. 15.9 ms/cm Sample Collection : start OD280 > 25mAU End start OD280 < 25mAU
Column: Mabselect Sure LX 0.66x15 cm x3 Linear Flow rate: 400 cm/hr (2.28mL /min ) Loading: 50% BT Sample: IgG # 2 (2.63 g/L, 3.64L ) Buffer A: 1X PBS pH 7.4 , cond. 16.9 ms/cm Buffer B : 0.1M NaCitric/ Citric pH 6.0 , cond. 16.1 ms/cm Buffer C : 0.1M NaCitric/ Citric pH 3.0 , cond. 3.8 ms/cm CIP: 0.1M NaOH pH adjusting 3M Tris pH8.8 (pH6-7) Sample Collection : start OD280 > 250mAU End OD280 < 250mAU
Run : 28 (57.7min/run)
Elution A Elution B Elution C
Case study
250
150
100
75
50
37
25
20
15
10
12% SDS Reducing Page
M
Operation of three-column PCC purification
Sample IgG #1 IgG #2 IgG #3 IgG #4
Column 0.66*15cm 0.66*15cm 0.66*15cm 0.66*15cm
Run 4 28 13 13
Total Mass 1.3 g 8.8 g 3.4 g 4.1g
Recovery 88.9% 92.0% 88.4% 84.3%
Recovery is around 80-90%
Microbial Expression System
• Escherichia coli
– Periplasmic
– Extracellular
• Pichia pastoris
– Extracellular
47
E. coli system/Fab
48
查核點 目前成果
41-4 run (48 h) 45-3 run (24 h)
菌體密度 (OD600) 達30以上 79.18 58.77
產率 (mg/L/OD600) ≥ 1.0 1.23 1.14
Table. 查核點及目前成果
Column: ACQUITY UPLC Protein BEH C4 Column, 300Å , 1.7 µm, 2.1 mm
X 150 mm
Elute A:H2O+0.1%FA
Elute B: ACN+0.1%FA
Intact mass of Lucentis
STD (commercial
Lucentis)
ID-8
45-3
STD (commercial Lucentis)
48379.00 Da
ID-8 48379.00 Da
45-3 48379.00 Da
Incomplete cleavage of signal peptide-A in scFv 2 peak1
peak2
28523 Da,
Cysteinylation
(+115Da)
27996.5 Da
Cysteinylation
(+115Da)
理論值 With signal peptide 28408 ± 1 Da
Without signal peptide 27881.74 ± 1 Da
Case study
Single band but 2 peak
in mass spectrum
50
Signal peptide toolbox to improve the cleavage
SP-C_ScFv2_3 H3
27882 27882
SP-B_ScFc2_5-D4
Glycation Glycation
Case study
51
E. coli and P. pastoris secretion expression system
Seed culture
52
Optimized induction conditions
→ Methanol fed medium → Methanol feeding rate
Off-line measure
Residues glucose concentration. Accumulate acetic acid concentration.
Mini fermentor system
Adjust glucose feeding pump rate
E. coli system P. pastoris system
Expression strains construction ↓
High through put screening ↓
High production strains
Microbial production process develop pipeline
53
Cell line generate group High through put screening
Top1~2 clone
Optimize fermentation parameter by Mini- fermentor system
Mini fermentor system
pH Dissolve oxygen
Feeding strategy
Medium
Temperature
Optimized fermentation parameter scale up to 5L fermentor
Scale up
Scale down
DASGIP Parallel Bioreactor Systems
For Cell culture (8X)
For Microbial Fermentation (8X)
55
Capabilities of antibody fragments by E. coli and P. pastoris in 250 ml fermentation process
0
20
40
60
80
100
120
Ver. 1 Ver. 2 Ver. 3 Ver. 4
Pro
du
ct y
ield
(m
g/L)
10
0
200
400
600
800
Ver. 1 Ver. 2 Ver. 3 Ver. 4 Ver. 5
Pro
du
ct y
ield
(m
g/L)
179
471 572
682
62
E. coli system
P. pastoris system
5 20
100
56
Compare Production Titer of Different Induction Strategy by using 5L fermentor
Methanol feedback control system
Dissolve oxygen feedback control system
The optical density profile of different
induction strategy by 5 L fermentation
Dissolve oxygen feedback control system Methanol feedback control system
Case study
Batch
Glycerol feeding
Methanol induction
57
Using 5L fermentor to compare different induction strategy by semi-quantitative SDS-PAGE analysis
The methanol feedback control system can achieve higher antibody production.
Dissolve oxygen feedback control system Methanol feedback control system
Case study
58
Antibodies production yield improvement from deep well, shake flask, 250 ml mini-fermentor to 5L fermentor
* The supernatant protein production yield.
The protein maxima concentration (mg/L)
Deep well Flask (50ml) Dasgip* 5L Fer. *
BsAb 0.04-0.24 0.32-5.00 131 54
scFv1 0.64-2.56 2.56-5.12 35-682 1200 - 1800
scFv A33 0.64 2.56 22 N.A.
Light chain 0.025 0.4 109 N.A.
scFv2/methanol
free 0.64 2.56 30 30
The improvement of production yield in E. coli and P. pastoris
P. pastoris:One of scFv production yield form deep well 1mg/L, 50 ml shake flask
stage 5 mg/L, 500 ml shake flask stage 20 mg/L , 250 ml mini-fermenter 600 mg/L and
5 L fermenter stage over 1700 mg/L.
Deep well screening 50 ml shake flask 250 ml mini-fermenter
限閱資料、禁止複製、轉載及外流
5L fermenter
1 mg/L 2-10 X 5 mg/L 10-100 X 600 mg/L 1-2.5 X 1700 mg/L
0.5 mg/L 10 X 5 mg/L 20 X 100 mg/L
E. coli:One of Fab production yield form deep well 0.5 mg/L, 50 ml shake flask stage 5 mg/L and 250 ml mini-fermenter stage over ~100 mg/L, with amplification
fold around 200 fold.
59
Microbial production platform
60
Scale E. oli
intracellular soluble
E. coli inclusion
body/refolding
E. coli secretion
Pichia secretion
(Methanol induction)
Pichia secretion
(Methanol free)
250 ml √ √ √ √ √
5 L √ √ √ √ √
20 L √
Production of 0.1 – 10 g scFv, Fab with 5 L scale
fermentor using microbial secretion system
Outline
•生技藥品簡介
•生技藥品產業
•新藥開發-DCB
•生技藥品生產技術 – case studies
• Q&A
61
From Bench Scale to GMP Manufacturing
62
Technology Transfer to CMO
Scale-up to 20~50 L
Toxicology Material Production
GMP Manufacture
Upstream scale-up Bioreactor • CHO cell • E.coli / Yeast Downstream scale-up • Continuous
centrifugation • Depth filtration • Column
chromatography • UF/DF
Establishment of 20-50L process flow Establishment of drug
substance specification Production of ~100g
toxicology material Stability of drug
substance
Transfer of analytical methods and reference standard Transfer of 20-50L
upstream and downstream process Scale-up to
100-200L
Establishment of Analytical SOP Establishment
of Production Batch Record Engineering Run GMP Production QC/QA Lot
release
One-Stop-Solution from DNA to Clinic
63
Pre-Clinical Research IND Clinical Study NDA Market
Protein Characterization
Biosafety Test (TFBS), Toxicology (QPS)
Quality Management
Cell Line Development
Process Development & Process Characterization(QbD)
Regulatory Support
Analytical Development
Documentation Support
CGMP Manufacturing (Mammalian cell & Microbial)
Core Service
• CMC service up to 500L-scale mammalian cell culture GMP production of Mabs for clinical trial
• CMC service up to 100L-scale microbial fermentation GMP production of recombinant proteins/DNA for clinical trial
• Separated mammalian cell culture and microbial fermentation GMP facilities
64
Capability/Cell Line
• Manufacturing cell line development – Cell line stability test up to 70 generations
– CHO (>2 g/L in bioreactor)
– PER.C6 (>1.5 g/L in bioreactor)
• Cell bank production and testing – Separated GMP cell banking facilities
– Mammalian cell • 200~300 vials
– Microbial cell • 300~400 vials
65
Capability/Process Development
• Upstream:
– Mammalian cell
• Cell testing, media screening, glycan profile adjusting, fed-batch development from shaker to 100L bioreactor
– Microbial cell
• Cell testing, media screening, and fed-batch development from shaker to 20L fermentor
• Downstream – Recovery, Affinity capture, Polishing, Viral Clearance
– Cell disruption, IB Refolding, Purifications, Pegylation
66
5 Liter scale 50 Liter scale
Current availability : 4 Future availability: additional 4 bioreactors will be installed in 2017
Future availability: one single use STR bioreactors will be installed in Q4/2017
Protein Expression from 5L to 50L
67
68
Considerations for cGMP Cell Line Development
• Productivity Target 3-5 g/L monoclonal antibody • Product characteristics e.g. glycosylation, de-amidation, aggregation, bioactivity, other PTMs • Clonality Demonstrate monoclonality e.g. formal cloning round(s) • No animal components e.g. chemically defined, animal component free • Scalability Consistent product characteristics, growth and productivity as manufacturing scale increases • Stability Consistent expression and product characteristics • SPEED Fast cell line construction
69
Cell Line Development
• 20 – 60 pcd (pg/cell/day)
• Serum free media pre-adapted host cell grown in suspension culture to high cell density (>10E6 cells/ml)
• Boehringer Ingelheim – CHO/BIHEX system, 3 – 7 g/L
• Lonza Biologics – CHO/GS system (no amplification), 0.8 – 8 g/L
• Crucell – PER.C6 (human cell line; no amplification), 1 - 8 g/L
mAb Structure
• Mass ~150 kDa
• 2 light chains (~25 kDa each)
• 2 heavy chains (~50 kDa
each)
• 16 disulfide bonds
• Glycans
70
71
Alain Beck, et al. Analytical Chemistry 2013,85, 715-736
IgG glyco-variants
Capability of Protein Characterization LC-MS/MS; UPLC;
N-terminal sequencing
LC-MS/MS;
Ellman’s assay
CE; UPLC; MS
LC-MS/MS; UPLC
SEC; SDS-PAGE;
Intact MS
UV; amino acid composition analysis
CE-IEF; CE-SDS
(reduced and
nonreduced);
SDS-PAGE; IEF
SEC; RP;
IEC; HIC
CD; DSC;
FT-IR
AUC; SEC-MALS
ELISA; qPCR; 2D gel;
LAL for endotoxin
▓: Outsourcing
▓: Not established
Physicochemical
properties
72
Structural Characterization and Confirmation
LC/MS/MS
• Peptide mapping • Glycomapping • Glycosylation site ID • Glycan structure ID
73
©財團法人生物技術開發中心版權所有 保留一切權利
Protein Characterization
HPLC-SEC-UV-MALLS-RI
CE SDS: IgG purity –reduced & nonreduced
cIEF
CE glycan analysis
74
Capability/Protein Characterization
75
• Primary structure analysis by HPLC and LC/MS/MS
– Peptide map; total sequence analysis
– N-/C-terminal variants
– Disulfide linkages
– Oxidation, deamidation and other post-modifications
• Glycan analysis
– N-, O-linked carbohydrates site and structure by LC/MS/MS
– N-glycan ratio by CE
• Monosaccharide analysis by HPIC
• Secondary and higher order structures
– CD
– DSC
– Fluorescence
• Heterogeneity analysis
– IEF/cIEF
– CIX
--
Capability/Analytical Development
76
• Identification – SDS-PAGE – Western blot – IEF/CIEF – Peptide mapping – HPLC – CE-Glycan profile
• Quantification – BCA/Bradford – A280
• Purity – SEC-HPLC – RP-HPLC – PAGE
• Potency/Activity – ELISA – Cell base assay
• Impurities – Host cell DNA – Host cell protein – ProA residue – Others
• Biological safety – Endotoxin – Bioburden – Microbial enumeration
Capability/GMP Manufacturing/ Mammalian
• Mammalian cell facility at 1st floor
– Two Upstream Suites
• 10/50/300L bioreactor
• 20/100/500L bioreactor
– Downstream
• Filtration/Ultrafiltration
• Chromatography skids
77
Track Record/ Mammalian Cell Projects
• Cell line experience including CHO, NS0, and PER.C6.
• Prior monoclonal antibody project experience, including
– Anti-IL 20 (CHO, PER.C6)
– Anti-HSV (CHO)
– Anti-TNFα (CHO)
– Biosimilars (CHO)
• Monoclonal antibody project for EU consultation
• GMP production of Fc-fusion protein (NS0) 500L scale, for US FDA pre-IND
78
Capability/GMP Manufacturing/ Microbial
• Microbial facility at 5th floor
– Upstream
• 20/100L fermenter
– Downstream
• Microfiltration/Centrifuge
• Homogenizer/Refolding
• Chromatography skid
• Ultrafiltration
79
Track Record/ Microbial Cell Projects
• r-protein complex (E. coli., soluble) for US FDA pre-IND and TFDA IND approvals (Mar. 2012)
• Customized Pegylated r-protein (E coli., IB)
– IND approval/Taiwan/USA/Canada
– Phase I (Canada) finished in 2010
– Phase II clinical material to client (Oct. 2011)
• Biosimilar IND filing (E coli., IB) in USA and Canada (Oct. 2011)
• Protein vaccine IND approved (PRP + E. coli. Soluble) in Taiwan (Sep. 2012)
• Production of plasmid DNA (E coli., soluble)
• Production of r-protein for animal studies (Pichia, soluble)
80
Formulation Development
Lead Selection
Characterization
Formulation
Screen
Forced
Degradation
pH Ionic Strength Excipients Concentration Vial/stopper
Optimization
/Selection
Understand product : IgG1 , 150 ~ 200 mg/ml
Formulation depends upon requirement: dose/frequency, route of delivery
Establish analytical techniques (stability indicating)
BI HEX Vector + Product
Selection marker
Product
Enhancer
Terminator
BI promotor
Titer
Medium & Feed
Promoter Terminator
Sequence
Marker &
TEs
Folding/ Transport
Ampli- fication
Clone Selection
Harvest
Ferm. Process
Case study 1. BI HEX® at a Glance
– Efficient vector systems with novel genetic
elements for generation and selection of high
producer clones
– Serum-free cloning procedures, transfection
and cultivation of suspension-adapted CHO
cells in chemically defined media
– Sophisticated design of integrated high
throughput screening platforms delivering
highly productive and robust clones suitable
for large scale commercial manufacturing
– Product quality as integral criterion for clone
screening and selection
BI HEX ® is Boehringer Ingelheims proprietary CHO based high expression
platform
Decades of scientific experience in represented in BI HEX ®
172 190
1929
1476
2350
1400
2540
1698
0
500
1000
1500
2000
2500
3000
Tit
er
mg
/L (
by
HP
LC
)
Growth curve
0
20
40
60
80
100
0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
2.5E+07
0 72 144 216 288 360
Via
bilit
y (
%)
VC
D (c
ells
/ml)
Culture time(hr)
B06 B15 FB03 FB10FB12 FB14 FB16 FB17
Productivity
-- Fed-batch process can increase productivity 7 - 13 fold to 1.5 – 2.5 g/L in 2-20L bioreactor.
-- DCB was able to develop robust upstream process based on general guideline in a short
period of time.
Batch and Fed-batch profile in bioreactor (2-5L, 20L bioreactor)
Analytical comparison of IgG1 sample from DCB with BI reference
SDS PAGE IEF SDS-PAGE (reduced with DTT)
SDS-PAGE, IEF: purity patterns of all pools are comparable to the standard material
Lane:
1 – Marker
2 –MabSelect SuRe elute, DCB
3 – Capto Adhere pool, DCB
4 – Before Capto Q, DCB
5 – After Capto Q, DCB
6 – BI-Std.
85
Case study 2. Crucell/DSM Per.C6 Technology Crucell/DSM’s PER.C6 R/D license on March 2007.
Successfully express Mab by PER. C6 cell line and get excellent
performance in both bioreactor fed-batch culture and XD process
355
1163
1029
0
500
1000
1500
batch 5L 20L
Tit
er
mg
/L (
EL
ISA
)
Growth profile
0.0E+00
1.0E+07
2.0E+07
3.0E+07
4.0E+07
0 72 144 216 288 360 432
Culture time(hr)
VC
D (
ce
lls
/ml)
0
20
40
60
80
100
Via
bilit
y (
%)
batch 5L fed batch 20L Fed batch
Titer
-- Fed-batch process can increase titer 3 fold compared to batch process (0.36g/L to 1.2g/L).
Maximal cell density can reach 10-30E6 cells/ml.
--Titer was determined by IgG/ELISA.
Batch and Fed-batch Profile in Bioreactor (5L, 20L bioreactor)
86
355
11631029
3865
0
500
1000
1500
2000
2500
3000
3500
4000
4500
batch 5L 20L XD
Tit
er
mg
/L (
EL
ISA
)
Growth profile
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
2.5E+08
3.0E+08
0 72 144 216 288 360 432Culture time(hr)
VC
D (
ce
lls
/ml)
0
20
40
60
80
100
Via
bilit
y (
%)
batch XD5L fed batch 20L Fed batch
Titer
-- XD® Mark I process can increase titer 3-6 fold compared to 5-20L fed-batch process
(~ 1.2g/L to 3.86g/L [in total broth] or ~ 1.2g/L to 7.1g/L [in supernatant]).
Maximal cell density of PER.C6/Ab3 in XD® Mark I process can reach 200E6 cells/ml.
Batch, Fed-batch and XD® Profile in Bioreactor (5L, 5-20L, 2L bioreactor)
87
N-Glycan Profile of PER.C6/Ab
88
Minutes
7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4
RF
U
0
1
2
3
4
5
RF
U
0
1
2
3
4
5
1
2
3
4
5
6
RF
U
Minutes
7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4
RF
U
0.0
0.5
1.0
1.5
2.0
2.5
3.0
RF
U
0.0
0.5
1.0
1.5
2.0
2.5
3.0
1 2
3
45
6
7
RF
U
Minutes
7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4
RF
U
0.0
0.5
1.0
1.5
2.0
2.5
3.0
RF
U
0.0
0.5
1.0
1.5
2.0
2.5
3.0
1
2
3
4
5
6
RF
U
Minutes
7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4
RF
U
0.0
0.5
1.0
1.5
2.0
2.5
3.0
RF
U
0.0
0.5
1.0
1.5
2.0
2.5
3.0
1
2
3
4
5
67
89
生技中心CGMP生技藥品先導工廠 DCB CGMP Biopharmaceutical Pilot Plant
Path Forward
DCB CGMP BPPF has supported various TFDA/US FDA/EMA Phase I/II IND projects during 2006 – Q1 2013.
The facility has been spun-off to form EirGenix, Inc. (台康生技)to further and expand the capabilities and capacities on April 1, 2013 in private sector.
DCB will continue to develop next generation bioengineering technologies with Omics related system biotechnology knowledge.
90
91
Future Trends • Cell line
– Development of Mab with appropriate amino acid sequence to avoid aggregation etc. undesirable properties
– No amplification, direct cloning (automation), screening relevant to platform production process
– Human cell lines (human like glycosylation structure)
• Upstream process – Platform production process – ~10 g/L for monoclonal antibodies
• Production scale – 20,000 L (0.1 – 1 g/L/14 – 21 days) 2,000 ~ 5,000L (1 10 g/L/12
- 14 days) -> 200 ~ 500 L (2-5 g/L/day @ Integrated Continuous Biomanufacturing (in 5 years?)
• Disposable technology – Single unit bioreactor (SUB) and downstream processing units
• Modular design for GMP production facilities – Flexibility and timeline - Space and cost reduction
What can we do on technology developments?
• Novel biologic molecule design (efficacy, long half life, low side effects etc.)
• Cell based screening, biopotency assay
• in vivo animal model
• PK of antibody, immunogenicity of therapeutic antibody, TCR (tissue cross reactivity)
• Producing cell engineering
• Expression vector design
• Rational cell line generation/screening
• Cell culture optimization through metabolic pathway
• Novel purification technologies 92
What can we do on technology developments?
Computer modelling, molecular biology, cell biology, genomics, bioinformatics, proteomics, metabolomics, cell culture, flow cytometry, PCR, genome editing, bioassay, animal model, bioreactor engineering, protein/sugar biochemistry, bioanalytical characterization, pharmacology, pharmacokinetics etc.
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Suggestions for Students Pursuing Bioindustry Position
• Good at what you have been doing (skillful, knowledgeable etc.)
• In-depth scientific knowledge
• Highly motivated
• Sound logical thinking
• Good presentation skills
• Good personality
• Can work in a team with good interactions with colleagues
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Thank You and Q&A
Wei-Kuang Chi Distinguished Scientist (特聘專家)
TEL:+886-2-2695-6933 EXT.2250
FAX:+886-2-6615-1110
EMAIL: [email protected]
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Some Thoughts
• Creation, Discovery and Innovation – Discovery: find something new from existing creation. – Innovation: a new medicine satisfying a patient’s need that was
unmet before (by Novartis)
• Innovative drug molecules – Taiwan has advantage in biologic drug innovation (21 in Phase I/II/III,
5 in Phase III) – Also work on small molecules and botanical drugs
• Smart biologics manufacturing technologies – Smart biologic manufacturing technology development is crucial to
the quality and cost of biologic drugs produced.
• Integration of Innovative biologic drug development and Smart biologics manufacturing is the key to success of Taiwan biopharmaceutical industry
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Some Thoughts
• Small and medium enterprise (SME) – Taiwan is good at SME; Biotech is SME by it’s nature – Biotech companies are more innovative than big pharma – Academia and R&D institutes conduct basic research and drug
discovery – Biotech industry carry out up to clinical Phase IIb proof of concept
studies – Develop drugs to approval in Taiwan, China and regional markets in
Asia – License out market territory (US, EU, Japan etc.) to internal big
pharma for Phase III studies and marketing
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Some Thoughts
• Future industry for Taiwan – innovation driven, high value, - low energy requirement - environmentally friendly - Biopharmaceutical Industry is one of the future industry for consideration - unlike ICT industry without innovation, even TSMC can only count on iPhone contracts, also need to invest NT3,000億 annually to upgrade their manufacturing equipments
• Education of biotech professional in Taiwan - need to view from biotech industry development - need to train professional that can create values in the biotech industry (innovation is the key)