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
1
 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

2
 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

3
 Major Classes of Biopharmaceutical Products
Biopharmaceuticals
Pharmaceutical Products Manufactured by Biotech Methods
(involving live organisms, bioprocessing)

Biopharmaceuticals
Non-rDNA Monoclonal Antibodies
Radio-immune Conjugates
Vaccines
rDNA Proteins
Toxins
rDNA Monoclonal Antibodies
Enzymes
Gene Therapy
Cultured Cells and Tissues
Blood Products, Human
Immune Globulins
Genetically Blood Products, Animal
Immune Globulins
Engineered Products
Non-recombinant DNA
Products
4
Source: BioExecutive International; ITIS Program, DCB
 The Emergence of Biotech Industry
1940 → 1950 → 1960 → 1970 → → →1980 → → → → →→ → 1990 → → → → 2000 → → →2010

1953 Herbert Boyer & Stanley Cohen 2008

DNA Double-helix Originators of Genetic Engineering Revenues of Publicly
Traded Biotechs Grew
1973 Robert Swanson & Herbert Boyer 12% to US$89.7 Billion
Recombinant DNA The father of the Biotechnology Industry
Technology
Genentech
1976 1980
Acquired by Roche
1975 Genentech Genentech
Hybridoma founded went public for US$46.8 Billion
Technology
1980 1983 1992 Amgen
1983 Amgen Amgen Listed in Revenue US$ 15 Billion
PCR founded went public Fortune 500 Ranked 11th
Among Pharmas
Technology
1982 1986 1989
Insulin Interferon EPO
launched launched launched

5
Source: DCB ITIS Program
 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
生技藥品優點 CAGR 2007～2011 2011～2016
 滿足unmet medical need 全球藥品市場 6.1% 4.75%
 安全性、臨床效果佳
全球生技藥品市場 10.0% 5.48%
 抗藥性較少

資料來源：IMS；生物技術開發中心產業資訊組整理
7
 生技藥品成長潛力大
生技藥品優點
 滿足unmet medical need
 安全性、臨床效果佳
 抗藥性較少
300 278 12

250 10
銷
售 200 187 8成
額 165
（ 長
十 150 6率
億 （
美 %
元 100 4.2 4 ）
）

50 2

0 0
2011 2012 2013 2014 2015e 2016f 2017f 2018f 2019f 2020f 年

資料來源：EvaluatePharma, Medtrack（2016.07）；財團法人生物技術開發中心(DCB)
8 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 2015銷售額排名 2015年
產品名 銷售額 主要適應症
– 標的明確、開發時程短、成功率高 生技藥品 整體藥品 （百萬美元）

– 臨床表現佳，能逐步擴充不同適應 1 2 Humira 14,950 類風濕性關節炎

2 3 Lantus 11,458 糖尿病

症
類風濕性關節炎、
乾癬性關節炎、
3 4 Enbrel 9,471
斑塊型乾癬、
僵直性脊椎炎
牛皮癬、關節炎、
克隆氏症、潰
4 6 Remicade 8,195
瘍性大腸炎、
僵直性脊椎炎
Rituxan/ 非何杰金氏淋巴
5 9 6,298
MabThera 瘤
轉移性結腸直腸
癌、多形惡性
神經膠質瘤二
6 10 Avastin 6,183
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

PhRMA: 907項

mAb銷售額占53%
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
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 2015年美國FDA核准上市之生技藥品
核准日期
產品名 主成分 廠商名 適應症 產品類別
（月.日）
CDER核准之生技藥品
Cosentyx secukinumab Novartis 斑塊型乾癬 01.21 單株抗體
parathyroid
Natpara NPS Pharma 副甲狀腺功能低下患者之低血鈣症 01.23 重組蛋白質
hormone
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
Diphtheria and Tetanus Toxoids and
預防百日咳、白喉、破傷風、脊髓
Quadracel Acellular Pertussis Adsorbed and Sanofi 03.24 疫苗
灰質炎
Inactivated Poliovirus Vaccine
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10
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
 11

2016 Biologics Approvals

12 biologics
3 biosimilars

Nature Biotech. 35: 108-112

 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
NCE BLA 生技藥品占比
50

45

40
6 11 12 35
5 31.8

藥 30 占
2
品 5 6 2 比
25
數 6 （
3 6 %
量 7 4 20
2 6 7 ）
2 33
31 33
30 15
27 25 1121
24 24
21 21 19 10 $Bn
17 18 16
18 15 15
5

0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
年
778

650

專利到期高峰
'22(f)

'16(e)
'08 30%
yr 29% 29% 30%
25% 26% 27% 28%
19% 19% 21% 23% 24%
18% 18% 生技藥物占比
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
 生物藥品占所有新藥研發比例持續增長
 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
2015年1月 2016年1月 成長率

1,800 41.8 45
1,597
天然物 其他 1,600 40
2.4% 1.7% 35
1,400
25.6 30 成
1,200
21.3 25 長
件 1,000
數 率
800 729 13.2 13.9 20 （
生物藥品 10.3 15 %
34.9% 600 469 437 432 10 ）
417 412 386 349 345
400 5
小分子藥品 200 0
61.1%
0 -5

13
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
 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

Biologics
33% SM Drugs

2008 2014 Biologics 2020(f)

44%
111bn SM Drugs
131bn 135bn
USD 62.2bn
USD USD Biologics
SM Drugs
64%
73%

2016～2020年間專利到期之全球暢銷生技藥品
14,950
2015年銷售額（百萬美元）

9,471
8,195

6,298
4,737

910

Humira Enbrel Remicade Rituxan/ Neulasta Gonal-f

adalimumab etanercept infliximab MabThera peg-figrastim follitroplin alfa
rituximab
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
 暢銷藥物專利到期吸引Biosimilars投入
生物相似性藥品的研發pipeline
EU expiry US expiry
Drug INN M A PA PIII P II PI PC R
date date
Rituxan,
14 1 1 8 4 11 7
MabThera
Humira Adalimumab 2018 2016
Avastin 5 2 7 4 14 10
Enbrel Etanercept 2015 2028 Herceptin 6 1 4 2 6 11 9
(extended)
Humira 3 1 2 9 1 3 12 8
Remicade Infliximab 2014 2018 Remicade 5 5 6 4
Lantus Insulin Glargine 2014 2014 Erbitux 3 1 5 6

Mabthera Rituximab 2013 2016 Xolair 1 4 3

Lucentis 2 1 3 1
Avastin Bevacizumab 2019 2017
Stelara 5 2
Avonex, Rebif Interferon β-1A 2012 Expired
Synagis 3 3
Actemra 4 2
Herceptin Trastuzumab 2015 2015
ReoPro 2 1 1
Novomix, Insulin Aspart 2014 2019 Simponi 2 2
Novorapid
Prolia 2 2
Copaxone Glatiramer Acetate 2017 2015 Cimzia 2 1
Campath,
1 2
MabCampath
Neulasta Pegfilgrastim 2015 2014
Perjeta 1 2
Lucentis Ranibizumab 2016 2016 註：M(Marketed); A(Approved); PA(Pending Approval);
PIII(Phase III); PC(PreClinical); R(Research)
銷售額～670億美元
資料來源：IMS Health, MedTrack（2017.03）；DCB產資組ITIS 研究團隊整理
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 單株抗體Biosimilars核准上市
EMA已核准18項Biosimilars上市
目前審核中有3項產品

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)

Product Authorization Co. name

INN Therapeutic Area
name Date (Country)
Remsima infliximab 僵直性脊椎炎、克羅恩病、乾癬性 2013.09.10 Celltrion
關節炎、牛皮癬、類風濕關節炎、 (Korea)
2009年
潰瘍性大腸炎
起進行
Inflectra infliximab 僵直性脊椎炎、克羅恩病、乾癬性 2013.09.10 Hospira 合作
關節炎、牛皮癬、類風濕關節炎、 (USA)
潰瘍性大腸炎
預估3～4年後，
資料來源：EMA, GaBi；生物技術開發中心ITIS計畫整理
40% EU Market，↓40% price
2013桃竹苗CEO策略餐會_131120_Copyright 2013 DCB
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 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
 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。
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

 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
進口值 出口值
產品分類
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 1,930.7 2,088.4 1,868.6 0 0 0
製劑
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

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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。

資料來源：財團法人生物技術開發中心(DCB) ITIS研究團隊整理 19
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

在國內外進行臨床階段之我國生物藥品 獲美國FDA許可進入臨床階段之我國生物藥品
25 12
11
20 10
20 中樞神經 10
自體免疫
16 自體免疫
件 8
件 15
數 血液 數 感染
11 6
10 感染
癌症
4
癌症
3
5
2
1
0 0
Phase I Phase II Phase III Phase I Phase II Phase II/III Phase III

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
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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

21
 DCB
 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.)

22
 Brief History
 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 Headquarters moved from Chang-Hsin to Xizhi &
Nankang
 2011 GLP Toxicology Lab acquired by QPS Taiwan on
Jan. 1st

23
 “Taiwan’s Firsts” by DCB
 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)

24
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 25
 Core Business

Lead Validation Preclinical IND Clinical Trials NDA Market

Discovery Optimization ADME Tox Phase I, II, III

Biopharmaceutical Development：

Biologic Drugs,
Small Molecule & Botanical Drugs
From Lead Optimization to IND

26
 建立「新藥產業化」之關鍵設施及能力

生技中心建立之平台

新藥探索 ● GLP Toxicology Lab (毒理試驗) 人體臨床

研發 試驗
Discovery ● DMPK Lab (藥品代謝及藥動) Clinical Trial

● 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

27
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
 藥品代謝及藥動(DMPK)實驗室
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) 。

29
 動物藥理實驗室
• 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。

30
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)
 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

31
Protein Drug / Safety Testing(2016/10起由啟宏經營)
GLP Testing Facility for Biological Safety
Certifications
Cell Bank Characterization TFDA GLP
TAF OECD GLP
Bulk & Lot Release Testing ISO
In vitro / In vivo Lab
P2+ Lab
Virus Clearance Validation P2 In vitro Lab
P2 Animal Facility
Bacterial Lab
Bioassay Development R&D Lab
Alliance Partners
Clinical Sample Analysis ATIT
Vitrology (Acquired by SGS)
University of Queensland
Recent Case : 510(K) Accreditation for Biogenic Technology, Inc.

32
32
 Preclinical Development /
Integrated Capability

Target & Lead Lead Preclinical IND Clinical Trials NDA Market
Discovery Optimization Evaluation Phase I, II, III Launch

• Drug Design & Synthesis  IND core-team

integrates chemistry,
• Animal Pharmacology biology, pharmacology,
toxicology etc.
• Formulation
 Receive drug leads from
• DM/PK (ADME) academia & industry and
optimize into drug-like
• Toxicity Testing candidates for IND filing
and entry into clinical
• GMP Production
stage
33
 Transferred Technologies/Products

Calcitonin Injection,1993
Granisetron Long-Acting Injection, 2008
Misoprostol Long-Acting,1994
Misoprostol Fast-Acting,1997

Calcitonin Nasal Spray, 1996, 2005 (R)-HPBA ACE Inhibitor Intermediate

(駿瀚 1998)

Fluconazole Synthetic Process

Bio-Bac Biological Pesticides (Matrix Lab.2002; USA Market)
(百泰 1995;Japan Market 2004)

mTOR Anticancer Injection, 2011

DCB-WH1
Botanical Drug, 2007 Anti-HSV mAb
Anti-HiB Vaccine
(聯亞生技, 2012) LT platform 2014
2012
Raf 2015
Anti-ENO-1 2015
34
 Antibody Engineering Platform
•Antibody engineering
-Humanization
-Affinity maturation
-Glyco-emgineered Ab
•Protein expression/ purification-
- Scale-down bioreactor process development (250 ml)
- Scale-up to 2 – 5 liter
- Purification process development

Antibody Lead Lead Candidate Preclinical IND

screening antibody optimization antibody development filing

• Phage display •High yield cell line screening • Biosafety test

• B cell/plasma cell (CHO, E. coli, Pichia) -Cell line identity characterization
• Automated library •Medium optimization -Process validation of impurity
screening system •Micro-bioreactor system -Bulk & lot release tests
-Clinical sample analysis
• Ab types- IgG, BsAb, -Cell-based bioassays
scFv, Fab, ADC •Tissue cross reactivity, TCR
• Extinction coefficient
• Antibody characterization
-physical, chemical, biological, formulation and stability studies

35
 36

Antibodies from Single B Cell/Plasma Cell

Medical Infectious disease and Cell sorter Single B cell

cancer patient

Antibody characterization :
• Affinity
• Epitope
• Potency Automation
screening
ADCC/CDC system
Animal studies

Antibodies expression and characterization Antibodies screening of single B-cell

Antibody Screening is Facilitated by Automation

Library Constructed Panning and Screening Full length

Mouse immunize >10,000 clones assay antibodies
Library Size > 10-9 Time: 1month >100 antibodies
Time: 3 month Time: 1month
Cell line – Bioreactor – Purification – Scale-up – Tox material production

38
Cell Line Development and Process Development
Cell line development Basal medium adaptation Feed supplement
evaluation
A B C
F1 F2 F3

D E F4 F5 F6
Batch &
Simple fed-batch F7 F8 F9 F10
Fed-batch

DO
pH Basal medium
Temp &
Agitation Feed medium
Aeration
Process validation Feeding strategies
Process development 39
 40

Enhanced Single Cell Screening by Automation

WL FITC Before After

Fluorescence intensity
 CHO scale-down process development
Titer optimization from shake flask, 250 ml mini-bioreactors
and 2 – 5 liter bioreactors

5L Bioreactor
250ml mini-bioreactor (DASGIP)

With ATF

41
 Case study
CHO scale-down process development with shake flask
cell line A cell line B cell line C
cell line A cell line B cell line C
cell line A cell line B cell line C
6000
60 100
90 4877
50 5000
80 3764
VCD 10X6 cells/ml

40 70 4000

Viability %
Titer mg/L
60
30 50 3000
40
20 30 2000

10 20
1000
10
0 0
0
0 3 4 5 6 7 8 9 10 11 12 13 14
6 7 8 9 10 11 12 13 14
Day
Day
Flask 250ml-mini bioreactor 50L single-use bioreactor
40
35.0 3500
35 3070

30 3000
2478
25 2500 2568
Titer mg/L
Qp

20 2000
13.5
15 1500
10 7.4 1000
5
500
0
6 7 8 9 10 11 12 13 14 0
Day 6 7 8 9 10 11 12 13 14
Day
 Case study
Antibody titer enhancement through Bioreactor with ATF
2-5L Bioreactor
AFT2

cell line A 651.03

FB cell lineA CFB cell linA
FB cell lineB CFB cell lineB 700.00

7000 6491 556.39

6187 600.00

6000 489.24
3.3 fold 500.00
5000

Titer (mg/L)
400.00
Titer mg/L

4000 2.7 fold

300.00
3000
2294
1942 200.00
2000

1000 100.00

0 0.00
7 8 9 10 11 12 13 14 5 6 7 8 9 10 11 12 13
Day Day

Concentrated fed-batch Continuous perfusion culture

Continuous purification of scFv, BsAb and Fab
Stirred tank reactor 5-50L
IgG: Purification yield is > 78 %

Batch purification Continuous purification

Continuous bioprocess
Advantages-
･ Constant product quality
Batch ･ Higher efficiency
bioprocess ･ High product titer
･ Cost down (about 1/10 – 1/20
resin)
 Case study
Operation of three-column PCC Purification
Elution A Elution B Elution C

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
Column: Mabselect Sure LX 0.66x15
Buffer Ccm: 50mMx3 Tris, 0.2M NaCl, 1M D-galatose
Linear Flow rate: 400 cm/hr (2.28mL /min pH 7.5
) , cond. 15.9 ms/cm
Sample Collection : start OD280 > 25mAU
Loading: 50% BT End start OD280 < 25mAU
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)
Operation of three-column PCC purification
12% SDS Reducing Page

M
250
150
100
75

50

37

25

20

15

10

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

Table. 查核點及目前成果

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
48
 Intact mass of Lucentis
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 ID-8

STD
(commercial
Lucentis)

45-3

STD 48379.00 Da
(commercial
Lucentis)
ID-8 48379.00 Da
45-3 48379.00 Da
 Case study
Incomplete cleavage of signal peptide-A in scFv 2
peak1

peak2

理論值 With signal peptide 28408 ± 1 Da

Without signal peptide 27881.74 ± 1 Da

Single band but 2 peak

in mass spectrum

28523 Da,
27996.5 Da
Cysteinylation
Cysteinylation
(+115Da)
(+115Da)

50
 Case study
Signal peptide toolbox to improve the cleavage

SP-C_ScFv2_3 H3 SP-B_ScFc2_5-D4

27882 27882

Glycation Glycation

51
E. coli and P. pastoris secretion expression system

Expression strains construction

↓ Seed culture
High through put screening
↓
High production strains

Adjust glucose
Mini fermentor
feeding pump rate system

E. coli system P. pastoris system

Off-line measure Optimized induction conditions
Residues glucose concentration. → Methanol fed medium
Accumulate acetic acid concentration. → Methanol feeding rate
52
Microbial production process develop pipeline

Optimize fermentation parameter

Cell line generate group
by Mini- fermentor system
High through put screening
Top1~2 clone
Dissolve
pH Medium
oxygen

Optimized fermentation parameter scale

up to 5L fermentor Feeding
Temperature
strategy
Scale up

Scale down

Mini fermentor system

53
 DASGIP Parallel Bioreactor Systems

 For Cell culture (8X)

 For Microbial Fermentation (8X)
Capabilities of antibody fragments by E. coli and P. pastoris
in 250 ml fermentation process
120
Product yield (mg/L) E. coli system
100
100
80
60
40
20 10
5 20
0
Ver. 1 Ver. 2 Ver. 3 Ver. 4

800
Product yield (mg/L)

P. pastoris system
600 682
572
400 471
200
62 179
0
Ver. 1 Ver. 2 Ver. 3 Ver. 4 Ver. 5
55
 Case study
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

Methanol feedback control system

Dissolve oxygen feedback control system

Batch
Glycerol feeding
Methanol induction
56
 Case study
Using 5L fermentor to compare different induction
strategy by semi-quantitative SDS-PAGE analysis
Methanol feedback control system Dissolve oxygen feedback control system

The methanol feedback control system can achieve higher antibody production.
57
Antibodies production yield improvement from deep well,
shake flask, 250 ml mini-fermentor to 5L fermentor

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
0.64 2.56 30 30
free

* The supernatant protein production yield.

58
 The improvement of
production yield in E. coli and P. pastoris
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.

0.5 mg/L 10 X 5 mg/L 20 X 100 mg/L

50 ml shake flask 250 ml mini-fermenter 5L fermenter

Deep well screening

1 mg/L 2-10 X 5 mg/L 10-100 X 600 mg/L 1-2.5 X 1700 mg/L

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.
59
限閱資料、禁止複製、轉載及外流
 Microbial production platform

Pichia Pichia
E. oli E. coli
E. coli secretion secretion
Scale intracellular inclusion
secretion (Methanol (Methanol
soluble body/refolding
induction) free)
250 ml √ √ √ √ √
5L √ √ √ √ √
20 L √

Production of 0.1 – 10 g scFv, Fab with 5 L scale

fermentor using microbial secretion system

60
 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

Scale-up to Toxicology Material Technology

20~50 L GMP Manufacture
Production Transfer to CMO

Upstream scale-up  Establishment Transfer of analytical Establishment

Bioreactor of 20-50L methods and reference of Analytical SOP
• CHO cell process flow standard Establishment
• E.coli / Yeast Establishment of drug Transfer of 20-50L of Production Batch
Downstream scale-up substance specification upstream and Record
• Continuous Production of ~100g downstream process Engineering Run
centrifugation toxicology material Scale-up to GMP Production
• Depth filtration Stability of drug 100-200L QC/QA Lot
• Column substance release
chromatography
• UF/DF

62
 One-Stop-Solution from DNA to Clinic
Pre-Clinical Research IND Clinical Study NDA Market
Regulatory Support

Cell Line Development

Process Development & Process Characterization(QbD)

Analytical Development

Protein Characterization CGMP Manufacturing (Mammalian cell & Microbial)

Biosafety Test (TFBS), Documentation Support

Toxicology (QPS)

Quality Management 63
 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
 67

Protein Expression from 5L to 50L

5 Liter scale 50 Liter scale

Current availability : 4 Future availability: one single

Future availability: additional 4 bioreactors use STR bioreactors will be
will be installed in 2017 installed in Q4/2017
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
68
 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
69
mAb Structure

• Mass ~150 kDa

• 2 light chains (~25 kDa each)
• 2 heavy chains (~50 kDa
each)
• 16 disulfide bonds
• Glycans

70
IgG glyco-variants

Alain Beck, et al. Analytical Chemistry

2013,85, 715-736

71
 72

Capability of Protein Characterization

▓: Outsourcing ELISA; qPCR; 2D gel; LC-MS/MS; UPLC;
▓: Not established LAL for endotoxin N-terminal sequencing

LC-MS/MS;
AUC; SEC-MALS Ellman’s assay

CD; DSC;
FT-IR Physicochemical CE; UPLC; MS
properties

SEC; RP;
IEC; HIC LC-MS/MS; UPLC

CE-IEF; CE-SDS SEC; SDS-PAGE;

(reduced and Intact MS
nonreduced);
SDS-PAGE; IEF UV; amino acid composition analysis
 73

Structural Characterization and Confirmation

LC/MS/MS
• Peptide mapping
• Glycomapping
• Glycosylation site ID
• Glycan structure ID

©財團法人生物技術開發中心版權所有 保留一切權利
 74

Protein Characterization
cIEF HPLC-SEC-UV-MALLS-RI

CE glycan analysis
CE SDS: IgG purity –reduced & nonreduced
 Capability/Protein Characterization
• 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 75
– CIX
 Capability/Analytical Development
• Identification • Potency/Activity
– SDS-PAGE – ELISA
– Western blot – Cell base assay
– IEF/CIEF • Impurities
– Peptide mapping
– Host cell DNA
– HPLC
– Host cell protein
– CE-Glycan profile
– ProA residue
• Quantification – Others
– BCA/Bradford • Biological safety
– A280
– Endotoxin
• Purity – Bioburden
– SEC-HPLC – Microbial enumeration
– RP-HPLC
– PAGE

76
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 Forced
Screen 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)
 Case study 1. BI HEX® at a Glance
BI HEX ® is Boehringer Ingelheims proprietary CHO based high expression
platform
– Efficient vector systems with novel genetic
elements for generation and selection of high Enhancer
Medium
producer clones BI promotor
& Feed
Ferm. Promoter

– Serum-free cloning procedures, transfection Process Terminator

and cultivation of suspension-adapted CHO Product

BI HEX Vector + Product
cells in chemically defined media Harvest Sequence

Titer
– Sophisticated design of integrated high
Terminator
throughput screening platforms delivering Clone
Marker
&
highly productive and robust clones suitable Selection
TEs

for large scale commercial manufacturing Selection marker

Ampli- Folding/
fication Transport

– Product quality as integral criterion for clone

screening and selection

Decades of scientific experience in represented in BI HEX ®

 Batch and Fed-batch profile in bioreactor
(2-5L, 20L bioreactor)

Growth curve Productivity

2.5E+07 100 3000
2540
2.0E+07 80 2500 2350

Titer mg/L (by HPLC)

VCD (cells/ml)

2000 1929

Viability (%)
1.5E+07 60
1698
1476 1400
1500
1.0E+07 40

1000
5.0E+06 20

500
0.0E+00 0 172 190
0 72 144 216 288 360
0
Culture time(hr)
B06 B15 FB03 FB10
FB12 FB14 FB16 FB17

-- 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.
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.
 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

85
 Batch and Fed-batch Profile in Bioreactor
(5L, 20L bioreactor)

Growth profile Titer

4.0E+07 100 1500

80 1163
3.0E+07
1029
VCD (cells/ml)

Titer mg/L (ELISA)

60 1000

Viability (%)
2.0E+07
40

1.0E+07
20 500
355

0.0E+00 0
0 72 144 216 288 360 432
Culture time(hr) 0
batch 5L fed batch 20L Fed batch batch 5L 20L

-- 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. 86
 Batch, Fed-batch and XD® Profile in Bioreactor
(5L, 5-20L, 2L bioreactor)
Growth profile Titer
3.0E+08 100 4500
3865
2.5E+08 4000
80
3500
VCD (cells/ml)

2.0E+08

Viability (%)
60

Titer mg/L (ELISA)

3000
1.5E+08
40 2500
1.0E+08
2000
5.0E+07 20
1500 1163
1029
0.0E+00 0 1000
0 72 144 216 288 360 432 355
Culture time(hr) 500

batch XD 0
5L fed batch 20L Fed batch batch 5L 20L XD

-- 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]). 87
Maximal cell density of PER.C6/Ab3 in XD® Mark I process can reach 200E6 cells/ml.
 N-Glycan Profile of PER.C6/Ab
5 5 3.0 3.0

2.5 2.5
4 4

1
1

2
2.0 2.0

3 3

1.5 1.5
3
RFU

RFU
RFU

RFU
RF
U

RF
U
2 2
1.0 1.0

6
3
4

1 1 0.5 0.5

4
6

7
5
5
2

0.0 0.0
0 0

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 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
Minutes Minutes

3.0 3.03.0 3.0

2.5 2.52.5 2.5

2.0 2.02.0 2.0

3

1.5 1.51.5 1.5

RFU

RFU
RFU

RFU
RF
U

1.0 1.01.0 1.0

4
4

0.5 0.50.5 0.5

5
2

7
0.0 0.00.0
1

0.0

6
1

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 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
Minutes Minutes

88
生技中心CGMP生技藥品先導工廠
DCB CGMP Biopharmaceutical Pilot Plant

89
 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
 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
91
– 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.

93
 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: weikchi@mail.dcb.org.tw

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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
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biopharmaceutical industry
 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)
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