1

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

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

4

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

5

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

9

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

10

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

11

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

13

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

17

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

18

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.

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 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)