20130521─生物技术开发中心《生技医药产业实务概论 14 生技实务─生物安全性检测》58页

生技實務--生物安全性檢測Wenlynn Su, PhDDirector, Testing Facility for Biological Safety 20130521@NYMUDevelopment Center for Biotechnology大綱vs. 藥物生物安全2¾實際案例¾生物/生技藥物¾生物安全檢測介紹¾產業現況及生技藥品檢驗中心介紹一隻病毒價值USD85,000,000！Genzyme Corp.at Allston, MA (Headquarter at Cambridge, MA)Photography from CP Liu3Genzyme Temporarily Interrupts Production at Allston Plant¾Genzyme Corporation (NASDAQ: GENZ) 公布在Allston Landing manufacturing facility 的生物反應槽檢出病毒(impairs cell growth in one of six bioreactors)；並決定暫停生物製劑生產，以全面進行設備消毒(Date: June 16, 2009)。

¾Vesivirus 2117, 未有感染人的報導；可能來源為營養添加劑；the company was able to detect the virus in this case using a highly specific assay, which was developed after standard tests failed to identify the cause of the previous productivity declines.¾此停產行動造成兩項罕病用藥短缺：Cerezyme ®和Fabrazyme ®；該公司預期可於當年九月恢復市場供應。

(Source: Genzyme Corp.)4How Fabry Disease Affects the body? 56Vesivirus 2117VirusVesivirus 2117Family Caliciviradea, VesivirusStrain vesivirus 2117Genome type (+) ss-RNA , linearGenome size 7.4-7.7 kb Envelopenon-enveloped Virion size(Diameter)32-40nm Virus replication site Cytoplasm Host OrganismCHOicosahedral symmetry;32 cup-shaped depressionson the surfaceProfit from Genzyme's Problems¾生產相似功能藥物的藥廠成為此事件的受益方。

¾Shire Pharmaceuticals 生產之Replagal，為Fabrazyme 的相似功能藥物，主要在美國以外地區販售。

¾Citigroup估計Shire Pharmaceuticals 因此次事件，可提高Replagal的市佔率，增加美金85,000,000營收。

Source: ShareCast, Jun.17, 20098Price of Sanitation by Genzyme9(source:Genzyme Corp.)9Duration: 2 months92,236 gallons of solution used91,488 cans of isopropyl alcohol for cleaning9Outside contractors worked more than 40,000 servicehours9More than 700 fluorescent light lenses were replaced93,253 valve diaphragms936,625 gaskets9267 HEPA filters9233 ball valves9358 rebuild kits大綱10¾實際案例¾生物/生技藥物vs. 藥物生物安全¾生物安全檢測介紹¾產業現況及生技藥品檢驗中心介紹生物/生技藥物11¾生物/生技藥物的生產或來源：動物細胞、細菌、酵母菌；動物及基因改造動物；人類的組織細胞、血液、體液。

¾生物/生技藥物的形式：蛋白質藥物、單株抗體、疫苗；血漿或體液製劑；組織、幹細胞；基因治療載體。

藥物生物安全12¾目的：管控生物藥物內可能含有潛在危害物質的風險。

¾潛在危害物質：(1)生物體內潛藏的致病微生物(2)具潛在危害性質的生物成分¾藥物生物安全檢驗：檢測及排除微生物及生物成份（包含宿主）污染。

¾各國政府藥品查驗機關皆有相關法規管理及規範。

管控生物藥物內存在生物性危害物質的風險生物安全檢測Biological SafetyTesting生物安全實驗室Biological Safety Laboratory目的確保生物藥品安全確保操作人員與環境安全下，實施生物藥品安全檢驗。

實施對象生物藥品及原料操作者及設施實施方法管控、檢測、篩檢、確效…操作人員適當穿戴防護裝備、定期接受生物安全教育訓練、設施定期維護…13以細胞生產生技藥品時可能發生的污染產品細菌黴漿菌病毒宿主的蛋白質宿主的核酸14生技藥品可能的病毒污染來源15BioReliance, PDA Virus Safety Forum, June 2008生技藥品常見的病毒污染16Cell or Products Viral Contaminant Source of Virus Various BVDV (non-cytopathic) Bovine serumBHK/CHO Reovirus Bovine serumCHO EHDV Bovine serumCHO Cache Valley Virus Bovine serumCHO Minute Virus of Mice Components of media Rhesus monkey kidney SV40Primary cell lineInsect Tn5 (High Five)Nodavirus Latent infection of cellBlood derived products HBV、HIV、HCV etc MaterialsApproaches to control potential viral contamination in biologics17¾Cell lines and other raw materials including mediacomponents are tested for absence of undesirableviruses, which may be infectious and/or pathogenicfor humans.¾Capability of the production processes are assessedto ensure the ability to clear infectious viruses.¾Product at appropriate steps of production is testedto ensure free of contaminating infectious viruses.大綱18¾實際案例¾生物/生技藥物vs. 藥物生物安全¾生物安全檢測介紹¾產業現況及生技藥品檢驗中心介紹生技藥品製程蛋白藥，單株抗體，疫苗…將基因送入生產細胞小規模製程大規模製程純化19Scientific issues that should be considered 20¾Production, identification and characterizationof the cell substrate;¾Validation of the manufacturing process forremoval and/or inactivation of adventitiousagents;¾Testing of the bulk and final product to assuresafety.藥物生物安全性測試技術21細胞庫安全性測試：檢測技術(體內或體外試驗)9檢測生產宿主、中間產物、最終成品9是否有其他生物性污染製程病毒清除確效：9排除生物體或細胞工廠或生產過程導入的病毒污染9製程須有效地去除潛在病毒或將病毒去活化生產生技藥品必須測試項目測試項目種原細胞庫工作細胞庫終點生產細胞粗產物(每批)產品(每批)臨床實驗用藥(每批)無菌測試999999黴漿菌測試9999999病毒抗體產生測試9外源病毒測試(體外) 999外源病毒測試(動物)999特定病毒測試99(9)細胞核酸測試99細胞蛋白質測試99熱原測試99反轉錄病毒測試2223¾There is an inherent risk of retrovirus transmissionin cells used to produce human biologics.¾Risks exist for both endogenous and exogenousretroviruses.¾Presence of some retroviruses do not prevent useof cells, but the risk must be quantified.¾Retroviruses tend not to produce CPE (exit cells bybudding) hence specific methods are required todetect their existence.C-type Retroviruses -TEM 25Virus particles 60,000x Budding 60,000xIn Vitro Adventitious Virus DetectionBT (Bovine)ST (Pig)MRC-5 (Human Diploid)Vero (Simian)HeLaSome Species and Tissue-SP/0, BHK, CHO, etc.Cytopathic effect (CPE)Haemadsorpation(HAD)Haemaggulation(HA)Immunoflueoscent(IFA)Reverse Transcriptase (RT)Electron microscopy (EM)Quantitative PCR (qPCR) Detector Cells Detection MethodsTest Article (±spiked virus); PC/NCpassaged at 7-d intervalsMonolayer cells Passaged cells14-28 Days26In Vitro --Detection Method 27¾Cytopathic Effect (CPE)9Cells die -plaques9Cells are transformed –foci9Cells do not look/behave normally -rounding, growth, etc.¾Haemadsorption(HAd)9Binding of red blood cells to viral glycoproteins on the cell surface ¾Haemagglutination(HA)9Formation of cell aggregates through cross linking by red blood ¾Immunofluorescent assay (IFA)9Virus specific antibody is conjugated with a fluorescent dye and used to detect the specific antigen or virus.¾qPCR9Analysis of cell lysate by PCR for viruses that do not produce CPE in standard detector cells, e.g. HBV, HCV, B-19 parvovirus.In Vitro --Detection Method 28HA HAdIn Vitro Adventitious Virus Detection¾Multiple samples can be handled concurrently¾Low Limit of Detection (LOD) ~ 1 TCID50¾Well established, accepted method ¾Can not detect all viruses 9Viruses that do not growwell in cells9Viruses that do not causecytopathic effect¾Only detects infectious virus¾Limitations in the types of used detector cells¾Cultures are susceptible to toxicity as well as viral effectsAdvantages Limitations 29休息一下 (30)9A test article should be inoculated into animals, including suckling and adult mice as well asembryonated eggs to reveal viruses that cannot grow in cell cultures.9Health of tested animals should be monitored; any abnormality should be investigated to establish the cause of illness.9Additional animal species may be used depending on the nature and source of the cell lines being tested, e.g. Guinea Pig, Rabbit.In Vivo Adventitious Virus Detection31In Vivo --Detection MethodsTest article (cell lysate: 107cell/ml)Adult miceSuckling miceGuinea Pigi.p., i.c., i.n. and/or oral i.p., i.c. and/or orali.p., i.c.Sacrifice@D28Sacrifice@D28Passage@D14Sacrifice@D14Animals are monitored during test periods…32Routes of Entryi.p.oral i.n.i.c.Intraperitoneal IntracerebralIntranasal33In Vivo --Embryonated EggsAllantoic (尿囊腔)1)Eggs are injected with TAand incubated for 3 days 2)Allantoic fluids are testedfor hemagglutinins 3)Fluids are pooled andpassaged to eggs 4)Fluid from passaged eggsare tested for hemagglutinins34In Vivo --Antibody Production Tests 35¾Species-specific viruses present in rodent celllines may be detected by inoculating test articleinto virus-free animals followed by examiningserum for antibody or enzyme activity after aspecified period.¾Examples of such tests include Mouse AntibodyProduction (MAP) test, Rat Antibody Production(RAP) test, and Hamster Antibody Production(HAP) test.In Vivo --Antibody Production Testsmicehamsterrati.p., i.c., oral, i.n. i.p., oral, i.n. i.p., i.n.Animals are observed for 4 weeksAnimals are euthanized and blood is collected by heart puncturingSera are evaluated for existence of antibody against specific virusTest article (cell lysate: 107cell/ml)36In Vivo Adventitious Virus Detection9Detects viruses of a broad-spectrum9Detects viruses that do not grow well in tissue culture9Many viruses were firstisolated from suckling mice,e.g. SARS, human corona-virus OC43, new humancardiovirus(SAF-V)9High sensitivity 9Toxic effects of pluronic acid (serum free media),adenovirus penton proteins 9Uses animals9Little formal validation studies 9Lengthy assay9CostAdvantages Limitations 37製程病毒清除確效39Control of Starting Materials¾Characterized cGMP cell banking system In-Process and Final Product Testing¾Batch release testing for unprocessed bulkand final productRobust Down Stream Process (DSP) Control¾Virus clearance study to demonstrate theDSP is capable of removal/inactivatingviruses if any is present¾Host DNA clearanceTypes of Down Stream Process Steps9Heat treatment–pasteurization–lyophilisation/dry heat9Solvent/Detergent9Low or high pH–column elution/sanitisation 9propiolactone/ UV light9irradiation9Inactines/ psoralens 9Precipitation–PEG, EtOH9Chromatography –ion exchange–affinity–hydrophobic9Nano-filtrationInactivation Partitioning 40VirusesVary in¾Size: 18 -300 nm¾Genome9DNA, RNA9single stranded, double stranded9linear, circular, segmented¾Structure9enveloped/non-enveloped 9shape (rod, helix, icosahedron, sphere)9uniformity41Selection of Model Virus for Validation Study 42¾Representative for potential virus contaminants –Expected to be present, e.g. endogenous CHO retrovirus-like particles–Potentially present¾Range of physico-chemical properties–Structure (enveloped, non-enveloped)–Genome (DNA / RNA and single strand /double strand)–Resistance to physico-chemical inactivationSelection of Model Virus for Validation Study 43¾Practical reasons–Titer; Assay; Risk to operator¾Requirements are based on Clinical Phaseof project¾Phase I/II–Duplicate study–2 virus required [MLV/HIV + Parvovirus]–Platform technology that may be acceptable¾Phase III–Duplicate study–Panel of viruses [typically 4 -5]製程病毒清除確效實施方法純化病毒Log reductionCell Culture Chromatography IChromatography IINanofiltrationPurified product> 3 logs>5 logs++<1 logs> 8 logs~ 2 logs> 10 logs44大綱45¾實際案例¾生物/生技藥物vs. 藥物生物安全¾生物安全檢測介紹¾產業現況及生技藥品檢驗中心介紹產業現況及『生技藥品檢驗中心』Testing Facility for Biological Safety(TFBS) 46產業概況479目前全球生技藥品市場超過145億美元；預計西元2015年可達167億美元。