A-Novel-Bacterial-Contamination-in-Cell-Culture-ManufacturingLeptospira-licerasiae-Anders-Vinther--P.pptx

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,#,#,Anders Vinther,Genentech,PDA WCC 19 July 2012,A Novel Bacterial Contamination in Cell Culture Manufacturing,Leptospira licerasiae,Anders Vinther,Ph.D.,VP,Biologics Quality,Genentech,Roche,Vinther.anders,PDA WCC,19 July 2012,2,Presentation Outline,Objective,How a TV Rerun Reminded of an Important Aspect of What We Do in Cell Culture Manufacturing,Summary of Contamination,Characteristics of the Contaminant,Root Cause Analysis,Impact Assessment,Risk Control Strategies ConsideredLessons Learned,Acknowledgements,3,Objective of this Presentation,To communicate Roche,s findings associated with a novel contamination investigation at a cell culture manufacturing site,House,Season 2,Episode 10,Failure to Communicate,Patient in limbo all tests give negative results,yet he continues to display strange neurological symptoms in which his speech is non-sensical,Dr.House hypothesizes the likely cause-,“,Get his blood on a slide.And,DO NOT,put it through a computer this time!,”,Finally putting the patient,s sample in front of a human for analysis cerebral Malaria is properly diagnosed,Dr.Eric Foreman,after finally making the diagnosis,“,If a human being had actually looked at his blood anywhere along the line,instead of just running tests through the computer,the parasites would have jumped right out at them,”,Dr.Allison Cameron responds,“,Price of the electronic age,”,Maybe we shouldn,t always accept all elements of the electronic age,9,CHO Cell Culture Process Flow Diagram,Thaw&Early Passage Spinners,Secondary Repeated Passages,20L,N-3,N-2,N-1,N,N-4,Inoculum Train Scaleup,Production,11,Summary of Contamination Events,Contamination in 20L seed train bioreactors(STB)observed during routine microscopic visual examination of the cell culture,No bacteria observed in the Gram stain,and bioburden testing showed no growth after 5 days incubation with standard plate count media,Bacterial DNA associated with(CHO)colonies on Blood Agar Plates purified and identified as,Leptospira licerasiae,by 16S DNA sequencing.This is a novel organism in our biological production network.,Neither cell culture parameters(i.e.pH,DO,2,and cell culture performance)nor QC standard confirmatory contamination testing detected this bacterium!,12,Summary of Contamination Events,cont,d,Organism could not be further cultured at that time,preventing additional studies to further identify root cause,Subsequently,a second contamination of the same organism occurred in another 20L bioreactor.This time,organism was successfully cultured,allowing additional studies to understand root cause and determine potential additional detection controls,The scope of the events impacted multiple production batches due to the inoculation of multiple productions runs from the seed train,Leptospira,are bacteria from the Order,Spirochaetales,Leptospira,are thin(0.1um in diameter),coiled/spiral,motilecannot be visualized by the conventional bacteriological Gram staining method,Leptospira,are able to,survive in soil and water,for long periods of time.They have ability to form biofilms.,Commonly found in animals,(rodents,dogs,horses,etc.)excreted in urine,numerous species are pathogenic,Leptospirosis is a re-emerging zoonotic disease,13,Leptospira,Overview,Leptospira licerasiae,contains atypical lipopolysaccharides(LPS)which are weakly reactive with typical LAL methods,Leptospira licerasiae,possesses a gene sequence coding for hemolysin(exotoxin),Leptospira licerasiae can pass through typical 0.1 um filters!,14,Leptospira,Overview,Leptospira,are slow-growing obligate aerobes,favor liquid environment,Have a nutritional requirement for long-chain fatty acids they,will not grow in typical CHO medium alone,but will grow in the presence of CHO cells,Leptospira,are not spore formers and,therefore,are not expected to be heat resistant,CHO,Cell,15,Growth Characteristic in CHO Process&Connection to Contamination Observations,Growth rate estimation based on laboratory co-cultivation with CHO,td 16 hr,Event(s)genealogies and visual observation LOD suggest:,First event(multiple bioreactors)likely originated in spinner flasks;second event likely originated at the 20L stage,Initial contaminant levels estimated to be very low,MVE,Claimed MVE LoD,Analysis by Jun Luo,16,Initial 0.1,m,m Filtration Studies with Leptospira,Case#,Cell Source,Filter*,Lab Visual*,EMJH media growth testing,1,Lepto containing CHO culture,0.1 um PVDF Type 1,+,+,Few motile Leptospira observed,2,0.1 um PVDF Type 2,+,+,Several motile Leptospira observed,3,0.1 um PVDF Type 2,(different lot),+,+,Several motile Leptospira observed,4,0.1 0.1 um,(PVDF Type 2),+,+,Several motile Leptospira observed,5(positive control)N/A,+,+,Many motile Leptospira observed,6(negative control),Lepto-free CHO culture,0.1 um PVDF Type 1,-,-,No Leptospira observed,7(negative control),0.1 um PVDF Type 2,-,-,No Leptospira observed,*All cases were first filtered through 0.45 um PVDF to remove CHO cells,*Samples were concentrated before observation.,“,+,”,Lepto observed,“,-,”,no Lepto observed.,Work done in collaboration with EMD Millipore(Joe Runner from GNE),0.1 um Nuclepore membrane,No wonder Lepto can penetrate!,Leptospira Morphology Dependent on Environment,Cultured 10 days in EMJH,Cultured 7 days in EMJH,3 days in Product C medium,SEM images on 0.1,m,m isopore membrane filters,Work done in collaboration with EMD Millipore,18,Investigation Actions Associated with Events,Successfully cultured,L.licerasiae,in Ellinghausen-McCullough-Johnson-Harris(EMJH)medium,enabling root cause investigation and detection methods evaluation,Implemented non-routine culture testing in EMJH medium to enhance detection in the following samples:,Aliquot from each Working Cell Bank(WCB)ampoule thaw,Pre-harvest Cell Culture Fluid(PHCCF),Optimized and implemented a commercial,Leptospira,-specific PCR assay to enhance detection sensitivityEstimated LOD to be 100 organisms/mL in PHCCF.,19,Investigation Actions Associated with Events,Estimated the LOD of microscopic visual examination to be 106 organisms/mL,Estimated,Leptospira licerasiae,doubling time in CHO cell culture to be 16 hours,Performed survey testing across the manufacturing network using PCR method to further confirm the scope of investigation,none detected,Performed global risk assessment of current upstream microbial control system in relation to this novel microorganism and root cause analysis,Global Risk Assessment,Fault Tree Analysis(FTA)was the risk assessment methodology,The failure was defined as,“,potential contamination of cell culture production with microorganisms that have limited detectability with the current control systems(,L.licerasiae,as a worst-case model),”,.The FTA evaluated the following six categories of potential failure pathways:,Raw material,Process,Equipment,Personnel,Environment,and Potential weaknesses in the current detection systems.,A total of 101 potential failure points were identified and evaluated;numerous actions were taken and activities performed based on this evaluation.,The current prevention and detection control systems were evaluated for all identified failure points with respect to their adequacy and the need for any additional controls.Recommendations for improvements were identified as an output of the Fault Tree Analysis.,21,Root Cause Analysis,The most probable root cause for the contaminations was determined to be the small volume media preparation process occurring in the Small Volume Media Preparation Area:,Based on PCR results,EMJH medium culture study results,and data review,it was concluded that low levels of contaminations likely occurred very early in the seed train,Leptospira licerasiae,mostly likely present in the pre-filtered small volume media since only single-organism contaminations were observed,and demonstrated that,L.licerasiae,can pass through 0.1,m,m filters,Potential source of,L.licerasiae?,22,Root Cause Analysis,cont,d,Potential source of,L.licerasiae,Raw Materials no evidence found,but very difficult to test conclusively,Environment(found spirochetes in untreated water source used in site cooling tower),Discontinued use of this water source,Personnel no evidence found,but personnel could be carrier from environment,Based on extensive testing(EMJH/PCR),concluded that the working cell bank is very unlikely to be the source of,Leptospira licerasiae,23,Impact Assessment,Product,pH inactivation,Virus filtration,Impurities clearance(endotoxin,exotoxin,etc),Drug Substance freeze/thaw process,Equipment and Facilities,pH inactivation cleaning effectiveness,Heat inactivation cleaning and sanitization,Disinfectant efficacy testing environmental control,24,pH Inactivation studies,pH,Time(min),Result,2.8,15 to 240,No growth observed for all exposure times,3.4,15 to 240,No growth observed for all exposure times,3.6,15 to 240,No growth observed for all exposure times,4.0,15 to 240,No growth observed for all exposure times,5.0,15 to 240,At 104/mL Growth at 15 min exposure;No growth for all exposures 30 min,At 108/mL Growth at up to 60 min exposure;No growth for all exposures 120 min,6.0,15 to 240,Growth observed under all exposure times,Leptospira licerasiae,at 104/mL and 108/mL exposed to differing pH and exposure times in conditioned affinity pool,Treated samples incubated in EMJH cultural test method,Typical low-pH hold step(not robust),Large virus,removal filter Product F,Retentive,Large virus removal filter Product G,Retentive,Four,“,typical,”,0.1 um sterilizing-grade filters failed to retain,Leptospira,!,Both large virus removal filters retained,L.licerasiae,.These are not likely feasible for media filtration,but indicate significant downstream clearance should a contamination go undetected in cell culture,Work done in collaboration with EMD Millipore(Joe Runner from GNE),27,Risk Control Strategies Considered,Detection,EMJH media culture testing for non-frozen pre-harvest cell culture fluid samples and for residual fluid from each Working Cell Bank ampoule,PCR testing on pre-harvest cell culture fluid samples across the network,Prevention,Upstream prevention(media prep procedures),Barriers to prevent entry of adventitious agents into process streams,Heat treatment,Filtration,UV-C,28,Lessons Learned,L.licerasiae,is a bacterium that can,pass through industry-standard 0.1um filters,and contaminate a CHO culture with,no direct evidence of its presence,!,Extend risk assessment to other potential sources of contaminants with similar characteristics,and,update control strategies based on new information,Enhanced upstream barriers may be warranted(e.g.,heat treatment or other virus barriers),Spirochetes are in the environment;current compendial test methods are,not able to detect,29,Lessons Learned,cont,d,Look,at your cell cultures,Had routine microscopic examination not been in place,the contaminations may have gone undetected!,Engaging in,timely communications,with Health Authorities and Commercial Partners is critical,Sharing this knowledge(lessons learned)with the industry is,important for patients and for the industry,to continually improve control mechanisms,Acknowledgements,(and many more),Harry Lam,Robert Kiss,Vickie Frydenlund,Patricia Lufburrow,Dave Peers,Joseph Chen,Kathey Hanley,Jesse Bergevin,Gordon Walker,Jun Luo,Todd Battistoni,Batu Berkok,Kevin McCarthy,Dana Thompson,Vaishali Shah,Joe Runner,Aimee Lehman,Keith White,Mark Pedersen,Kevin Johnson,Rich Clements,Meliana Ratna,Mark Smith,Robb Shawley,Tom Stapp,Domenic Matthews,Robel Tezare,David Traub,Marcia Coyne,Louisa Gingery,Peter Hawkins,Ivar Kljavin,Mireille Methlin,Holger Kavermann,Tobias Manigold,Vijay Palsania,Emma Ramnarine,Mark Skoog,Friedrich von Wintzingerode,Nathan McKnight,Adeyma Arroyo,Bold,indicates presentation preparation,