A Novel Bacterial Contamination in Cell Culture Manufacturing Leptospira Licerasiae

A Novel Bacterial Contamination in Cell Culture Manufacturing Leptospira licerasiae

Anders Vinther, Ph.D. VP, Biologics Quality, Genentech, Roche [email protected] PDA WCC, 19 July 2012 • • • • • • • • • Risk Control Strategies Considered Control Risk AssessmentImpact Analysis Cause Root Contaminant the of Characteristics Contaminationof Summary What We Do in Cell Culture Manufacturing Cell in We Culture Do What of Aspect Important an of Reminded a RerunTV How Objective Lessons Learned Acknowledgements Presentation Outline Presentation

Anders Vinther, Genentech, PDA WCC 19 July 2012 manufacturing site a novel contaminationinvestigation at a cell culture ToRoche communicate’ Presentation this of Objective s findings associated with 3

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Anders Vinther, Genentech, PDA WCC 19 July 2012 CHO Cell Culture Process Flow Diagram

Thaw & Secondary Repeated Passages Early 20L Passage Spinners

N-4

Inoculum Train Scaleup Production

N-3 N-2 N-1 N • • • • Summary of Contamination Events network. production as Agar and on identified Blood withPlates (CHO) Bacterial purified associated DNAcolonies days incubationwithstandard media platecount No bacteria in observed the Gram stain, and culture cell of the examination Contamination in 20L train seed (STB) bioreactors visual during observed routine microscopic bacterium! this detected testing contamination confirmatory DO pH, (i.e. parameters culture cell Neither Leptospira

licerasiae by 16S DNA sequencing. This is a novel organism in our biological biological This a in is DNAnovel organism our 16S sequencing. by 2 bioburden and cell culture and cell performance)standard nor QC testing showed no growth after 5

Anders Vinther, Genentech, PDA WCC 19 July 2012 • • •

Summary ofSummary Contamination Events, cont train seed the fromruns productions of multiple inoculation the to due batches production multiple impacted events of the scopeThe to understand root cause and potentialdetermine additional controlsdetection studies allowing additional cultured, successfully was organism time,This bioreactor. in 20Lanother occurred organism same of the contamination second a Subsequently, cause root identify further Organism notcould be at further cultured that additional preventing time, to studies 12

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Anders Vinther, Genentech, PDA WCC 19 July 2012 • 13 • • • • Leptospira Leptospira zoonotic emergingdisease - re a pathogenic, Leptospirosis numerous are is urine,species in time. Theyhave abilityformto biofilms. Leptospira visualized theby conventional bacteriological Gram staining method Commonly found inCommonly animals found are thin (0.1um in diameter),(0.1um are thin in coiled/spiral, are bacteria from Order the are able to are able Leptospira survive in soil and water survivesoilin and water (rodents, horses, etc.) dogs,

Overview Spirochaetales

for long periods of motile

– , cannot be ,

excreted

Anders Vinther, Genentech, PDA WCC 19 July 2012 • • • • • • • 14 resistant therefore, are not expected to be heat cells CHO presence of mediumCHO alone fatty acids fatty acids

aerobes, favor liquid environment aerobes, favorliquid

Leptospira licerasiae can pass throughtypical um0.1 filters! (exotoxin) Leptospira licerasiae are weakly reactive with typical LAL methods Leptospira licerasiae Leptospira Have a nutritionalHave a requirement for long- Leptospira

– are not spore formers spore are not and, are are slow

they Leptospira will not grow in typical in grow not will - , but will grow in the, ingrow but will growing growing obligate possesses possesses gene sequence a coding for hemolysin contains atypical lipopolysaccharides (LPS) which

Overview chain chain

Cell CHO

Anders Vinther, Genentech, PDA WCC 19 July 2012 – –

Event(s) genealogies and visual observation LOD LOD suggest: observation and visual genealogies Event(s) td CHO, with -cultivation co on laboratory based estimation rate Growth

Estimated Lepto Conc. in 20L STB (lepto/mL) • • Initial contaminant levels estimated to be very low very be to estimated levels contaminant Initial 20Lthe at likely originated stage event second spinner flasks; in likely originated bioreactors) (multiple event First 1.E+01 1.E+03 1.E+05 1.E+07 1.E+09 Growth ConnectionCharacteristicContamination to Process& CHO Observationsin 1.E-05 1.E-03 1.E-01 0 Leptospira Calculation Growth hr) (DT=16 MVE 20

Days fromDays Thaw 40 15

60 Claimed MVE LoD MVE Claimed

Thaw 3 Visual 3 Thaw Visual 2 Thaw Visual 1 Thaw t=18d at /mL 1 Lepto t=0 at 1 Lepto/mL t=0 at Lepto/mL 100 t=0 at Lepto/mL 10000 Analysis by Jun Luo Jun by Analysis

~16 ~16 hr

Anders Vinther, Genentech, PDA WCC 19 July 2012 Initial 0.1 0.1 Initial ** Samples** were concentrated before observation. through remove to filtered um PVDF cells 0.45 CHO were first cases All * control) 5 (positive 4 3 2 1 control) 7 (negative control) 6 (negative

Case #

containing containing Lepto Work done in collaboration with EMD Millipore Millipore GNE) (Joe from Work Runner collaboration EMD in done with Source culture culture Lepto Lepto CHO CHO CHO CHO Cell

free -free µ

m Filtration Studies with LeptospirawithStudiesm Filtration (PVDF Type2) 0.1 um PVDF Type0.1 1 0.1 (different lot) um PVDF Type0.1 2 um PVDF Type0.1 2 0.1 um PVDF Type0.1 2 N/A 0.1 um PVDF Type0.1 1 No wonder Lepto can penetrate! can Leptowonder No 0.1 um Nuclepore membrane 

0.1 um 0.1 Filter *

Visual** “ + Lab Lab ” + + + + + - - , Lepto observed,

, No Leptospira observed No -, observed Leptospira No -, Leptospira motile Manyobserved+, Leptospira motile Severalobserved+, Leptospira motile Severalobserved+, Leptospira motile Severalobserved+, Leptospira motile Fewobserved+, testing growth media EMJH

“ - ”

no Leptono observed.

Anders Vinther, Genentech, PDA WCC 19 July 2012 µ on 0.1 images •SEM Leptospira Morphology Dependent on Cultured 10 daysEMJH in Work done in collaboration with EMD Millipore EMD withcollaboration in done Work Environment m isopore membrane filters membrane isopore m

3 days in Product C medium Cultured 7 days in EMJH,

Anders Vinther, Genentech, PDA WCC 19 July 2012 Investigation ActionsAssociated Events with • • • to enhance detection in the following samples: following the in detection enhance to non- Implemented cultured Successfully in PHCCF. LOD organisms/mL to be 100 Estimated PCR sensitivity. assay specific to detection enhance a commercial implemented and Optimized evaluation methods and detection investigation cause - McCullough • •

Pre Aliquotfrom Working each Bank (WCB) Cell ampoule thaw - harvest Cell Culture Fluid (PHCCF) FluidCulture harvest Cell - Johnson routine culture testing testing in EMJH culture medium routine L. licerasiae licerasiae L. Harris (EMJH) medium, enabling root root enabling medium, (EMJH) Harris - in Ellinghausen

- Leptospira

Anders Vinther, Genentech, PDA WCC 19 July 2012 Investigation ActionsAssociated Events with • • • • culture to be ≥16 hours ≥16 be to culture Estimated Estimated organisms/mL 10^6 be to examination visual microscopic of LOD the Estimated analysis cause root and microorganism system to control in relation this novel microbial upstream of current assessment risk global Performed investigation of scope the confirm further to method PCR using network the manufacturing across testing survey Performed Leptospira licerasiae licerasiae Leptospira –

none detected none detected

doubling time in CHO cell CHO in time doubling

Anders Vinther, Genentech, PDA WCC 19 July 2012 • methodology assessment (FTA) the risk was Analysis Tree Fault • • evaluated the following six categories of potential failure pathways: failure of potential six categories the following evaluated L. licerasiae current licerasiae controlsystems(L. the with detectability limited have that microorganisms with production as defined was failure The identified as an output of the Fault Tree Analysis. Tree Fault the of anoutput as identified werefor improvements Recommendations controls. any additional and the need for to adequacy their withrespect points failure identified all for evaluated were systems control and detection prevention The current • •

evaluation. onthis based performed and activities taken were actions numerous andevaluated; identified were points failure A of total 101 potential systems. detection current the in weaknesses Potential and Environment, Personnel, Equipment, Process, material, Raw Global Risk Assessment Risk Global

potential contamination of cell culture culture cell of contamination “potential as aworstas case model) -case

. The FTA FTA The ”.

Anders Vinther, Genentech, PDA WCC 19 July 2012 • • occurring occurring in the Small Volume Media Preparation Area: process preparation volumemedia to bethesmall determined was contaminations for the cause probable root most The

Potential source ofL. source Potential • • through 0.1 demonstratedobserved, and were that small volumemedia since onlysingle- Leptospira occurred likely veryearlythe intrain seed data review, concluded itwas thatoflevels contaminationslow results,Based on PCR EMJH medium culture study results, and

Analysis Cause Root licerasiae µ m filters ?? licerasiae

mostlypresent likelythe in - pre organism contaminations L. L. licerasiae

can pass pass can filtered 21

Anders Vinther, Genentech, PDA WCC 19 July 2012 • • licerasiae Leptospira of source to be the is very unlikely bank cell the working that / concluded (EMJH PCR), testing on extensive Based Potential source of of source Potential • • • Root Cause Analysis, cont Analysis, Cause Root from environment Personnel in sitein cooling tower) untreated used Environmentsource waterspirochetes (found in conclusively MaterialsRaw –

no evidence no found, but personnel could be carrier –

no evidence found, but very difficultto test

L. licerasiae licerasiae L. –

Discontinued use of this water source this water of use Discontinued

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Anders Vinther, Genentech, PDA WCC 19 July 2012 • • Product Equipment andFacilities • • • • • • • testing efficacy Disinfectant inactivation Heat pH inactivation process freeze/thaw Substance Drug exotoxin, etc) (endotoxin, clearance Impurities filtration Virus pH inactivation

Impact Assessment

–

– cleaning effectiveness

and sanitization cleaning –

environmental control environmental

Anders Vinther, Genentech, PDA WCC 19 July 2012 •

inactivation should a contamination go undetected in cell culture cell in undetected go contamination a should inactivation Typical low • • pH 4.0 3.6 6.0 3.4 2.8 5.0 Treated culturalsamples EMJH testmethodincubated in differing pH and exposure timesconditioned in affinitypool Leptospira licerasiae

pH Inactivation studies Inactivation pH - Time (min) pH hold step (<4) forMab production effective at 15 to 240 15 to 15 to 240 15 to 15 to 240 15 to 15 to 240 15 to 15 to 240 15 to 15 to 240 15 to

Work by Gordon WalkerGordon Work by microbiologygroup times exposure all for observed growth No times exposure all under observed Growth exposures times exposure all for observed growth No At 10^8/mL ≥ times exposure all for observed growth No At 10^4/mL No growth No observed exposure for all times 30 min at 10^4/mLat 10^8/mL and exposed to

≥ 120 min – –

Growth at up to 60 min exposure; No growth for for all growth No exposure; min up 60 to at Growth Growth at 15 15 at min No for exposure; exposures Growth all growth

Result

Anders Vinther, Genentech, PDA WCC 19 July 2012 • • • L. licerasiae L. licerasiae or the facility (cleaning and sanitization efficacy) and sanitization (cleaning facility the or equipment process in persist unlikely to is organism the that assurance good Provides at mild veryconditions effective Heat treatment at inactivation 102 Heat InactivationHeat Studies method test ≥10^6/mL,Treatedat cultural in EMJH incubated samples (std media HTST) media (std Temp (C) Temp (C) 100 Work by Gordon Walker microbiology group and Joe Runner WalkerGordon Joe Work by microbiology and group 85 65 65 45 Pilot - Scale Scale Continuous Flow Exposure(HTST)

Lab-

Scale Batch Exposure Batch Scale 5, 10, 30, 60 30, 10, 5, 60 30, 10, 5, 5, 10, 30, 60 30, 10, 5, 60 30, 10, 5, Time (sec) Time (sec) 5, 10, 5, 5, 10, 20 10, 5,

Result (Growth / / (Growth Result Result (Growth / / (Growth Result No Growth)No No GrowthNo GrowthNo No GrowthNo No Growth)No No GrowthNo No GrowthNo Growth

Anders Vinther, Genentech, PDA WCC 19 July 2012 Four • culture cell in undetected go should acontamination clearance downstream indicate significant but filtration, media retained filters removal virus large Both 0.1 um 0.1 um 0.1 um 0.1 Large virus removal filter Product G Product filter removal virus Large Large virus Large 0.1 um 0.1 Filter Type removal) for(designed improved mycoplasma “ typical um sterilizing um Work done in collaboration with EMD Millipore Millipore GNE) (Joe from Work Runner collaboration EMD in done with

sterilizing sterilizing sterilizing sterilizing

”

removal filter Product F Product filter removal 0.1 um sterilizing um 0.1 Filtration RemovalStudies grade Product D Product -grade C Product -grade B Product -grade grade Product E E Product -grade A Product -grade - grade filters failedto retain

. These are not likely feasible for feasible likely not are. These L. licerasiae

retentive Not retentive Not retentive Not retentive Not Retentive Retentive pmax Result Retentive under vmax under Retentive , conditions (=> not robust) not (=> conditions ,

, but ,

Leptospira! 26

not not

Anders Vinther, Genentech, PDA WCC 19 July 2012 • Detection • • Risk Control Strategies Considered network the across samples fluid culture cell -harvest on pre testing PCR Bank ampoule Cell from Working each fluid andfor residual samples fluid culture cell -harvest pre non-frozen for testing culture media EMJH

• Prevention

• • Upstreamprevention (media prep procedures) streams adventitiouspreventofprocessBarriers entry agentsinto to • • • UV Filtration treatment Heat

- C

Anders Vinther, Genentech, PDA WCC 19 July 2012 • • • are methods test compendial current environment; the in are Spirochetes treatment barriers) virus or other treatment (e.g., heat bewarranted may barriers upstream Enhanced information new on based strategies control and characteristics, similar with contaminants of sources to potential other assessment risk Extend culture with with culture industry licerasiae L. - standard 0.1um filters filters 0.1um standard no direct evidence of its ! presence its of evidence no direct

pass through passthrough can that is abacterium Lessons Learned not able to detect detect to able not

and contaminate a CHO a CHO and contaminate

update update 28 –

Anders Vinther, Genentech, PDA WCC 19 July 2012 • • • Look Engaging in goneundetected! have may the contaminations in place, been not examination Authorities and Commercial Partners Partners is critical andCommercial Authorities mechanisms control improve continually is industry the with learned) (lessons knowledge this Sharing important for patients and for the industry the andfor patients for important at your cell cultures – cultures cell your at Lessons Learned, cont Learned, Lessons timely communications communications timely

Had routine microscopic microscopic routine Had ’ with Health Health with d

to to 29

Anders Vinther, Genentech, PDA WCC 19 July 2012

Kathey Joseph Dave Lufburrow Patricia Vickie Robert Kiss Harry Lam Dana Thompson McCarthy Kevin Berkok Batu Todd Battistoni Jun Walker Gordon Jesse Bergevin Acknowledgements Acknowledgements Luo Peers Frydenlund

Hanley Chen

Bold

indicates presentation preparation presentation indicates

Adeyma Nathan McKnight Wintzingerode von Friedrich Mark Emma Vijay Palsania Tobias Manigold Holger Ivar Peter Hawkins Gingery Louisa CoyneMarcia David Mireille

Kljavin Skoog Traub Kavermann Ramnarine

Methlin Arroyo

(and many more…) many (and

Vaishali Robel Matthews Domenic Tom Robb Shawley Mark Smith Meliana Clements Rich Johnson Kevin Pedersen Mark White Keith Lehman Aimee Runner Joe Stapp

Tezare

Shah Ratna

Anders Vinther, Genentech, PDA WCC 19 July 2012