Development of Production and Purification Platformform for Influenza Vaccine

Priyabrata Pattnaik, PhDDirector, Head of Biologics Operations – Asia Pacific

Development of Production and Purification Platformfor Influenza Vaccine

2

Presentation Outline

Introduction

Upstream and virus production

Residual DNA removal by Benzonase® treatment

Virus inactivation by Formaldehyde treatment

Conclusions

Development of Production and Purification Platform for Influenza Vaccine | Priyabrata Pattnaik | 02 Nov 2016

Annual Vaccine HA protein (most common) Typically 3-4 varieties (valences) Dosage info (0.5mL/dose)− HA: 15μg of each HA protein− Formaldehyde: ≤ 200ppm− Endotoxin: < 100 IU/dose− Other protein: < 6 x HA content (< 300 μg/dose)− DNA: < 10ng/dose− Purity: 95% by gel (Coomassie blue)

BackgroundInfluenza

3

80-120 nm enveloped virus

By National Institutes of Health; originally uploaded to en.wikipedia by TimVickers (25 October 2006), transferred to Commons by Quadell using CommonsHelper. (California Department of Health Services) [Public domain], via Wikimedia Commons



Observations

Large number of unit

operations

Cumbersome

Multiple opportunities

for process

improvements

Application of

technologies developed

for small proteins – not

optimized for large

molecule separations

Generic Process for Cell Culture Based Influenza Vaccine

Cell culture and virus production


Modes of Influenza Vaccine Production

Egg Based Cell Culture Based2D Adherent 3D Stirred Tank

• Production is slow and subject to avian flu outbreaks

• Improved supply chain robustness• Rapid response to address pandemics• Industrial and regulatory drive for cell based processes

• Labor intensive• Large footprint to support equipment

• Reduced process steps• Easy to scale up

31,000 eggs ≈ 1000 L of culture!

7

Process Schematic

Process Step DetailsCell Selection MDCK (ATCC® CCL-34)

Cell Expansion (2-D Culture) Seed at 1 x 105 cells/mLPassage 3 - 4 daysGrowth Media:DMEM 10% FBS, 4.5 g/L Glucose, 2.25 g/L NaHCO3, 4mM L-Glutamine, 1 X NEAA, 1 x NaPyr

Bioreactor Culture(3-D Culture)

2 – 3e5 cells/mLCytodex® microcarriers (4 g/L)Cell Counts : NC-100™ Nucleocounter®

Bioreactor Infection / Media Exchange

Influenza A/WS/33Infection MediaDMEM 4.5 g/L Glucose, 2.25 g/L NaHCO3, 4mM L-Glutamine, 1 X NEAA, 1 x NaPyr

Virus QuantificationHemagglutination

Dilution-based assay

Viral protein binds to RBCs


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Cell attachment

Initial batch volume

Agitation speed

Sparging

Process Scale-up

Bioreactor Process Development


9

Cell Attachment in Mobius® 3 L Bioreactor with Continuous Mixing

Cells efficiently attach to microcarriers while continuously mixed at 75 rpm

Day 0 Day 1 Day 2

Day 3 Day 4


10

Optimize Mobius® 3 L Bioreactor Initial Volume

Bioreactor Starting Volume:

Uneven distribution of cells on microcarriers

Even distribution of cells on microcarriers

1.0L 1.6L 2.0L


Guidance from microcarrier manufacturer suggests starting bioreactors at partial volume with concentrated microcarriers and cells. Then feed to full volume after cells attach.

Mobius® 3L bioreactor capable of operating between 1.0 – 2.4 L

11

Optimize Starting Volume for Mobius® 3 L Bioreactor

• Cell growth performance comparable in the conditions tested

Bioreactors can be batched at full working volume eliminating additional feed step


12

Selection of optimal agitation to suspend microcarriers and attached cells Just suspended mixing speed (Njs) Homogenous suspension

Provide adequate mixing and aeration to support cell growth

What scaling factor do we use for agitation? Agitation Speed (rpm) Tip speed (cm/s) Power / volume (W/m3)

Optimize Agitation Speed in Mobius® 3 L Bioreactor


13

Optimize Agitation for Cell Growth in Mobius® 3 L Bioreactor

• Fully confluent microcarrierstend to settle near the bottom with slower agitation speeds with the increased biomass

• Sheer stress at higher speeds may slow cell growth

Using agitation speed of 90 rpm provides the best growth

90 rpm

Agitation Speed (rpm) Tip Speed (cm/s) Power Input (W/m3)

75 29.9 0.7

90 35.9 1.3

150 59.8 6.0


14

Sparging considerations

Growth Sparging needs to be able to supply enough oxygen to support viable cell growth Excessive sparging will impart shear stress on culture

Foaming Foaming an issue with cultures containing FBS Increased foam and shear from bubbles rupture cells at air surface interface Cell and microcarriers will tend to get trapped in foam layer Microspargers tend to produce more foam than open pipe

Evaluate Oxygen Sparge Strategies to Support Cell Growth and Minimize Foaming


15

Visual Inspection of Attached Cells On Microcarriers Using Open Pipe and Microsparger In Mobius® 3 L Bioreactor

Significant amount of cell debris 2.5 cm foam layer on surface

Selected O2 open pipe sparger

Open Pipe Oxygen Microsparger O2/Air Microsparger O2

No cell debris Minimal foam


16

Mobius® 3 L Bioreactor Performance Compared to Glass 10 L Bioreactor

• Comparable performance observed in Mobius® 3 L Bioreactor and Glass 10 L Bioreactor • Average pre-infection cell density ~ 3 x 106 cells/mL• Harvest virus titer ~3.5 x 104 HAU/mL


17

Process scale-up from Mobius® 3L Bioreactor to Mobius® 50 L Bioreactor Mobius® 50 L Process Compared to Mobius® 3 L

Scaled process from 3L to 50L vessel applying best practices from 3L process

Performed cell growth step in 50L and parallel 3L satellite

Infection of cultures performed in 3L satellites

Mobius® 50 L Mobius® 3 L

4 Days

Day 0

4 Days

Infection with A/WS/33


Comparable growth observed between Mobius® 50 L and 3L bioreactors

Residual DNA Removal by Benzonase®

Endonuclease

19

• Mg2+ (1-2mM) required for enzyme activity

Benzonase® Endonuclease

Genetically engineered endonuclease that cleaves all forms of DNA and RNA.

Origin: Serratia marcescens

Expression: E.coli K -12 mutant

Molecular mass: ca. 30 kD (subunit, exist as dimer)

Isoelectric point (pI): 6.85

Functional in pH range: 6–10

Temperature: 0 - 42ºC

One unit of Benzonase® Endonuclease degrades approximately 37µg DNA in 30 min to as low as 3-8 base pairs (<6 kDa).


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Parameters influencing activity of Benzonase® Endonuclease

Influenza / MDCK Process

Where in the process? Semi-purified feed (post

inactivated/TFF) Non-clarified bioreactor feed

Conditions? Concentration Time Temperature Magnesium / alternative metal

ions Impact of process step on

conditionsDevelopment of Production and Purification Platform for Influenza Vaccine | Priyabrata Pattnaik | 02 Nov 2016

21

Scoping studies in semi-purified feed: PicoGreen® Analysis

~4-fold reduction in DNA with 50 U/mL Benzonase® Endonuclease at 24 hr

Though DNA quantity and size are reduced, the PicoGreen® assay detects small fragments (~10bp fragments)

Illustrate the value of the holistic approach using multiple analytics: gel analysis indicate 0.5 U/mL while

PicoGreen® analysis indicates 50 U/mL is better

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 U/mL 0.5 U/mL 50 U/mL 300 U/mL MDCK gDNA MDCK gDNA +20U/mL

DN

A C

once

ntra

tion

(ug/

mL)

Concentration of Benzonase®

MDCK DNA Concentration Post Benzonase® Treatmentin semi-purified feed

Control

Incubation Time (hours at 37 °C): 4, 8, 24

Benzonase® Conc. (U/mL): 0, 0.5, 5, 10, 50, 100, 200,

300

Analytics: Agarose gels PicoGreen® Assay


0.0

0.2

0.4

0.6

0.8

1.0

DNA

Conc

entra

tion

(µg/

mL) MDCK DNA Concentration at Process Steps

qPCR PicoGreen

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Benzonase® digestion scale up studies (10L) in semi-purified feed

Combination of clarification, ultrafiltration & Benzonase® treatment removes nucleic acid As previously shown, there is value in using orthogonal approaches to assess residual nucleic acid


23

Scoping study: Is Benzonase® treatment in the bioreactor a viable option?

BioreactorNucleic Acid

Digestion

Incubation Time: 33 °C for 24 hours

Benzonase® Concentration (U/mL): 0.5, 50, 300

Analytics: PicoGreen® Assay qPCR Assay Capillary electrophoresis

®


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Scoping study Benzonase® Treatment in non-clarified bioreactor feed

Benzonase® Endonuclease active at 33oC in complex matrix

Very low nucleic acid levels after 24 hrs with Benzonase® Endonuclease

non-clarified 33oC control (0 U/mL)

non-clarified 33oC 24hr with 50 U/mL Benzonase

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 0 0.5 50 300 U/mL

4°C 33°C

Non-Clarified Feed

DN

A Co

ncen

trai

on (u

g/m

L)

MDCK DNA Concentration Post Benzonase in Non-Clarified Bioreactor Feed

qPCR

PicoGreen

®

®

®

Only 2X improvement in digestion with 10 fold increase in Benzonase® concentration

Virus Inactivation

Methods of Inactivation

Viral inactivation eliminates the virus’ ability to infect and propagate Inactivation should occur as early in the process as possible to reduce operator risk (Ph.Eur.) Must retain virological and immunological properties Validation of inactivation is a necessity

Formaldehyde

Most common method for seasonal influenza inactivation Must not exceed concentrations

of 0.2 g/L (Ph.Eur.) at any time Removal is critical to ensure

patient safety

Beta-Propiolactone

2nd most common chemical inactivating agent Carcinogenic but rapidly

undergoes hydrolysis in water

Alternatives

Heat Hydrogen Peroxides Gamma irradiation UV irradiation

1 2 3


RT-qPCR - measures viral genomes, but cannot distinguish between infectious & non-infectious virus. The TCID50 assay, used in conjunction with qPCR, confirms inactivation and the presence of virus particles

Hemagglutinin assay – measures intact virus particles and is rapid assay that is commonly used to determine HA titers. However, the assay has 50% variability

Infectivity (TCID50) – measures infectious virus particles, used for assessing the inactivation of infectious virus particles, but sample matrix present challenges which can be overcome by dilution, dialysis or ultrafiltration

Neuraminidase assay - measures functional NA. However, post-inactivation the assay no longer applies


Assays leveraged with virus inactivation

Assays

NO CPE CPE

Full factorial design 3 factors: [formaldehyde], incubation time and

temperature Varying levels of each factor 5 x 2 x 4 factorial

Experimental setup Clarified MDCK-based influenza feedstream Shaker flasks with agitation

Responses Infectivity assay (TCID50) performed for confirmation

of inactivation of Influenza Influenza RT-qPCR analysis conducted for presence of

viral genomic RNA before and after inactivation HA assay performed for hemagglutinin titer (Assay

variability of 50%)

Experimental Design and Responses


RT = 22°C-23°C


With up to 24 hours of incubation− 1-log reduction in infectivity titer seen

with room temperature − 3-logs reduction in infectivity titer

observed at 32°C Overall, no loss in HA titer − Note: Area between red horizontal lines

correspond to 50% assay variability

Effect of Temperature on Influenza Inactivation

More pronounced effect of incubation at 32°C on virus inactivation than room temperature

50%

var

iabi

lity

in H

A t

iter


5-logs reduction in infectivity titer with 0.02% formaldehyde and 4 hour incubation at room temperature No loss in HA titer overall − Even with increasing [formaldehyde] and

extended incubation time− Note: Area between red horizontal lines


Effect of Room Temperature and Formaldehyde on Influenza Inactivation

Influenza inactivation achieved with lowest [formaldehyde] and shortest incubation time

50%

var

iabi

lity

in H

A t

iter


6-logs reduction in infectivity titer with 0.02% formaldehyde and 4 hour incubation at 32°C Detrimental effect on HA titer with highest

[formaldehyde] and prolonged incubation time − 0.2% formaldehyde for 24 hours− 0.2% and 0.1% formaldehyde for 48 hours− Note: Area between red horizontal lines


Effect of 32°C Temperature and Formaldehyde on Influenza Inactivation

Influenza inactivation achieved with lowest [formaldehyde] and shortest incubation time

50%

var

iabi

lity

in H

A t

iter


Large Scale Virus Inactivation and Impurities

0.02% Formaldehyde Temp: 22°C & Agitation

Sample Collection at 4 and 24 hrs

Clarified Influenza Feed

HCP ELISA & DNA quantification vis Picogreen assay

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Conclusions

3

2

1Cell Culture and Influenza Virus Production Successfully cultured MDCK cells and produced virus in Mobius® 3 L Bioreactor

Cell attachment under continuous agitation conditions Batch inoculation at full working volume to ensure homogenous attachment of cells to

microcarriers Agitation at 90 rpm provides adequate mixing for cell attachment and growth Use of open pipe sparging to support cell growth and minimize foam accumulation

Demonstrated comparable process performance in Mobius® 3 L and 50 L Single-use Bioreactors

Benzonase Treatment Optimization Studied impact of Benzonase concentration, time and temperature on DNA digestion Studied successful removal of DNA by orthogonal processing methods Explored possibility of using Benzonase directly in bioreactor

Inactivation of Influenza virus Optimized inactivation using formaldehyde to ensure complete virus inactivation Limited impact on virus antigenicity and analytical assays. DOE derived process parameters were successfully verified at larger scale demonstration

of influenza inactivation with no negative impact on HCP and DNA quantification


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Acknowledgements

Upstream Process DevelopmentMichael McGlothlenPaul HatchMichael PhillipsChris Martin

Downstream Process DevelopmentChristopher GillespieSonal PatelJeff CaronLori MullinKrista CunninghamMichael Bruce

Vaccine ProgramAlex Xenopoulos

Vaccine Learning Center

www.merckmillipore.com/vaccines

Thank You

Priyabrata Pattnaik, [email protected]

@pattnaik_p

https://sg.linkedin.com/in/priyabratapattnaik

https://plus.google.com/109816383630328905377

Science

Development of Production and Purification Platformform for Influenza Vaccine