Constraints and Complexityfplreflib.findlay.co.uk/images/pdf/bioproduction/Tony-D... · 2018-02-12 · Unique Aspects of Vaccines •Vaccines are biologicals •products derived from

Tony D’Amore, VP, Product Research & Development17th Global Bioproduction Summit | 5-6 February 2018 | San Diego | USA

Advances & Challenges in Vaccine Development and Manufacture

Advances and Challenges in Vaccine Development and Manufacture

• Review of the constraints and complexity of vaccine product

development and manufacture

• Evolution of bioprocess and analytics innovation and technologies

to overcome these challenges

• Strategy and leveraging innovation and technology for rapid

product development (examples)

• What does the future look like?

2

Constraints and Complexity

Unique Aspects of Vaccines

• Vaccines are biologicals

• products derived from living organisms

• Vaccines are complex entities

• both in terms of their components and the technology required to produce them

• Vaccines (those receiving the vaccine)

• are generally healthy (for prophylaxis or preventative)

• whereas persons receiving drugs (for treatment) often have medical conditions

for which the drugs are prescribed

• risk: benefit requires high bar for safety

• Unlike biologics and drugs,

• vaccines are hard to make in a generic form and modernize

• so they retain their commercial value (no patent cliff)

4

Centers for Disease Control and Prevention. Principles of Vaccination.

http://www2a.cdc.gov/nip/isd/immtoolkit/content/products/pinkbook.pdf.

Accessed May 11, 2010.

Vaccines are Manufactured viaa Wide Variety of Cell Substrates

Novel antigens

often require novel

cell substrates

CHO E. coli

P. pastoris

S. cerevisiae

Per.C6 P. fluorescensN. meningitidisVero

MDCK SF9

SF21

S. pneumoniae

Hi-5

MAMMALIAN INSECT MICROBIAL FUNGAL

5

Vaccines Are Diverse

6

Polysaccharide

DNA plasmid

Virus Like Particles

Protein complexes

Viruses

Combinations

Plus Chemistry

Inc

rea

sin

g c

om

ple

xit

y Wide

Variations

in properties

Product

“characterize-

ability”

There are few

platform processes

Vaccine Industry Environment

Business Environment- High cost / high risk- Low investment- Few Suppliers / Fragile supply

Scientific / Medical Environment- New vaccine targets / technologies- Compliance revolution- Ever increasing safety expectations

Competitive Environment- Other big pharmaceuticals

Sanofi Pasteur, GSK,

Merck, Pfizer

- New biotech companies- New technology

Traditional Vaccine Environment- Regulatory environment- Static products- Static processes

7

Vaccine Product Development

8

is a Highly Regulated, Complex & Competitive Industry

Many Years 2-4 years 6-8 years 1 year 2 years continue

LaunchPre-clinical Proof

of Concept (POC) File

EXPLORATORY PRE-CLINICAL CLINICAL DEVELOPMENT REGISTRATION LCM

Desired outcomes

•Relentless cost pressure- return on investment

•Increasing competition- get to the clinic as soon as possible (minimize time)

•Localized manufacturing

•Quality Requirements are rigorous from the start of phase I

Industrial Challenges / Pressures

•Higher productivity

•Higher plant utilization: multi-products, different scales, flexible manufacturing

•Higher efficiency: less variability, failures, and waste

•Higher manufacturing quality standards

Desired outcomes

Safety Requirements Continue to Increase

9

Number of people Enrolled in Phase III Studies

A Glance at the History of Biotechnology

10

Data source: A.S. Verma “Biotechnology in the Realm of History” J Pharm Biollied Sci. 2011; 3(3): 321–323

Mab (1975)

PCR (1979)

Transgenic (1981)

Cloning (1982)

Human genome project (1990)

DNA structure (1953)

Pre-1800 1800 to ~mid of 20th

century

From post 2nd world war to present

The 80’s

Booming of biotechnology

Domestication of plants and animals

Fermentation

Ancient Classical Modern

Where will it end ?

Genetics, vaccines and antibiotics (from 1911- )

Constraints and Moving Forward

• The Challenge of Production

• Anticipation, quantity, quality

• Need

• Accelerate time to POC and focus portfolio

• Reduce cost, increase flexibility and quality

• How

• Leverage the evolution of bioprocess and analytics innovation and technologies to overcome these challenges

• Single use technologies, high throughput screening and innovation in analytics

11

Evolution and Innovation

Single Use & Disposable Technology Has Entered the Mainstream of Bioprocessing, including Vaccines

13

BioreactorClarification & concentration

Downstream processing

Formulation & filling

13

Single Use Bioreactors: Market Review

Wave bioreactor (GE) NucleoTM Single-Use Bioreactor (Pall)

Single use Bioreactor (Thermo Fisher)

Air-wheel bioreactor (PBS)

XDR bioreactor (Xcellerex/GE)

Biostat STR (Sartorius)

Mobius CellReady (Millipore)

14

Depth filters for Clarification: Market Review

Clarisolve (Millipore) Sartopure PP3 (Sartorius)

Diatomaceous Body Feed Filtration

(Sartorius)

Seitz V100P (Pall)

Zeta Plus (3M) Emphaze™ AEX Hybrid Purifier (3M)

15

Single Use Centrifuge for Cell Harvest & Clarification

16

UnifugeKsep

Single Use Chromatography: Market Review

17

Chromatography Columns

Chromatography Systems

Ready to Process (GE):

1, 2.5, 10 & 20LChromabolt (Millipore):

10, 20 &32cm ID

Opus columns (Repligen):

1.2-60cm ID

GoPure (Life

Technologies):

10, 20 &30cm

ID

MaxiChrom

(Atoll):

10, 20 &30cm ID

ÄKTA ready

(GE Healthcare)

Mobius FlexReady

(Millipore)

Limitation: All SU Chromatography flow paths and columns on the market are not sterile

Single Use Application – From Bench Top to 100-L Scale Process for Serum-Free Live Viral Product

18

Disposable Bioreactor

Disposable Mesh Bag (ThermoFisher

Scientific)

Disposable Mixing System

(Pall Life Science)

Disposable 20 µm / 3 µm/

0.65 µm depthfiltration system

Disposable Fluid Path HF TFF Manifold

(Spectrum Laboratory)

Disposable Mixing System

(Pall Life Science)

Disposable Fluid Path HF TFF Manifold

(Spectrum Laboratory)

Sterile Bulk Drug

Substance Bags

0.65 µm

3 µm

20 µm

1) Virus Release &

Microcarrier Separation2) Clarification Filtration by

Depth Filtration3) UF/DF by

TFF

4) DNA

Digestion

5) UF/DF

by TFF

Summary of Purification Process for Serum-Free Live Viral Product

• Successful demonstration of:

• Robust purification process with the following results:

• The virus yield was significantly increased from 20-40 doses /L to 400-500 dose /L – 10 fold increase

• The Vero DNA was significantly reduced to 1.35 ng/dose

• The purification process shortened from 3 to 2 days of operation

• Easy operation (no freeze-thaw, no centrifugation)

• Reduced number of production run (due to high yield)

• Reduced production cost

• Large scale (i.e. 100-L scale) purification in a closed disposable system

• Sterility assured

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Conclusions and Points to Consider

• Completed multiple demonstration / material supply runs, and five

GMP runs with single use technology and closed system

• All products passed sterility testing

• Process scale up ongoing

• Industrial Challenges

• Developing quality processes while driving down operational costs

• Quality Requirements are rigorous from the start of phase I

• Always a balancing act, Cost and Time

• Application of single use technology provides opportunities

• Reduced fixed costs and equipment validation

• Increasing facility and process flexibility

• Accelerating process development

20

Cutting-Edge Technologies (1/2)

21

State-of-the-art filling line (isolator technology)Mathematical modeling & scale down model to solve scale up challenges

Cutting-Edge Technologies (2/2)

22

A completely closed system from manufacturing to formulation with single use technologies High throughput process development

Analytical New TechnologiesReal Time PCR

DNA Detection

Ion Torrent/ NGS

Genetic Stability

Bioinformatics

Computer Cluster

Illumina

Ad Agents/ NGS

Nucleic Acid Detection

Homogenous Time

Resolved Fluorescence

Biacore

Fortebio Multiplex MSD

Antigencity & Affinity

23

Hydrogen deuteriumexchange (HDX) MS

Quantitative MS

Protein

Sequencing

Mass SpectrometryDynamic Light

ScatteringMicro Flow Imaging

MasterSizer Coulter Counter

Particle Sizing

+

+

+

N C

HCl and microwave irradiation

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dde

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Deconvolution

Ion c

ounts

N-terminal sequence

A2 A3 A4 …. A7 …. An-3 …. An +

+

+

C-terminal sequence

An-1 …. An-3 …. A7 …. A4 A3 A2

m/zm/z

Ion c

ounts

Mass spectrometry

+

+

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A1A2A3A4………………………………….An-1An

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Deconvolution

Ion c

ounts

N-terminal sequence

A2 A3 A4 …. A7 …. An-3 …. An +

+

+

N-terminal sequence

A2 A3 A4 …. A7 …. An-3 …. An +

+

+

C-terminal sequence

An-1 …. An-3 …. A7 …. A4 A3 A2

m/zm/z

Ion c

ounts

Mass spectrometry

+

+

+

N C

HCl and microwave irradiation

A1A2A3A4………………………………….An-1An

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C-t

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dde

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Deconvolution

Ion c

ounts

N-terminal sequence

A2 A3 A4 …. A7 …. An-3 …. An +

+

+

C-terminal sequence

An-1 …. An-3 …. A7 …. A4 A3 A2

m/zm/z

Ion c

ounts

Mass spectrometry

+

+

+

N C

HCl and microwave irradiation

A1A2A3A4………………………………….An-1An

++

C-t

erm

inal la

dde

r °°°

N-te

rm

inal la

dd

er •••

m/z

Deconvolution

Ion c

ounts

N-terminal sequence

A2 A3 A4 …. A7 …. An-3 …. An +

+

+

N-terminal sequence

A2 A3 A4 …. A7 …. An-3 …. An +

+

+

C-terminal sequence

An-1 …. An-3 …. A7 …. A4 A3 A2

m/zm/z

Ion c

ounts

Mass spectrometry

Mass Spectrometry (MS): Now and in the future

• Applications in Sanofi Pasteur Analytical R&D

• Highly accurate determination of protein intact mass

and AA sequence by LC-MS and LC-MS/MS to ID

target protein, HCP impurities, and truncations or other

covalent modifications

• Key challenges and future opportunities

• Quantitative MS - a single technology to assess antigen

content (surrogate of vaccine potency) at multiple

product stages

• Expand in-house capabilities beyond LC-MS through

acquisition of CE-MS instrumentation and development

of workflows/methods

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Waters Synapt G2-S LC-MS with HDX module

Mascot MS databaseinfrastructure

Capillary Electrophoresis: Now and in the future

• Applications in Sanofi Pasteur Analytical R&D

• Purity and quantitation analysis of vaccine antigen protein(s) by SDS-CGE

• Quantitative stability-indicating assay for vaccine antigen protein

• Used or quantitation of MAbs

• Screening as replacement for SDS-PAGE for commercialized products

25

Protein antigen purity analysis:

SDS-CGE vs SDS-PAGE

Linearity of quantitative SDS-CGE

stability-indicating assay

HTS Platforms within Sanofi Pasteur

• Focus

• Prevent a circovirus-like crisis by detecting a broader range of adventitious agents

• Implement HTS as an alternative to in vivo (animal) and in vitro adventitious agents testing

• In 2011, began further exploring various HTS technologies and started development of an automated analysis pipeline - PhyloID™

“Cataloguing the Taxonomic Origins of Sequences from a Heterogeneous Sample using Phylogenomics: Applications

in Adventitious Agent Detection”, R.L. Charlebois, S.H.S. Ng, L. Gisonni-Lex and L. Mallet, PDA J Pharm Sci and

Tech, 68: 602-618, 2014

26

Illumina HiSeq 1500Illumina HiSeq 3000NextSeq500Ion Torrent PGM

Applications of High Throughput in Process Definition Studies

27

Conventional approach High throughput system

Column chromatography

TECAN/ Atoll mini columnAKTA Explorer & columns

• DoE studies were performed in parallel using the conventional approach and the high throughput system

• Generated fit model in JMP DoE program for prediction and design space• Comparative data analysis between the two systems

Analysis

SDS-PAGELabchip GXII (96 well) Electropharogram & virtual gel

ForteBio Octet RED96Western blot

HTPD & Automation Capability At Sanofi Pasteur

High throughputin sample

preparation(96 well format)

High throughput in sample analysis(96 well format).

Timely feedback to process

Method scouting, DoE studies,

high throughput sample

generation

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TECAN Freedom EVO150

ForteBio Octet RED96 System

Zephyr Compact liquid handling

Microfluidic electrophoresis Caliper LabChip GXII

High throughput process development will only

become a reality when high throughput in-process test

capabilities are established

A

complete

high

throughput

platform

High Throughput System - Outcome • Comparable results were obtained between the conventional approach and high throughput

system (Identified similar trends for critical operating parameters between the two approaches)

• Significant time and material saving were achieved using the high throughput approach

• Suitable for purification process definition studies of soluble expressed proteins

29

Days required Conventional approach

(24 studies)

High throughput system

(24 studies)

For Experimental runs 50 days 6 days

For sample analysis 8 days 2 days

Total # of Days required 58 days 8 days

Equipment used Conventional approach High throughput system

Chromatography AKTA Explorer, 50 ml column TECAN, 600 µl column

Analysis SDS-PAGE, HPLC Labchip GXII (96 well), UPLC

What Does the Future Look Like – Opportunities/Challenges

• Continuous manufacturing

• Transformative technologies- e.g., mRNA

• Big data management

• Artificial intelligence, machine learning

• Scale-down modeling

• Molecular imprinting

• Novel adjuvants

• Immunomodulation

• Novel expression systems

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Next Generation for MAb manufacturing platform

• Integrated and continuous process

• High volumetric productivity

• Minimal scale up

• Smaller facilities

• Disposables

• Limitation of Prot A media capacity

• Steady state high product quality

• Short cycle time / Minimal hold time

• Chemically defined media

• High degree of automation

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Example:

Purified

ProductMedia

Generic platform facilitates the transposition in

continuous mode

Continuous Chromatography: Simulated Moving Bed Technology

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www.chromacon.ch www.sembabio.com www.novasep.com

www.tarponbiosystems.com

www.ge.com

✓Closed system (zero biobuden) design

✓Fully SIPable✓4-column system✓Delta UV based control built in✓The 1st multi-column system at

GMP scale for biotech

Rahul Godawat, Late Stage Process Development, Genzyme, May 22nd 2014

Periodic counter-current chromatography (PCC)

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