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Shelly A. Parra Sr. Manager, Global Field Applications

Enabling technologies for efficient downstream processing of biosimilars, vaccines and gene therapy vectors

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• Case Studies • Biosimilars & Recombinant Proteins • Virus Like Particles • Gene Therapy

• New products on the horizon

• Closing

Overview

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Monoclonal antibodies

• mAb-based therapeutics continue to dominate even with new cell lines emerging

• More Mab Fragments, Fabs, ADCs • Dramatic increases in cell expression levels • Interest in continuous processing

Vaccines • Processes redesigned to be more responsive, more reproducible, with higher yields

Plasma • Interest in modernizing legacy processes

Biosimilars • More drugs coming off patent, need for shorter time to end-user

Gene Therapy • Growing interest in the area and the market expected to grow 15% CAGR

First in human • Speed to clinic is main product development driver

Ease of use • Major shift to disposables in downstream purification

Quality and efficiency

• Continued demand for highest quality resolution, capacity, salt tolerance and operation speed

• Consistent, reproducible material from batch to batch • Supply risk mitigation, cost minimization

Industry Trends Driving Growth in Downstream Purification

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BioProduction Solutions for Downstream Processing

POROS™ Ion Exchange • High performance cation and anion exchange

resins • Differentiated surface chemistries provide

unique selectivity

CaptureSelect™ Products and Services • Unique, tunable affinity ligands / resins • High purity in a single step

POROS™ Resins •High performance Ion Exchange resins

Simplifying biomolecule purification and reducing COGs

Cell Culture Clarification

Capture/Affinity Chromatography

Viral Inactivation

Polish/Non-Affinity Chromatography

Viral Filtration

Formulation

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Protein purification challenges

• Conventional purification processes require multiple chromatographic steps (IEX, HIC, size exclusion) to effectively separate the target protein from the feedstock contaminants

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Protein purification challenges

• Conventional purification processes require multiple chromatographic steps (IEX, HIC, size exclusion) to effectively separate the target protein from the feedstock contaminants

50% product loss

• Limit the number of steps in a purification procedure

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Purification Can Be More Efficient

Non-Affinity Capture

Polish 1

Polish 2

Polish 3

Polish 4

Polish 5

Affinity Capture

Polish 1

Polish 2

80% reduction in purification time

Cell culturefeed

Purifiedproduct

CaptureSelect™ solution • Enabling a platform approach

for biomolecules through selectivity /specificity • High purity in single step

• Reduction of process steps • Higher yields, reduced costs

• Mild elution conditions • To retain biological activity of

target

• Efficient clearance of HCP, DNA, virus • High selectivity in capture step

Simplify recombinant protein

purification

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Broad range of target molecules covered (RUO and cGMP)

• Antibodies • Human IgG, IgA, IgM • Multi-species IgG • Human specific • Mouse and Rat specific • Fab fragment

− CH1 − LC-kappa/lambda

• Fc fragments and Fc-fusions

• Non-Antibody targets (recombinant and plasma derived) • Blood factors: FI, FII, FVII, FVIII, FIX, vWF, • Proteins: HSA, AAT, tPA, ATIII, TF, Fib, ApoA1, ApoH • Hormones; G-CSF, FSH, hGH, hCG, GM-CSF, Insulin • Viruses: AAV serotypes, Adenovirus, Influenza • Affinity Tags: C-Tag (EPEA)

CH3

CH1

CH2

VH

CL

VL

CH3

CH1

CH2

VH

CL

VL

CH4

CH1

CH2

VH

CL

VL

CH3

CH3

CH1

CH2

VH

CL

VL

www.thermofisher.com..captureselect-affinity-products.html

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• Case Studies • Biosimilars & Recombinant Proteins • Virus Like Particles • Gene Therapy

• New products on the horizon

• Closing

Overview

10

CaptureSelect™ bioprocess cGMP resins: proteins and viruses

Product Target applications Uniqueness

CaptureSelect™ FSH

Human follicle stimulating hormone Binds only intact FSH due to alpha/beta chain epitope

CaptureSelect™ HSA Human serum albumin, albumin fusion proteins

Mild elution conditions

CaptureSelect hCG rec Human chorionic gonadotropin Binding rec hCG with cross binding to LH and FSH

POROS® CaptureSelect™ AAV9 Adeno-associated virus type 9 (AAV9) Highly selective for AAV9 with high capacity (1e14 VG/ml)

POROS® CaptureSelect™ AAV8 Adeno-associated virus type 8 (AAV8) Selective for AAV8

Caution: For manufacturing, processing, or repacking.

FSH HSA AAV

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CaptureSelect™ FSH Affinity Matrix Mild eluting affinity resin for intact follicle stimulating hormone

Binding specificity

Human intact FSH

Matrix and particle size

Aldehyde-activated agarose, 65 μm

Dynamic binding capacity

~3 to 5 g/L of matrix (depends on flow rate, column height, and contact time)

Elution buffer • 20 mM Tris, 2.0 M MgCl2 pH 7.0

• Mild elution, retaining

biological activity • Binding intact FSH

(conformational epitope) • Excellent scalability • Non-animal-derived

Caution: For manufacturing, processing, or repacking.

Load

2x Elutie frac003:10_UV2_280nm 2x Elutie frac003:10_Cond% 2x Elutie frac003:10_pH 2x Elutie frac003:10_Fractions

-50

0

50

100

150

200

250

mAU

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 ml2B3 2B5 2B7 2B9 2B11 2C1 2C3 2C5 2C7 2C9 2C11 2D1 2D3 2D5 2D7 2D9 2D11 2E1 2E3 2E5 2E7 Waste 2E9 2E11 2F1 2F3 2F5 2F72F9 Waste

Elution MgCl2

Strip pH 2.0

Chromatogram of the purification of FSH from clarified cell culture harvest using CaptureSelect FSH.

Red line: OD 280 nm, blue line: conductivity, grey line: pH

0.0

0.3

0.5

0.8

1.0

1.3

1.5

1.8

2.0

FSH intact alpha-chain beta-chain

Ligand 1Ligand 2Ligand 3Ligand 4anti intact FSHanti beta FSHanti hCG (alpha)

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Case Study

Providing solutions for biosimilar development

• Follicle Stimulating Hormone (FSH) is one of the top non-mAb biosimilars (market of $1.26bn in 2013 expected to grow to ~$2bn in 2024*)

• Original process contains multiple steps, up to 7 purification steps have been reported, and has a low overall process yield

• Need for shorter and more cost effective manufacturing process

• Development program to develop an affinity resin specifically binding to FSH

• Ligand would ideally bind only the intact form of FSH

• Mild elution to contain the activity of FSH

• Collaborated with a well known biosimilar manufacturer in Egypt

• Affinity resin suitable for commercial manufacturing developed

• Binds a conformational epitope only present on intact FSH

• Increase in yield (over 3-fold has been reported)

• Decrease in number of process steps and overall COGs

SITUATION OUR RESPONSE VALUE DELIVERED

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HSA - high capacity, mild eluting resin for HSA fusion proteins

Binding specificity

Human albumin from recombinant sources, plasma, and serum

Matrix and particle size

Aldehyde-activated agarose, 65 μm

Dynamic binding capacity

~12 g Albumin/L of matrix (5% breakthrough at 150 cm/h)

Elution buffer • Neutral: 20 mM Tris, pH 7.4, plus one of the following – 2.0 M MgCl2 – 1 M NaCl, 50%(v/v) propylene glycol, pH 7.4 – 1 M NaCl, 0.5 M L-Arginine, pH 7.4 • Acidic: Glycine, pH 3.0

• Highly selective for human

albumin • Unique elution characteristics

allowing for gentle elution of albumin fusion proteins

• Excellent scalability • Non-animal-derived

Equilibration/wash buffer: PBS, pH 7.4 Load: Feedstock of recombinant albumin fusion production Elution buffer: 20 mM Tris, 2.0 M MgCl2, pH 7.4 Flow: 200 cm/h

0

2

4

6

8

10

12

14

16

0 50 100 150 200 250 300

DBC

at C

5 (g

/L m

atrix

)

Flow (cm/h)

The dynamic binding capacity of the CaptureSelectTM albumin affinity matrix at 5% breakthrough (C5) as a function of the linear flow rate on a 5x20 mm column.

Lower flow rates during sample loading drive the dynamic binding capacity

• CaptureSelect Human Albumin

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Pipeline of affinity products

CaptureSelect™ affinity products for purification of: Therapeutic proteins (non-mAb) & viruses Antibody types

Bioprocess cGMP

FSH, Human Albumin, hCG AAV8, AAV9,

KappaXL FcXL

IgG-CH1

Stage 5: RUO products

hGH, tPA Antithrombin III, Fibrinogen,

Transferrin, ApoH, C1-Inh

IgA, IgM CH1-XL

Stage 4: Lead Selection

Prothrombin, GM-CSF Exotoxin A (PE),

IgG-CH2

Stage 3: Prototype Resins

Insulin, EPO Adenovirus (Adv5), Flu (HA)

IgE, Free LC-kappa

Stage 2: Lead Screening

TSH, IFNa/b, hIL2, Protein C, FV, FX, FXI, FXII, FXIII, FH, vWF,

Lentivirus (VSV-G)

Rabbit IgG, Anti-Idiotypes

Stage 1: Library Construction DNAse Free LC-lambda,

scFv, IgY, Mouse IgG

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Purification of Large Biomolecules: Viruses, VLP, Vectors, Plasmid DNA, IgM, Fusion Proteins

• Why is POROS well suited for these applications? • Unique pore morphology which increases surface area

rendering large biomolecules accessible to charged functional groups

• Ultimately increasing capacity for large biomolecules • Ability to flow at high flow rates makes the process more

flexible, scalable and productive while maintaining resolution • Easy to use and pack • Reproducible

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POROS™ Chromatography Resin

• Backbone : Polymerized polystyrene - divinylbenzene • Unique base beads are used for different chemistries

Base Bead Average Pore Size*

(Angstroms)

Average Particle Size

(µm) Functional Chemistries

R150 2000 50 HQ, PI, IgM R250 1100 50 HS, D, HE, AAV8/9

R450 1000 45 MabCapture A/

MabCapture A Select R550 1100 50 XS, XQ

* Average pore size determined by water pore asymmetry on the base bead prior to coating and functionalization.

• Smallpox 200-400nm in diameter • Adenovirus 70-90nm in diameter • Adeno-associated virus 20nm in diameter • Papilloma virus 40-55nm in diameter

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POROS Applications: Purification of Viruses, Virus-Like Particles (VLPs), Vectors, and Plasmid DNA

• Known applications for large biomolecule purifications center around 6 POROS Chemistries: HS50, HQ50, D50, PI50, XQ, Heparin

• Particles bind tightly as elution is typically at 0.4->1.0M NaCl • Can be employed as capture or polish chromatography step • High purity achieved in one chromatographic step • Applications in Foot & Mouth Disease, Rabies, Polio, Flu, etc

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Case Study: POROS HS in HPV VLP Process

• Cook, James C. et al, “Purification of Virus-Like Particles of Recombinant Human Papillomavirus Type 11 Major Capsid Protein L1 from Saccharomyces cerevisiae”, Protein Expression and Purification, 1999, V 17, Pages 477-484

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Case Study: Purification of HPV VLPs

• Single Step Chromatographic Purification

Reprinted from Cook, J., Protein Expression and Purification, 1999, V17, 477-484

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• Advantages of POROS HS50 • Pore Structure

• “The large throughpores of POROS 50HS resin were probably accessible to the VLP,…”

• “Small pores would physically exclude the VLP and restrict binding to the outer surface of the beads, thus leading to low binding capacity.”

• Selectivity and Recovery • “Excellent selectivity of the

chromatography made it possible to achieve 98% purity” with a 2 step purification process: microfiltration and CEX Chromatography

• POROS HS50 was “the best for selectivity and recovery for this particular application”

Case Study: Purification of HPV VLPs

Reprinted from Cook, J., Protein Expression and Purification, 1999, V17, 477-484

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Enabling Technology – POROS CaptureSelect AAV Resins

Gene Therapy

• Growing interest – lack of industrialized purification platform to meet market needs

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• Increased interest and focus in gene therapy applications over the past decade

• Gene therapies promise versatile treatment of long-term and potentially curative benefits to patients with genetic or acquired diseases

• Of recent, > $1.2B has been secured for gene therapy companies via IPOs and VC financings

Gene Therapy – A Field that is Evolving

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Clinical Trial Information

• United states, has the highest number of approved clinical trials (63%) taking place, however no approved product in the market

• Most of the clinical trials are in early phase development

• Successes in the space of rare genetic diseases

• The patient population afflicted by these diseases are small

• Clinical success in Phase I/II may be enough to proceed towards commercialization

Country No. of Trials

Approved Products

USA 1312 0

UK 206 1

Germany 83 0

France 51 0

China 37 2

Key Players in the Field

Interest from Pharma Companies

CMOs

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• The Ideal Vector for Gene therapies: Cost effective to manufacture with a simple vector design

Highly precise and specific target cell delivery

Cause no damage to target cell

Effectively carry the desired genetic load

Have low immunogenicity

Capability for longer lasting expression if indications require this criteria

• Adenoassociated virus (AAV) have emerged as the vector of choice for many therapies

• AAV can effectively target most cell types

• Mediate long-term tissue-specific gene expression with low immunogenicity

Adeno-associated Virus (AAV) for Gene Therapy

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AAV and Chromatography Solutions for Purification

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Serotype Heparin Anion Cation AVB AAV8 AAV9

AAV1 No Yes Yes Yes No No

AAV2 Yes Yes Yes Yes No No

AAV3 Yes Yes Yes Yes No No

AAV4 No Yes Yes No No No

AAV5 No Yes Yes Yes No No

AAV6 Yes Yes Yes No No No

AAV7 No Yes Yes No No No

AAV8 No Yes Yes No Yes No

AAV9 No Yes Yes No No Yes

AAV and Chromatography Solutions for Purification

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Harvest Centrifugation Concentration Filtration Size exclusion

Ultra Centrifugation

Ion Exchange

Diafiltration

Current Gene Therapy Purification Strategies

• Current Paradigm • Multiple chromatography

steps & concentration • Long process

development lead-times • More expensive process • Cumulative yield losses

20% product

yield

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Enabling a Paradigm Shift in Viral Vector Purification

Harvest Centrifugation Concentration Filtration Size exclusion

Ultra Centrifugation

Ion Exchange

Dia-Filtration

Clarification Affinity Concentration Filtration

• CaptureSelect™ Paradigm • Affinity capture and

concentration only • Process simplified • Lower cost & complexity • Speed to market • Less steps = higher yield

Customer testimonial: “Process yield improvement from 20% to 60% & cost reduction by a factor of 6”

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Enabling Affinity Resin for AAV8 and AAV9 Viral Vectors

Selectivity Ligand

Binding Capacity (vg/mL)

pH Stability

POROS™ CaptureSelect™

AAV9 High > 1014 pH range 1- 10

POROS™ CaptureSelect™

AAV8 High > 1013 pH range 1- 10

12-15kDa

AAV9

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POROS™ CaptureSelect™ AAV8 and AAV9 – Performance Overview

Purity

Equivalent purity profile as conducting 3 steps of IEX Chromatography No residual proteins were detected Improved yield, ≥ 80% vector recovery

Process Yield

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Process Optimization–Washing Away Cellular Debris

• Transfection reagents • Additives • Helper viruses

• Host Cell Proteins • Host Cell DNA • Plasmid DNA • Media

Components

AAV replicates in the nucleus of the cell and is not released until the cell undergoes apoptosis or is disrupted, additionally releasing:

32

Process Optimization – Secondary wash recommendations

Washing Agents: • 1M NaCl at various pH • MgCl2 • Chaotropic Salts • Arginine • Ethanol (phosphate buffers should be avoided) • Detergents • Glycol • Organics

33

POROS™ CaptureSelect™ AAV8 and AAV9 – Enabling Scale-Up

Low back pressure - <3.0 bar at 700 cm/H in 22 cm length column

Linear and predictable scalability

Manufacturing of AAV8 and AAV9 vectors feasible at large scale (confirmed up to 200L scale)

Affinity chromatography allows a simple, efficient and high yield process

Process Yield

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• Affinity resins that offer high selectivity and capacity in the purification of viral vectors AAV8 and AAV9 for production of clinical grade gene therapy molecules

• A fast, robust, high yield and purity, from crude material in one step, purification process

• Significant improvement to the downstream processing, by reducing the purification steps and maximizing productivity

• Offer scalability and processing consistency

• Unique products designed specifically for AAV8 and AAV9 subtypes supported with Regulatory Support Packages.

POROS™ Affinity Resins - Gene Therapy Applications

Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only.

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Polish Chromatography–Ion Exchange Solutions

pH 3

pH 9

pI

Anion exchange (AEX)

Cation exchange (CEX)

Full Empty Size 20 nM (200Å) 20 nM (200Å)

Density 1.40 g/cm3 1.32 g/cm3

average pI 5.9 6.3 Qu W, Wang M, Wu Y, Xu R. Scalable downstream strategies for purification of recombinant adeno-associated virus vectors in light of the properties. Curr Pharm Biotechnol. 2015;16(8):684-95.

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Empty Capsid Removal Using POROS™ AEX Resins

• Empty capsids are a byproduct of vector biosynthesis

• Full vectors bound more tightly to the AEX resin

• All AEX POROS™ resins (XQ, HQ, D, and PI) have been shown to bind and elute the desired AAV capsid

• Superior resolution and capacity

0

200

400

600

800

1000

mAU

10.0 15.0 20.0 25.0 ml

Volume (ml)

Abs

orba

nce

280n

m (m

AU

)

____POROS HQ

____Q Sepharose FF

0

200

400

600

800

1000

mAU

10.0 15.0 20.0 ml

Volume (ml)

Abs

orba

nce

280n

m (m

AU

)

____POROS HQ

____Fractogel TMAE 650

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Empty Capsid Removal Using POROS™ AEX Resins

• Slightly different anionic behavior was shown with empty capsids and full vectors using POROS™ AEX

• POROS™ HQ was optimized to efficiently separate empty capsids

G. Qu et al. / Journal of Virological Methods 140 (2007) 183–192

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• Case Studies • Biosimilars & Recombinant Proteins • Virus Like Particles • Gene Therapy

• New products on the horizon

• Closing

Overview

39

POROS™ HIC Voice of Customer – Most important design features

Selectivity/Improved Resolution

Consistency of Performance

High Performance at Lower Salt

Concentration

Current focus of development

Incorporated in current prototypes

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Prototype Design: Novel chemistries targeting best-in-class capacity and resolution

Increasing Hydrophobicity

41

High dynamic binding capacity

Stationary Phase Lysozyme DBC (C5) mg/mL

Leading commercially available ethyl 0.9

POROS HIC-1 7.0 Leading commercially

available low sub phenyl 13.5

POROS HIC-3 30.1 Leading commercially

available high sub phenyl 25.8

POROS HIC-5 37.2

Study Format: Column Dimensions: 0.66 cmD X 20 cmL Load Buffer: 1.5 M ammonium sulfate in 50 mM sodium phosphate (pH 7) Lysozyme Concentration: 1.5 mg/mL Flow rate: 300 cm/hr

42

Hydrophobicity effects resolution- A range is needed for varying applications

Inc

reas

ing

Hyd

roph

obic

ity

Study Format: Column Dimensions: 0.66 cmD X 20 cmL Load Buffer: 1.7 M ammonium sulfate in 50 mM sodium phosphate (pH 7) Buffer Gradient: Load buffer to 50 mM sodium phosphate (pH 7) in 10 CVs Flow Rate: 100 cm/hr Protein Mixture: Ribonuclease A, Lysozyme and Chymotrypsinogen & Chymotrypsin

Abso

rban

ce a

t 280

nm

(mAU

)

43

Aggregate Removal from a mAb

Load in PBS at pH 7.5 Column loaded at

100 mg mAb/mL of resin

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Conclusion - Your Partner for Purification

• Performance-

• High specificity and capacity resins offering high purity in a single step

• Our solutions provide an industrialized purification platform for AAV production to support market needs

• Productivity - Our purification solutions improve process flow and reduce process time

• Capabilities- State-of-the-art manufacturing and robust quality

• Support- Technical engagement & global response

Enabling, purification solutions that simplify processes and reduce COGs

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• Multi-function calculator, converter, and reference tool for process scientists

• https://www.thermoscientific.com/content/tfs/en/about-us/general-landing-page/mobile-apps.html

A New Tool for Chromatography Process Scientists…

Chromatography PRO

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Chromatography PRO: Key Calculators

Column Calculator • Converts flow rates to linear

velocity from L/min, as well as calculating column sizes and cross sectional area.

Process Modeling • Models a given downstream

unit operation to understand column volumes, timing, throughput, etc.

Symmetry Calc • Calculates peak asymmetry,

number of theoretical plates, and HETP from volume, bed height, and peak calculations

Buffer Calculator • Calculates amount of buffer

required from molar solution and final volume

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A solution that’s fits your needs

PHARMACEUTICAL GRADE REAGENT. FOR MANUFACTURING AND LABORATORY USE ONLY.

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