APPROACHES TO LIGAND

DESIGN

2

Contents

• About Us

• Affinity Chromatography

• Ligand Design

• Library Synthesis

• Library Screening

• Case Study One – Albumin Fusion Protein

• Case Study Two – Insulin Precursor

• Case Study Three – Transferrin Fusion Protein

• Case Study Four – Antibody Binding Ligands

• Conclusions

• Contact Us

3

ABOUT US

ProMetic BioSciences Ltd (PBL): Affinity / Purification / Solutions

• PROTEIN PURIFICATION

• CONTAMINANT REMOVAL

• PATHOGEN REDUCTION

• CUSTOM ADSORBENT DEVELOPMENT

• DOWNSTREAM PROCESS DEVELOPMENT

4

We deliver solutions that enable our global clients to

produce safe and economical therapeutic products -

ProMetic BioSciences Ltd: Locations (North America, Canada)

• Corporate HQ – Montreal, Canada

• Sales & Marketing – New York, US Los Angeles, US

5

PBL UK Head Office: Horizon Park, Comberton Cambridge United Kingdom

PBL Manufacturing Site: Ballasalla Isle Of Man British Isles

ProMetic BioSciences Ltd: Locations (UK, British Isles)

6

ProMetic BioSciences Ltd: Our Expertise

• Proven Mimetic Ligand™ technology

• Full range of chromatography products

• Custom designed chromatography adsorbents

• 25 years experience and success

• Fourteen affinity products used in licensed production processes

• Global supply chain and support

• Technical knowledge & in-house training

• Ability to tailor processes using adsorbent development & chromatography optimization

7

8

• Complete affinity adsorbents (ligands & support matrix)

• Large scale manufacturing ability

• ISO 9001 accredited quality management system

• cGMP-compatible manufacture

• Clean room manufacture of products

• Comprehensive Regulatory Support Files (RSF’s)

• PBL adsorbents form key parts of manufacturing processes for many regulated biopharmaceutical and biomedical products

ProMetic BioSciences Ltd: Manufacture

9

ProMetic BioSciences Ltd: Partner to Mitigate Risk

• Secure supply chains

• Long term product availability

• Multiple end users in industry

• Single batches produced up to 275 litres

• Synthetic, non-toxic, non-animal derived products

• Highly reproducible batch-to-batch manufacture

• High purity, chemically defined ligand structures

10

AFFINITY CHROMATOGRAPHY

Affinity Chromatography: Need for DSP Performance Improvements

• Improved separation technologies appropriate for large-scale manufacturing (especially product capture from culture media/fermentation broth)

• Cost of goods pressure (yield improvements/cost reductions)

• Product safety (increased purity/contaminant removal)

• Limited biomass availability (yield improvements)

• Follow-on biologics (process improvements/cost reductions)

• New biological products in development

11

ADSORPTION

DESORPTION

WASH

Affinity Chromatography: Synthetic vs Biological Ligands

Criterion Synthetic/biomimetic ligand Biological/specific ligands

Cost Inexpensive Usually expensive, e.g. monoclonal

antibodies/Protein A

Availability Scaleable organic synthesis Biological origin, e.g. cell culture,

fermentation etc

Synthesis Facile Often complex and purification needed

Specificity Moderate to high Usually high

Capacity High (up to 40 mg protein/mL adsorbent) Often Low (1 – 10 mg/mL for MAb ligands)

Scale-up Large scale use: columns at

>100 litre scale Limited applications (Protein A)

Sterilization High Mostly low or not sterilizable

12

Affinity Chromatography: Advantages

Affinity chromatography offers the user several major advantages compared to other protein purification techniques –

• selective binding and elution

• very pure product in a single unit operation

• high yields of purified product

• greatly reduced processing time

• cost reduction with economical affinity adsorbents

• high concentrations of material leaving the column

• large scale use

• stabilization of bound protein

13

Pro

du

ct P

uri

ty

Time/cost

} Yield

• Compounds identified from nature (co-factors, substrates, inhibitors, antigens etc.)

• Peptides

• Antibodies (monoclonal, polyclonal)

• Engineered proteins (synthetic antibodies)

• Synthetic (chemical) ligands

14

Development of new affinity adsorbents

Development of new affinity ligands =

Affinity Chromatography: Challenges

15

Affinity Chromatography: Optimization of Mimetic Ligands™ (albumin purification)

O NH

2

O NH

N N

NN

N

Cl

SO3-

SO3-

H H

SO3-

C.I. Reactive Blue 2 Mimetic Blue® SA

Optimization of ligand structure and coupling chemistry

16

Affinity Chromatography: Purification of Human Serum Albumin from Plasma

Ab

sorb

ance

28

0n

m

Volume (litres)

0 0.25 0.5 0.75 1.00 1.25 1.50 2.00

Non-bound Pool

Albumin Pool

CIP Pool

• Column: 250 mL radial flow column

• Flow rate: 27 mL/min

17

Affinity Chromatography: Purification of Human Serum Albumin from Plasma

SAMPLE PROTEIN (g)

Diluted Plasma 5.26

Albumin Pool 2.70

HSA recovery = 100%

IgG/IgA/IgM undetectable in HSA pool

18

Affinity Chromatography: Optimization of Mimetic Ligands™ (albumin purification)

O NH2

O NH

N N

NN

N

Cl

SO3-

SO3-

H H

SO3-

19

Affinity Chromatography: Mimetic Ligand™ Discovery Phase

Library Synthesis

Verification Chromatography

Library Screening

Identification of binders

Ligand Synthesis & Scale-up

Ligand Design

20

LIGAND DESIGN

1. Active ligands known:

Modeling and optimization of existing ligands (analogue synthesis)

2. Active site known:

Modeling of complementary ligand structures (rational design)

3. Neither active site or ligand known:

Ligand screening (systematic screening of ligand arrays)

21

Ligand Design: Different Approaches

• Many of the techniques used for the design of new ligands are analogous to those used in the development of drug compounds.

• The important factors that must be considered when designing a selective ligand are:

1. An affinity ligand is constrained in space by attachment to a large solid support,

2. An affinity ligand should not bind the target too tightly,

3. The immobilized ligand must be available to the protein binding site.

22

Ligand Design: Different Approaches

• Where a suitable crystal structure is available workstation or web based algorithms have been applied to identify possible functional sites for targeting.

• However, when dealing with novel therapeutic proteins, information on ligand binding sites may not be available.

• Techniques that can be used when the crystal structure is available include:

1. Blind docking,

2. Virtual screening.

23

Ligand Discovery: Rational Design

• X-Ray crystallographic data for transferrin iron carrier proteins was analysed using the ConSurf server programme to identify conserved and variable residues. The higher the level of conservation, the greater the likelihood of functional importance and applicability as a target site for affinity ligand design.

• Blind docking was used to computationally “roam” ligands over the entire protein surface and identify minimum energy binding sites.

24

Ligand Design: Rational Design

• Molecular docking was applied to prioritize ligands for screening using a technique known as virtual screening (VS).

• VS algorithms place a ligand into a binding site and then “score” the resulting pose to allow screened ligands to be ranked.

• Methods were developed to select out docked poses to remove those that would be disallowed for an immobilized ligand.

25

Ligand Design: Virtual Screening

• Using the AutoDock algorithm binding energies can be calculated.

• These binding energies can be used as a surrogate for experimentally determined binding affinities.

• Docking of a small, diverse library into a potential binding site can be extended to the rest of the virtual library using quantitative structure activity relationship (QSAR) modeling to quickly rank the remaining compounds.

26

Ligand Design: Compound Prioritization

* Morris et al (1998) J.Comp.Chem, 19(14), 1639-1662

• Over 100,000 triazine based compounds are available in our Chemical Combinatorial Library CCL®.

• Calculating similarity metrics has been used for:

1. Diverse combinatorial selection,

2. Focused selection based on early activity,

3. Exploration of hit chemical space,

4. Compound prioritization.

27

Ligand Design: Navigating Chemical Space

• In addition to the design of general diverse libraries, combinatorial libraries can be developed based on early screening hits.

• The hits present a ‘cherry picked’ selection of candidates from which a new combinatorial library is developed to explore the surrounding chemical space.

28

Ligand Design: Combinatorial Design

29

Ligand Design: Decision Process

Target protein structure know and available?

Known ligand binding site?

Known ligands or inhibitors?

Docking/vHTS Binding site

determination or blind docking

Similarity search or pharmacophore

search

Diverse library design and HTS

Yes

Yes Yes

No

No No

30

LIBRARY SYNTHESIS

31

Library Synthesis: Schematic Overview of 2D Ligand Synthesis

N

N

N

Cl

Cl Cl

N

N

N

Cl

NH

Cl

Activated Dichlorotriazine

PuraBead® PuraBead®

Base Matrix

Triazine scaffold

N

N

N

Cl

NH

Cl

N

N

N

NH

Cl

N

N

N

NH

HN

H2N

NH

H2N

HN

Cl-Cl

-

Activated Dichlorotriazine

PuraBead®

Monochlorotriazine Disubstituted product 2D ligand

32

Library Synthesis: Base Matrix Technology

Agarose 6XL PuraBead® 6XL PuraBead® 6HF

Abbreviated name

A6XL P6XL P6HF

Mean particle size

~90 µm ~100 µm ~90 µm

Matrix

Cross-linked 6% agarose

Cross-linked near-monodisperse 6%

agarose

Highly cross-linked near-monodisperse 6%

agarose

33

Library Synthesis: Automated Library Synthesis

34

Library Synthesis: Step 1 - Addition of first amines to columns 1-8

PuraPlate™ - 96 well, fritted block

Reactor vessels in the robot

Intermediates -

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

N N

N N H

Cl

R 1 _ 8

35

Library Synthesis: Step 2 - Addition of second amines to rows A-H

1 2

A

B

C

D

E

F

G

H

Left blank for standard curves

N N

N N H

N H

R 1 _ 8

R A - H

3 4 5 6 7 8

Final 2D Ligands -

36

LIBRARY SCREENING

37

Library Screening: PuraPlate™ (PBL Library Block)

Adsorbent Drainage Point

Preservative

Duoseal

Frit

• PuraPlate™ layout – 96 individual columns, each with a separate drainage point

• Column volume (CV) – 0.25 mL of adsorbent (0.5 cm bed height)

PuraPlate™

(1 library = 8x8 array = 64

adsorbents)

Electronic Pipette

(gravity fed)

96 Well Collection

Plate

38

Library Screening: Typical Screening Conditions

Application Description Volume Dispense

Speed (µL/sec)

Equilibration Conductivity and pH similar to load 3 x 1.0 mL (12 CV) 250

Load Usually untreated feedstock ------- 70

Wash Equilibration buffer 4 x 0.75 mL (12 CV) 150

Elution

Change in pH, conductivity and addition of excipients (e.g.

chatropes, solvents, detergents, PEGs)

2 x 0.75 mL (6 CV) 250

Clean in Place (CIP) Usually 0.5 M NaOH 2 x 0.5 mL (4 CV) 250

39

Library Screening: Automated Liquid Handling Systems

• High throughput 96 well plate sample analysis

40

Library Screening: Example of Activity Patterns

1 3

2 4

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

A

B

C

D

E

F

G

H

A

B

C

D

E

F

G

H

Activity Patterns

active

inactive

non-specific or v poor binding

41

Library Screening: Chemical Combinatorial Libraries CCL® Strategy

Diversity SAR SAR SAR Screen Screen Screen

General Library

Sub Library 1

Sub Library 2

Sub Library 3

Analysis

Analysis

Analysis Virtual Library

42

Albumin Fusion Protein

In collaboration with Novozymes Biopharma UK

CASE STUDY ONE

43

Albumin Fusion Protein: Project Description

• Novozymes Biopharma UK have developed a

platform technology (Albufuse® and Albufuse®Flex) that fuses a therapeutic peptide or protein to albumin or albumin variant

• Many different albumin fusion proteins have been developed

• Fusion to albumin extends the circulatory half life of the target protein

• PBL and Novozymes have developed a selective, high capacity adsorbent for the capture and purification of albumin fusion proteins - AlbuPure®

Structure of recombinant albumin

Curry et al. (1998) Nature Structural Biology 5, 827-835

44

Albumin Fusion Protein: Design Approach – Navigating Chemical Space

45

Albumin Fusion Protein: Primary Screening Data Evaluation

Flow through fraction

Elution fraction

1 2 3 4 5 6 7 8

A 93 97 82 92 103 121 28 73

B 107 12 91 92 96 102 93 93

C 99 79 79 80 109 101 35 64

D 85 71 71 70 98 88 31 60

E 108 93 109 106 96 109 87 88

F 98 106 90 88 101 117 50 86

G 80 30 70 74 75 104 33 58

H 68 63 86 60 89 90 24 66

Screening samples

1 2 3 4 5 6 7 8

A 0 0 0 0 0 0 54 0

B 0 88 0 0 0 0 2 0

C 0 0 0 0 0 0 48 6

D 0 0 0 0 0 0 38 0

E 4 6 0 0 0 0 6 0

F 0 0 0 2 0 0 31 0

G 15 53 11 7 0 0 58 30

H 0 0 0 0 0 0 65 5

Screening samples

• Lead candidate – Library 23 B2, from >800 candidate ligands

46

Albumin Fusion Protein: Purification of IL-1ra Albumin Fusion Protein

Lane Sample Load

1 Load 1/100

2 Load 1/1000

3 Flow Through Neat

4 Wash 1 Neat

5 Wash 2 Neat

6 Wash 3 Neat

7 Wash 4 Neat

8 Eluate 1/100

9 Eluate 1/1000

10 rHA 1 µg

24052002001:1_UV1_254nm 24052002001:1_Cond 24052002001:1_pH 24052002001:1_Fractions

0

500

1000

1500

2000

2500

mAU

0 20 40 60 80 100 120 140 160 min

F3 1 Waste 2 Waste 3 Waste 4 Waste 5 Waste

47

Albumin Fusion Protein: Purification of scFv Albumin Fusion Fermentation Supernatant

Conditions

Capacity (mg/mL matrix)

pH 4.5, 240 cm/hr 38.4

pH 4.5, 420 cm/hr 26.2

pH 4.5, 600 cm/hr 21.4

pH 6.0, 240 cm/hr 19.9

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 10 20 30 40 50 60

Bre

akth

rou

gh

Matrix Loading (mg/mL matrix)

pH 4.5, 240cm/hr pH 6.0, 240cm/hr

pH 4.5, 600cm/hr pH 4.5, 420cm/hr

• Column: 15 cm bed height

• Breakthrough measured as percentage of load by GP-HPLC

48

Albumin Fusion Protein: Purification Process Comparison

Standard Process AlbuPure® Process

Fermentation

Cell Separation

Ion Exchange

Ion Exchange

Blue Affinity

Fermentation

Cell Separation

AlbuPure®

Ion Exchange

49

Albumin Fusion Protein: Ion Exchange vs AlbuPure® Process

• Yield ~50% greater from 2 step AlbuPure® process than standard 3 step process with equivalent HCP levels

Start Step 1 Step 2 Step 3

Yeas

t H

CP

Lev

els

(lo

g sc

ale)

Standard Process AlbuPure® Process

50

Albumin Fusion Protein: AlbuPure® Applications

Fusion Partner

Approx. Fusion Partner MW

Fusion Tested

HIV Peptides 5 kDa C & N Terminal

IL-1ra 18 kDa C & N Terminal

Endostatin 20 kDa C & N Terminal

Prosaptide 2.5 kDa C Terminal

Kunitz Domain 7 kDa C Terminal

scFv 30 kDa C & N Terminal and Bivalent

dAb 13 kDa N Terminal

Nanobody 14 kDa N Terminal

vNAR 13 kDa N Terminal

51

Insulin Precursor

In collaboration with Novo Nordisk and University of Cambridge

CASE STUDY TWO

52

Insulin Precursor: Project Description

• Insulin analogues are recombinant proteins produced in expression

systems such as E.coli

• Analogue insulin is generally either faster acting or longer acting than human insulin

• Insulin and insulin analogues are commonly purified using a combination of process steps such as IEX and SEC

• To reduce the number of process steps an affinity adsorbent was designed and developed

53

Insulin Precursor: Design Approach – Rational Design and Navigation of Chemical Space

54

• Column: XK16 packed to 2.6 cm bed height (5 mL column volume (CV))

• Equilibration: 0.1 M sodium acetate, pH 5.4

• Load: 40 mL of pre-conditioned yeast cell supernatant insulin precursor material – loaded at 1 mL/min (5 minute residence time)

• Wash: 0.01 M sodium acetate, pH 5.4

• Elution: 0.3 M acetic acid

• Strip*: 20% ethanol, 1.0 M acetic acid

*Note: 0.5 M NaOH is recommended for CIP

Insulin Precursor: Chromatography Conditions

Insulin purification semi pure 271011:1_UV1_280nm

0

500

1000

1500

2000

2500

3000

3500

4000

mAU

0 20 40 60 80 100 ml

55

Insulin Precursor: Chromatography Conditions - Chromatogram

Load Wash Elution Strip

Absorbance (AU)

Volume (mL)

56

Insulin Precursor: Chromatography Conditions - SDS-PAGE

• Non-reduced SDS-PAGE, 4-12% Bis-Tris, MOPS running buffer

1 2 3 4 5 6 7

Lane 1 – Mw marker Lane 2 – Purified human insulin Lane 3 – Load Lane 4 – Flow through Lane 5 – Wash Lane 6 – Elution (1 in 5 dilution) Lane 7 – Strip

188 kDa

98

62

49

38

28

17

14

6

Insulin target protein (~6 kDa)

57

Insulin Precursor: Performance Consistency – Breakthrough Curves

• CG725 – pre-validation material • FA0321 and FA0325 – commercial samples • #1 and #2 in each case represent different feedstock batches

58

Insulin Precursor: Performance Consistency – Performance Data

Insulin Precursor Concentration (mg)

Purification Run 1 Purification Run 2

Load 734 787

Flow through 0.2 0

Wash 58 33

Elution (Pool 1) 600 689

Elution (Pool 2) 27 27

CIP 1 0.2 0

CIP 2 0.2 0

Yield 81% 87%

Purity 92% 92%

Mass Balance 95% 97%

59

Transferrin Fusion Protein

In collaboration with Pfizer Inc.

CASE STUDY THREE

60

Transferrin Fusion Protein: Project Description

GLP mimic

Transferrin Scaffold

• Proteins and peptides are commonly fused to larger protein scaffolds in order to increase the half life of the target protein

• One such scaffold is transferrin:

• Glycoprotein

• Molecular weight is ~ 80 kDa

• pI ~ 5

• In this case the fusion protein proposed indication was for Type 2 diabetes:

• GLP-1 mimic – activates GLP-1 (glucagon like peptide) receptor to stimulate insulin production and reduce blood glucose levels

• Half-life is extended to >3 hours than GLP-1 (<2 minutes)

61

Transferrin Fusion Protein: Design Approach

• Identification of binding sites (rational design)

• Exploration of chemical space

Front view Side View

* Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, Liang J. (2006) CASTp:Computed

Atlas of Surface Topography of Proteins with Structural and Topographical Mapping of Functionally Annotated residues. Nucl. Acids Res., 34 W116-W118.

62

Transferrin Fusion Protein: Primary Screening Data Evaluation

• The best performing candidates score a 4 in each category

• 29 ligand absorbents chosen where > 80% target protein bound

• Results confirmed by rescreening and elution of bound protein

63

Transferrin Fusion Protein: Chromatographic Performance

Load PBL 1025

PBL 1026 PBL 1027

Binding capacity – 14 g/L Purity – 96% Recovery – 98%

Binding capacity – 19.2 g/L Purity – 99.5% Recovery – 90%

Binding capacity – 16.8 g/L Purity – 96.5% Recovery – 110%

Target

64

Transferrin Fusion Protein: Structure Activity Relationship (SAR)

Binding Free Energy = - 8.89 kcal

Binding Free Energy = -6.68 kcal

PBL-1027

PBL-1022

65

Transferrin Fusion Protein: Lead Candidate Performance

• Free energy of binding vs binding capacity for the top six ranked ligands

R2 = 0.696

0

5

10

15

20

25

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0

Free Energy of Binding (-kcal/mol)

Bin

din

g C

ap

acit

y (

mg

/ml)

66

Transferrin Fusion Protein: SDS-PAGE Analysis

Lane Sample

1 Blank

2 MW Marker

3 Purified control

4 PBL-1026 Pre-Peak

5 PBL-1026 Main Peak

6 PBL-1027 Pre-Peak

7 PBL-1027 Main Peak

8 PBL-1025 Pre-Peak

9 PBL-1025 Main Peak

10 MW Marker

11 Blank

12 Blank

67

Transferrin Fusion Protein: Conclusions

• Project Timeline = 4+ months

• DBC = 13-14 g/L with clarified harvest, expect 2-3 fold increase with ligand density and spacer arm optimization

• Significant binding differences seen between purified transferrin fusion protein and clarified harvest transferrin fusion protein

• Correlation observed between free energy of binding and adsorbent binding capacity for the six ligands tested

• Eluate purity > 95%

• Binding is to the transferrin carrier protein = Platform Purification Step

68

Antibody Binding Ligands

CASE STUDY FOUR

69

Antibody Binding Ligands: Project Description

• Monoclonal antibodies (Mabs) represent approximately 35% of the

market for biotherapeutics

• Protein A is the most commonly used affinity adsorbent for Mab purification

• Advantages:

1. Selectivity for most full chain IgG’s

2. High capacity

3. Well established in regulated processes

4. Re-usable

5. Enables a platform approach to DSP

70

Antibody Binding Ligands: Project Description

• Disadvantages:

1. Adsorbent cost

2. Limited resistance to NaOH

3. Requires chromatography steps to remove potential Protein A leachates

4. Not applicable to IgG fragments lacking the Fc region

71

Antibody Binding Ligands: Rational Design (Synthetic Affinity Ligands for IgG Purification)

Interaction of Protein A with IgG

72

Antibody Binding Ligands: Rational Design (Synthetic Affinity Ligands for IgG Purification)

Protein A

Phe 132 Tyr 133

N H

O

O

OH

N H

Protein A Mimic (19/11)

N N

N N H

H N

N H 2

OH

N H

73

Antibody Binding Ligands: Purification of IgG from Plasma using Protein A Mimic (19/11)

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0

9

18

27

36

45

54

63

72

81

90

99

108

117

126

135

144

153

162

171

180

189

198

207

0

2

4

6

8

10

12

14

280nm

pH

A280

74

Antibody Binding Ligands: MAbsorbent® A2P Binding Site

• Crystals of the IgG Fc domain complexed with A2P (PBL) and PAM (Xeptagen) ligands were prepared and analyzed by X-ray crystallography

• Bujacz and Redzymia, Institute of Technical Biochemistry, Technical University of Lodz, Poland

• A2P ligand (blue)

• PAM ligand (pink)

75

Antibody Binding Ligands: MAbsorbent® A2P Typical Column Conditions

• Load: Neutral pH (pH 6.0 – pH 8.0); 0 - 0.25 M NaCl; 100 – 250 cm/hr

• Wash: 25 mM sodium phosphate, pH 7.0

• Elution: 50 mM sodium citrate, pH 3.5 (NB: optimal pH for elution may vary in range pH 2.0 – pH 4.0)

• Regeneration: 50 mM citric acid or 0.2 M NaOH/30% isopropanol

• Sanitization: 0.5 M sodium hydroxide

76

Antibody Binding Ligands: Purification of hIgG from Plasma

Lane 1 - MW Marker Lane 2 - Load (HSA depleted Plasma) Lane 3 - Flow-through Lane 4 - Wash Lane 5 - Elution Lane 6 - 1 M NaOH

Sample Volume (mL)

Protein (mg)

HSA depleted plasma

IgG pool 225

130

1200

264

1 2 3 4 5 6

77

Antibody Binding Ligands: IgG from albumin-depleted plasma

• Column: 1.6 cm Ø x 9.4 cm bed height,

19 mL CV

• Load: flow-through from Blue SA1

• Loaded protein: 27.8 mg/mL resin

• IgG capacity: 12.5 mg/mL

• Equilibration/wash: 0.05 M NaAc, 0.15 M NaCl, pH 7.0

• Elution: 0.05 M Na citrate, 0.01 M glycine, pH 3.0

• Sanitization: 0.5 M NaOH

Relative ratio of IgG subclasses determined by Nephelometry -

Subclass IgG1 IgG2 IgG3 IgG4

II + III Paste extract 64.1% 29.3% 2.9% 3.7%

MAbsorbent® A2P elution 62.8% 29.2% 3.1% 5.0%

78

Antibody Binding Ligands: Comparisons between MAbsorbent® A1P, A2P and rProtein A at pH 3.5

% Aggregate by SEC

20.4

4.1 4.0

9.4

0

5

10

15

20

25

IEX A1P Eluate A2P Eluate rPA Eluate

% Total Half Antibody by NR Gel Chip

10.7 9.8 9.5

17.5

0

5

10

15

20

IEX A1P Eluate A2P Eluate rPA Eluate

A “half antibody” variant was cleared more efficiently by the synthetic affinity ligands compared to rProtein A resin

Data courtesy of Biogen Idec

Aggregate level with A2P or A1P was much lower than that of rProtein A eluate

79

CONCLUSIONS

Conclusions: Benefits of Affinity Technology

• Increased yields More product units from the same amount of expressed protein

• Reduced cost of goods Increased margins / more competitive product pricing

• Increased purity Safer products / fewer side-effects

80

Conclusions: Mimetic Ligands™ Currently Developed for -

• Albumin

• Albumin-fusion proteins

• IgG

• Antibody fragments

• Cytokine binding protein

• Insulin & Insulin Analogues

• rFactor VII

• rFactor VIII

81

• Alpha 1-Antitrypsin

• Fibrinogen

• Plasminogen

• tPA

• rtPA-Urokinase

• Alkaline Phosphatase

• Endotoxin

• Prions

Contact Us: Global Support

82

VISIT OUR WEBSITE

Please do not hesitate to contact us for any product specifications, sales information or technical support. Also let us know if you would like us to organise any in-house training or seminars in your area.

For sales: sales-pbl@prometic.com

For tech support: techsupport-pbl@prometic.com

For more information on any of our products, services, latest company news or to view our ‘on-line shop’ visit our website: www.prometicbiosciences.com

SALES & TECHNICAL SUPPORT

EUROPE / REST of the WORLD

Horizon Park Barton Road, Comberton Cambridge

CB23 7AJ, UK Tel: +44 (0) 1223 420300 Fax: +44 (0) 1223 420270

1330 Piccard Drive Suite 201 Rockville, Maryland 20850 USA Tel: +1 301 251 8821 Fax: +1 301 251 8826

NORTH AMERICA

PuraPlate™, Mimetic Ligand™, ProMetic BioSciences Ltd and the PBL Logo are trademarks of ProMetic BioSciences Ltd. PuraBead®, Mimetic Blue®

and MAbsorbent® are registered with the U.S. Patent & Trademark Office. Chemical Combinatorial Library CCL® is registered with the U.K. Patent

& Trademark Office. AlbuPure® and Albufuse® are registered trademarks of Novozymes Biopharma UK Ltd.

© ProMetic BioSciences Ltd 2012. Issue – 25/02/14 82