Approaches to Ligand Design (Issue - 250214)
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Transcript of Approaches to Ligand Design (Issue - 250214)
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
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© ProMetic BioSciences Ltd 2012. Issue – 25/02/14 82