Alternatives for affinity adsorbent development for downstream bioprocessing B. Dawson, Astrea Bioseparations Ltd, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
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Abstract
From ligand discovery to cGMP manufacturing at scale
A successful affinity adsorbent demonstrates high selectivity, reversible binding and high binding capacity, in addition to being robust, resistant to fouling, affordable and scalable. These qualities are very dependent on the target protein, the individual feedstock and upstream processing. As the probability of a good quality hit in ligand screening is dependent on the number of ligands screened, it is important to have access to large library of potential ligands with different properties.
Phase 1
Here we showcase examples from our Synthetic Ligand and Affimer® Ligand discovery platforms, demonstrating the process from ligand discovery to adsorbent development.
Synthetic Ligand Discovery and Adsorbent Development 2
Routes to discover new synthetic affinity ligands
• Base matrices • Ligand density • Spacer arms and attachment chemistry • Ligand synthesis method
Ligand Screening
Chromatography Development/Optimisation
Yes
Binding site determination or blind docking
Docking/vHTS
Phase 4
Regulatory Support
Three Verification Batches
Extractables & Leachates
One TT Batch
Stability
Three Validation Batches
Toxicology
Affimer® Ligand Discovery and Adsorbent Development 5
Affimer® affinity Ligand discovery for Human Factor IX
No
Similarity or pharmacophore searches
Phase 3
Tech Transfer & Validation
There are 4 modular phases involved in the development of a GMP ready affinity chromatography adsorbent. Here we demonstrate the process from ligand discovery (Phase 1) to adsorbent development (Phase 2) for our synthetic and Affimer® programmes.
Deselection targets: Factor II and Factor VII 167 clones selected
Known ligand or inhibitors?
No
• Performance assessment • Load, Elution, Wash conditions • CIP conditions • Initial evaluation of re-use
Verification Chromatography
Target: Factor IX
No
Known ligand binding site?
Yes
Adsorbent Development/Optimisation
Ligand Synthesis
Target protein structure known and available?
Yes
Adsorbent & Process Development
Ligand Design
Astrea has 2 ligand discovery platforms. Our synthetic Chemical Combinatorial Library CCL® is highly diverse, comprising of >100,000 triazine based ligands. These libraries are highly stable, and non-toxic/non mutagenic. The adsorbents are straightforward to scale-up, inexpensive and with an excellent track record of use in regulated downstream processes. Alternatively, the Affimer® Ligands, developed by Avacta, and licensed by Astrea have exquisite selectivity, good chemical and thermal stability, broad tolerance to organic solvents and wide range of pH values. Again, the library is highly diverse with 1010 ligands.
Phase 2
Ligand Discovery
All clones were assayed in the iQue primary screen
Target Protein QC
Primary screening
Phage Display
Sequencing
42 positive clones were sequenced
Sub-cloning and colony picking
Subcloning & Expression
5 Affimers were subcloned
Small scale Expression
Pulldown & Characterization
Selection of lead affimer
Diverse library design and HTS
Computational techniques are used in several stages in the Affinity ligand design process, as well as to give an understanding of the target protein and adsorbent interactions.
This schematic describes the development of an Affimer® ligand for Human Factor IX, a treatment used in haemophilia. The target for phage display selection was Factor IX, and the deselection targets for phage display were Factor II and Factor VII.
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Client Project A: Purification performance of a lead synthetic ligand candidate
After primary and secondary screens for Client Project A, 3 candidates were scaled up for testing. The lead candidate, showed significant gains in purity and recovery of the target protein. Client Project A
Number of ligands tested
PRIMARY SCREENING
768
SECONDARY SCREENING
29
SCALE-UP SYNTHESIS
The lead Affimer® candidate was immobilised onto PuraBead® P6HF. Triplicate chromatography runs were performed using a Factor IX containing feedstock and analysis of fractions carried out by coagulometry, SDS PAGE, HTRF and BCA total protein assay.
Target protein
Results demonstrated 90% recovery of purified Factor IX with 58 fold increase in specific activity. Minimal optimization was required to achieve these results.
Load Total protein titre: 1.8 mg/mL Target protein titre: 0.7mg/mL Purity = 39%
Specific activity (IU/mg total protein)
Target protein
3
Ave
2.71
156.23
SD
0.08
9.83
Fold increase 57.55 2.00
Active FIX recovery
Load
Elution
%
Ave
134.29
120.69
89.88
SD
2.08
5.99
4.26
Human Factor IX Affimer® adsorbent performance: Breakthrough capacity
The binding capacity at 10% was 2.65 mg/mL adsorbent, and the sharp curve was typical for an affinity adsorbent.
Final adsorbent - pilot scale
Ligand Candidate
Binding Capacity (g/L)
Yield (%)
Purity (%)
HMW (%)
Dimers (%)
1
2.4
56
81
0.5
15.2
2
3.1
68
82
0
10
3
2.3
73
75
3
19.9
4
2.1
55
89
0
11.8
Binding Capacity (g/L)
Yield (%)
Purity (%)
HMW (%)
Dimers (%)
Lead candidate 2
16
>95%
>92
0
<5
Improvement
>5 fold change
25%
10%
-
50%
Ligand
The performance lead candidate for Client Project B, in yellow, was improved after an adsorbent and chromatography development programme. This examined factors such as ligand densities and attachment chemistries (e.g. spacer arms) and process conditions, to optimized to maximize product purity and yield.
Search: Astrea Bioseparations Affimer® is a registered trademark of Avacta Life Sciences Ltd. Purabead® is a registered trademark of Astrea UK Services Ltd. Astrea Bioseparations and the Astrea logo are trademarks of Astrea UK Services.
Elution
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Client project B: Adsorbent development leads to substantial improvement in lead ligand candidate performance Secondary screening stage
Purity
Load
FIX activity (IU/mg FIX)
Lead adsorbent Elution fraction Binding Capacity = 17mg/mL Recovery = 96% Purity = 90%
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Human Factor IX Affimer® adsorbent development: Purification performance
Summary
By using multiple ligand discovery platforms, affinity adsorbents for a diverse range of target molecules from complex feedstocks have been developed. The Synthetic Library and Affimer® library technology provide different advantages that can be exploited to meet diverse ligand criteria required for successful affinity adsorbent development.