iTRAQ® Reagents Amine-Modifying Labeling Reagents - AB Sciex
Affimer® reagents facilitate affinity chromatography ... · chromatography purification of IgG...
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0 5 10 15 20 mL 60 40 20 0 200 150 100 50 0 30 20 10 0 100 80 60 40 20 0 mAU mAU mAU mAU IgG1 kappa Free kappa LC F ab kappa F c UB UB UB UB El El El El 0 5 10 15 20 mL 0 5 10 15 20 mL 0 5 10 15 20 mL A C B 10 atom spacer b-turn 3 C-terminus Binding loops POS013 Geoffrey Platt For further information regarding Affimer technology, please contact [email protected] or visit www.avacta.com Summary Affimer reagents in affinity resins Figure 1A. Structure of the Affimer scaffold protein with the two regions containing variable amino acid loops in grey. 1B. Schematic showing conjugation of Affimer protein to agarose resin. 1C. Column loaded with affinity resin. Affimer® reagents facilitate affinity chromatography purification of IgG Affimer reagents are proteins which present high affinity binding surfaces for specific interactions with a wide range of targets. The scaffold is based on the cystatin fold and permits the presentation of two variable binding loops (Fig 1). The Affimer reagents are small (~12-14 kDa), easy to manufacture in bacterial expression systems and biophysically stable over a wide range of pH conditions. As both the scaffold and the randomised binding loops are engineered to lack cysteines, the introduction of this residue using basic molecular biology methods provides a straightforward route for immobilising the protein to surfaces in a defined orientation via thiol chemistry (Fig 1). agarose • The high specificity achieved by Affimer protein-based affinity resins suggests potential applications in pharmacokinetic studies and bioprocessing platforms. • Flexible reformatting provides a route to further optimisation - improvements in performance may be possible by development of the resin, cysteine location, multimer options, linker lengths and coupling methods. • Affimer resins are robust to a wide range of pH conditions enabling a high level of reproducibility and ability to endure cleaning routines. Affimer D11 binds the F c domain of human IgG (hIgG) and exhibits high target specificity in the presence of complex mixtures and when challenged with mammalian homologues (Fig 2). High specificity has enabled the use of this resin for pharmacokinetic studies of hIgG-drug conjugate stability in mouse plasma (data not shown). Affimer clones that bind different regions of IgG molecules were immobilised on highly cross-linked 4 % agarose beads via iodoacetyl chemistry and the resins packed on columns (bed height 31 mm, column volume 0.6 mL). The characteristics of the affinity resins were studied for target specificity, binding capacity, reproducibility and stability. Binding capacity Affimer D11 was engineered to include extra residues (Gly-Gly-Ser) that extend the length of linker to the agarose bead. The dynamic binding capacities of columns packed with Affimer resin containing standard and extended linkers were measured. Extension of the linker produced a resin with increased binding capacity (Fig 3). Figure 5. Example traces produced upon loading pure IgG species across Affimer H2 resin, suggesting this Affimer clone is capable of recognizing and enriching assembled IgGs and F ab fragments. Affimer H2 binds intact F ab domains allowing purification of whole IgGs and F ab s from contaminants such as free light and heavy chains (Fig 5). The novel ability of Affimer H2 resin to bind intact F ab suggests it could be useful in bioprocessing and analytical applications where discrimination of intact F ab s from free light chains (LCs) is required. In this respect the Affimer-based resin would be complementary to protein L, which bind kappa LCs. Indeed it displays more efficient elution of targets at pH 3 than commercially available protein L resins. 0 10 20 30 40 mL 2000 1000 0 mAU C/C 0 Standard linker Extended linker (GGS) 0.8 0.6 0.4 0.2 0.0 Saturated column capacity (mg mL -1 ) Dynamic binding capacity (mg mL -1 ) Extended linker Standard linker Extended linker Standard linker Standard linker Extended linker (GGS) 0 5 10 15 20 25 30 mL A C B D Load | Wash | Elute Figure 3. Dynamic binding during continual loading of 0.5 mg mL -1 pure hIgG over Affimer D11 resins with a column residence time of 3 min. 3A. Full traces showing load, wash and elution phases. 3B. Normalised breakthrough curves. 3C. The amount of target protein recovered during elution, normalised to column volume. 3D. The dynamic binding capacity at 10 % breakthrough, normalised to column volume. Fragment Binder Figure 4A. An Affimer D11 column demonstrated high levels of reproducibility over 81 consecutive purification cycles. Inset: Example traces for a section of the experiment. 4B. Example western blot indicating no detectable leaching during elution, pure Affimer protein is included for reference. Stability and reproducibility The reproducibility and stability of an Affimer-based resin was measured by repeated injection and elution of pure hIgG (81 runs), including 19 clean-in-place (CIP) cycles (100 mM NaOH, 10 min). The data indicate consistent capture performance over the course of this study (Fig 4A). No leaching from Affimer columns was detected by western blotting of concentrated elution fractions (Fig 4B). Pure affimer 160 ng 16 ng Eluted fractions (240 x conc) A B 0 20 40 60 80 Run 100 98 96 94 92 90 % Area in eluted peak CIP CIP mAU Conductivity % Elution buffer CV Unbound (UB) Eluted (El) mL % area in eluted peak 100 80 60 40 20 0 A B Human IgG Dog IgG Rat IgG Mouse IgG Figure 2A. Traces produced upon loading IgG-depleted human serum spiked with known concentrations of hIgG onto Affimer D11 resin. The wash buffer used is PBS (pH 7.4) and elution buffer is glycine, NaCl (pH 3.0). Inset: SDS-PAGE (under reducing conditions) confirms specificity of binding and elution. 2B. % Area of elution peak after injection of 1 mg of pure immunoglobulins. Specific target binding and elution
Transcript of Affimer® reagents facilitate affinity chromatography ... · chromatography purification of IgG...
0 5 10 15 20 mL
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Fab kappa Fc
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POS013
Geoffrey Platt
For further information regarding Affimer technology, please contact [email protected] or visit www.avacta.com
Summary
Affimer reagents in affinity resins
Figure 1A. Structureof the Affimerscaffold protein withthe two regionscontaining variableamino acid loops ingrey. 1B. Schematicshowing conjugationof Affimer protein toagarose resin.1C. Column loadedwith affinity resin.
Affimer® reagents facilitate affinity chromatography purification of IgG
Affimer reagents are proteins which present high affinity binding surfaces for specific interactionswith a wide range of targets. The scaffold is based on the cystatin fold and permits thepresentation of two variable binding loops (Fig 1). The Affimer reagents are small (~12-14 kDa),easy to manufacture in bacterial expression systems and biophysically stable over a wide range ofpH conditions.
As both the scaffold and the randomised binding loops are engineered to lack cysteines, theintroduction of this residue using basic molecular biology methods provides a straightforwardroute for immobilising the protein to surfaces in a defined orientation via thiol chemistry (Fig 1).
agarose
• The high specificity achieved by Affimer protein-based affinity resins suggests potential applications in pharmacokinetic studies and bioprocessing platforms.• Flexible reformatting provides a route to further optimisation - improvements in performance may be possible by development of the resin, cysteine location,multimer options, linker lengths and coupling methods.
• Affimer resins are robust to a wide range of pH conditions enabling a high level of reproducibility and ability to endure cleaning routines.
Affimer D11 binds the Fc domain of human IgG (hIgG) and exhibits high target
specificity in the presence of complex mixtures and when challenged with
mammalian homologues (Fig 2). High specificity has enabled the use of this
resin for pharmacokinetic studies of hIgG-drug conjugate stability in mouse
plasma (data not shown).
Affimer clones that bind different regions of IgG molecules were immobilised on highly cross-linked4 % agarose beads via iodoacetyl chemistry and the resins packed on columns (bed height 31 mm,column volume 0.6 mL). The characteristics of the affinity resins were studied for target specificity,binding capacity, reproducibility and stability.
Binding capacityAffimer D11 was engineered to include extra residues (Gly-Gly-Ser) that extendthe length of linker to the agarose bead. The dynamic binding capacities ofcolumns packed with Affimer resin containing standard and extended linkerswere measured. Extension of the linker produced a resin with increased bindingcapacity (Fig 3).
Figure 5. Example traces produced upon loading pure IgG species across Affimer H2 resin,
suggesting this Affimer clone is capable of recognizing and enriching assembled IgGs and
Fab fragments.
Affimer H2 binds intact Fab domains allowing purification of whole IgGs and Fabs
from contaminants such as free light and heavy chains (Fig 5). The novel ability
of Affimer H2 resin to bind intact Fab suggests it could be useful in bioprocessing
and analytical applications where discrimination of intact Fabs from free light
chains (LCs) is required. In this respect the Affimer-based resin would be
complementary to protein L, which bind kappa LCs. Indeed it displays more
efficient elution of targets at pH 3 than commercially available protein L resins.
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Load | Wash |
Elute
Figure 3. Dynamic binding during continual loading of 0.5 mg mL-1 pure hIgG over AffimerD11 resins with a column residence time of 3 min. 3A. Full traces showing load, wash andelution phases. 3B. Normalised breakthrough curves. 3C. The amount of target proteinrecovered during elution, normalised to column volume. 3D. The dynamic binding capacityat 10 % breakthrough, normalised to column volume.
Fragment Binder
Figure 4A. An Affimer D11 column demonstrated high levels of reproducibility over 81consecutive purification cycles. Inset: Example traces for a section of the experiment. 4B.Example western blot indicating no detectable leaching during elution, pure Affimerprotein is included for reference.
Stability and reproducibilityThe reproducibility and stability of an Affimer-based resin was measured byrepeated injection and elution of pure hIgG (81 runs), including 19 clean-in-place(CIP) cycles (100 mM NaOH, 10 min). The data indicate consistent captureperformance over the course of this study (Fig 4A). No leaching from Affimercolumns was detected by western blotting of concentrated elution fractions(Fig 4B).
Pure affimer
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Eluted (El)
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% a
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A B
Human IgG Dog IgG Rat IgG Mouse IgG
Figure 2A. Traces produced upon loading IgG-depleted human serum spiked with known
concentrations of hIgG onto Affimer D11 resin. The wash buffer used is PBS (pH 7.4) and
elution buffer is glycine, NaCl (pH 3.0). Inset: SDS-PAGE (under reducing conditions)
confirms specificity of binding and elution. 2B. % Area of elution peak after injection of 1 mg
of pure immunoglobulins.
Specific target binding and elution
