A two-step process for recombinant insulin purification using Insulin Adsorbent & SP PuraBead® HF Astrea Bioseparations, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
3
Insulin analogues are recombinant proteins which are often produced in microbial expression systems. The analogues are developed by varying the amino acid sequences of natural insulin, which alters the adsorption, distribution, metabolism, and excretion characteristics in glycaemic control. Insulin analogues offer advantages for the management of diabetes mellitus over natural insulin; these are generally commercially available as ‘rapid-acting’ or ‘long-acting’. Rapid-acting insulin analogues start to act immediately after injecting offering a better postprandial profile. Long-acting insulin analogues are designed to provide a longer, more uniform duration which provides confidence in overnight control.1 Insulin and insulin analogues are commonly purified using a combination of downstream process steps such as reverse-phase HPLC, ion exchange, or size exclusion chromatography which require multiple steps. Due to the growing demand to optimize the process time and cost of purifying insulin analogues, Insulin Adsorbent, a synthetic ligand affinity chromatography adsorbent, has been developed to support large-scale process applications.
Polishing with SP PuraBead® HF
Insulin Adsorbent eluate was purified with SP PuraBead® HF using the following conditions: Description (Polishing step) SP PuraBead® HF
Platform:
Automated chromatography platform
Column parameters:
5 mL CV (1 cm diameter, 6.3 cm bed height)
Operational flow rate:
Insulin Adsorbent provides >95% purity for recombinant single-chain insulin in a single direct capture step. The data below demonstrates a two-step process using Insulin Adsorbent to capture & SP PuraBead® HF to polish recombinant single-chain insulin using a safe, non-toxic, caustic stable adsorbent through multiple cycles to deliver ≥99% pure insulin.
Chromatogram for the purification of recombinant single-chain insulin using Astrea Bioseparations SP PuraBead® HF (post-Astrea Bioseparations Insulin Adsorbent)
Condition Adsorbent:
75 cm/hr
Equilibration buffer:
50 M sodium citrate, pH 3.0
Load:
Astrea Bioseparations Insulin Adsorbent elution (no adjustment required)
Elution buffer:
50 mM sodium citrate, 0-1.0 M NaCl gradient, pH 3.0
Clean-in-place (CIP):
0.5 M NaOH
Conductivity 300
250
Absorbance (mAu)
Abstract
Clean-in-place 200
Gradient elution 150
100
Non-bound 50 0
0
50
100
150
Volume (mL)
Recombinant insulin
Insulin Adsorbent
1
kDa
2
3
4
5
6
7
Non-reduced SDS page: Lane 1: MW marker
188
Lane 2: Load Lane 3: Flow through
1
98
Column packing using Insulin Adsorbent
Insulin Adsorbent comprises a novel synthetic ligand coupled to PuraBead® prioprietary agarose-based matrix technology. The PuraBead® process provides uniform near-monodisperse beads with excellent flow properties, which is capable of supporting high flow rates making Insulin Adsorbent an easy-to-pack chromatography resin. The adsorbent was packed into a 10 cm diameter column, 17.6 cm bed height, and has been demonstrated to produce asymmetry results within the range of 0.8 to 1.2. Packing results for Astrea Bioseparations Insulin Adsorbent (10 cm diameter column, 17.6 cm bed height).
Lane 7: CIP
28
17 14
6
700
Flow rate (cm/hr)
Absorbance (mAu)
80
Lane 6: Strip
38
Pressure vs. flow results for Astrea Bioseparations Insulin Adsorbent (10 cm diameter column, 17.6 cm bed height), using 0.2 M sodium acetate, pH 5.5 packing buffer.
HETP: 0.041 Plates/m: 2435 Asymmetry: 0.98
Lane 5: Elution (dilution 1 in 3)
49
800 100
Lane 4: Post-load wash
62
60
40
600 500
4
400 300
Toxicological studies
Insulin Adsorbent synthetic ligand shows no toxicity or mutagenic activity demonstrated by a range of toxicity studies.
200 20 100 0
0 0
500
1000
1500
SP PuraBead® HF achieved 99% purity while successfully removing residual nucleic acid materials and residual yellow chromophore. The removal was observed as a peak on the chromatogram at 450 nm in the non-bound fraction, which was absent in the elution gradient fraction.
0
2000
2
4
6
8
10
12
14
16
Pressure (psi)
Volume (mL)
Ames test (S.typhimurium TA) OECD/EEC/US EPA
Insulin Adsorbent provides excellent flow properties and can be processed at operational flow rates of up to 600 cm/hr with low column back pressures.
Soluble ligand
Soluble adsorbent extract
5000 mg/plate – no toxicity
5000 mg/plate – no toxicity
No mutagenic activity
No mutagenic activity
50-500 µg/kg/day
60-600 µg/kg/day
No evidence of toxicity
No evidence of toxicity
>500 mg/kg/day
>600 mg/kg/day
Rat IV dose study
2
Capture step using Insulin Adsorbent Condition
Description (Capture Step)
Adsorbent:
Insulin Adsorbent
Platform:
Automated chromatography platform
Column parameters:
5 mL column volume (CV) (1.6 cm diameter, 2.5 cm bed height)
Equilibration buffer:
0.1 M sodium acetate, pH 5.4
Load:
Pre-conditioned yeast cell culture supernatant containing recombinant single-chain insulin
Elution buffer:
0.3 M acetic acid
Strip:
1 M acetic acid, 20% ethanol
Clean-in-place (CIP):
0.5 M NaOH
ETD
Recombinant single-chain insulin from yeast cell culture was loaded into a 1.6 cm diameted column, 2.5 cm bed height under the following conditions: Non-bound
4000
Insulin Adsorbent provides an easy-to-pack chromatography adsorbent with excellent column packing results and can be operated at flow rates of up to 600 cm/hr with low column back pressures.
3500
Absorbance (mAu)
3000 2500
Recombinant single-chain insulin was purified through a two-step process, achieving ≥99% purity and high yield using Insulin Adsorbent in combination with a polishing step with SP PuraBead® HF, this provided effective removal of a yellow chromophore and residual nucleic acids.
Elution
2000 1500
Strip 1000 500 0 0
50
100
150
200
Volume (mL)
Recombinant insulin 1
2
3
4
5
6
kDa
7
Non-reduced SDS page: Lane 1: MW marker Lane 2: Load
191 97
Lane 3: Flow through Lane 4: Post-load wash Lane 5: Elution (dilution 1 in 3)
The chromatogram and non-reduced SDS-page demonstrate a 95% purity in the process fractions containing the purified recombinant single-chain insulin. The load containing the recombinant single-chain insulin displays a yellow chromophore, traces of which can be seen remaining in the Insulin Adsorbent elution fraction after the initial capture step. 1
2
3
4
5
Lane 6: Strip 64
Lane 7: CIP
51
39 28 19 14 6
Sample 1: Load Sample 2: Astrea Bioseparations non-bound Sample 3: Astrea Bioseparations Insulin Adsorbent elution Sample 4: Astrea Bioseparations Insulin Adsorbent strip Sample 5: Purified recombinant insulin
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Summary
Insulin Adsorbent synthetic ligand shows no toxicity or mutagenic activity demonstrated by a range of toxicity studies. Insulin Adsorbent provides a robust, easy to pack, high-performance chromatography adsorbent for the capture and purification of insulin and insulin analogues, with binding capacities of up to 25 g/L of adsorbent (depending on feedstock), excellent flow properties, and can be cleaned using NaOH allowing multiple cycles.
References: •
1
http://www.ncbi.nlm.nih.gov/pubmed/11853368