A two-step process for recombinant insulin purification using Insulin Adsorbent & SP PuraBead® HF
Abstract
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.
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.
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).
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.
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.
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:
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.
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
Summary
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.
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.
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.