Skip to main content

Article Filters

Clear Filters
Finish

Capture and purification of recombinant albumin-fusion proteins using AlbuPure®

Published date: 08 December 2023

Back to Article Listing

Human serum albumin (HSA) has a vast array of applications within the BioPharmaceutical industry including; plasma expansion, formulation excipient, drug delivery, wound healing as well as extending the half-life of a protein drug as a fusion partner. A wide range of albumin-fusion proteins (Albufuse®) have been produced to extend the circulatory half-life of the fusion partner whilst maintaining it’s activity.

Veltis® developed by albumin-fusion leaders Albumedix is a single step expression platform for the production of proteins with extended half-life eliminating lengthy and minimising costly post-production processing, providing clear patient benefits and reduced healthcare costs.

In order to support the rise in the production of recombinant albumin-fusion proteins, an affinity matrix is required that is highly stable, robust, non-toxic and provides high binding capacity, recovery and purity.

AlbuPure®, developed in collaboration with Albumedix and manufactured exclusively by Astrea Bioseparations is produced in a controlled environment to ISO 9001 quality standard and is designed for use in cGMP manufacturing of biological molecules.

The AlbuPure® adsorbent comprises a novel synthetic triazine ligand derived from Astrea Bioseparations' Mimetic Ligand™ technology and coupled to Astrea Bioseparations' proprietary PuraBead® 6HF base matrix. This proven technology provides a stable ligand and attachment chemistry with low ligand leakage, enables the use of 1 M NaOH for sanitisation and provides excellent adsorbent lifetime.

In this blog, the capture and purification of recombinant albumin-fusion proteins using AlbuPure® is described.

Materials & Methods

Purification of different albumin-fusion proteins (1) IL-1ra and (2) anti-HIV gp41 were performed using AlbuPure® on an automated chromatography workstation. Both columns were packed, equilibrated and operated as summarised in Table 1.

The Albumedix yeast based high expression system was used to produce cell culture supernatant (CCS) containing either (1) IL-1ra or (2) gp41 albumin-fusion proteins. Each material was pre-conditioned prior to loading.

Non-bound and loosely bound proteins were removed with equilibration buffer. Bound non-target proteins were subsequently washed from the adsorbent using a series of buffers with increasing pH steps.

The albumin-fusion proteins were selectively eluted using buffer containing sodium octanoate.

AlbuPure® is hydroxide stable and was cleaned with 0.5 M NaOH.

 

Results & Discussion

IL-1ra Albumin-Fusion Protein

 The IL-1ra albumin-fusion protein (~85 kDa) was captured by the AlbuPure® adsorbent with the non-target proteins present in the column flow through (FT). Minor host cell proteins were removed using a series of wash steps with buffers of increasing pH. The albumin-fusion protein was then selectively eluted from the column using a buffer containing sodium octanoate (Figure 1). The process fractions were analysed by SDS-PAGE (Figure 2).

 

SDS-PAGE analysis (Figure 2) indicates high target protein binding with non-target material present in the flow through and post load wash (lanes 2 and 3). The increasing pH wash steps (lanes 4 to 6) highlights significant clearance of non-target protein (with minor loss of target). The elution fraction shows high IL-1ra albumin-fusion protein purity and recovery (lanes 7 and 8).

Anti-HIV gp41 Albumin-Fusion Protein

 The anti-HIV gp41 albumin-fusion protein (~72 kDa) was captured and purified using AlbuPure®. Non-target proteins were present in the column flow through (FT) and post load wash, host cell proteins were removed using a series of wash steps with buffers of increasing pH. The gp41 albumin-fusion protein was selectively eluted from the column with sodium octanoate (Figure 3); process fractions were analysed by SDS-PAGE (Figure 4).

 

 

SDS-PAGE analysis (Figure 4) indicates high target protein binding with non-target material present in the flow through and post load wash (lanes 2 and 3). The increasing pH washes (lanes 4 to 6), indicates significant clearance of bound host cell protein, although minor levels of target is observed. High anti-HIV gp41 albumin-fusion protein purity and recovery is observed in the elution fraction (lanes 7 and 8).

Conclusions

AlbuPure® can be used to capture and purify recombinant albumin-fusion proteins from cell culture supernatants with high purity and recovery, as shown in this application note.

The AlbuPure® adsorbent can be used to purify a variety of different albumin-fusion proteins (ranging from 2.5 to 30 kDa) including; anti-HIV peptides, IL-1ra, endostation, prosaptide, Kunitz domain, scFv, dAb, nanobody and vNAR.

 In combination with Albumedix’ Veltis® technology, AlbuPure® is able to process complex feedstocks at flow rates up to 600 cm/h. As a result, the AlbuPure® adsorbent can significantly reduce manufacturing costs by shortening processing times due to its increased operational flow rates coupled with the ability to clean and re-use the adsorbent over many cycles.

References

 Data courtesy of Albumedix, reference: ‘Use of a Novel Affinity Matrix as a Platform Purification for Albumin Fusions’, Recovery of Biological Products XIV Conference presentation, Lake Tahoe, California, August 1st – 6th 2010.

Your basket

Your basket is empty. Continue shopping to add products to your basket.

Search our catalogue

Call Centre

Request Quote

Please enter the quantity of products you want a quote for

Please enter the date you need these products byPlease enter a date in the format DD/MM/YYYY

View Content

Please complete the form below to receive the requested content from Astrea Bioseparations. We will use the data you provide to send you relevant updates, special offers, and product-related information. See our Privacy Notice for details on how we use personal data.

If you have an account please log in to gain access to content. You can apply for an account here

Please enter a First Name

Please enter a valid First Name, the maximum length is 50 characters.

Please enter a Last Name

Please enter a valid Last Name, the maximum length is 50 characters.

Please enter a Company Name

Please enter a valid Company, the maximum length is 100 characters

Please enter a valid Email AddressPlease enter a valid Email AddressThe Email Address entered is already registered, please sign in with the Email Address or enter a different one

Please select a valid Telephone Number, the maximum length is 30 characters

Please enter a valid Telephone Number consisting only of the following characters and spaces ( ) + 0 1 2 3 4 5 6 7 8 9

Please select a State / Province / Territory

Please select a valid State / Province / Territory, the maximum length is 150 characters

Please select a Country

Data entered into form will be stored in your session (not in a cookie) to avoid having to re-enter in future form submissions.

  • Home
  • |
  • Resources
  • |
  • Blog
  • |
  • Capture and purification of recombinant albumin-fusion proteins using AlbuPure®

Capture and purification of recombinant albumin-fusion proteins using AlbuPure®

Published date: 08 December 2023

Back to Article Listing

Human serum albumin (HSA) has a vast array of applications within the BioPharmaceutical industry including; plasma expansion, formulation excipient, drug delivery, wound healing as well as extending the half-life of a protein drug as a fusion partner. A wide range of albumin-fusion proteins (Albufuse®) have been produced to extend the circulatory half-life of the fusion partner whilst maintaining it’s activity.

Veltis® developed by albumin-fusion leaders Albumedix is a single step expression platform for the production of proteins with extended half-life eliminating lengthy and minimising costly post-production processing, providing clear patient benefits and reduced healthcare costs.

In order to support the rise in the production of recombinant albumin-fusion proteins, an affinity matrix is required that is highly stable, robust, non-toxic and provides high binding capacity, recovery and purity.

AlbuPure®, developed in collaboration with Albumedix and manufactured exclusively by Astrea Bioseparations is produced in a controlled environment to ISO 9001 quality standard and is designed for use in cGMP manufacturing of biological molecules.

The AlbuPure® adsorbent comprises a novel synthetic triazine ligand derived from Astrea Bioseparations' Mimetic Ligand™ technology and coupled to Astrea Bioseparations' proprietary PuraBead® 6HF base matrix. This proven technology provides a stable ligand and attachment chemistry with low ligand leakage, enables the use of 1 M NaOH for sanitisation and provides excellent adsorbent lifetime.

In this blog, the capture and purification of recombinant albumin-fusion proteins using AlbuPure® is described.

Materials & Methods

Purification of different albumin-fusion proteins (1) IL-1ra and (2) anti-HIV gp41 were performed using AlbuPure® on an automated chromatography workstation. Both columns were packed, equilibrated and operated as summarised in Table 1.

The Albumedix yeast based high expression system was used to produce cell culture supernatant (CCS) containing either (1) IL-1ra or (2) gp41 albumin-fusion proteins. Each material was pre-conditioned prior to loading.

Non-bound and loosely bound proteins were removed with equilibration buffer. Bound non-target proteins were subsequently washed from the adsorbent using a series of buffers with increasing pH steps.

The albumin-fusion proteins were selectively eluted using buffer containing sodium octanoate.

AlbuPure® is hydroxide stable and was cleaned with 0.5 M NaOH.

 

Results & Discussion

IL-1ra Albumin-Fusion Protein

 The IL-1ra albumin-fusion protein (~85 kDa) was captured by the AlbuPure® adsorbent with the non-target proteins present in the column flow through (FT). Minor host cell proteins were removed using a series of wash steps with buffers of increasing pH. The albumin-fusion protein was then selectively eluted from the column using a buffer containing sodium octanoate (Figure 1). The process fractions were analysed by SDS-PAGE (Figure 2).

 

SDS-PAGE analysis (Figure 2) indicates high target protein binding with non-target material present in the flow through and post load wash (lanes 2 and 3). The increasing pH wash steps (lanes 4 to 6) highlights significant clearance of non-target protein (with minor loss of target). The elution fraction shows high IL-1ra albumin-fusion protein purity and recovery (lanes 7 and 8).

Anti-HIV gp41 Albumin-Fusion Protein

 The anti-HIV gp41 albumin-fusion protein (~72 kDa) was captured and purified using AlbuPure®. Non-target proteins were present in the column flow through (FT) and post load wash, host cell proteins were removed using a series of wash steps with buffers of increasing pH. The gp41 albumin-fusion protein was selectively eluted from the column with sodium octanoate (Figure 3); process fractions were analysed by SDS-PAGE (Figure 4).

 

 

SDS-PAGE analysis (Figure 4) indicates high target protein binding with non-target material present in the flow through and post load wash (lanes 2 and 3). The increasing pH washes (lanes 4 to 6), indicates significant clearance of bound host cell protein, although minor levels of target is observed. High anti-HIV gp41 albumin-fusion protein purity and recovery is observed in the elution fraction (lanes 7 and 8).

Conclusions

AlbuPure® can be used to capture and purify recombinant albumin-fusion proteins from cell culture supernatants with high purity and recovery, as shown in this application note.

The AlbuPure® adsorbent can be used to purify a variety of different albumin-fusion proteins (ranging from 2.5 to 30 kDa) including; anti-HIV peptides, IL-1ra, endostation, prosaptide, Kunitz domain, scFv, dAb, nanobody and vNAR.

 In combination with Albumedix’ Veltis® technology, AlbuPure® is able to process complex feedstocks at flow rates up to 600 cm/h. As a result, the AlbuPure® adsorbent can significantly reduce manufacturing costs by shortening processing times due to its increased operational flow rates coupled with the ability to clean and re-use the adsorbent over many cycles.

References

 Data courtesy of Albumedix, reference: ‘Use of a Novel Affinity Matrix as a Platform Purification for Albumin Fusions’, Recovery of Biological Products XIV Conference presentation, Lake Tahoe, California, August 1st – 6th 2010.

Call Centre Product Compare