Purification of alpha-1 antitrypsin using an antibody based affinity chromatography medium Ulrika Meyer a, Hanna Wlad a, Sven Blokland b, Frank J.M. Detmers b and Henrik Ihre a a GE Healthcare Bio-Sciences AB, R&D, Björkgatan 30, SE-751 84 Uppsala, Sweden b BAC BV, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands First presented at the Plasma Product Biotechnology meeting 2011, Cyprus, May 2011.
Introduction A new method for purification of alpha-1 antitrypsin (AAT) is presented. An agarose matrix functionalized with recombinant single domain antibody fragments recognizing AAT was shown useful for capture of AAT from crude plasma but also for improvement of product purity of a registered pharmaceutical AAT drug. SDS-PAGE analysis using Coomassie Brilliant Blue (CBB) staining and Western Blot proved efficient enrichment of AAT from crude plasma in the 1 st capture step and also high specificity of the towards AAT. The total binding AAT capacity of the new was in the range 9-11 mg AAT/ml medium. 2 /Purification of alpha-1 antitrypsin using
Characteristics of Alpha-1 Antitrypsin Select 3 /Purification of alpha-1 antitrypsin using
Antibody fragment as affinity ligand The AAT affinity ligand is a single domain antibody fragment derived from a heavy chain antibody of a Llama. For the discovery and selection process of this affinity ligand, an antibody expression library was constructed that represented the single domain antibody repertoire of a Llama immunized with human alpha-1 antitrypsin. This library was screened, at monoclonal level, for target specific ligands that meet defined process conditions. The robustness of the screening process allows incorporation of key features that relate to the final purification process, like specific elution conditions at neutral ph. 4 /Purification of alpha-1 antitrypsin using
Antibody fragment as affinity ligand The DNA code of the selected affinity ligand (i.e., the heavy chain antibody) was amplified using PCR and thereafter the gene was cloned into a yeast cell expression system (Saccharomyces cerevisiae). Hence the ligand production is a non-animal process. General information on the principle of the affinity ligand development is found in reference 1 and 2. References 1. ten Haaft M, Hermans P, Dawson B. Separations in Proteomics: Use of Camelid Antibody Fragments in the Depletion and Enrichment of Human Plasma Proteins for Proteomics Applications. Separation Methods in Proteomics. CRC Press. 29-40, 2006. 2. Detmers F, Hermans P, ten Haaft M. Novel affinity ligands for bioprocessing. Innovations in Pharmaceutical Technology 2007, 23, 53-54, 56. 5 /Purification of alpha-1 antitrypsin using
Antibody fragment as affinity ligand 6 /Purification of alpha-1 antitrypsin using
Alpha-1 Antitrypsin Select: the Chromatography Medium The primary amines of the AAT ligand is covalently attached to NHS activated matrix forming chemically stable amide linkages (Figure 2). 7 /Purification of alpha-1 antitrypsin using
Alpha-1 Antitrypsin Select: the Chromatography Medium The spacer arm between the base matrix and ligand facilitates effective binding of the target AAT molecule. Figure 3. The high/flow agarose base matrix of Alpha-1 Antitrypsin Select displays superior open/bed pressure/flow properties compared to the base matrix of Sepharose 6 Fast Flow. Running conditions BPG 300 column open bed with water at 20 ºC at a settled bed height equal to 20 cm. 8 /Purification of alpha-1 antitrypsin using
Alpha-1 Antitrypsin Select: the Chromatography Medium Alpha-1 Antitrypsin Select is based on highly rigid spherical agarose particles. The rigid base matrix offers outstanding pressure and flow properties (Figure 3), which are key attributes for costeffective, large-scale use. Alpha-1 Antitrypsin Select permits a wide working range of flow velocities, bed heights, and sample viscosities, all of which have a positive impact on processing costs. 9 /Purification of alpha-1 antitrypsin using
Enrichment of Alpha-1 Antitrypsin from Human Plasma 10 /Purification of alpha-1 antitrypsin using
Enrichment of Alpha-1 Antitrypsin from Human Plasma Figure 4. UV 280 absorbance curve for plasma loading and elution of AAT using Alpha-1 Antitrypsin Select as the first capture step for purifying AAT from plasma. Including a wash step prior to the first elution would improve the purity of the elution peak even further. 11 /Purification of alpha-1 antitrypsin using
Enrichment of Alpha-1 Antitrypsin from Human Plasma The protein content of the starting material (plasma), the flow through fractions and 2 elution fractions are analyzed by gel electrophoreses and visualized by Coomassie Brilliant Blue (CBB) staining and Western Blot (WB, using a goat anti/ human AAT antibody). Figure 5. CBB staining and WB of the start material (human plasma), the non-retained fractions and the elution fractions. Lane 14 shows AAT from the first elution peak. 12 /Purification of alpha-1 antitrypsin using
Enrichment of Alpha-1 Antitrypsin from Human Plasma Alpha-1 Antitrypsin Select is able to deplete AAT from the crude plasma and the AAT is eluted in the 1 st elution (Figure 5) at neutral ph. The extra signals visible in the WB are highly likely IgG due to cross-reactivity of the used antibodies. The SDS-PAGE analysis proves that the media has a high specificity towards AAT and that enrichment was achieved. Enrichment of AAT from crude human plasma in a 1 st capture step Effective elution at neutral ph 13 /Purification of alpha-1 antitrypsin using
Purification of commercial AAT drug 14 /Purification of alpha-1 antitrypsin using
Purification of commercial AAT drug Figure 6. Loading of pharmaceutical AAT drug at ph 7.4 followed by elution using magnesium chloride at the same ph. 15 /Purification of alpha-1 antitrypsin using
Purification of commercial AAT drug During loading of the pharmaceutical drug an increased absorbance was detected in the chromatogram already from the loading volume of 1 ml (Figure 6). This level of absorbance was constant until the breakthrough was seen from approximately 8 ml of loading. This result was somewhat unexpected since impurities were not expected from a drug of pharmaceutical quality. To investigate this further, and exclude leakage of AAT from the column already from the beginning of the loading, the starting material and fractions from the chromatogram were collected and analyzed using SDS PAGE. 16 /Purification of alpha-1 antitrypsin using
Purification of commercial AAT drug Figure 7. SDS PAGE gradient gel on AAT drug before additional purification, flow through fractions and fractions from elution together with molecular mass protein markers. 17 /Purification of alpha-1 antitrypsin using
Purification of commercial AAT drug In addition to the expected AAT band at 52 kda, the starting material showed weak bands on the SDS page gel corresponding to proteins of approximately 40 kda and 10 kda in molecular weight (Lane 2, 3 and 4). The flow through fraction showed the same bands at 40 kda and 10 kda but also some in the region of 50-60 kda (Lane 5). Finally, the elution fractions showed the typical AAT band and no other bands (Lane 6 and 7). Improvement of the purity of an AAT drug in a polishing step 18 /Purification of alpha-1 antitrypsin using
Conclusions Enrichment of AAT in a 1st capture step from human plasma Mild elution conditions at neutral ph Improvement of the purity of a commercial available and registered AAT drug Outstanding pressure and flow rates reduce process time and improve process economy 19 /Purification of alpha-1 antitrypsin using
Acknowledgments www.gelifesciences.com GE, imagination at work, and GE Monogram are trademarks of General Electric Company. Sepharose, BPG and Tricorn are trademarks of GE Healthcare Companies. All third party trademarks are the property of their respective owners. A non-exclusive license from BAC BV, Huizerstraatweg 28, 1411 GP Naarden, The Netherlands, is neededfor the use of Alpha-1 Antitrypsin Select for production of clinical materialand for commercial manufacturing. Certain preparations may require a license under European patent no. EP991666. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. 2011 General Electric Company - All rights reserved. GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden 20 /Purification of alpha-1 antitrypsin using