Plasma Protein Purification

The production of Human Plasma-based products differs from the typical biotechnology or pharmaceutical product because variable concentrations of multiple proteins of interest are purified from the same source. This presents unique challenges. Sterogene Bioseparations has proven itself in several FDA-approved processes in the production of human plasma-based pharmaceuticals. Our resins are in use by leading international plasma fractionation companies for the production of several plasma-derived proteins and have been the mainstay of our product line for 25 years.  Drug Master Files are on file at the FDA and other regulatory agencies. 

Affinity chromatograpy resins are used in the purification of plasma proteins, such as Factors VIII, IX, and X, Antithrombin III, von Willebrand Factor (vWF) and Complement protein C1 inhibitor.  Other resins, for example, our ActiClean Etox for endotoxin removal and our IonClear BigBeadTM, are applicable for the purification of any therapeutic protein. 

Our core resins Actigel ALD and Actigel Superflow can be linked to numerous ligands other than those specifically mentioned here.  Actigel ALD is used in immunoaffinity processes for the production of plasma proteins. It has a highly stable coupling chemistry with virtually no leaching of ligands. For example, Hyaluronic Acid can be linked to our resins using our proprietary chemistries for the production of fibrinogen.  

Plasma, including cryo-poor plasma, is a turbid liquid due to lipoprotein content. During the course of plasma fractionation, these may denature and precipitate, resulting in clogged resins unless pre-filtration is done. Sterogene’s CellthruTM resins eliminate the need for filtration. This Direct CaptureTM technology replaces the need for filtration and allows very high flow rates resulting in reduced processing time, cycle time and increased yield.

 Sterogene Bioseparations’ CellthruTM BigBeads can be used in plasma fractionation. The resin allows the free flow of debris and precipitates through a column without sacrificing binding of the protein of interest.  The resin has the additional property of being resistant to sanitization with 0.5M NaOH.  Ligands other than those shown below have been successfully immobilized to this resin, but those with direct application to plasma fractionation are as follows:

Heparin Actigel has been used in the fractionation of plasma products for manufacture of licensed therapeutic proteins for many years. Several companies use Heparin chromatography in the manufacture of plasma proteins such as Factor VIII, IX, vWF and ATIII.  

Heparin Actigel resin is recommended for intermediate flow rate purifications, since some proteins react more slowly with heparin than others.  The upside of Heparin Actigel is that the beads have a 10% higher binding capacity than our higher flow rate Heparin Superflow. The resins are stable in 0.5M NaOH, and chaotropes like 6M guanidine HCl or 8M urea, and some organic solvents.  Drug Master Files have been filed with the FDA and other regulatory agencies.

Sterogene also manufactures resins, such as DS-Superflow and DS-Cellthru™ with a Dextran sulfate ligand for the purification of Factor IX from plasma. These resins are also used in the purification of recombinant plasma proteins.

Sterogene Bioseparations is one of the leading manufacturers of resins with chelating moieties, such as Chelating Superflow and Chelating Cellthru™. Our resins are in several OEM products on the market.  Immobilized Metal Affinity Chromatography (IMAC) is used in the production of several plasma proteins, such as Factors IX and X, alpha1-proteinase inhibitor as well as C1 Inhibitor.

Benzamidine Superflow ligand is p-aminobenzamidine (the inhibitor of trypsin-like serine proteases), which covalently attached to a 6% agarose. Trypsin and trypsin-like serine proteases bind to Benzamidine resin and can be used for purification and/ or removal of these proteins. Benzamidine affinity resin is used in the purification of native plasminogen and plasmin.

Lysine Superflow is L-lysine immobilized to 6% highly crosslinked agarose. Lysine resin is an affinity adsorbent and can be used for isolation of plasminogen and plasminogen activator, and alpha1-proteinase inhibitor.

Lectins are proteins that interact specifically and reversibly with certain sugar residues on proteins. Their specificity enables binding to polysaccharides and glycoproteins. Immobilized lectins, such as Con A Superflow and Jacalin Superflow can be used for the purification of several plasma proteins, such as alpha1-proteinase inhibitor and C1 Inhibitor.

The reduction of ionic strength of plasma can be carried out by ultrafiltration, gel filtration, or capturing the salt on a mixed bed resin.  Ultrafiltration requires expensive membranes and equipment, and standard gel filtration leads to protein losses, dilution, and is time consuming.  For large-scale reduction of ionic strength, Sterogene Bioseparations offers IonClear BigBeadsTM, which are compatible with many column geometries and flow rates.

IonClear BigBeadsTM are both cation and anion exchangers, so the resin efficiently removes dissolved salts.  The beads are both mechanically and chemically stable, 500 – 1,100 mm in diameter, and can withstand very high flow rates.  The resin is stable at extreme pH (1-14) and can be used with many organic solvents.  For many processes, IonClear BigBeadsTM represents a rapid, cost effective resin for desalting large volumes of solutions with physiologic salinity.

Immunoaffinity technology is used in the purification of plasma proteins. Actigel ALD is available, along with a coupling solution that is free of sodium borohydride, for the immobilization of proteins, peptides, or other ligands containing primary amines.  The coupling is gentle on the ligand, extremely efficient, and scales up reproducibly.  The resulting secondary amine is stable, even in 0.15 M NaOH, providing the ligand can withstand those conditions.

It is used in the production of licensed biopharmaceutical, including plasma proteins.  Leaching from the resin is negligible, and Sterogene has a Drug Master File with the regulatory agencies.

Endotoxin level has to be low in all pharmaceutical injectables and ActiClean Etox  can be used to remove endotoxin from plasma proteins, such as albumin and other plasma proteins below the limits of detection. ActiClean Etox can be sanitized through several cycles using 0.5M NaOH.  ActiClean Etox efficiency has been documented in the depyrogenation of numerous plasma proteins, including human serum albumin, Immunoglobulin G, and Coagulation Factors VIII and IX.  ActiClean Etox has been used in the manufacture of licensed therapeutics and has a Drug Master File at the regulatory agencies.

We have 25 years of manufacturing experience and a proven track record with international regulatory agencies.  Whether your process needs a standard resin, such as heparin or IMAC, or a proprietary ligand you want conjugated for your bioprocess, Sterogene Bioseparations should be your first choice because of our

  • Unique Resins
  • Experience with custom coupling
  • Experience with regulatory hurdles
  • All of our manufacturing is performed in-house according to cGMP
  • We are ISO 9001-2008 certified

 

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