Applications

Monoclonal antibodies are an important segment of the biopharmaceutical industry. Seven out of the 15 best-selling drugs are monoclonal antibodies. Within the protein-based drugs, they are the market leader in terms of sales and annual revenues. Almost all therapeutic antibodies are produced by mammalian cell culture and downstream processing is used to produce a safe and highly purified product for human therapeutic applications.

Cell culture productivity has improved over the last decade, concentrations of 1-5 grams of antibody per liters of cell culture is typical. Chromatography is the workhorse of downstream processing due to its high resolution and reproducibility using simple with bind-wash-elute cycles. Initial recovery is the critical step in determining the economy of a downstream process. The protein of interest needs to be efficiently captured and a majority of the impurities removed to increase the efficiency of further downstream. Protein A affinity resins typically represent the first step of monoclonal antibody purification.

Protein A resin provides an excellent concentration step, and typically removes 95-98% of the contaminating DNA, CHOP, virus and small molecules. Further purification and virus removal are achieved by adding polishing steps consisting of ion exchange and HIC resins.

Clarification of the feed stream can become a bottleneck in harvesting monoclonal antibodies from a fermentation broth. By selecting our Direct Capture CellthruTM resins, the purification process complexity is reduced and yield is improved. In the current downstream process, cells are lysed under high sheer conditions resulting in contamination with DNA, CHOP, and other cellular components. Using our Protein A Cellthru™ Direct Capture™ resin, the protein of interest is bound under low shear conditions with a decreased level of contamination by cellular components.

Chelating affinity chromatography, such as Chelating Superflow and IDA Chelating CellthruTM has been also used for the purification of monoclonal antibodies from cell culture supernatant and from Pichia pastoris. High selectivity and high purification factors can be achieved for IgG. The monoclonal antibodies can be eluted using mild conditions causing no loss of antigen binding capacity.

Sterogene Bioseparations’ affinity resins are in the production of several FDA-approved human plasma-based pharmaceuticals. Our resins are in use by leading international plasma fractionation companies. 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.

Actigel ALD is used in immunoaffinity processes using monoclonal antibody for the production of plasma proteins. Actigel ALD 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.

Heparin-Actigel ALD, used in plasma protein purification, is resistant to sanitization with 0.5M NaOH. Heparin Actigel has been used in the fractionation of plasma products for manufacture of licensed therapeutic proteins for many years. Companies use Heparin chromatography in the manufacture of plasma proteins such as Factor VIII, IX, vWF and ATIII. The resin is 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 with a Dextran sulfate ligand for the purification of Factor IX from plasma. DS-Superflow and DS-Direct Capture 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 (IDA-Superflow and NTA-Superflow) for Factors IX and X, alpha1-proteinase inhibitor as well as C1 Inhibitor, etc.

Benzamidine Superflow ligand is p-aminobenzamidine (the inhibitor of trypsin-like serine proteases), which is covalently attached to our Superflow base resins. Trypsin and trypsin-like serine proteases bind to Benzamidine resin is used for purification and/ or removal of these proteins, such as the purification of native plasminogen and plasmin.

Lysine Superflow is L-lysine immobilized to our Superflow base resin. Lysine resin is an affinity adsorbent and 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. Lectins, such as Con A Superflow and Jacalin immobilized to our Superflow resin used for the purification of several glycoproteins including plasma proteins, such as alpha1-proteinase inhibitor and C1 Inhibitor.

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 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 Factor VIII and IX. ActiClean Etox has been used in the manufacture of licensed therapeutics and has a Drug Master File at the regulatory agencies.

High value proteins are frequently produced using recombinant DNA technologies.  Different expression systems can be used to allow expression of secreted proteins, while others require lysis  of the cells to allow purification of the protein of interest. Recombinant expression allows the incorporation of tags (His-tag, MBP, GST, Strep-tag II, c-myc, FLAG, etc.) to facilitate and improve purification.

The E. coli expression system is the most widely used, however the probability of successfully expressing a protein decreases considerably as molecular weight approaches 60 kDa. Approximately 30% of proteins cannot be produced in soluble form when. Common alternatives are the baculovirus expression system in insect cells, Pichia pastoris and Saccharomyces cerevisiae, human cells, or cell-free systems using prokaryotic or eukaryotic extracts.

Sterogene offers several resins for:

• the purification of tagged and untagged recombinant proteins, ( Chelating Superflow, Gluthatione Superflow )
• the immobilization of capture ligands, including monoclonal antibodies, ( Actigel ALD, Actigel ALD Superflow )
• the removal of endotoxin from recombinant proteins, especially proteins produced in E. coli expression systems ( ActiClean Etox )
• quick desalting ( IonClear BigBeadsTM )

Extracorporeal devices are mechanical organs used in blood purification such as apheresis, hemodialysis, hemofiltration, plasma-pheresis, or extracorporeal membrane oxygenation. Extracorporeal affinity adsorption devices, which are aimed at the removal of substantial amounts of compounds that participate in the pathogenesis of diseases in humans, provide effective therapeutic intervention. Several of these devices are on the market and are used successfully. New technologies such as antibody-coated adsorbents and microsphere-based detoxification are being introduced that are aimed at specific therapeutic targets. These devices must possess certain requirements including: anti-allergenic, anti-carcinogenic, resistance to microorganisms, antibacterial, non-toxic, and the ability to be sterilized. The function and performance of these devices depend on the adsorbent used.

Sterogene has developed special particles and surface chemistries specifically address these requirements. Our particles have been characterized extensively for TOC and other leachables. They are nontoxic, autoclavable, and are already a part of an FDA-approved device.

Endotoxin

Endotoxins, also called lipopolysaccharides (LPS), can be a major impurity of proteins or biologically active substances. The presence of small amounts of endotoxin in a recombinant protein can cause serious side effects in patients including endotoxin shock, tissue injury, and death. Due to these side effects, it is essential that endotoxin is removed from drugs, injectables, and other biological and pharmaceutical products. It is advantageous to remove endotoxin from research samples to insure accurate screening data.

Efficient and cost-effective removal of endotoxins from pharmaceutical and biological preparations can be challenging. Ion exchange chromatography during downstream processing is frequently used, however affinity chromatography such as ActiClean Etox may also be used. ActiClean uses a high affinity ligand for endotoxin removal and helps to dissociate protein-endotoxin interactions, leading to a more efficient recovery and higher yields of protein recovery.

DNA removal

Mammalian cell culture-derived therapeutics has the risk of cellular DNA contamination. Regulatory authorities have set strict standards for DNA contamination and require reduction by orders of magnitude from the fermenter to the final product. The FDA recommends the DNA in the final product be less than 100 pg/dose.

Most lysis methods cause the release of large quantities of nucleic acids. These contaminants must be removed because they can increase the viscosity of the feed stream as well as interfere with subsequent chromatographic steps.

Cell culture clarification processes, centrifugation or tangential flow filtration are primarily used as clarification steps prior to chromatography. These technologies operate under high shear conditions and can increase cell disruption, resulting in increased contamination by host cell DNA that can be carried through the purification steps. Direct CaptureTM of proteins using CellthruTM resins can reduce the DNA load of downstream purification. The cells pass through the resin bed of large beads with lower shear forces and less disruption of cellular integrity.

Different methods exist to remove DNA from protein preparations. Affinity chromatography using Cellthru™ Protein A resin typically removes 95-98% of contaminating DNA, CHOP, virus and other cellular debris. Ion exchange chromatography can also be used although affinity chromatography typically gives a higher log reduction of DNA contamination. Heparin affinity chromatography has also been used for DNA removal as heparin binds DNA very efficiently.

Contaminating Host Cell Proteins (CHOP)

Using some of the expression systems proteins are secreted into the culture media leading to a reduced HOP contamination. Cell culture clarification systems, centrifugation or tangential flow filtration are used as a primary clarification steps prior to chromatography. These technologies produce high shear forces and can increase cell disruption, resulting in increased host cell protein contamination, which can be carried through the downstream purification. Direct CaptureTM of proteins from cell culture using CellthruTM resins can reduce the DNA load on downstream purification as the cells gently pass through between the large beads with less disruption while protein is captured on the beads.

Virus removal

The downstream processing of any biologically-derived therapeutics requires effective viral clearance. Biological products have the potential risk of carrying and amplifying adventitious viruses that may be present or introduced into the materials. Typically a number of processing steps are necessary to provide adequate viral clearance. Some methods such as virus removing filters and chromatography provide physical means to separate and remove the. Other methods include irradiation, high heat, or chemical treatment to inactivate the virus. Nanofiltration can remove almost all viral species, while exposure to a low pH or detergents are also robust steps for inactivating enveloped viruses. Chromatographic or ion exchange resins show greater variances and decreased capacity in the removal of virus. Several of our stable affinity chromatography resins allow inactivation of viruses and may be sanitized with sodium hydroxide.