14 Second, the use of high concentrations of salts is highly undesirable in any manufacturing process because it can cause corrosion of stainless steel tanks. This strategy has been particularly popular as a polishing step in antibody processes since aggregates are usually more highly retained on HIC. ![]() 13 To circumvent this issue, HIC is sometimes used in the flowthrough mode in which the product of interest flows while the more hydrophobic impurities remain bound to the column. 10, 11 Resin vendors have lately tried to optimize the pore size and ligand density in an effort to maximize capacity 12 however, 10% breakthrough capacities of > 40 mg/mL of resin have not yet been reported. In general, binding capacity has been traditionally limited on HIC, especially in comparison to ion exchange chromatography (IEX). 9 Elution is usually facilitated by decreasing salt concentration or by use of organic mobile phase modifiers.ĭespite its orthogonal selectivity, the use of HIC in any purification process presents two primary challenges. 8 Kosmotropic salts interact with water molecules to reduce solvation of protein molecules in solution and expose their hydrophobic patches to promote binding. 7 Interactions of proteins on HIC are usually promoted by kosmotropic salts, e.g., ammonium sulfate, sodium citrate, potassium phosphate. 3 - 6 HIC is based on interactions between hydrophobic (aliphatic or aromatic) ligands on the stationary phase with hydrophobic patches on the surface of proteins. 1, 2 This mode of chromatography is particularly useful for aggregate removal, and it provides good clearance of other process-related impurities such as host cell protein (HCP), leached Protein A and endogenous viruses. Hydrophobic interaction chromatography (HIC) occupies a unique niche as a polishing step in many monoclonal antibody (mAb) purification processes. Performance was comparable to that observed using conventional HIC conditions with high salt. ![]() This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity. Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase. These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product. HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. Your use hereof is hence at your own risk and does not relieve you of your own responsibility for checking the suitability of our products for your intended use.Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes. The information may change as additional facts come to light, or as the circumstances warrant. Signature: Date: 08 December 2016 This document has been electronically produced and is valid without a signature Name: Virve Inglis Title: Regulatory Support Manager The information herein is provided to the best of our knowledge and belief, based on the information we have been given and the information available to us at the time that this document was issued. Testing for possible contamination of aflatoxins in our final products is not performed. It is hereby stated that: None of the ingredients used in the manufacturing of bioprocess chromatography resin products listed in the attachment, are known sources for aflatoxin. The US Food and Drug Administration (FDA) has issued regulatory guidance, 21 CFR Parts 109 and 509, 55 FR for three mycotoxins (aflatoxin, deoxynivalenol (vomitoxin), and fumonisin) that may be present in raw grains, animal feed ingredients, and finished feed. The European Union introduced measures to minimize the presence of aflatoxins in different foodstuffs and maximum levels of aflatoxins B1, B2, G1, G2, and M1 are laid down in Commission Regulation (EC) No 1881/2006 as amended by Commission Regulation (EU) No 165/2010. ![]() 1 To whom it may concern Regarding: Aflatoxin in chromatography resins Aflatoxins are mycotoxins produced by two species of Aspergillus and are known to be genotoxic and carcinogenic.
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