Use and application of hydrophobic interaction chromatography for protein purification.

The objective of this section is to provide the reader with guidelines and background on the use and experimental application of Hydrophobic Interaction chromatography (HIC) for the purification of proteins. The section will give step by step instructions on how to use HIC in the laboratory to purify proteins. General guidelines and relevant background information is also provided.

Mitigation of chromatography adsorbent lot performance variability through control of buffer solution design space.

The separation of undesired product-related impurities often poses a challenge in the purification of protein therapeutic species. Product-related impurity species, which may consist of undesirable isoforms, aggregated, or misfolded variants of the desired monomeric form of the product, can be challenging to remove using preparatory scale chromatographic techniques. When using anion exchange chromatography to remove undesirable product-related impurities, the separation can be highly sensitive to relatively small changes in the chromatography operating conditions, including changes to buffer solution pH, buffer solution conductivity protein loading, and operating temperature.

Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes.

For the manufacturing of recombinant protein therapeutics produced from mammalian cell culture, demonstrating the capacity of the purification process to effectively clear infectious viruses is a regulatory requirement. At least two process steps, using different mechanisms of virus removal and/or inactivation, should be validated in support of the regulatory approval process. For example, exposure of the product stream to low pH, detergents or solvent/detergent combinations is commonly incorporated in protein purification processes for the inactivation of lipid-enveloped viruses.

Theory and use of hydrophobic interaction chromatography in protein purification applications.

Hydrophobic interaction chromatography (HIC) is a valuable tool used in protein purification applications. HIC is used in the purification of proteins over a broad range of scales-in both analytical and preparatory scale applications. HIC is used to remove various impurities that may be present in the solution, including undesirable product-related impurities.

Application of a novel affinity adsorbent for the capture and purification of recombinant factor VIII compounds.

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product.

Modeling the effects of column packing quality and residence time changes on protein monomer/aggregate separation.

The packing quality of chromatography columns used for the purification of protein therapeutics is routinely monitored to ensure consistent and reproducible performance. In this work, we used established chromatography models to determine the effect of column packing quality and fluid residence time on the separation of protein therapeutic monomer and aggregate species using a hydrophobic interaction chromatography adsorbent (Phenyl Sepharose Fast Flow). The relationship between the number of theoretical plates, fluid residence time, and column separation performance was quantified using modeling simulations.

Effect of phenyl sepharose ligand density on protein monomer/aggregate purification and separation using hydrophobic interaction chromatography.

In the large-scale manufacturing and purification of protein therapeutics, multiple chromatography adsorbent lots are often required due to limited absorbent batch sizes or during replacement at the end of the useful column lifetime. Variability in the adsorbent performance from lot to lot should be minimal in order to ensure that consistent product purity and product quality attributes are achieved when a different lot or lot mixture is implemented in the process. Vendors of chromatographic adsorbents will often provide release specifications, which may possess a narrow range of acceptable values.

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF.

Use of an alternative scale-down approach to predict and extend hydroxyapatite column lifetimes.

Ceramic hydroxyapatite (CHT) chromatography offers unique selectivity for protein purification. However, columns composed of CHT, a crystalline form of calcium phosphate, often suffer from short column lifetimes, particularly under acidic operating conditions. In this paper, CHT was used under slightly acidic conditions (pH 6) for the production scale purification of a recombinant protein.

Application of a two-dimensional model for predicting the pressure-flow and compression properties during column packing scale-up.

A two-dimensional model was formulated to describe the pressure-flow behavior of compressible stationary phases for protein chromatography at different temperatures and column scales. The model was based on the assumption of elastic deformation of the solid phase and steady-state Darcy flow. Using a single fitted value for the empirical modulus parameters, the model was applied to describe the pressure-flow behavior of several adsorbents packed using both fluid flow and mechanical compression.

Evaluation of protein-A chromatography media.

In process-scale antibody purification, protein-A affinity chromatography is commonly used as the initial purification step. In this paper, two different protein-A media were evaluated. These adsorbents have a porous glass backbone with different pore sizes: 700 A and 1000 A.