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Bio-Rad's mixed-mode chromatography resins offer unique separation properties and unparalleled selectivity and resolution for a variety of molecules. Multimodal chromatography combines the properties of cation exchange media and metal affinity media, often enabling the separation of biomolecules that appear homogeneous using other chromatographic methods. These mixed-mode media can be used at any stage in a purification process from initial capture to final polishing.
This whiteboard animation video provides a brief introduction to Bio-Rad’s innovative mixed-mode chromatography media, CHT Ceramic Hydroxyapatite. The unique separation properties and selectivity of CHT Media enable you to maximize purity and recovery in a single step of downstream purification for the development of biotherapeutics.
Developing an effective and robust method with Nuvia cPrime media is simple. Below is general information about the binding and elution mechanisms and an approach to guide method development; results will vary depending on the protein of interest and the feed composition.
The binding and elution mechanisms of Nuvia cPrime media are determined chiefly by pH and salt. The high salt tolerance of the media often allows for direct loading at high conductivity. An increase in pH will in most cases achieve elution. Conductivity is another way to achieve and/or optimize elution and the final method is often a combination of an increase in pH and/or an increase/decrease in salt concentration. In some cases, the use of an elution buffer modifier or a different salt in the elution buffer may be required for optimal elution, recovery, and resolution.
The schematic below outlines a recommended approach to method development. In most cases, conducting a few simple experimental designs to identify optimal binding and elution conditions will yield an effective, robust, and scalable method.
1. Load feed or eluate from previous step directly without dilution onto the Nuvia cPrime media column. To elute, use an increasing pH gradient. If satisfactory elution and recovery are achieved, refine and/or make a step gradient to complete the step (pH 4–8, depending on protein).
2. If elution is not satisfactory after step 1, run a salt gradient to disrupt electrostatic or hydrophobic interactions that may be preventing elution or broadening the peak. Use the pH where there was best elution (from step 1). The direction of this salt gradient (increasing or decreasing) can be easily assessed and will depend on the relative contributions of ionic vs. hydrophobic interactions involved in binding.
3. If elution is still unsatisfactory after step 2 of this process, consider using a modifier such as propylene glycol, urea, or arginine to disrupt any remaining interactions. Other modifiers may also be used; in some cases changing to another salt may also be required.
Packing and Slurry-in-Place Procedures for CHT Ceramic Hydroxyapatite Using a Bio-Rad InPlace Process-Scale Chromatography Column
Slurry valve on Bio-Rad InPlace column. The separated inlet and washing manifolds make it easy and convenient to fill and unload the chromatography media in the contained system and to sanitize the manifold. The valve body allows buffer to pass behind the valve when it is closed.
Ceramic hydroxyapatite requires special consideration during process-scale chromatography packing primarily due to its high specific gravity and rapid settling rate. Ceramic hydroxyapatite media have a bulk density of 0.63 g/ml and a free setting velocity of 35–125 cm/hr for 40 µm and 125–275 cm/hr for 80 µm. Additionally, ceramic hydroxyapatite media are sensitive to mechanical shear, which can fracture the particles and produce fines.
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Advances in Separation & Purification: Purifying Monoclonal Antibodies
The affinity capture paradigm that dominates industrial IgG purification has proven unsuitable for IgMs because, in most cases, they are affected adversely by harsh elution conditions. The large size of IgMs is also a challenge because it limits the operating conditions and performance of traditional porous-particle-based chromatography media. This article describes how these challenges can be overcome with available technology to develop effective manufacturing procedures for IgM monoclonal antibodies.
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Workflow for mAb purification with UNOsphere SUPrA affinity, UNOsphere Q, and CHT Type 1 media.
Purification of IgG using CHT Ceramic Hydroxyapatite. Read more.
Plasmid Purification Using CHT Ceramic Hydroxyapatite Support
Purification of plasmid DNA on CHT II support. Buffer A: 10 mM sodium phosphate + 1 mM EDTA, pH 7.0 Buffer B: 0.4 M sodium phosphate +1 mM EDTA, pH 7.0 Flow rate: 1.5 ml/min Gradient: 0–100% buffer B for 10 column volumes Fraction size: 2.0 ml
Plamid DNA is being used successfully as a gene delivery vector in a variety of clinical application (Smith et al. 1999). Plasmids for gene therapy are usually produced in an E.coli host. One of the technical challenges associated with producing plasmid DNA of gene therapy grade is the removal of contaminants such as bacterial chromosomal DNA, RNA, host proteins, and endotoxin.
CHT Ceramic Hydroxyapatite Mechanism
Hydroxyapatite contains two types of binding sites, positively charged calcium and negatively charged phosphate groups. These sites are distributed regularly throughout the crystal structure of the matrix. Solute species dominantly interact through cation exchange via the phosphate groups and/or metal affinity via the calcium atoms.
Cation exchange occurs when protein amino groups interact ionically with the negatively charged phosphates. The amino groups are similarly repelled by the calcium sites. Binding depends upon the combined effects of these interactions. These ion exchange interactions can be disrupted by adding neutral salts such as sodium chloride or buffering species such as phosphate to the mobile phase. Cation exchange interactions also weaken with increasing pH. Hence, the addition of salt or phosphate, or an increase in pH, can be used to weaken the interaction. Studies with model proteins have demonstrated that anion exchange, which might be expected from interactions of negatively charged surface residues with calcium, does not make a significant contribution.
Calcium affinity occurs via interactions with carboxyl clusters and/or phosphoryl groups on proteins or other molecules (e.g., nucleic acids); these groups are simultaneously repelled by the negative charge of the CHT phosphate groups. The affinity interaction is between 15 and 60 times stronger than ionic interactions alone and, like classical metal-affinity interactions, is not affected by increasing ionic strength using typical elution ions (e.g., chloride). Species binding through calcium affinity may adsorb more strongly as the ionic strength increase due to ionic shielding of the charge repulsion from the CHT phosphate sites. Metal affinity interactions can be dissociated by phosphate in the mobile phase.
Most large proteins bind by a combination of mechanisms:
Dominantly acidic proteins, such as albumin, bind chiefly by metal affinity interactions. Sodium chloride at 1.0 M reduces retention time by approximately 10% in the presence of phosphate gradients, indicating a minor contribution by cation exchange. To elute acidic proteins, phosphate buffers are required.
Dominantly basic proteins, such as IgG, bind chiefly by cation exchange interactions. Sodium chloride reduces retention time in the presence of phosphate gradients, indication a minor contribution by metal-affinity. Basic proteins may be selectively eluted with either phosphate or salts.
Nuvia aPrime 4A is a hydrophobic AEX resin engineered with distinctly balanced modes to optimize biomolecule interactions. It offers a wide design space and straightforward method development delivering both high purity and yield.
Nuvia cPrime mixed-mode media is designed for versatile capture and high recovery across a wide range of salt concentrations and pH in process-scale purification processes for biotherapeutic applications.
CHT XT Media is engineered with advanced technology, resulting in unique selectivity and robust, uniformly-sized beads with excellent pressure-flow properties. It is fine-tuned for the removal of aggregates and process impurities.
CHT Ceramic Hydroxyapatite and Bio-Gel® Crystalline Hydroxyapatite Mixed-Mode Media offer unique selectivities and can be used at any stage in a chromatographic workflow from initial capture to final polishing.
MPC Ceramic Hydroxyfluoroapatite (Ca10(PO4)6(OH)1.5(F)0.5) is a mixed-mode chromatography resin, a composite of hydroxyapatite and fluoroapatite, with unique separation properties and unmatched selectivity and resolution in the purification of biomolecules.
CFT Ceramic Fluoroapatite (Ca10(PO4)6F2) mixed-mode media is a rigid, spherical macroporous support used in the purification of biologically active compounds.
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