Nuvia™ cPrime™ Media

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Overview

General Approach to Method Development Using Nuvia™ cPrime™ Media

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.

Simple Method Development

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.

Learn more

Tutorial: Using HT Methods — Case Study

Using High-Throughput (HT) Methods to Accelerate DOE

High-throughput experimentation (HTE) was used in this case study in concert with design of experiments (DOE) methodology to study the mass recovery of a basic therapeutic protein (pI 9.2–9.5) using a hydrophobic cation exchange mixed-mode resin.

Nuvia cPrime hydrophobic cation exchange media are a new addition to Bio-Rad's family of mixed-mode purification products. These media are designed for process-scale purification of a wide variety of therapeutic proteins. Nuvia cPrime media's unique selectivity allows method developers to use hydrophobic and cation exchange interaction modes to achieve effective purification. More importantly, the media have a large design space for binding and elution, allowing for the development of highly robust methods in a commercial manufacturing setting. Nuvia cPrime media are built on a rigid, mechanically and chemically stable macroporous base matrix with a particle size optimized to deliver exceptional flow properties, fast mass transfer, and stability.

Nuvia cPrime delivers value by providing:

New and unique selectivity
Salt tolerant media
Simple method development
Large design space for capture and elution of a variety of biotherapeutic proteins
High recovery of target protein
Mechanical and chemical stability

Functional group	Hydrophobic weak cation exchange
Base matrix composition	Macroporous highly crosslinked polymer
Particle size	70 µm ± 10 µm
Dynamic binding capacity* (hlgG)	>40 mg/ml
Dynamic binding capacity (lactoferrin)	>60 mg/ml
Ligand density	55–75 µeq/ml
Recommended linear flow rate range	50–600 cm/hr
Pressure vs. flow performance**	Under 2 bar at a flow rate of 600 cm/hr
pH stability short-term	2–14
pH stability long-term	3–13
Chemical stability	1.0 N NaOH, 1.0 N HCl, 25% HOAc, 8 M urea, 6 M GuHCl, 6 M KSCN, 3 M NaCl, 1% Triton X-100, 2% SDS + 0.25 M NaCl, 20% EtOH, 70% EtOH, 30% IPA
Shipping solution	20% ethanol
Storage conditions	20% ethanol
Shelf life in 20% ethanol	5 years at room temperature
* At 10% breakthrough, 300 cm/hr.
** 20 cm × 20 cm packed bed (1.17 comp. factor).