Protein Purification, all you need to know about CEP Strategy

Protein purification, a process which can be as easy as complex

Protein purification looks simple. For a Histidine or a GST tagged protein as well as for an Immunoglobulin one single affinity step followed by a polishing step is enough to get a pure protein. For all the other proteins it is much more complex. The CEP (Capture – Enhance – Polishing) strategy is a key to solve your purification problem.

For a top level purification some key points have to be kept in mind :

• Protein stability
• What will be the use of the column
• Purification tools
• Proteins characteristics
• Purification planning

Protein stability

Prior any proteins purification we have to keep in mind the proteins could lose their activity entirely or partly. This is due to:

• Inhibitors
• Proteolytics enzymes
• Structure loss
• Aggregation/precipitation
• Their lost (non-specific adsorption)
• Chemical modifications

To minimize these risks, we have to avoid :

• Multi-step purifications
• Long term storage
• Repeated freeze /thawing steps

and we have to :

• Optimize the purifications conditions
• Work at low temperature
• Optimize the protein concentration
• Eliminate proteases
• Add stabilizers and co-factors

What will the protein be used for?

Following the future use of the protein purifications strategy are different

Purification tools

Following the proteins quantity needed purification tools are different

Chromatography technics that can be used

Protein characteristics

All the purification technics are usable since all the proteins own negatively charged portions, positively charged portion, hydrophilic, hydrophobic and affinity zones.

 

Planning

 

1/ Defining the goal

• Targets
• Trial conditions

2/ Describe

• The protein to purify
• The starting material (proteic medium)

3/ Analytical conditions to develop

 

CEP strategy BioWorks

The CEP strategy: how and which separative technics can be combined

• Combine complimentary technics
• Reduce as much as possible the sample manipulation

3 purification steps must be a maximum. If more, we lose too much target protein. The ratio Purity/cost is no more acceptable.

The purification steps:

“Don´t waste clean thinking on dirty enzymes” Efraim Racker

 Keep it simple!

CEP strategy summary

In the Bio-Works CEP strategy the planning will define precisely the purification way of the starting material:

• In which medium is the protein to purify?
• What is the protein (Antibody, recombinant protein, Enzyme…)
• What will the protein be used for?
• Which purifications technics are the more relevant?

The different development steps are used to validate the purification technics, the purification resin and to optimize the protocol (pH, Ionic force, temperature, linear flow rate…)