Optimizing protein recovery in the purification step using Cytiva™ Protein Select™ resin technology

Introduction

Achieving high protein recovery during purification is critical for downstream success in recombinant protein production. Cytiva™ Protein Select™ resin, designed for tag-based purification, offers a streamlined workflow that combines affinity capture with self-cleaving tag removal—eliminating the need for proteases or harsh elution conditions. To fully unlock its potential, optimizing each step is essential to minimize protein loss and maximize recovery.

In this article, we will outline practical strategies to troubleshoot issues, improve recovery, and finetune your purification protocol.

The key factor: Cleavage kinetics

Recovery with Cytiva™ Protein Select™ resin can be influenced by many parameters, but the amino acid sequence at the N-terminus of your protein often has the largest impact. The initial amino acids play a role (with the first two having the greatest impact) in how efficiently the tag cleaves.

This means that recovery is largely shaped by cleavage kinetics, and while optimizing process conditions (such as pH, temperature, flow rate, and buffer choice) can improve results—these adjustments generally finetune recovery as opposed to fully overcoming N-terminal amino acid sequence limitations.

In other words, the sequence sets the foundation, while process optimization helps you get closer to the best possible recovery. Read more about cleavage kinetics, click here.

Understanding the workflow

The purification process involves four key steps:

1. Binding and wash
2. Hold step (cleavage)
3. Elution
4. Regeneration/cleaning in place (CIP)

Fig 1. The four key steps of purification.

Each step presents opportunities for optimization to improve your overall recovery. If you experience low recovery after purification, it's essential to pinpoint where in the workflow the losses are occurring. We suggest you first optimize the hold-step time, which can significantly impact protein stability and recovery. Following this, optimize the sample loading and washing conditions. Furthermore, temperature and buffer conditions can influence recovery and should be optimized for maximum yield.

Step-by-step optimization

1. Hold step

The hold step is a critical phase in the purification workflow using the resin—it's during this pause in flow that the major self-cleaving of tagged protein occurs. For initial trials, we recommend a 4 h hold time to allow sufficient cleavage and testing the protein cleavage kinetics. However, for proteins with slow or moderate cleavage kinetics, extending the hold duration—such as overnight incubation—can significantly improve recovery. Table 1 lists some possible causes of low recovery and troubleshooting tips.

Table 1. Possible causes of low recovery and troubleshooting tips

Cause	Remedy
Hold time too short	Optimize the hold-step time, using serial step elution method on one column, or parallel screening method on several columns.
Cleavage kinetics too slow	Alter the first two amino acids on the N-terminus of the target protein.
Cold temperature and/or low pH causing slow cleavage	Increase pH and/or temperature to increase the cleavage rate​.

2. Binding and wash

The cleavage will start once the tagged protein is bound to the resin. This is why we recommend the sample application including wash step to be completed within 30 min. Balancing the flow rate, temperature, and volume is essential for keeping proteins bound and cleaved efficiently. Table 2 lists some possible causes of low recovery and troubleshooting tips.

Table 2. Possible causes of low recovery and troubleshooting tips

Cause	Remedy
Sample volume too large causing increased loading time	Increase flow rate Concentrate the sample
Flow rate too high during loading causing loss of tagged protein	Decrease flow rate for loading while maximizing flow rate for washing to keep the total time of loading and washing within 30 min.
Flow rate too low during loading causing loss of cleaved protein	Maximizing flow rate for washing to keep the total time of sample loading and washing within 30 min.
Temperature too high causing loss of cleaved protein	Decrease the temperature during sample loading and washing.
Batch purification	We recommend purification using a packed column as batch purification is slower and may cause more protein loss.
Buffers	Use a separate buffer for wash and elution with higher pH than the binding buffer. Avoid using high concentration of denaturing substances which will prevent purification.
Resin overloaded	Screen different sample loads to avoid overloading.
Insufficient regeneration in the previous cycle	Follow the regeneration/CIP protocol in the instruction for use (available at the bottom of this page) and optimize based on your specific sample.

3. Elution

Cytiva™ Protein Select™ resin works with mild, protein-friendly conditions, but buffer choice still matters for recovery. The protein will elute in the buffer that was used for washing after sample loading. There is not a dedicated elution buffer; the binding buffer is used as the elution buffer. Table 3 lists optimization and troubleshooting tips.

Table 3. Possible causes of low recovery and troubleshooting tips

Cause	Remedy
The protein of interest is still bound to the resin due to too short hold-step time	Optimize hold-step time and/or temperature in the previous hold step. Use a separate buffer for wash with higher pH than the binding buffer in the previous step.

4. Regeneration/CIP

During this step, the tag, noncleaved protein and tightly bound or precipitated substances are removed and therefore the resin can be reused. See Table 4 below for optimization and troubleshooting tips.

Table 4. Possible causes of low recovery and troubleshooting tips

Cause	Remedy
Decreased binding capacity due to insufficient regeneration/CIP in the previous purification run	Follow the regeneration/CIP protocol in the instructions for use (available at the bottom of this page), and optimize based on your specific sample to improve the recovery.

Conclusion

Optimizing protein recovery is a multistep process that requires attention to protein sequence, timing, buffer conditions, and resin handling. By applying these best practices using Cytiva™ Protein Select™ resin, you can achieve more consistent and higher-yield purifications.

For detailed protocols and product support, visit Cytiva™ Protein Select™ resin page.

CY55836

Related resources

• Data file for Cytiva™ Protein Select™ resin
• Tag sequence for Cytiva™ Protein Select™ tag
• Product page for Cytiva™ Protein Select™ resin and HiTrap™ Protein Select™ column

Additional practical tips

• Factors affecting the cleavage efficiency when using Cytiva™ Protein Select™ technology
• AxiChrom™ column packing with Cytiva™ Protein Select™ resin
• Design flexibility of protein construct with Cytiva™ Protein Select™ tag
• Integrating Cytiva™ Protein Select™ tag into an expression vector
• Instructions for use for Cytiva™ Protein Select™ resin
• Instructions for use for HiTrap™ Protein Select™ columns