Standard Purification Protocol for protein fragments

Optimising the final purity
When the Standard Protocol does not provide satisfactory purity, optimisation of the individual steps must be carried out. However, optimisation should advance only to the stage where the purification goals are fulfilled. The optimisation protocol therefore deals with optimisation parameters in the order of decreasing influence on the final purity (see table below!) and stops when satisfactory results are reached. It is often difficult to judge the overall effect of any single optimisation effort by just looking at individual steps, since it is the contaminants that are not removed by "later steps" that need to be better resolved from the target peptide.
The success of optimisation measures should therefore be judged by their effect on the whole protocol, i.e. the final result.

Selectivity optimisation
Measures influencing selectivity, such as the combination of techniques, pH and the choice medium in RPC have the strongest impact on the final purity and should be dealt with first.
The effect of running pH, however, has already been taken into account in the standard protocols.
Adding an extra step will certainly affect the overall selectivity.

Optimisation measure Effects Comments 1. Screen for optimal RPC medium. Influences elution order and spacing of peaks Type of base matrix and the ligand fixation chemistry are more important than the choice of ligand type. 2. Scout for optimal gradient slope. Influences mainly the spacing of peaks Reducing the gradient slope will broaden peaks 3. Add a new step. (CIEX or SEC) Changes overall selectivity. May require the removal of ACN. 4. Scout for optimal flow rate Decreases peak width Only limited effect on final purity

• Adjust the sample conditions to match eluent A.
• Filter or spin the sample to remove any particulate matter

• Use the recommended values for gradient and flow rate for each column to be test

• Prepare the originally used eluents.

Trouble-shooting
When a chromatogram shows unexpected deviations such as non-symmetrical or excessively broadened peaks, when recovery suffers, or when the system pressure deviates from normal, trouble-shooting has to be performed. The strategy outlined below starts with the perhaps most common and obvious source of malfunction, faulty eluents. Then make sure that the system performs normally to avoid the risk of misjudging column function tests. Finally, test sample behaviour.

General trouble-shooting strategy

I. Check the eluents - Prepare fresh solutions. Cause Results not OK II. Check solvent delivery - Run installation test. Results OK. Results not OK Refer to instrument manual. III. Check the column - Run a known sample. Results OK Results not OK. Column malfunction Refer to trouble-shootingguide below. Sample incompatible with experimental conditions Refer to trouble-shooting guide below.

The following table lists symptoms of malfunction.
The actions recommended are intended as "first aids".

Trouble-shooting guide

Symptom Cause Action I. Pressure - Low Air in the pump heads. Worn-out piston seals. Leaks. Purge the pump with MeOH. Replace piston seals. (Refer to instrument manual) - High Clogged tubing or column. Watch pressure while removing column and tubing in the "upstream" direction until theclogged part is identified. - Irregular Solvent delivery malfunction. Re-run with fresh eluentsPurge the pump with MeOHl. Replace piston seals.(Refer to instrument manual) II. Aberrant peak shapes Always check column first by running a known sample. -Triangular with sharp front Overload. Reduce sample load. -Excessively broad Overload. Column bed irregularities. Air in the column. Run column with 2 column volumes of MeOH to remove any trapped air. Perform recommended CIP. -Tailing Column bed irregularities. Air in the column. Non-specific adsorption. Run column with 2 column volumes of MeOH to remove any trapped air. Perform recommended CIP. Try additives like ACN or MeOH. -Fronting Channels or air in the column bed. Run column with 2 column volumes of MeOH to remove any trapped air. -Split peaks Partially blocked frit.Column bed irregularities. Air in the column. Run column "backwards" with 2 columnvolumes of MeOH to remove any particles inthe frit or trapped air. -Irregular Solvent delivery malfunction. Sample incompatible with experimentalconditions. Run Installation Test. Run sample stability test. Try additives like ACN or MeOH,chaotropic salts. Change conditions, type of column or technique. - Ghost peaks(Peaks appearing even inblank runs) Column regeneration incomplete. Quality of eluent chemicals and /orsolvent too low. Aged eluents. Increase eluting component concentration in eluent B. Use HPLC quality chemicals and solvents only. Unexpectedly low or missingPeaksLow recovery of activity. Non-specific adsorption. Sample incompatible with experimental conditions. Run sample stability test. Try additives like ACN or MeOH chaotropic salts. Try to block non-specific binding sites by injecting e.g. albumin. Change conditions, type of column or technique. Base line instability - Irregular Air in the detector cell. Detector problem. Check constrictor function.Run Installation Test. - Drifting Detector problem. Bleeding of precipitated ornon-specifically adsorbed material. Faulty or aged eluents. Run Installation Test.Perform specified CIP procedure. Re-run with fresh eluents.

Alternative Technique I, Cation Exchange

Sample preparation

• Adjust sample conditions to match those of eluent A.
• Filter or spin the sample to remove any particulate matter

• Select a Mono S or a RESOURCE S column to match the amount of sample

• Use the recommended values for gradient and flow rate for the column selected

• Eluent A: 5 mM phosphate pH 3.0 in water containing 30% acetonitrile (final conc.).
• Eluent B: 5 mM phosphate pH 3.0 in water containing 30% acetonitrile and 1.0 M NaCl (final conc.).

Alternative Technique II, Size Exclusion Chromatography

Sample preparation

• Adjust sample conditions to match those of eluent A.
• Filter or spin the sample to remove any particulate matter

• Select a Superdex peptide column for target peptides with Mr <3 000.
• Select a Superdex 75 column for target peptides with Mr >3 000.

• Use the recommended flow rate for the column selected.

• 50 mM phosphate, pH 7.0 in water containing 30% acetonitrile (final conc.).
• 0.1% TFA in water containing 30% acetonitrile (final conc.).
• 70% formic acid in water (applicable to very hydrophobic peptides).
• 70% acetonitrile in water (applicable to very hydrophobic peptides).