MiniBeads: Purification or Analysis of Microgram-Milligram Quantities with Highest Resolution

Extracted from Ion Exchange Chromatography & Chromatofocusing, GE Healthcare, 2007

Use MiniBeads for purification and analysis of proteins, peptides or oligonucleotides.

Use MiniBeads for polishing steps at microscale when highest resolution is essential and the capacity of the prepacked column is sufficient.

Use MiniBeads for intermediate purification if only microgram – milligram quantities are required, if there is no requirement for scale-up and if the capacity of the prepacked column is sufficient. Note that, to avoid column blockage, it is especially important to remove particulate matter before using MiniBeads.

Use MiniBeads for faster, higher resolution separations, when compared to MonoBeads, if the capacity of the prepacked column is sufficient.

Run MiniBeads on systems such as ÄKTAdesign, FPLC™ System and HPLC. Appendix 4 gives guidance on how to select the most suitable ÄKTAdesign system.

MiniBeads are based on a non-porous, monodispersed matrix of rigid, hydrophilic polymer particles, substituted with quaternary amino (Q) or methyl sulfonate (S) groups. The very small size (3 μm), uniformity and physical rigidity of the particles create ideal conditions for extremely high resolution ion exchange separations at relatively high flow rates and low back pressures (non-uniform, porous particles would create higher back pressures, reduce flow rate and impair achievable resolution). Such high resolution is essential for successful separation of complex samples in the pg to μg scale. The strong ion exchange groups (Q and S) maintain their charge over a broad pH range, allowing selection of the most suitable pH for each application.

Purification options

Mini Q and Mini S™ media are available prepacked in Tricorn™ (4.6/50 PE) and Precision (PC 3.2/3) columns.

Figure 27. Mini Q and Mini S™ media are available prepacked in Tricorn™ (4.6/50 PE) and Precision (PC 3.2/3) columns.

 

Product, column volume Binding capacity,
per column
Maximum flow Recommended work flow Working pH range Maximum operating back pressure ** (MPa/psi)/1 MPa=10 bar
Strong anion exchangers
Mini Q PC 3.2/3, 0.24 ml*** 1.44 mg (a-amylase, Mr 49 000)
1.44 mg (trypsin inhibitor, Mr 20 100)
1 ml/min 0.1–1.0 ml/min 3–11 10/1450
Mini Q 4.6/50 PE, 0.8 ml 4.8 mg (a-amylase, Mr 49 000)
4.8 mg (trypsin inhibitor, Mr 20 100)
2 ml/min  0.5–2.0 ml/min 3–11  18/2600
Strong cation exchangers
Mini S PC 3.2/3, 0.24 ml*** 1.2 mg (ribonuclease, Mr 13 700)
1.2 mg (lysozyme, Mr 14 300)
1 ml/min 0.1–1.0 ml/min 3–11 10/1450
Mini S 4.6/50 PE, 0.8 ml  4 mg (ribonuclease, Mr 13 700)
4 mg (lysozyme, Mr 14 300)
2 ml/min 0.5–2.0 ml/min 3–11 18/2600

*Working pH range refers to the pH interval where the medium binds protein as intended or as needed for elution without adverse long term effects.
**Maximum operating back pressure refers to the pressure above which the medium begins to compress.
***Requires a Precision Column Holder for attachment to ÄKTApurifier™ and other HPLC systems.

Purification examples

Fast separations at high resolution

Separation of a protein mixture on Mini S 4.6/50

Figure 28. Separation of a protein mixture on Mini S 4.6/50.

Mini S PC 3.2/3 gives fast, high resolution separation

Figure 29. Mini S PC 3.2/3 gives fast, high resolution separation.

Purity check

Purity check of 5'-biotinylated synthetic oligonucleotide 20-mer on Mini Q 4.6/50 PE before and after purification on a RESOURCE RPC column

Figure 30. Purity check of 5'-biotinylated synthetic oligonucleotide 20-mer on Mini Q 4.6/50 PE before and after purification on a RESOURCE RPC column.

Long term reproducibility

Chromatograms from the 1st, 5th and 201st separation of a series run on the same Mini S PC 3.2/3 column

Figure 31. Chromatograms from the 1st, 5th and 201st separation of a series run on the same Mini S PC 3.2/3 column. The same consistent reproducibility has been confirmed on Mini Q PC 3.2/3 (data not shown).

Performing a separation

Guidelines for selection of media, buffer, pH and ionic strength conditions and method optimization are given in Chapter 2. Use the instructions given here as a basis from which to optimize a separation.

Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any deterioration in column performance, especially when using small particles such as MiniBeads. Samples must be fully dissolved and free from particles or other material likely to interfere with the separation. Refer to Chapter 2 and Appendix 1 for recommendations and advice on sample preparation.

Filter buffers after all salts and additives have been included. Use high quality water and chemicals. Filter solutions through 0.22 μm filters. To avoid formation of air bubbles in a packed column, ensure that column and buffers are at the same temperature when preparing for a run.

The pH of the start buffer should be at least 0.5–1 pH unit above the pI of the target substance when using an anion exchanger (Q) and 0.5–1 pH unit below the pI of the target substance when using a cation exchanger (S). See Appendix 2 for recommendations on volatile and non-volatile buffer systems for anion and cation exchangers.

For samples with unknown charge properties, try the following:

  • anion exchange (Q)
        start buffer: pH 8.0
        elution buffer: start buffer including 1 M NaCl, pH 8.0
  • cation exchange (S)
        start buffer: pH 6.0
        elution buffer: start buffer including 1 M NaCl, pH 6.0

Users of ÄKTAdesign systems with BufferPrep functionality can select one of the buffer recipes recommended for anion exchange chromatography at pH 8 or cation exchange chromatography at pH 6.

First time use or after long term storage

  1. To remove ethanol, wash with 4 column volumes of distilled water at 0.5 ml/min. This step ensures removal of ethanol and avoids the risk of precipitation if buffer salts were to come into contact with the ethanol. The step can be omitted if precipitation is not likely to be a problem.
  2. Wash with 4 column volumes of start buffer at 0.8 ml/min.
  3. Wash with 4 column volumes of elution buffer at 0.8 ml/min.
  4. Wash with 4 column volumes of start buffer at 0.8 ml/min.
  5. Run a blank elution before applying sample.

Separation by gradient elution

Flow: 0.4 ml/min (PC columns) or 0.8 ml/min (PE columns). Collect fractions throughout the separation.

  1. Equilibrate column with 5–10 column volumes of start buffer or until the baseline, eluent pH and conductivity are stable.
  2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column.
  3. Wash with 5–10 column volumes of start buffer or until the baseline, eluent pH and conductivity are stable i.e. when all unbound material has washed through the column.
  4. Begin elution using a gradient volume of 10–20 column volumes and an increasing ionic strength up to 0.5 M NaCl (50%B).
  5. Wash with 5 column volumes of 1 M NaCl (100%B) to elute any remaining ionically-bound material.
  6. Re-equilibrate with 5–10 column volumes of start buffer or until eluent pH and conductivity reach the required values.

Separation by step elution

Although separations by step elution (see Chapter 2, page 19) can be performed using MiniBeads, gradient elution is recommended in order to achieve the highest possible resolution.

If ionic detergents have been used, wash the column with 5 column volumes of distilled water, followed by 2 column volumes of 2 M NaCl. Re-equilibrate with at least 10 column volumes of start buffer until the UV baseline, eluent pH and/or conductivity are stable. Organic solvents such as ethanol can be used to remove non-ionic detergents. When selecting an organic solvent, check the chemical stability of the medium to determine a suitable concentration.

Refer to Chapter 2 for advice on optimizing the separation. Check column performance regularly by determining column efficiency and peak symmetry. See Appendix 3.

Cleaning

Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order to remove contaminants.

It is recommended to reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire length of the column. The number of column volumes and time required for each cleaning step may vary according to the degree of contamination.

The following procedure should be satisfactory to remove common contaminants:

  1. Wash with 2 column volumes of 2 M NaCl at 0.2 ml/min.
  2. Wash with 4 column volumes of 1 M NaOH at 0.2 ml/min.
  3. Wash with 2 column volumes of 2 M NaCl at 0.2 ml/min.
  4. Rinse with at least 2 column volumes of distilled water at 0.2 ml/min until the UV-baseline and elutent pH are stable.
  5. Wash with at least 4 column volumes of start buffer or storage buffer at 0.2 ml/min until pH and conductivity values have reached the required values.

To remove precipitated proteins, lipids, hydrophobically bound proteins or lipoproteins, refer to Appendix 1.

Media characteristics

Composition: rigid, non-porous matrix of monodisperse, hydrophilic polymer particles (3 μm) substituted with quaternary amino (Q) or methyl sulfonate (S) groups.
 

Product Functional group pH stability* Mean particle size
Mini Q -CH2N+(CH3)3 Long term: 3–11
Short term: 1–14
3 μm (monosized)
Mini S -CH2SO3 Long term: 3–11
Short term: 1–14
3 μm (monosized)

*Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on the chromatography performance.
Short term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
All ranges are estimates based on the experience and knowledge within GE Healthcare.

Chemical stability

For daily use, MiniBeads are stable in all common aqueous buffers in the range pH 3–11 and in the presence of additives such as denaturing agents (8 M urea or 6 M guanidine hydrochloride), non-ionic or ionic detergents and up to 30% acetonitrile in aqueous buffers. Note that aqueous solutions of urea, ethylene glycol and similar compounds will increase the back-pressure due to increased viscosity.

MiniBeads can be used with organic solutions such as dimethylsulfoxide, dimethylformamide or formic acid, but the separation properties of the media will change.

Avoid anionic detergents with Mini Q. Avoid cationic detergents with Mini S. Avoid oxidizing agents.

Storage

For column storage, wash with 4 column volumes of distilled water followed by 4 column volumes of 20% ethanol. Degas the ethanol/water mixture thoroughly and apply at a low flow rate to avoid over-pressuring the column. Store at room temperature or, for long periods, store at +4° C to +8° C. Whenever possible, use the storage and shipping device if supplied by the manufacturer. Ensure that the column is sealed well to avoid drying out. Do not freez

Materials