MEP Resin

Sample Prep

Protein-free cell culture supernatant
– The synthetic iron carriers present in protein-free cell culture media may cause the resin to take on a significant brown color during loading. If this occurs or if IgG recovery is low, 10 mM EDTA may be added to the sample to reduce the formation of iron complexes. 200 mM sodium citrate may alternatively be used.

– If samples are concentrated (e.g. using UF/TFF) prior to column loading, high molecular weight iron carrier, often present in protein-free cell culture media,
can aggregate and accumulate on the retentate side of the UF/TFF membrane. Such aggregates can bind strongly to MEP HyperCel resin and lead to column
fouling. The addition of 10 mM EDTA to the sample prior to concentration will limit the accumulation of these aggregates.

– Tween and Triton should not be included in sample or buffers. Surfactants can interfere with the binding of protein to MEP HyperCel resin. Tween and
Triton can be removed from sample by using a Pall SDR HyperD resin Column (PN 20033-C001) or Pall SDR HyperD resin.

– Lipid supplements added to the culture medium may compete for the MEP ligand, fouling the resin and reducing binding capacity. Contact Pall technical
service for assistance

Addition of modest concentrations of sodium chloride (< 1.3 M) or other lyotropic salts (e.g., ammonium sulfate) may be required as salts increase hydrophobic interaction. In such cases, the salt concentration required will be significantly less than that employed in traditional hydrophobic interaction chromatography. Elution is accomplished by reducing the pH and/or salt.

The column must be stored at 2-8 °C (36-46 °F) and cannot be frozen. Between runs, store the column in neutral buffer containing 1 M NaCl and
20% (v/v) ethanol to prevent bacterial growth.

Chromatography

1. Attach column to pre-primed system. To prevent air from getting into the column, fill the neck of the column dropwise while system is running very slowly.
Allow the buffer to flow through the column until all bubbles at the bottom of the column have been evacuated.
2. Equilibrate with 5-10 CV of loading buffer.
3. Load the sample. A flow rate of 0.2 -1.0 mL/min should be used
4. Wash with at least 5 CV of loading buffer or until the OD280 returns to baseline level.
5. Elute with 5-10 CV chosen elution buffer, stepwise, or gradient. Collect eluate in appropriately sized fractions.
6. If the column is to be reused, strip with 5-10 CV of pH 3 buffer.
7. Re-equilibrate with 5 CV loading buffer

Optimization

Antibody Purification – General Considerations
• Antibody binding and optimization
– Antibody-containing sample can be loaded directly onto MEP HyperCel AcroSep columns without preliminary desalting or concentration. Capacities of ≥ 20 mg/mL are generally obtained. Prior to sample loading, equilibrate the column with PBS or buffer, with pH and salt concentration equivalent to sample.
– During preliminary studies, it is recommended that loading be conducted at a low to moderate flow rate of 0.2-0.5 mL/min. Maximum capture efficiency is
achieved when the residence time is around 5-7 min.
– Check the pH. For most antibodies, maximum capacity is obtained when pH is between 7.4 and 9.5. Binding capacity is generally independent of pH in this
range
– Salt concentration. Although the majority of antibodies do not require the addition of salt beyond physiologic concentrations (~150 mM, Figure 2) for
good binding, this is not true for all antibodies. If binding is below the stated capacity and increased capacity is required, consider adding an additional
200-400 mM salt to promote binding. There will be a concomitant increase in impurity binding as well, but the increase in antibody binding is generally larger Elute bound antibody using a dilute buffer at pH 4. Buffers typically used include 50 mM sodium acetate or 20 mM sodium citrate (2-10 CV). For highly
pH sensitive proteins, pH up to 5.8 can be used, but elution may be less efficient. Elution will be more efficient in the absence of salt.
– All eluted antibodies should be neutralized as quickly as possible after elution in order to maintain the structural integrity.
– To improve purity of eluted antibody, elution optimization is recommended. A step-elution sequence using buffers of decreasing pH (e.g. 5.5, 5.0, 4.5, 4.0,
and 3.0) is effective. 50 mM sodium acetate or 20 mM sodium citrate are typically used as elution buffers. Elution at each step should be continued for
2-4 CV, or until complete elution of an observed peak is accomplished. Based on analysis of collected fractions, a second step-elution experiment at narrower pH increments or at alternative values may be useful. Ultimately, the elution procedure would be simplified to a three-step sequence. First, at the highest pH, elution of basic impurities would be accomplished. Second, at the optimized, intermediate pH, the target protein would be recovered. Fin
ally, at the lowest pH in the sequence, acidic impurities, aggregates, and misfolds would emerge. Depending on the characteristics of the acidic and hydrophobic impurities, this final step may be conducted at pH 3.0. It is useful to conduct a final wash at pH 3.0 to elute any remaining impurities, if the column is to be regenerated for use with additional samples.

Cleaning and Maintenance

In order to avoid frequent regeneration, it is advisable to introduce into the column only samples and buffers that are clear and previously filtered. Make sure that changes in pH and ionic strength prior to loading or during elution do not cause precipitation of sample components. The following regeneration procedures are recommended for general and specific cleaning challenges.

General cleaning-in-place

Wash with 0.5-1 M sodium hydroxide, 30-60 min contact time.

Adsorbed contaminants
Wash with 6 M guanidine (2-3 CV) or 2 M urea

If you are working with protein-free cell culture media and the addition of EDTA to the sample is not feasible, resin performance can be preserved by use of an EDTA solution during cleaning. Following antibody elution, and before the column is washed with NaOH, wash the column with 100 mM EDTA (adjusted to pH 7.2). Alternatively, 200 mM sodium citrate, pH 3.0 may be used.

When using NaOH during the clean-in-place procedure, MEP HyperCel resin may become colored. This does not affect the chromatographic performance of the resin and is due to the cyclic group on the 4-MEP.

Storage

• The column must be stored at 2-8 °C  and cannot be frozen.
• Between runs, store the column in neutral buffer containing 1 M NaCl and 20% (v/v) ethanol to prevent bacterial growth.