RPC Purification Options and Scale up with SOURCE™ Media

  • Use SOURCE™ RPC media for purification and analysis of proteins, peptides and oligonucleotides.

     

  • Use SOURCE™ RPC as an alternative to silica-based matrices when separations must be performed above pH 8 or when requiring different selectivity or higher capacity.

     

  • Use SOURCE™ 30RPC for polishing stages of industrial processes requiring high flow rate and low back pressure.

     

  • Use SOURCE™ 15RPC for polishing steps in laboratory or large-scale applications that require highest resolution and fast separation (flows up to 1800 cm/h).

     

  • Use SOURCE™ 5RPC for highest-resolution, analytical separations as required for peptide mapping and LC-MS techniques.

     

  • Run SOURCE™ RPC columns on systems such as ÄKTAdesign, FPLC System and HPLC. Appendix 3 gives guidance on how to select the most suitable ÄKTAdesign system.

SOURCE™ media are based on a matrix made from monodispersed, rigid, polystyrene/divinyl benzene (Figure 80). The media demonstrate extreme chemical and physical stability and, unlike silica-based media, can be used at extremes of pH. A range of particle sizes (30 μm, 15 μm or 5 μm) enables SOURCE™ RPC to be used from large-scale purification through to high-resolution analysis. The uniformity and stability of SOURCE™ particles ensures high flow rates at low back pressure. Such high flow rates are useful for speeding up cleaning and re-equilibration steps. Flow rates are more likely to be limited by the equipment available and the eluents used rather than the physical properties of the media.

Scanning electron micrograph of SOURCE™ 15RPC shows the uniform size distribution.

Fig 80. Scanning electron micrograph of SOURCE™ 15RPC shows the uniform size distribution.

Purification options

Table 23. RPC media based on SOURCE™ matrices are available in prepacked columns and as media packs.

Product Dynamic binding capacity per column* Recommended working flow† Maximum flow Maximum operating back pressure‡ (MPa/psi) 1MPa= 10 bar
SOURCE™ 5RPC ST 4.6/150, 2.5 ml ~ 80 mg bacitracin 1 ml/min n.d. 40/5800
SOURCE™ 15RPC ST 4.6/100, 1.7 ml ~ 17 mg BSA
~ 85 mg insulin
0.5–2.5 ml/min 5 ml/min 4/580
RESOURCE™ RPC, 1 ml ~ 10 mg BSA
~ 30 mg bacitracin
~ 50 mg insulin
1–5 ml/min 10 ml/min 4/580
RESOURCE™ RPC, 3 ml ~ 10 mg BSA
~ 30 mg bacitracin
~ 50 mg insulin
1–5 ml/min 10 ml/min 4/580
SOURCE™ 15RPC ~ 10 mg BSA
~ 30 mg bacitracin
~ 50 mg insulin
150–900 cm/h 1800 cm/h 4/580
SOURCE™ 30RPC ~ 14 mg BSA
~ 23 mg bacitracin
~ 72 mg insulin
100–1000 cm/h 2000 cm/h 1.5/220

*Determined at 10% breakthrough by frontal analysis. The dynamic binding capacity of an RPC medium is dependent on several parameters including the properties of the target molecule, the selectivity and pore size of the medium, eluent conditions and flow rate.

Capacities given here were determined using 0.1% TFA at a flow rate of 300 cm/h.

† See Appendix 3 to convert linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa. Flow rate used will depend also on the pressure specification of the chromatography system, the eluents used and the column bed height.

‡ Maximum operating back pressure refers to the pressure above which the medium begins to compress.

  • In RPC many parameters, such as properties of the protein, flow rates and selectivity of the medium play a significant role in the determination of binding capacity. Final capacity must be determined by experimentation.
  • Use RESOURCE™ RPC 1 ml for rapid screening and method development. Transfer RESOURCE™ RPC 3 ml column for higher resolution and method development on a 10 cm bed height.
  • Use SOURCE™ 5RPC columns for highest resolution as required, for example, for peptide mapping.

Table 24. Packing volumes and bed heights for SOURCE™ media for RPC.

  Volume Bed height
Tricorn 10/100 Up to 8 ml up to 10 cm
Tricorn 10/150 Up to 12 ml up to 15 cm
Tricorn 10/200 Up to 16 ml up to 20 cm

Select a production column such as FineLINE for larger volumes.

Purification examples

Capture and purification of a synthetic peptide

Figure 81 shows the purification and mass spectrometric analysis of a crude mixture of synthetic amyloid-β 1–42. This peptide is generated from a large transmembrane precursor protein by proteolytic cleavage and may polymerize to form rigid, linear, non-branching fibrils building up to the senile plaques that represent one of the characteristics of Alzheimer's disease. Amyloid-β 1–42 was synthesized for use in studies of the mechanisms underlying fibril formation. However, while readily soluble under alkaline conditions, the peptide is virtually insoluble under conditions so that traditional purification by RPC at low pH cannot be used. The wide pH stability of SOURCE™ media is therefore well suited for such a purification challenge.

Purification and MS analysis of a synthetic peptide

Fig 81. Purification and MS analysis of a synthetic peptide.

Purification at high pH

Figure 82 shows the successful purification of beta-lipotropin fragment 1–10 (MW 950, Sigma) achieved by using high pH conditions (pH 12), possible only on polymer-based media. At pH 2 the contaminants are eluted in the beta-lipotropin peak.

Purification at high pH

Fig 82. Purification at high pH

Polishing step and scale-up

Figure 83 shows a high-resolution preparative separation of recombinant human epidermal growth factor (EGF) expressed in yeast. Most impurities have been removed by an initial hydrophobic interaction chromatography step on Phenyl Sepharose 6 Fast Flow (high sub) followed by ion exchange on Q Sepharose High Performance. The final polishing step on SOURCE™ 15RPC was optimized on a RESOURCE™ 3 ml column before scale-up to a pilot-scale column.

Figure 83 shows a high-resolution preparative separation of recombinant human epidermal growth factor (EGF) expressed in yeast. Most impurities have been removed by an initial hydrophobic interaction chromatography step on Phenyl Sepharose 6 Fast Flow (high sub) followed by ion exchange on Q Sepharose High Performance. The final polishing step on SOURCE™ 15RPC was optimized on a RESOURCE™ 3 ml column before scale-up to a pilot-scale column.

Preparative separation of a recombinant protein

Fig 83. Preparative separation of a recombinant protein.

Scaling up

Figure 84 shows the excellent scalability of SOURCE™ 30RPC. The medium is easily packed and maintains its performance during scale-up. In this example, the separation of a model protein mixture was scaled up by a factor of 400 from a 24 ml column to a 10 liter FineLINE 200L column.

Scalability of SOURCE™ 30RPC

Fig 84. Scalability of SOURCE™ 30RPC.

Materials