Products

Arg-SEC Mobile Phase

Size-Exclusion Chromatography (SEC) is an essential technology for developing biopharmaceuticals. SEC is normally carried out in aqueous solution so that the mobile phase has a minimal impact on the target biopharmaceutical proteins, e.g., not disrupting the protein structure. However, such mobile phase is not often optimal for the performance of SEC. Proteins often bind to the SEC column surface, leading to a compromised protein elution and peak resolution. Such binding can cause incorrect determination of the size of the eluted proteins and the amount of the aggregated species. To overcome these problems, arginine-based mobile phase* has been developed. This mobile phase reduces non-specific protein binding to many SEC columns, while not affecting the protein structure. Depending on the strength of non-specific binding, there are 3 mobile phases: strong, standard and weak.

*Arginine is effective in suppressing non-specific molecular interactions.

This product is manufactured with permission from Ajinomoto Co., Inc. based on the patent 4941882.

Product Information

Features:

  • Increased recovery of proteins and peptides that are more hydrophobic and have stronger tendency to stick to the columns.
  • Correct determination of protein aggregates that have stronger tendency to stick to the columns than the monomeric species.
  • Optimal for quality control.
  • Effective separation of protein conjugates, e.g., ADC and sticky cytokines.

Advantages of using this product:

  • Quantitative analysis of protein (in particular sticky protein) samples.
  • Applicable to many commercial SEC columns.
  • Reduce lot-to-lot variability due to non-specific protein binding.
  • Less non-specific protein binding increases the life time of the columns.
  • Reduce the time required to condition the columns.
  • Increase the purification yield as a result of less binding.

Arg-SEC Mobile Phase Series

Product IDApplicationExample
Standard First choice, all proteins IgGs
Strong More hydrophobic, sticky proteins TGF-β3, Aggregates
Mild Oligomeric proteins with weak subunit-subunit interaction Hemoglobin*

Filtered through 0.22 µM filter, ready to use, pH 6.8, contains arginine.
*Such a weakly associating oligomeric protein as hemoglobin may dissociate to monomers in the presence of arginine.

Caution

Arginine has moderate absorbance in far UV region and thus care should be exercised when using 220 nm. Using UV 280 nm is recommended.

Comparison 1

Data 1: Aggregate content of hIgG4 mAb determined by the standard strength Arg-SEC mobile phase in comparison with NaCl-based SEC mobile phase.

A mAb, hIgG4, containing aggregated species, was analyzed by SEC using our standard Arg-SEC mobile phase and NaCl-based 100 mM phosphate. Although the monomer content was identical between these 2 mobile phases, the aggregate content was much higher with our Arg-SEC mobile phase. This is due to suppression of non-specific adsorption of the aggregates to the SEC column by Arg-SEC mobile phase, but not the NaCl-based mobile phase.

Aggregate Content of Antibody Sample

Arg_Com1.png

Data courtesy of Ajinomoto Co., Inc.

Condition
Column TSKgel G3000SWXL
Column size 7.8mmI.D.-300mm
Flow Rate 0.8 ml/min
Temperature Ambient
Detection UV 280 nm
Sample hIgG4, with aggregates
Injection Vol. 10µl

Data 2: Comparison of Mild, Standard and Strong all showed comparable aggregate recovery and monomer peak size.
For this particular analysis, any Arg-SEC mobile phase can be used.

Aggregate Content of Antibody Sample

Arg_Com1-2.png

Data courtesy of Ajinomoto Co., Inc.

Condition
Column TSKgel G3000SWXL
Column size 7.8mmI.D.-300mm
Flow Rate 0.8 ml/min
Temperature Ambient
Detection UV280nm
Sample hIgG4, with aggregates
Injection Vol. 10µl

Data 3: Comparison of Mild, Standard and Strong showed significant differences for the recovery of TGF-β3. Such a stocky protein as TGF-β3 cannot be recovered with the NaCl-based mobile phase nor with Arg-SEC Mild, but can be recovered by Standard and Strong. A much greater recovery was obtained with the Strong.

Purification of Crude TGF-β

Arg_Com1-3.png

Data courtesy of Ajinomoto Co., Inc.

Condition
Column Superdex75 10/300 GL
Column size 10mmI.D.-300mm
Flow Rate 0.8 ml/min
Temperature Ambient
Detection UV280nm
Sample CrudeTGF-†3
Injection Vol. 10µl

Comparison 2

Arg-SEC Standard mobile phase works on any column and gives a consistently higher aggregate recovery than NaCl based mobile phases:

Arg-SEC can overcome differences in non-specific characteristics of three commercial columns. With the NaCl-based mobile phase, 3 columns gave a low and variable aggregate content due to diferent surface properties of the columns and non-specific binding. On the contrary, Arg-SEC mobile phase suppressed non-specific binding on all 3 columns and gave a consistent and expected aggregate content. Such effects should help minimize the lot-to-lot or column-to-column variation of non-specific protein binding.

Column A

Arg_Com2-1.png

Data courtesy of Ajinomoto Co., Inc.

Condition
Column TSKgel G3000SWXL
Column size 7.8mmI.D.-300mm
Flow Rate 0.8 ml/min
Temperature Ambient
Detection UV280nm
Sample mIgG1, with aggregates
Injection Vol. 10µl

Column B

Arg_Com2-2.png

Data courtesy of Ajinomoto Co., Inc.

Condition
Column AdvanceBIO SEC 300Å, 2.7µm
Column size 7.8mmI.D.-300mm
Flow Rate 0.8 ml/min
Temperature Ambient
Detection UV280nm
Sample mIgG1, with aggregates
Injection Vol. 10µl

Column C

Arg_Com2-3.png

Condition
Column COSMOSIL 5Diol-300-II
Column size 7.8mmI.D.-300mm
Flow Rate 0.8 ml/min
Temperature 30°C
Detection UV280nm
Sample mIgG1, with aggregates
Injection Vol. 10µl

Applications

Arg-SEC can give correct estimate of minute amount of aggregate content. Arg-SEC mobile phase gave a slightly, but significantly, higher aggregate content (both panels) and a higher protein recovery (bottom panel) than NaCl-based mobile phase.

Aggregate Content of Antibody Sample

Arg-App1.png

Data courtesy of Ajinomoto Co., Inc.

Condition
Column TSKgel G3000SWXL
Column size 7.8mmI.D.-300mm
Flow Rate 0.8 ml/min
Temperature Ambient
Detection UV280nm
Sample IgG4 monomer
Injection Vol. 10µl
Aggregate Content of Antibody Sample

Arg-App2.png

Data courtesy of Ajinomoto Co., Inc.

Condition
Column Superdex200 Increase
Column size 10mmI.D.-300mm
Flow Rate 0.8 ml/min
Temperature Ambient
Detection UV280nm
Sample mIgG1 (15µg)

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Ordering Information

Product NameGradeStorageProduct No.Price
Arg-SEC Mobile Phase(Standard)
SP Refrigerate
after opening
16999-31 e-Nacalai
Arg-SEC Mobile Phase(Strong) 17000-51 e-Nacalai
Arg-SEC Mobile Phase(Mild) 16998-41 e-Nacalai