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Arg-SEC Mobile Phase

Effective separation of protein conjugates, e.g., ADC and sticky cytokines / Universal mobile phase for size-exclusion chromatography

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*1 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 Mild.

This product is manufactured with permission from Ajinomoto Co., Inc. based on the patent US 7501495*2.

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

*2 JP: 4941882, US: 7501495, EP: 1698637

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 arginine-based mobile phase

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

Arg-SEC Mobile Phase Series

 

Product IDApplicationExample
StandardFirst choice, all proteinsIgGs
StrongMore hydrophobic, sticky proteinsTGF-β3, Aggregates
MildOligomeric proteins with weak subunit-subunit interactionHemoglobin*

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.

Reference: 

Mobile Phase Containing Arginine Provides More Reliable SEC Condition for Aggregation Analysis
Journal of Pharmaceutical Sciences 99(2), 618-620 (2010)

Arginine as an effective additive in gel permeation chromatography
Journal of Chromatography A 1094(1-2), 49-55 (2005)

Comparison with 100 mmol/Phosphate buffer + 200 mmol/NaCl, pH6.8

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.

Condition
ColumnTSKgel G3000SWXL
Column size7.8mmI.D.-300mm
Flow Rate0.8 ml/min
TemperatureAmbient
DetectionUV 280 nm
SamplehIgG4, with aggregates
Injection Vol.10 µl

 

Data courtesy of Ajinomoto Co., Inc.


 

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 data) and a higher protein recovery (Data 2) than NaCl-based mobile phase.

Data 1

Data 2

Difference between Standard, Mild and Strong

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.

Data courtesy of Ajinomoto Co., Inc.

Condition
ColumnTSKgel G3000SWXL
Column size7.8mmI.D.-300mm
Flow Rate0.8 ml/min
TemperatureAmbient
DetectionUV280nm
SamplehIgG4, with aggregates
Injection Vol.10 µl

 


 

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.

Data courtesy of Ajinomoto Co., Inc.

Condition
ColumnSuperdex75 10/300 GL
Column size10mmI.D.-300mm
Flow Rate0.8 ml/min
TemperatureAmbient
DetectionUV280nm
SampleCrudeTGF-b3
Injection Vol.10 µl

Applicable to three commercial columns

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.


 

TSKgel G3000SWXL

Condition
ColumnTSKgel G3000SWXL
Column size7.8mmI.D.-300mm
Flow Rate0.8 ml/min
TemperatureAmbient
DetectionUV280nm
SamplemIgG1, with aggregates
Injection Vol.10 µl


Courtesy of Ajinomoto Co., Inc.


 

AdvanceBIO SEC 300A, 2.7 µm  

Condition
ColumnAdvanceBIO SEC 300Å, 2.7µm
Column size7.8mmI.D.-300mm
Flow Rate0.8 ml/min
TemperatureAmbient
DetectionUV280nm
SamplemIgG1, with aggregates
Injection Vol.10 µl

Data courtesy of Ajinomoto Co., Inc.


 

Cosmosil 5Diol-300-II

Condition
ColumnCOSMOSIL 5Diol-300-II
Column size7.8mmI.D.-300mm
Flow Rate0.8 ml/min
Temperature30°C
DetectionUV280nm
SamplemIgG1, with aggregates
Injection Vol.10 µl

Ordering Information

Product Cat.No. Storage Quantity Price(US$)  
Arg-SEC Mobile Phase, Mild 16998-41 Room Temp. 1 L 150.00 Buy
Arg-SEC Mobile Phase, Standard 16999-31 Room Temp. 1 L 150.00 Buy
Arg-SEC Mobile Phase, Strong 17000-51 Room Temp. 1 L 150.00 Buy