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Home / Applications / Analysis of Aggregates in Antibodies by Size Exclusion Chromatography (SEC)

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Analysis of Aggregates in Antibodies by Size Exclusion Chromatography (SEC)

By DJ Tognarelli

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June 6, 2023

Introduction

LC-4000 SeriesIgG, which is a type of antibody, is used as a drug substance for antibody drugs. It is known that IgG may form dimer, trimer, and aggregates due to mechanical stress in each process during manufacturing and environmental factors during transportation and storage. Since aggregates in antibody drugs can reduce drug efficacy and cause side effects, it is necessary to evaluate monomer, multimers, and aggregates for quality control. In 2014, the U.S. Food and Drug Administration (FDA) has provided guidance on the evaluation of immunogenicity of therapeutic protein products and recommended that the validity of each analysis result should be judged while comparing the results using multiple methods (Orthogonal Method) with different measurement principles for protein aggregates contained in biopharmaceuticals. This time, we show the results of measuring human serum-derived IgG reagent by size exclusion chromatography (SEC), which is one of the analytical methods for aggregates, and separating and detecting monomer, multimers, and aggregates.

 

Experimental

PumpPU-4080i
AutosamplerAS-4050i*
Column ovenCO-4060
UV detectorUV-4075*
* with option units
ColumnTSKgel G3000SWXL (7.8 mmI.D. x 300 mmL, 5 µm)
Eluent0.2 mol/L sodium phosphate buffer (pH 6.7)
Flow rate0.8 mL/min
Column temp25 ºC
Wavelength220 nm
Injection volume10 µL
StandardProtein standard mix 15-600 kDa (Merck)
30 mg/mL in water
SampleIgG from human serum (Merck)
1 mg/mL in water

Keywords

Antibody, IgG, Antibody Drug, Biopharmaceutical, Aggregate, TSKgel G3000SWXL, SEC, UV Detector

Results

Figure 1 shows a UV chromatogram of a protein standard sample. As reference data, an RI chromatogram is also shown, which was measured by connecting an RI detector (RI-4030) downstream of the UV detector.

Fig. 1 Chromatograms of protein standard sample (A) : UV chromatogram, (B) : RI chromatogram 1: Thyrogrobulin (MW 670,000), 2: γ-globulins (MW 150,000), 3: Ovalbumin (MW 44,300), 4: Ribonuclease A (MW 13,700), 5: p-aminobenzoic acid (MW 137)

 

Figure 2 shows a UV chromatogram of human serum-derived IgG reagent. The molecular weight calibration curve is overwritten on this figure. In addition, Table 1 shows the calculation results of the peak-top average molecular weights (Mp) of human serum-derived IgG and the area ratio of each section, and Figure 3 shows the molecular weight distribution curves of each section. From the Mp in Figure 3 and Table 1, it was estimated that peak 4, peak 3, and peaks 2 and 1 correspond to an IgG monomer, dimer, and aggregates with a higher molecular weight, respectively. In addition, based on the ratio of each section area, the human serum-derived IgG reagent measured in this experiment was found to contain approximately 79% of monomer (peak 4) and approximately 21% of aggregates including multimers (peaks 1 to 3).


Fig. 2 UV chromatogram of human serum-derived IgG reagent

 

Peak No.Section Range (min)Baseline Range (min)tR (min)MpSection AreaArea %
15.967-7.3775.967-14.5237.1278350152557202.44
27.377-8.3005.967-14.5238.0175489394265214.07
38.300-9.3735.967-14.5238.810362067149515714.28
49.373-14.1275.967-14.52310.227154964829253879.2
Fig. 3 Molecular weight distribution curves for each section of human serum-derived IgG reagent
(A) : Peak 4, (B) : Peak 3, (C) : Peak 2, (D) : Peak 1

System Configuration

Part NumberModelDescriptionQuantity
7002-J012APU-4080iInert Pump (Base Unit)1
7018-J002AAS-4050iInert Autosampler1
7061-J002ATC-4000-1Sample Temperature Control Unit1
7021-J002ACO-4060Column Oven1
7026-J002AUV-4075UV/VIS detector1
7025-H136AUV-4000 Inert flow cell unit1
7139-J002ABS-4500-4Bottle stand1
Local SupplyTSKgel G3000SWXL (7.8 mmI.D. x 300 mmL, 5 µm) SEC column manufactured by TOSOH1
6688-H564ALC-NetCGLC-Net CG cable3
7059-J012AChromNAV Ver. 2Chromatography Data System1
7059-J016AChromNAV-GPC2GPC Calculation Program1
7001-H402BHPLC PEEK Start Up Kit for LC-40001
7001-H405BMaintenance tool kit1
0410-0170Screw Thread Vial, 2 mL, PP, 100 pcs./set1
0402-0094Open top Screw Cap with PTFE/Silicon Septa (Pre-Slit), 100 pcs./set1

References

U.S. Food and Drug Administration, “Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products.”, https://www.fda.gov/media/85017/download, (2014).

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About the Author

DJ Tognarelli received his B.S. from the University of Richmond where he studied Analytical Chemistry. He is a Chromatography Applications Scientist at JASCO.

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