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

About the Author

Introduction

IgG, 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

Pump	PU-4080i
Autosampler	AS-4050i*
Column oven	CO-4060
UV detector	UV-4075*
	* with option units

Column	TSKgel G3000SWXL (7.8 mmI.D. x 300 mmL, 5 µm)
Eluent	0.2 mol/L sodium phosphate buffer (pH 6.7)
Flow rate	0.8 mL/min
Column temp	25 ºC
Wavelength	220 nm
Injection volume	10 µL
Standard	Protein standard mix 15-600 kDa (Merck) 30 mg/mL in water
Sample	IgG from human serum (Merck) 1 mg/mL in water

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)	Mp	Section Area	Area %
1	5.967-7.377	5.967-14.523	7.127	835015	255720	2.44
2	7.377-8.300	5.967-14.523	8.017	548939	426521	4.07
3	8.300-9.373	5.967-14.523	8.810	362067	1495157	14.28
4	9.373-14.127	5.967-14.523	10.227	154964	8292538	79.2

**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

Keywords

747022G, Antibody, IgG, Antibody Drug, Biopharmaceutical, Aggregate, TSKgel G3000SWXL, SEC, UV Detector

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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