Monoclonal Antibody Titer Determination Using aChromolith^® WP 300 Protein A Wide PoreMonolithic Silica HPLC Column

Analyzing Cetuximab, Trastuzumab, and Universal Antibody Standard by HPLC-UV

Gisela Jung, R&D Scientist, Benjamin Peters R&D Lab Head, Cory Muraco, Biomolecule Workflows Product Manager

MilliporeSigma

Article from Analytix Reporter - Issue 16

Analyzing Cetuximab, Trastuzumab, and Universal Antibody Standard by HPLC-UV

• Introduction
• Experimental
• Analysis of Universal Antibody Standard, Human
• Analysis of Cetuximab
• Analysis of Trastuzumab
• Conclusion

Introduction

Monoclonal antibod (mAbs) manufacturing is  often done by fermentation processes where the amount and quality of the expressed product are essential and are monitored via affinity chromatography. This antibody titer determination can be carried out by using a Chromolith^® WP 300 Protein A HPLC column.

In this work, bind and elute experiments are shown for three different monoclonal antibodies: cetuximab, trastuzumab, and the universal antibody standard, human, on a Chromolith^® WP 300 Protein A column (Figure 1).

Figure 1. Schematic of the antibody interaction in the bimodal pore structure of the Chromolith^® WP 300 Protein A column.

All three immunoglobulin variants were analyzed with the addition of a matrix-standard to demonstrate the ability of this column to handle high matrix load.

 

Experimental

Three mAbs in standard solutions and three simulated matrix samples were prepared using SILu™ Lite SigmaMAb standards and the matrix standard, SigMatrix Serum diluent, a 6% recombinant HSA (Human Serum Albumin) in phosphate-buffered saline (PBS) solution, pH 7.4, that can be used as a blank or matrix/diluent for proteinaceous analytes in LC-MS/MS calibrators, controls, or samples. The standard solutions and matrix samples were analyzed on a 25 x 2 mm I.D. Chromolith^® WP 300 Protein A column under the conditions shown in Table 1.

Table 1. Conditions for titer determination of mAbs by HPLC-UV

LC Conditions
Column:	Chromolith® WP 300 Protein A 25 x 2 mm (1.52358)
Mobile phase:	[A] 100 mM Disodium hydrogen phosphate pH 7.4; [B] 100 mM Disodium hydrogen phosphate pH 2.5
Gradient:	Time (min)	A%	B%
	0	100	0
	0.50	100	0
	0.55	0	100
	2.60	0	100
	2.65	100	0
	5.00	100	0
Flow rate:	0.38 mL/min
Pressure:	As indicated
Temperatures:	Column: 25 °C Autosampler: 10 °C
Detector:	UV @ 280 nm (analytical flow cell; 10 µL)
Injection:	1.0 µL or as indicated for linearity determination
Samples
Universal Antibody Standard, Human	Sample without matrix (5 mg/mL): 1 mg SILuTMLite SigmaMAb Universal Antibody Standard, human in 200 µL water Sample with matrix (1 mg/mL): 1 mg SILuTMLite SigmaMAb Universal Antibody Standard, human in 200 µL water + 800 µL 6% SigMatrix Serum diluent
Cetuximab	Sample without matrix (5 mg/mL): 0.5 mg SILu™Lite SigmaMAb Cetuximab Monoclonal Antibody in 100 µL water Sample with matrix (1 mg/mL): 0.5 mg SILu™Lite SigmaMAb Cetuximab Monoclonal Antibody in 100 µL water + 400 µL 6% SigMatrix Serum diluent
Trastuzumab	Sample without matrix (5 mg/mL): 0.5 mg SILu™Lite SigmaMAb Trastuzumab Monoclonal Antibody in 100 µL water Sample with matrix (1 mg/mL): 0.5 mg SILu™Lite SigmaMAb Trastuzumab Monoclonal Antibody in 100 µL water + 400 µL 6% SigMatrix Serum diluent

Results

Analysis of Universal Antibody Standard, Human

Figure 2. Analysis of SILu™Lite SigmaMAb Universal Antibody Standard, human, in matrix on a Chromolith^® WP 300 Protein A (25 x 2 mm).

Table 2. Chromatographic data for analysis of SigmaMAb Universal Antibody Standard, human, in matrix

Peaks	Compound	Retention Time (min)	Peak width at 50 % (min)	Peak area (mAU*min)	Tailing factor	Column backpressure (bar)
1	SigMatrix Serum diluent	0.22	-	-	-
2	SILuTMLite SigmaMAb Universal Antibody Standard, human	2.20	0.023	4.33	2.24	13

Figure 3. Linearity, Universal Antibody Standard, human, peak area vs injected amount of mAb.

Table 3. Linearity data for Universal Antibody Standard, human, using different injection volumes

Concentration (µg/µL)	Injection volume (µL)	Amount (µg)	Mean Area (mAU*min)
5.00	0.05	0.25	0.66
5.00	0.10	0.50	1.55
5.00	0.20	1.00	3.31
5.00	0.50	2.50	9.06
5.00	1.00	5.00	18.53

Figure 4. Linearity, Universal Antibody Standard, human, peak intensity with different injection volumes vs retention time.

Figure 5. Analysis of Universal Antibody Standard, human (1), in matrix on a Chromolith^® WP 300 Protein A (25 x 2 mm) – multiple injections.

Table 4. Chromatographic data for multiple injections of Universal Antibody Standard, human, in matrix solution

Injection #	Retention Time (min)	Peak width at 50 % (min)	Peak area (mAU*min)	Recovery rate (%)	Column backpressure (bar)
1	2.20	0.023	4.28	99	13
10	2.20	0.024	4.16	96	14
20	2.20	0.023	4.34	100	16
30	2.21	0.024	4.22	97	18
40	2.21	0.024	4.25	98	19
50	2.21	0.024	4.29	99	21
60	2.21	0.025	4.23	98	22

Analysis of Cetuximab

Figure 6. Analysis of SILu™Lite Cetuximab in matrix on Chromolith® WP 300 Protein A (25 x 2 mm).

Table 5. Chromatographic data for analysis of cetuximab in matrix

Peaks	Compound	Retention Time (min)	Peak width at 50 %(min)	Peak area (mAU*min)	Tailing factor	Column backpressure (bar)
1	SigMatrix Serum diluent	0.21	-	-	-
2	SILuTMLite SigmaMAb Cetuximab	2.22	0.023	2.16	1.68	15

Figure 7. Linearity cetuximab peak area vs injected amount of mAb.

Table 6. Linearity data for cetuximab using different injection volumes.

Concentration (µg/µL)	Injection volume (µL)	Amount (µg)	Mean Area (mAU*min)
5.00	0.10	0.50	0.77
5.00	0.20	1.00	2.34
5.00	0.50	2.50	7.31
5.00	1.00	5.00	16.04

Figure 8. Linearity cetuximab peak intensity with different injection volumes vs retention time.

Figure 9. Analysis of cetuximab (1) in matrix on a Chromolith^® WP 300 Protein A (25 x 2 mm) - multiple injections.

Table 7. Chromatographic data for multiple injections of cetuximab in matrix solution.

Injection #	Retention Time (min)	Peak width at 50 % (min)	Peak area (mAU*min)	Recovery rate (%)	Column backpressure (bar)
1	2.23	0.024	2.17	99	15
10	2.22	0.024	2.14	98	15
20	2.22	0.024	2.11	96	16
30	2.22	0.024	2.21	101	19
40	2.22	0.024	2.39	109	20

Analysis of Trastuzumab

Figure 10. Analysis of SILu™ Lite Trastuzumab in Matrix on Chromolith® WP 300 Protein A (25 x 2 mm).

Table 8. Chromatographic data of analysis of trastuzumab in matrix.

Peaks	Compound	Retention Time (min)	Peak width at 50 % (min)	Peak area (mAU*min)	Tailing factor	Column backpressure (bar)
1	SigMatrix Serum diluent	0.22	-	-	-
2	SILuTMLite SigmaMAb Trastuzmab	2.18	0.028	2.96	4.47	16

Figure 11. Linearity trastuzumab peak area vs injected amount of mAb.

Table 9. Linearity data for trastuzumab using different injection volumes.

Concentration (µg/µL)	Injection volume (µL)	Amount (µg)	Mean Area (mAU*min)
5.00	0.05	0.25	0.33
5.00	0.10	0.50	0.82
5.00	0.20	1.00	2.17
5.00	0.50	2.50	6.18
5.00	1.00	5.00	12.93

Figure 12. Linearity trastuzumab peak intensity with different injection volumes vs retention time.

Figure 13. Analysis of trastuzumab (1) in matrix on a Chromolith^® WP 300 Protein A (25 x 2 mm) - multiple injections.

Table 10. Chromatographic data for multiple injections of trastuzumab in matrix solution.

Injection #	Retention Time (min)	Peak width at 50 % (min)	Peak area (mAU*min)	Recovery rate (%)	Column backpressure (bar)
1	2.18	0.027	2.83	94	16
4	2.18	0.029	3.06	102	16
8	2.18	0.029	2.99	99	17
12	2.18	0.028	3.01	100	18
16	2.18	0.029	3.04	101	19
20	2.18	0.029	3.01	100	20

Conclusion

It could be shown that all three antibodies (universal antibody standard, human, cetuximab, trastuzumab) can be analyzed reproducibly using the Chromolith^® WP 300 Protein A column.

A fast separation, within about 2 min, can be achieved, with high linearity values, for a broad range of injected sample amounts.

Furthermore, it was demonstrated that the column can handle a high matrix load for multiple injections. A standardized matrix was used for this study (SigMatrix Serum diluent).