Biosimilar Comparability Workflow for Middle-Up Mass Analysis Using Trastuzumab as Model Protein

Geoffrey Rule¹, Sundaram Palaniswamy², Uma Sreenivasan³, Paresh Tank⁴, Fatima D’Souza⁴

¹Bellefonte, PA, USA, ²Bangalore, India, ³Round Rock, TX, USA, ⁴ZRAS, Zelle Biotechnology Pvt. Ltd., Thane, India

Introduction

Biosimilar mAb drugs have surged in the last decade due to patent expiration of innovator drugs, offering affordable care to patients. Biosimilars, while sharing amino acid sequences with innovators, can differ structurally due to post-translational modifications (PTMs) resulting from changes in manufacturing processes. These variations, like N-glycosylation and alterations to termini, pose challenges in proving comparability.¹

Thorough characterization of therapeutic mAbs is vital for safety and efficacy and establishing critical quality attributes (CQAs) are essential for both innovator and biosimilar drugs.^{2, 3} Characterization generally involves chromatography (e.g., SEC, RP, HILIC) combined with mass spectrometry (MS).

Trastuzumab received approval for medical usage in the United States in September 1998 and in the European Union in August 2000. At present, five biosimilars have gained approval in both the European Union and the United States.^{4, 5}

This note describes the application of a reducing RPLC-MS workflow for middle-up comparison of trastuzumab innovator and biosimilar. The application note also includes the correlation of de-charged masses of respective heavy and light chains with theoretical masses.

A recombinant human antibody standard, SILu^TMLite SigmaMAb (MSQC4), is utilized as a reference and assay control sample. Here, this is referred to simply as SigmaMAb although several other SigmaMAb standards are also available commercially.

• General Procedures - Sample and Reference Preparation and System Setup
• Results
• Conclusion
• References

Workflow for Middle-Up Mass Analysis of Trastuzumab

Antibody reduction (optional)

Chromatography

Measurement and Analysis

An RPLC-MS workflow has been developed to simplify mass analysis of reduced monoclonal antibodies (mAbs) for biosimilarity assessment.

In detail, it includes:

• Antibody reduction procedures
• Mass spectrometer calibration
• System suitability test utilizing a recombinant human monoclonal antibody reference
• An RPLC-MS method compatible with sample separation and analysis of reduced monoclonal antibodies

General Procedures - Sample and Reference Preparation and System Setup

Antibody Reduction Procedure

All mAb samples were solubilized in 100 μL water at a concentration of 1 mg/mL.

Full reduction of samples is performed by this protocol:

1. Prepare a 100 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in 6 M aqueous guanidine hydrochloride by dissolving 2.87 g of TCEP and 57.32 g of guanidine hydrochloride in 100 mL of water (if less solution is needed, scale down accordingly).
2. Combine 30 μL of the resulting solution with 10 μL of sample.
3. Incubate for two hours at 37 °C.

Note: Full reduction is performed in denaturing conditions, where both the inter-chain and intra-chain disulfide bonds are broken.  For a partial reduction procedure see the application note Protocol for Purification, Optional Reduction, and SEC-MS Analysis of a Monoclonal Antibody.⁶

Instrument Calibration

The Waters QToF Xevo G2XS mass spectrometer was calibrated in a mass range of 400 – 4000 m/z with a 10 μL/min infusion of 0.4 mg/mL of sodium iodide in water. Calibration can also be performed with a 10 μL/min infusion of 0.4 mg/mL cesium iodide and polyalanine in water, prior to running the samples.

System Suitability

Reduced SigmaMAb antibody reference (formulated at 2 mg/mL and further diluted to 1 mg/mL) was analyzed alongside samples to determine system suitability.

RPLC-MS System Setup and MS Data Analysis

RPLC-MS System Setup

Essential settings of the UHPLC-PDA chromatography system and qToF mass spectrometer are listed in Tables 1 and 2 below.

Table 1.UHPLC-PDA settings

LC  Conditions
Instrument:	WatersTM H-Class Acquity UPLC Chromatography System
Software:	UNIFI
Column:	BIOshell™ A400 Protein C4, 100 x 2.1 mm I.D., 3.4 μm (66825-U)
Mobile phase:	[A] Water + 0.1% FA; [B] acetonitrile + 0.1% FA
Gradient:	See Table 1b
Flow:	0.4 mL/min
Column temp:	80 °C
Autosampler temp:	8 °C
Run time:	20 min
Detector:	PDA 280 nm and MS TOF (see Table 2 for conditions)
Flow divert:	1.0 – 20.0 min
Loop volume:	10 μL
Injection volume:	2 μL

Table 1b.Gradient table for reduced mass analysis

Time (min)	%A	%B	Curve
0.00	75.0	25.0	Initial
1.00	75.0	25.0	6
8.00	65.0	35.0	6
11.00	40.0	60.0	6
14.00	5.0	95.0	6
14.90	5.0	95.0	6
15.00	75.0	25.0	6
20.00	75.0	25.0	6

Table 2.qToF-MS settings

MS Conditions
Instrument:	WatersTM QToF Xevo G2X2 Mass Spectrometer
Software:	UNIFI
Capillary (V):	3,000
Sample cone (V)	40
Extraction cone (V):	3
Ion guide (V):	3
Desolvation temp (° C):	100
Source temp (° C):	120
Scan range (Da):	400 – 4,000
Desolvation gas (L/h):	800
Cone gas (L/h):	50
Collision energy (V):	5
Pusher (V):	930
RF Setting:	Autoprofile

MS Data Analysis

Data were processed using the MaxEnt1 module within the UNIFI software to generate deconvoluted mass spectra. In general, a summed spectrum was created from the total ion chromatogram (TIC) of the heavy chain (HC), or light chain (LC), subunits and the spectrum then processed by MaxEnt1; detailed parameters in Table 3. Glycoforms were matched by the software and HC glycans are listed in the Results section. Relative abundance data were tabulated based on peak intensities of the coeluting glycoform species. 

Table 3.Deconvolution parameters

Reduced
m/z Range:	750 – 2,250
Mass range (Da):	LC 20,000 - 25,000; HC 45,000 - 55,000
Output resolution:	1 Da
Peak width model:	tof
TOF Resolution:	10,000
Minimum intensity ratios, L and R (%) :	30
Iterations:	21

Results

Intact Mass Analysis of Reduced Trastuzumab

The objective of this analysis was to compare the molecular weight of trastuzumab innovator and biosimilar, light, and heavy chains, using an RPLC-MS, middle-up approach.

System Suitability Test Results

Four microliters of reduced SigmaMAb^TM reference sample was injected onto the RPLC-MS system. The observed heavy chain glycoforms matched the expected glycoform masses of SigmaMAb^TM, as listed in Table 4. Discrepancies between the observed masses and the theoretical values for four glycoforms are all within 0.003% mass error. Figure 1 illustrates the UV (280 nm) and TIC chromatograms of the SigmaMAb^TM reference, while Figure 2 shows deconvoluted mass spectra of the light and heavy chain glycoforms.

Figure 1. Photodiode array (280 nm, left) and TIC trace (right) of reduced SigmaMAb^TM reference.

Figure 2. Deconvoluted mass spectra of light and heavy chains (left and right, respectively) of fully reduced SigmaMAb^TM reference.

Table 4.Theoretical and observed masses of reduced SigmaMAb^TM reference light and heavy chain glycoforms

Species	Molecular Formula	Theoretical Mass (Da) Fully Reduced*	Intra Disulfide Bonds	Theoretical Mass (Da) Partially Reduced*	Observed Mass (Da)	Error (%)
Light chain	C1006H1555N267O333S7	22,942.2	2 (-4 Da)	22,938.2	22942	-0.001
Heavy Chain/G0F	C2237H3485N591O702S16	50,403.2	4 (-8 Da)	50,395.2	50403	-0.000
Heavy Chain/G1F	C2243H3495N591O707S16	50,565.3	4 (-8 Da)	50,557.3	50565	-0.001
Heavy Chain/G2F	C2249H3505N591O712S16	50,727.5	4 (-8 Da)	50,719.5	50726	-0.003
G0F: GlcNAc2Man3GlcNAc2Fuc G1F: GalGlcNAc2Man3GlcNAc2Fuc G2F: Gal2GlcNAc2Man3GlcNAc2Fuc *Masses based on NIST Physical Reference Data

Reduced Sample Results

The fully reduced trastuzumab innovator and biosimilar samples were also analyzed using RPLC-MS. The corresponding photodiode array (280 nm) traces, TICs, and the summed and deconvoluted MS spectra of the samples are shown in Figures 3 and 4. The measured masses of reduced forms correlated well with the calculated masses as shown in Table 5 (mass error 0.001%, or less, for innovator and 0.003%, or less, for biosimilar). 

Figure 3. Comparative photodiode array (280 nm, left) and TIC trace (right) of fully reduced trastuzumab. Red: Innovator; Blue: Biosimilar.

Figure 4. Comparative summed (top) and deconvoluted (bottom) mass spectra of light and heavy chains (left and right, respectively) of fully reduced trastuzumab. Red: Innovator; Blue: Biosimilar.

Table 5.Theoretical and observed masses of reduced trastuzumab light chain and heavy chain glycoforms (*theoretical mass calculated based on NIST Physical Reference Data)

Species	Theoretical Mass (Da) Fully Reduced*	Intra Disulfide Bonds	Theoretical Mass (Da) Partially Reduced*	Observed Mass (Da)		Error (%)
				Innovator	Biosimilar	Innovator	Biosimilar
Light chain	23442.8	2 (-4 Da)	23,438.8	23443	23443	+0.001	+0.001
Heavy chain/G0F	50601.2	4 (-8 Da)	50,593.2	50601	50601	+0.000	+0.000
Heavy chain/G1F	50763.4	4 (-8 Da)	50,755.4	50763	50763	-0.001	-0.001
Heavy chain/G2F	50925.5	4 (-8 Da)	50,917.5	50925	50927	-0.001	+0.003
G0F: GlcNAc2Man3GlcNAc2Fuc G1F: GalGlcNAc2Man3GlcNAc2Fuc G2F: Gal2GlcNAc2Man3GlcNAc2Fuc

Conclusion

Ensuring regulatory approvals of biosimilar mAb drug products requires meticulous comparability assessment to guarantee safety and efficacy. This application note utilizes reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) at the subunit level for assessing comparability between originator trastuzumab and its biosimilar counterpart. An RP method suitable for reduced mAbs was established using a wide-pore, superficially porous C4 column.

The workflow uses a mAb reduction procedure, performed with full reduction so that all disulfide bonds are broken. Solutions of sodium iodide, or cesium iodide, were used for mass spectrometer calibration.

Upon analyzing the reduced SigmaMAb^TM reference sample, observed glycoform masses of the heavy chain were consistent with anticipated masses with the LC and three HC glycoforms all within a 0.003% mass error threshold. Similarly, the measured masses of reduced innovator trastuzumab, and biosimilar, exhibited close alignment with theoretical masses (mass error of 0.003%, or below).

These findings affirm the versatility of the RPLC-MS workflow for analyzing reduced monoclonal antibody samples, delivering accurate results for distinct identification of multiple glycoforms.

See more applications on the Biopharmaceutical Characterization page.

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References

1. Review and Approval. 2022 Dec 13 . [Internet]. U.S. Food and Drug Administration. Available from: https://www.fda.gov/drugs/biosimilars/review-and-approval

2. Sandra K, Vandenheede I, Sandra P. 2014. Modern chromatographic and mass spectrometric techniques for protein biopharmaceutical characterization. Journal of Chromatography A. 133581-103. https://doi.org/10.1016/j.chroma.2013.11.057

3. Berkowitz SA, Engen JR, Mazzeo JR, Jones GB. 2012. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov. 11(7):527-540. https://doi.org/10.1038/nrd3746

4. Biosimilars approved in the US. Gabionline.net. [Internet]. Available from: https://www.gabionline.net/biosimilars/general/biosimilars-approved-in-the-us

5. Biosimilars approved in Europe. Gabionline.net. [Internet]. Available from: https://www.gabionline.net/biosimilars/general/biosimilars-approved-in-europe

6. Protocol for purification, optional reduction, and SEC-MS analysis of a monoclonal antibody. [Internet]. Sigmaaldrich.com. Available from: /US/en/technical-documents/protocol/pharmaceutical-and-biopharmaceutical-manufacturing/biologics-biosimilars-characterization/protocol-for-analysis-of-a-monoclonal-antibody