Monoclonal antibody (mAb) drugs are one of the fastest-growing biotherapeutics in the pharma market. The majority of mAbs are for the treatment of cancers.1 The investment during the discovery, development, manufacturing, and clinical trials is huge for innovator mAb drugs. As a result, the cost of innovator drug treatment is usually high for patients. Therefore, more affordable generic versions of innovator drugs, called biosimilars, are in high demand. The first biosimilar was approved for the European market in 2006, and the U.S. market opened nine years later after the introduction of the Affordable Care Act in March 2010. The development of biosimilars is gaining traction due to the patent expiry of innovator molecules.
For biosimilars to be approved by regulatory agencies, manufacturers need to demonstrate that there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency.2 A critical part in this process is an extensive comparative analytical study to understand the physicochemical similarities between the innovator and biosimilars. Aggregates, truncation, and other modified forms (deamidation, isomerization, and so forth) are product‑related impurities that arise during the manufacturing process or storage. Their presence in the drug negatively impact drug stability, activity, and efficacy. Therefore, they are usually considered CQAs and are closely monitored and tested throughout the manufacturing process.
This Application Note uses two analytical workflows to demonstrate a comparison between two biosimilars of rituximab and their reference innovator in terms of aggregate and charge variant profiles. Rituximab is a well-known biotherapeutic drug for the treatment of rheumatoid arthritis, lupus, vasculitis, and dermatomyositis. The two biosimilars were obtained from two manufacturers in different geographical locations. Both workflows are based on the 1260 Infinity II bio-inert LC system together with advanced Bio columns and OpenLab CDS. Charge variants were separated on a weak cation exchange (WCX) column, while aggregates were separated on a size exclusion (SEC) column. Figure 1 shows the two workflow details. Good reproducibility on intraday and interday results ensured reliability of the workflows and demonstrated clear similarities or differences between the innovator and biosimilars.
The systems were composed of the following modules:
- Agilent 1260 Infinity II Bio-inert Pump (G5654A)
- Agilent 1260 Infinity II Bio-inert Multisampler (G5668A) with sample cooler
- Agilent 1260 Infinity II Multicolumn Thermostat (G7116A) with bio-inert heat exchanger
- Agilent 1260 Infinity II Diode Array Detector WR (G7115A) with bio-inert flow cell
- Agilent 1260 Infinity II Bio-inert MultiDetector Suite (MDS) (G7805A) featuring dual-angle static and DLS detection (G7809A)
- Agilent Bio mAb, nonporous, 2.1 × 250 mm, 5 µm HPLC, PEEK (p/n 5190-2411) for charge variants analysis
- Agilent AdvanceBio SEC 300Å, 7.8 × 300 mm, 2.7 µm (p/n PL1180‑5301) for aggregation analysis.
- Agilent OpenLab CDS Version 2.3
- Agilent Buffer Advisor A.01.01 
- Agilent Bio-SEC Software version A.02.01 Build 9.34851
LC instrument control as well as LC data analysis was carried out using Agilent OpenLab CDS Version 2.3. It provides a smooth user interface with customized and interactive reporting with drag-and-drop template creation. The peak explorer feature of the software was used to compare the results between the innovator and biosimilars.
Chemicals and samples
All solvents used were LC grade. Fresh ultrapure water was obtained from a Milli-Q Integral system equipped with a 0.22 µm membrane point-of-use cartridge (Millipak). Sodium phosphate monobasic, sodium phosphate dibasic, and sodium chloride were purchased from Sigma-Aldrich, St. Louis, USA. The mAb drugs, including the innovator and two biosimilars, were purchased from a local distributor. Before analysis in the DLS system, the mobile phase was triple filtered through a 0.1 μm hydrophilic PTFE membrane filter (Merck Millipore). Samples were taken from the original container and centrifuged at 13,000 g for two minutes. Supernatant was aliquoted to an LC sample vial for analysis.Please Download Full Document