Chromatography
High Resolution Separation of Charge Variant Profi les of Monoclonal Antibodies: Rituximab Innovator and Biosimilar
Suresh Babu C.V., Agilent Technologies India Pvt Ltd, Bangalore, India
This article describes the high-resolution separation of charge variants of innovator and biosimilar rituximab using a Bio-inert Quaternary LC, and OpenLAB ChemStation software. A weak cation exchange (CEX) , 4.6 X 250 mm, 5 um PEEK ion exchange column was used to obtain a separation. The column features a unique resin designed for the charge-based separation of monoclonal antibodies (mAbs). The optimised salt gradient revealed differences in acidic and basic charge variant profi les of innovator and biosimilar rituximab. Precision of retention time, peak height, and peak area of the charged isoforms were well within the acceptable range. C-terminal digestion by carboxypeptidase B (CPB) revealed the major lysine variant peaks in biosimilar rituximab.
Recombinant monoclonal antibodies (mAbs) are important biotherapeutics with a wide range of diagnostic and clinical applications. Recently, biosimilar products are increasing in popularity in biopharmaceuticals. MAbs can undergo various post-translational modifi cations (PTMs) including lysine truncation, deamidation, oxidation, glycosylation, and so forth, becoming heterogeneous in their biochemical and biophysical properties. Due to these modifi cations, charge variants can affect the effi cacy, activity, and stability of mAbs as biotherapeutics. Hence, it is important to characterise the charge heterogeneity during drug development, which serves as a quality control (QC) step for the biopharmaceutical industry. In addition, precise bioanalytical methods are necessary to demonstrate the similarity between a biosimilar and the innovator product.
Cation-exchange chromatography (CEX) is the gold standard for charge-sensitive antibody analysis [1,2]. This article describes a salt-gradient method [1, 3] for separating the charge variants of innovator and biosimilar rituximab. The method compares the CEX profi les of innovator and rituximab biosimilar. Precision of retention time, peak height, peak area and quantifi cation of acidic, basic, and main forms was determined. Carboxypeptidase B (CPB) digestion was performed to study the contribution of C-terminal lysine variants.
Equipment
Instrumentation An Agilent 1260 Infi nity Bio-inert Quaternary LC, with diode array detector fi tted with a bio-inert fl ow cell, operating to a maximum pressure of 600 bar, was used. The Agilent 1260 Infi nity Bio-inert Quaternary LC system is a dedicated solution for large bio-molecule analysis. Solvent delivery was free of any stainless steel or iron components. All the capillaries and fi ttings throughout the autosampler, column compartment and detector are metal free so that bio-molecules come in contact with ceramics or PEEK. This will ensures the integrity bio-molecule, minimises unwanted surface interactions and avoid the pitfalls of peak tailing, low recovery, and decreased column life. The column was an Agilent Bio MAb, 4.6 × 250 mm, 5 µm PEEK.
Software
Agilent OpenLAB CDS ChemStation Edition, revision C.01.06, and Agilent Buffer Advisor, revision A.01.01, software for instrument control and data analysis was used.
Reagents, samples, and procedure
Innovator and biosimilar rituximab were purchased from a local pharmacy and stored according to the manufacturer’s instructions. Sodium phosphate dibasic, sodium phosphate monobasic, sodium chloride, hydrochloric acid, and sodium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO). All the chemicals and solvents were HPLC grade, and distilled water (18 M-ohm grade) was from a Milli Q water purifi cation system (Millipore Elix 10). Carboxypeptidase B was also purchased from Sigma-Aldrich.
Ion-exchange chromatography parameters
Table 1 shows the chromatographic parameters for ion-exchange chromatography. Rituximab (innovator and biosimilar) were diluted to 1 mg/mL in water and the elution was monitored at 280 nm. Retention time (RT), area, and percent area were used to calculate standard deviation (SD) and relative standard deviation (RSD%) values. Relative percent area was used to quantify the charge variants of mAbs.
Table 1. Chromatographic parameters used for IEX chromatography Parameter
Conditions
Mobile phase A Water Mobile phase B NaCl (850.0 mM) Mobile phase C NaH2 Mobile phase D Na2
PO4 HPO4 Gradient
0 min 2 min 8 min
20 min 21 min
Injection volume 5 µL Flow rate
0.75 mL/min
Data acquisition 280 nm/4 nm, Ref.: 360 nm /100 nm Acquisition rate 5 Hz TCC
Ambient
Sample thermostat 5°C Post run time
10 min
(41.0 mM) (55.0 mM)
Time (min) Mobile
phase A (%) 30.3 26.0 21.5 13.3 30.3
Mobile
phase B (%) 0
5.0
10.0 19.0 0
Mobile
phase C (%) 59.6 56.9 54.9 51.9 59.6
Mobile
phase D (%) 10.1 12.1 13.6 15.8 10.1
Figure 1. Charge-variant profi les of innovator (A) and biosimilar (B) rituximab using an Bio MAb 5 µm column. (C) Overlay of innovator and biosimilar rituximab. Peaks 1 and 2 are acidic variants, peak 3 is the main form, peaks 4, 5, 6, and 7 are basic variants.
Figure 1: Charge-variant proles of innovator (A) and biosimilar (B) rituximab using an Bio MAb 5 μm column. (C) Overlay of innovator and biosimilar rituximab. Peaks 1 and 2 are acidic variants, peak 3 is the main form, peaks 4, 5, 6, and 7 are basic variants.�
Biosimilar rituximab
(B)
Innovator rituximab
(A)
Innovator rituximab Biosimilar rituximab
(C) Main Peak
Basic variants Acidic variants
INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2016
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