13 Carboxypeptidase B digestion
Biosimilar and innovator rituximab were diluted to 1 mg/ml using 10 mM sodium phosphate buffer, pH 7.5. CPB (0.25 units) was added and incubated at 37ºC. At the various time points (0 min, 30 min, 1 hr, 2 hr and overnight), the reaction mixture was aliquoted and quenched with acetic acid before analysis.
(A)
Innovator rituximab (B)
11.44 0.09
367.94
1.31
Figure 2. Overlay of fi ve replicates of innovator (A) and biosimilar (B) rituximab on an Bio-inert Quaternary LC using an Bio Mab, 4.6 × 250 mm, 5 µm PEEK column.
Untreated Results and Discussion
The Buffer Advisor Software is an ideal tool to generate pH or ionic strength gradients for protein charge-variant separations. The Buffer Advisor software enables the development of a robust method through design-of experiment principles. The automatic blending facilities and dynamic mixing of solvents from stock solutions simplifi es IEX workfl ows and can save considerable time and solvent cost. In this study, a series of method development scouting runs were carried out using Buffer Advisor for optimal mAb charge-variant separation. Figure 1 shows the charge-variant profi les of innovator and biosimilar rituximab on the Bio mAb PEEK column, demonstrating high-resolution separation of charge variants in 20 minutes with three distinct peaks in biosimilar (buffer 30 mM, pH 6.3, and NaCl 0 to 161.5 mM). The Bio MAb column contains a highly uniform, densely packed, weak cation-exchange resin. Early and late-eluting peaks were called acidic and basic variants, respectively. The peak at 11.4 min was designated as the main peak. The overlay of fi ve replicates of innovator and biosimilar rituximab shows excellent separation reproducibility (Figure 2). The average RTs, and area RSDs for the main peak are shown in the Figure. The RSDs are within the acceptable range, which demonstrates the precision of the system.
The high-resolution separation of mAbs facilitated the quantifi cation of charge variants using peak areas. Table 2 summarises the area percent of charge variants of fi ve consecutive analyses. There was a signifi cant difference in the area percent of the charge variants between two mAbs. The main form in the innovator rituximab was found to be 93.21% and 29.78% in biosimilar rituximab. The major charge variant in biosimilar rituximab was 69.46% basic variants compared to the innovator product (3.22%). The difference in amount of acidic and basic variants between innovator and biosimilar may affect effi cacy profi les.
To further to characterise the basic variants peaks, both mAbs were subjected to carboxypeptidase B digestion. Figure 3a and 3b show the overlay of the IEX profi les before and after C-terminal cleavage of innovator and biosimilar rituximab, respectively. The disappearance of basic variant peaks after carboxypeptidase B treatment confi rmed that the peaks corresponded to lysine variants. Figure 4 shows the overlay of the IEX profi les of biosimilar rituximab after CPB treatment and innovator rituximab without CPB treatment, revealing the charge-variant similarity between the mAbs.
Table 2. Charge-variant quantifi cation by area %, n = 5
Innovator – Ristova RT (min)
Acidic variant Main peak
Basic variant
Acidic variant Main peak
Basic variant 10.84, 11.21 11.9, 12.7
Area% 3.56
11.44 93.21 3.22
Biosimilar – Reditux 10.73, 11.22
11.87, 12.15, 12.59, 13.1, 13.77
0.76
11.45 29.78 69.46
Table 2: Charge-variant quantication by area%, n = 5
Figure 3. Characterisation of basic charge variants. Separation of carboxypeptidase-treated (overnight) and untreated, of innovator (A) and biosimilar (B) rituximab on an Bio-inert Quaternary LC using an Bio Mab, 4.6 × 250 mm, 5 µm PEEK column.
Figure 3: Characterisation of basic charge variants. Separation of carboxypeptidase-treated (overnight) and untreated, of innovator (A) and biosimilar (B) rituximab on an Bio-inert Quaternary LC using an Bio Mab, 4.6 × 250 mm, 5 μm PEEK column.�
Figure 4. Overlay of innovator rituximab without carboxypeptidase treatment (red) and biosimilar rituximab after carboxypeptidase treatment (blue).
Figure 4: Overlay of innovator rituximab without carboxypeptidase treatment (red) and biosimilar rituximab after carboxypeptidase treatment (blue).
Conclusions
The salt-gradient method described demonstrates the high-resolution separation of charge-variant profi les of mAbs. The innovator and biosimilar rituximab had different separation profi les with different degrees of acidic and basic variants. Carboxypeptidase B digestion confi rmed that the major basic variant peaks in biosimilar corresponded to lysine variants. The columns used and the reproducible method make this solution suitable for the QA/QC analysis of mAbs for the biopharmaceutical industry.
References
1.Szabolcs Fekete et al. Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: Salt gradient approach. J. Pharm. Biomed. Anal. 102, 2015, 33–44.
2.Szabolcs Fekete et al. Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach. J. Pharm. Biomed. Anal. 102, 2015, 282-289.
3.Szabolcs Fekete et al. Ion-exchange chromatography for the characterization of biopharmaceuticals. J. Pharm. Biomed. Anal. 113, 2015, 43–55.
Innovator rituximab Biosimilar rituximab
CPB treated Biosimilar rituximab
11.45 0.05
95.07
0.42 Biosimilar rituximab (B) Innovator rituximab (A)
CPB treated
Untreated
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