Rituximab is a chimeric monoclonal antibody employed for the treatment of CD20-positive B-cell non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, granulomatosis with polyangiitis and microscopic polyangiitis. It binds specifically to the CD20 antigen expressed on pre-B and consequently on mature B-lymphocytes of both normal and malignant cells, inhibiting their proliferation through apoptosis, CDC, and ADCC mechanisms. The immunomodulatory activity of rituximab is closely related to critical quality attributes that characterize its chemical composition and spatial configuration, which determine the recognition of CD20 and the binding to receptors or factors involved in its effector functions, while regulating the potential immunogenic response. Herein, we present a physicochemical and biological characterization followed by a pharmacodynamics and immunogenicity study to demonstrate comparability between two products containing rituximab. The physicochemical and biological characterization revealed that both products fit within the same response intervals exhibiting the same degree of variability. With regard to clinical response, both products depleted CD20+ B-cells until posttreatment recovery and no meaningful differences were found in their pharmacodynamic profiles. The evaluation of anti-chimeric antibodies did not show differential immunogenicity among products. Overall, these data confirm that similarity of critical quality attributes results in a comparable immunomodulatory activity.
Rituximab is a chimeric monoclonal antibody (mAb) approved by the FDA on 1997 as single agent for the treatment of relapsed or refractory, low-grade or follicular CD20-positive B-cell non-Hodgkin’s lymphoma (NHL) and later, in 2006, as a treatment in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or other anthracycline-based chemotherapy regimens for patients with diffuse large B-cell lymphoma (DLBCL). In both cases it increases the response rate, diminishes disease progression events, and augments patients survival [
The molecular weight of rituximab is 144,544 Da and is constituted of 1328 aa. As an IgG isotype 1/
Rituximab mechanisms of action comprise the binding of its Fab domain to CD20+ B-lymphocytes for the induction of apoptosis, either directly or throughout the recruitment of immune effector functions by its Fc domain, thus mediating B-cell lysis through complement-dependent cytotoxicity mechanism (CDC), after binding to C1q, or antibody-dependent cellular cytotoxicity mechanism (ADCC) once is recognized by the Fc
Besides, the current knowledge concerning monoclonal antibodies (mAb) permits us to correlate the immunomodulatory activity of a mAb to critical quality attributes (CQAs) that depict its chemical composition and spatial configuration. On this regard, rituximab CQAs are associated with the appropriate recognition of CD20+ B-cells and the achievement of effector functions. Nevertheless, rituximab is subject to posttranslational modifications that can be acquired during its lifecycle, which provides an inherent physicochemical heterogeneity that could impact on its functionality [
Charge and glycosylation heterogeneities are relevant modifications that influence the immunomodulatory activity of mAbs. It is reported that acidic and basic isoforms, coming mainly from oxidation, deamidation, isomerization, amination, cyclization, glycation, and the presence of C-terminal lysines [
Regarding its immunogenicity, rituximab is considered as a low risk molecule although potentially immunogenic, since it does not exhibit cross-reactions with endogenous antibodies or autoimmunity induction; however, due to its chimeric nature, the production of human anti-chimeric antibodies (HACAs) may lead to the loss of efficacy in certain cases. Consequently, to discard any differential immunogenic response of a biosimilar rituximab, the comparability of its chemical composition (i.e., sequence and posttranslational modifications) should be demonstrated [
In this work, we conducted a comprehensive characterization followed up by a pharmacodynamics-immunogenicity clinical study of two products containing rituximab. The characterization exercise is focused on the comparison between the CQAs associated with the pharmacodynamic profile (PD) and the potential immunogenicity of rituximab such as protein identity (amino acid sequence), charge and glycosylation heterogeneity, aggregates content, and binding affinity to Fc
Dibasic sodium phosphate heptahydrate (Na2HPO4·7H2O), monobasic sodium phosphate monohydrate (NaH2PO4·H2O), sodium chloride (NaCl), Tris-hydrochloride (NH2C(C2OH)3·HCl), and sodium hydroxide (NaOH) were obtained from J. T. Baker (Center Valley, PA). Sodium azide (NaN3), ammonium formate (CH5NO2), RPMI-1640 medium, fetal bovine serum (FBS), and formic acid were acquired from Sigma-Aldrich (St. Louis, MO). 2-Aminobenzamide (2-AB) was obtained from ProZyme Inc. (Hayward, CA); PNGase F was purchased from New England Biolabs (Woburn, MA) and Human IgG-Fc antibody from Bethyl Laboratories Inc. (Montgomery, TX). Tetramethylbenzidine (TMB) substrate was obtained from Thermo Scientific (Waltham, MA). ADCC Reporter Bioassay Kit and CellTiter 96 MTT were purchased from Promega (Madison, WI). Water was obtained from a Millipore Milli-Q Biocel system (Billerica, MA). All solutions were filtered through 0.2
MS analyses were performed on a SYNAPT G2 HDMS (Waters Corp.; Manchester, UK) coupled to an ACQUITY UPLC H-Class Bio System (Waters Corp., Milford, MA) using an ESI source. Data was analyzed using BiopharmaLynx software (Waters Corp., Milford, MA) according to reported conditions [
Capillary isoelectrofocusing (cIEF) was performed as we described in a previous report [
Glycan release and derivatization were performed as previously described [
Rituximab purity was assessed on a 4.6 mm × 300 mm ACQUITY Ethylene Bridged Hybrid 200 analytical column with particle and pore diameters of 1.7
Affinity constants under equilibrium (
WIL2-S cell line (ATCC: CRL-8885) that expresses the CD20 antigen was incubated in the presence of different concentrations of rituximab in RPMI-1640 medium with 10% FBS for 2 h at 37°C. A secondary antibody (anti-human IgG-Fc) coupled to a radish peroxidase was added to detect the rituximab-WIL2-S complex after 1 h of incubation at 37°C, using TMB as substrate for 30 min at room temperature. Absorption was acquired at 450 nm. The test results were expressed as the relative percentage of the EC50 from the concentration-response curve of Kikuzubam with respect to the reference product.
CD20 positive cells (WIL2-S, ATCC CRL-8885) were incubated in RPMI 1640 media with 10% of FBS with different concentrations of rituximab and complement human serum for 4 h at 37°C and 5% CO2. Then MTS substrate was added to each well with a further incubation of 2 h at the same conditions. The result of the assay was expressed as % relative potency, which is obtained comparing to the EC50 of the dose-response curve of Kikuzubam with respect of the EC50 of the dose-response curve of the reference product.
The ADCC Reporter Bioassay Kit from Promega (Madison, WI) was used according to manufacturer instructions. CD20 positive cells (WIL2-S, ATCC CRL-8885) were incubated with different concentrations of test antibody and a specific concentration of Jurkat transformed cells expressing CD16. Then a luminescent substrate was added with further incubation of 20 min. The result of the assay was expressed as % of relative potency of Kikuzubam with respect to the reference product.
A double-blind, randomized, three-arm, and prospective study was designed. Two arms (1 and 2) were crossed after three cycles of treatment in order to review the expected use conditions of Kikuzubam and the possible impact on its efficacy as suggested by the Mexican health authorities.
The study protocol was approved by the IRB/IEC (Institutional Review Board/Independent Committee) of the participating research centres and by the Mexican health authorities (study protocol codes CAS/OR/01/CMN/08330041
The aim of the study was to evaluate the biological effects and safety of Kikuzubam compared to the reference product during six treatment cycles with CHOP therapy. Patients received either Kikuzubam or the reference product in each cycle, according to their treatment group, at a dose of 375 mg/m2 every 14 days by IV infusion. 59 patients diagnosed with moderate to high degree diffuse CD20+ B-cell non-Hodgkin lymphoma were randomly assigned into three groups. Group 1 was treated with Kikuzubam during the first three cycles and subsequently with the reference product for the remaining three cycles. Group 2 was initially treated with the reference product for three cycles and then with Kikuzubam for the next three cycles. Group 3 was treated with Kikuzubam throughout six cycles. All patients received concomitant CHOP chemotherapy for the six cycles. A 12-month observational period was included after the completion of the treatments.
Blood samples were collected from all patients for the determination of CD20+ B-cells levels as the PD endpoint on visits 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 using a CD20 Becton Dickinson FITC Labelling Kit in a EPIC XL Beckman Coulter Inc. (Brea, CA) flow cytometer. Additionally, levels of serum human anti-chimeric antibodies (HACAs) were determined using the Human Antirituximab (HADA/HACA/HAMA/HAHA) IgG ELISA Kit for Human from Alpha Diagnostics (San Antonio, TX). The assay precision was determined from the graphs obtained with serum samples, resulting in a coefficient of variation (CV) lower than 10% with accuracy ranging from 90 to 110%.
Analysis of covariance was performed to evaluate the effect of both treatments (Kikuzubam and the reference product) on the number of CD20+ B-cells relative to basal values (covariable). The ANOVA test was evaluated with a significance level of 0.05.
To avoid the effect of crossing treatments, the CD20+ B-cells depletion analyses were performed considering only the results from the first three cycles of treatment with either Kikuzubam or the reference product, in order to compare the response between treatments in a parallel design.
The physicochemical properties of rituximab are discussed according to its impact on PD and immunogenicity potential. Identity, heterogeneity, purity, and biological activity CQAs were studied by comparing several batches of Kikuzubam and the reference product.
The identity of both products was verified by its tryptic peptide chromatographic profiles followed by MS/MS analyses matched with the theoretical sequence of rituximab (Figure
Chromatographic profiles of tryptic peptide mappings followed by MS/MS analyses of Kikuzubam (up) and the reference product (down).
The theoretical sequence was obtained by reverse engineering, comprising a
For both products sequence verification, expressed as MS/MS sequence coverage, exceeded the accepted consensus value of 90%, being 98.7% and 98.6% for Kikuzubam and 98.7% and 97.2% for the reference product of their heavy and light chains, respectively (Figures
Sequence coverage of the heavy and light chain of Kikuzubam.
Sequence coverage of the heavy and light chain of the reference product.
In order to confirm the identity of Kikuzubam, exact mass of the whole deglycosylated molecule, coming uniquely from the amino acid sequence, was determined (Table
Analysis of the exact mass of Kikuzubam and the reference product.
Product | Batch | Experimental mass (Da) |
---|---|---|
MabThera | B60480 | 144190.99 |
B60711 | 144190.04 | |
B6084 | 144190.96 | |
|
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Kikuzubam | RPPP11003 | 144191.29 |
RPPP11014 | 144191.40 | |
RPPP12015 | 144191.93 |
The glycosylation heterogeneity of Kikuzubam and the reference product was also evaluated as a relevant CQA on the immunomodulatory activity of rituximab. Table
Glycosylation microheterogeneity obtained by HILI-UPLC. Variation is presented as confidence interval at 95% (
Product | Batch | Nonfucosylated (%) | Hybrid (%) | Sialylated (%) | Galactosylated (%) | High mannose (%) |
---|---|---|---|---|---|---|
MabThera | H0605 | 1.81 ± 0.04 | 5.00 ± 0.32 | 0.72 ± 0.08 | 43.34 ± 1.42 | 4.37 ± 0.42 |
N3518 | 1.69 ± 0.11 | 3.48 ± 0.24 | 0.98 ± 0.22 | 46.21 ± 1.54 | 3.19 ± 0.54 | |
B62222 | 1.89 ± 0.11 | 2.91 ± 0.18 | 0.82 ± 0.12 | 45.58 ± 0.47 | 3.03 ± 0.17 | |
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Kikuzubam | 5445130608 | 0.65 ± 0.21 | 2.72 ± 0.39 | 0.80 ± 0.21 | 57.08 ± 8.52 | 3.25 ± 0.43 |
5445131216 | 0.65 ± 0.21 | 2.80 ± 0.08 | 0.71 ± 0.13 | 56.35 ± 0.64 | 3.31 ± 0.16 | |
5445131015 | 0.67 ± 0.05 | 3.19 ± 0.25 | 0.77 ± 0.13 | 54.26 ± 3.02 | 3.51 ± 0.23 |
Nonetheless, the glycan heterogeneity of a biosimilar must correspond to the reference product. In this analysis, both products revealed similar glycan heterogeneity, which is consistent with the presence of the same glycoforms observed by the MS analyses of the whole molecule [
Regarding charge heterogeneity, changes higher than 1.0 units in the isoelectric point (pI) of a mAb could affect its therapeutic activity [
Isoelectric point by cIEF. Variation is presented as confidence interval at 95% (
Product | Batch | Main isoform (pI units) | Most acidic variant |
Most basic variant |
Global pI |
---|---|---|---|---|---|
MabThera | M0605 | 9.31 ± 0.00 | 8.68 ± 0.00 | 9.49 ± 0.00 | 9.07 ± 0.06 |
N3518 | 9.31 ± 0.00 | 8.68 ± 0.00 | 9.49 ± 0.00 | 9.09 ± 0.06 | |
B62222 | 9.31 ± 0.00 | 8.68 ± 0.00 | 9.49 ± 0.00 | 9.09 ± 0.06 | |
|
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Kikuzubam | 5445130608 | 9.30 ± 0.00 | 8.64 ± 0.00 | 9.42 ± 0.00 | 9.03 ± 0.07 |
5445131015 | 9.30 ± 0.01 | 8.64 ± 0.01 | 9.42 ± 0.00 | 9.02 ± 0.08 | |
5445131216 | 9.29 ± 0.01 | 8.63 ± 0.01 | 9.42 ± 0.00 | 9.01 ± 0.08 |
Another relevant CQA related to the immunomodulatory activity of rituximab is the aggregation level, which involves the irreversible interaction of two or more denatured protein molecules revealing new epitopes that could stimulate the immune system. A positive correlation between protein aggregation and immunogenicity has been reported for therapeutic proteins, as well as affectations on the biological activity, either directly or indirectly through the formation of neutralizing or binding antibodies. Thus, the evaluation of aggregates is an important component of the analytical comparability assessment of therapeutic proteins. The aggregates content of Kikuzubam was comparable to the reference product (Table
Aggregates content obtained by SE-UPLC. Variation is presented as confidence interval at 95% (
Product | Batch | Aggregates (%) |
---|---|---|
MabThera | B62222 | 0.11 ± 0.01 |
H0605 | 0.07 ± 0.01 | |
N3518 | 0.09 ± 0.02 | |
|
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Kikuzubam | 5445131015 | 0.13 ± 0.03 |
5445130608 | 0.10 ± 0.05 | |
5445131216 | 0.25 ± 0.03 |
In addition to the physicochemical analyses, an extensive biological characterization to assess comparability of the functions (mechanisms of action) described for the reference product and Kikuzubam was performed through
The main mechanism of action of rituximab is binding to CD20 [
(a) Relative binding affinity against CD20, (b) affinity constants towards Fc
(a) ADCC and (b) CDC
Rituximab also can induce the death of CD20+ B-cells by activating effector cells such as natural killer cells (NK), monocytes, and macrophages through the binding of the Fc
The described Fc and Fab affinities further modulate CDC and ADCC mechanisms of rituximab [
An abbreviated study conducted on CD20+ non-Hodgkin’s lymphoma patients was designed to confirm that the physicochemical and functional characteristics of Kikuzubam are adequate to exhibit the same PD profile as the reference product.
CD20 was used as the main endpoint. During the treatment, CD20 B-cells were depleted to serum levels lower than 20 cell/mL in the three arms of the study (Figure
(a) Serum concentrations of CD20+ B-Lymphocytes measured along the PD study from patients of all groups. (b) Comparison of mean serum concentrations of CD20+ B-lymphocytes from all patients treated with Kikuzubam against the reference product.
In order to determine the comparability of the primary endpoint, statistical analyses for the three arms before crossing were performed. Shapiro-Wilk test revealed departures from normality of the data (
Comparability of the primary endpoint between treatments.
Patients |
Arms | Student’s |
Wilcoxon |
---|---|---|---|
10 versus 13 | 1 versus 2 | 0.5114 | 0.5558 |
38 versus 13 | 1 and 3 versus 2 | 0.5742 | 0.6421 |
37 versus 13 | 1 and |
0.8603 | 0.7401 |
The production of antichimeric human antibodies (HACAs) as a result of the loss of tolerance to rituximab by the immune system was evaluated on the three study arms.
On arm 1, two patients showed positive results for the screening test of HACAs right after the shift from Kikuzubam to the reference product on visit 5 (Table
HACAs determination in the immunogenicity study.
Arm | Number of patients | Positive HACAs patients | Positive HACAs patients (%) |
---|---|---|---|
1 | 15 | 2 | 13 |
2 | 13 | 2 | 15 |
3 | 31 | 3 | 10 |
Likewise, three patients from arm 3 presented positive results for the screening HACAs test (Table
The hematologic recovery after R-CHOP cycles, even in patients positive to HACAs screening test for both Kikuzubam and the reference product, was accomplished within the expected period reported in studies with chemotherapy; thus, it can be inferred that neither the HACAs developed by Kikuzubam nor the reference product had a negative effect on the hematologic recovery of patients included in the study.
The comprehensive physicochemical, biological, and
The authors are employees of Probiomed S.A. de C.V., which is developing, manufacturing, and marketing biosimilar products. All authors are involved in the development of biosimilar products for Probiomed.
This work was supported by CONACYT, Mexico, Grant PEI-CONACyTC 134016. The authors thank José Luis Olguín-García for his valuable contribution in the statistical analyses for this paper.