Published on USP Medicines Compendium ( )
Rituximab
Final Authorized Version 1.0
RITUXIMAB HEAVY CHAIN
QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS AASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS
SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKAE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT
CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K
RITUXIMAB LIGHT CHAIN
QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKPG SSPKPWIYAT SNLASGVPVR FSGSGSGTSY SLTISRVEAE DAATYYCQQW TSNPPTFGGG TKLEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC
C 6416H 9874N 1688O 1987S 44 Molecular weight, approx. 144,187 Da
Immunoglobulin G 1 (human-mouse monoclonal IDEC-C2B8 γ1-chain anti-human antigen CD 20), disulfide with human-mouse monoclonal IDEC-C2B8 κ-chain, dimer;
Immunoglobulin G 1 (human-mouse monoclonal IDEC-C2B8 γ1-chain anti-human antigen CD 20), disulfide with human-mouse monoclonal IDEC-C2B8 κ-chain, dimer [174722-31-7].
Rituximab is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. This IgG1 kappa antibody contains murine light and heavy chain variable regions, and human gamma 1 heavy chain and kappa light chain constant regions. Rituximab is composed of two heavy chains each having 451amino acids and two light chains each having 213 amino acids.
Rituximab is a clear colorless liquid.
Performance-Based Monograph
(Contains tests, procedures, and acceptance criteria for the material under test. It also includes the criteria-based procedures to demonstrate that an Acceptable Procedure is equivalent to the Reference Procedures .)
DEFINITION
Rituximab contains recombinant chimeric murine/human monoclonal antibody directed against the CD20 antigen in a sterile solution having measured potency of NLT 80.0% and NMT 125.0% of the stated potency.
IDENTIFICATION
• A. B IOASSAY
Standard solution: USP Rituximab RS in an appropriate diluent
Sample solution: Rituximab diluted in an appropriate diluent similar to that of the Standard solution .
System performance requirements and Analysis: Proceed as directed in the Assay for Potency .
Acceptance criteria
Measured potency: 80.0%–125.0% of the stated potency
• B. P EPTIDE M APPING北医三院
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Use a chromatographic system. (Proceed as directed in Biotechnology Derived Articles—Peptide Mapping <1055>.)
Analyze the material to be tested by a chromatographic technique capable of resolving peptides generated from a Trypsin digest. The digest is carried out under reducing conditions which provides NLT 80% digestion. The test procedure used provides a
minimum of 90% coverage of the protein sequence.
Standard solution: Digest and dilute a portion of USP Rituximab RS in an appropriate diluent.
Sample solution: Digest and dilute a quantity of Rituximab in an appropriate diluent to obtain a nominal concentration of
Rituximab similar to that of the Standard solution.
Control solution: Digest and dilute a portion of an appropriate control (non-Rituximab monoclonal antibody) in an appropriate diluent to obtain a nominal concentration of the control that is similar to that of Standard solution. [N OTE—The digests described in the Standard solution, Sample solution, and Control solution are conducted at the same time, using the same stock and
concentration of reagents.]
Analytical system: Use a procedure validated as described in MC general chapter Assessing Validation Parameters for Reference and Acceptable Procedures <10>.
System performance requirements
Specificity: The profile obtained from the Standard solution, CDR regions should be identified using a suitable procedure. The peptide profile obtained from the Control solution is distinctly different from that obtained from the Standard solution.
Analysis
Samples:Standard solution and Sample solution
The peptide profiles obtained from the Standard solution are visually compared to the Sample solution.
Acceptance criteria: The profile obtained from the Standard solution is similar to the profile obtained from the Sample solution.
The retention times of the peaks from the Sample solution differ from retention times of the corresponding peaks in the Standard solution by NMT ± 0.2 min.
• C. C APILLARY I SOELECTRIC F OCUSING
Analyze Rituximab using a capillary isoelectrophoretic focusing procedure using a broad range ampholyte (isoelectric point range of 3.0–10.0).
Standard solution: USP Rituximab RS in an appropriate diluent
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Sample solution: Dilute a quantity of Rituximab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution.
Control solution: Dilute a quantity of a non-Rituximab protein (such as Trastuzumab) in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution.
Analytical system: Use a validated procedure. (See Biotechnology-Derived Articles—Capillary Electrophoresis <1053>.)
System performance requirements
Specificity: The isoelectric point obtained from the Standard solution is between 9.1 and 9.5. The isoelectric point obtained from the Control solution should be different from the isoelectric point obtained from the Standard solution.
Analysis
Samples:Standard solution and Sample solution
Compare the isoelectric point from the Sample solution and the Standard solution.
Acceptance criteria: The isoelectric point (pI) of the main peak obtained from the Sample solution differs by NMT ± 0.2 pI units from the corresponding peak from the Standard solution.
ASSAY
• P OTENCY [C OMPLEMENT D EPENDENT C YTOTOXICITY (CDC) A SSAY]
Determine the potency using a suitable CD20 positive cell line (similar to WIL2-S) in a CDC assay with a suitable read out. Perform a comparison of a dilution series of the Sample solution with a dilution series of the Standard solution.
Standard solution: USP Rituximab RS in an appropriate diluent
Sample solution: Rituximab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution Control solution: Dilute a quantity of an appropriate protein (such as non-specific monoclonal antibody) in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution.
Analytical system: Use a validated procedure. (See Biotechnology-Derived Articles—Biological Assay Validation <1033>.)
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System performance requirementsSpecificity: The Standard solution provides a significant dose response and the Control solution shows no response.
Precision
Repeatability: NMT 10.0% RSD
Intermediate precision: NMT 15.0% RSD
Linearity: Plot the measured potency versus expected potency. The R2 is NLT 0.95. [N OTE—The slope should be NLT 0.80.] Accuracy: Spike recovery 85.0%–115.0%
Range: Should be wider than 80.0% and above 125.0%.
Analysis
Samples: Standard solution and Sample solution
双灵固本散
The potency of the Sample solution is calculated relative to the Standard solution using a suitable parallel line method.(Proceed as directed in Analysis of Biological Assays <1034>.)
Calculate 95.0% confidence limits for each independent determination of the measured potency.
Acceptance criteria
95.0% Confidence limits for independent determination: 74.0%–136.0%
Mean measured potency: 80.0%–125.0% of the stated potency obtained as a mean of a minimum of three independent determinations.
95% Confidence limits of the mean measured potency: 85.0%–115.0%. [N OTE—Measure as many independent Sample solutions as necessary to achieve the 95.0% confidence limits.]
• G LYCAN P ROFILING
Use a procedure that is capable of separating all glycans.
Standard solution: USP Rituximab RS in an appropriate diluent
Sample solution: Rituximab in an appropriate diluent
Resolution solution: Use Standard solution
Control solution: Use a non-glycosylated protein in an appropriate diluent.
Analytical system: Use a procedure validated as described in MC general chapter Assessing Validation Parameters for Reference and Acceptable Procedures <10>. (See Glycoprotein and Glycan Analysis—General Considerations <1084>.)
System performance requirements
[N OTE—Identify the peaks using commercial glycan standards of G0, G0F, G1, G1F, G1F', G2, and G2F, or any other suitable procedure.]
Specificity
Peak profile: The profile obtained from the Resolution solution shows prominent peaks for G0, G0F, G1, G1F, G1F', G2, and G2F.
Resolution: NLT 1.0 between G0–G0F and G2–G2F glycan peaks from the Resolution solution.
Negative control: The Control solution shows no glycan peaks.
[N OTE—The following criteria are with respect to the G0F peak in the Standard solution.]
Precision
Repeatability: NMT 4.0% RSD
Intermediate precision: NMT 5.0% RSD
Accuracy: Probability NLT 0.95 for 90.0%–110.0%
Linearity: R2 NLT 0.99
Range: 80.0%–120.0% of the Acceptance criteria set for oligosaccharides with galactose
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of oligosaccharides with galactose in the Sample solution.
Acceptance criteria
Sum of all oligosaccharides with galactose: NLT 35.0%
• C ATION E XCHANGE C HROMATOGRAPHY(AFTER C ARBOXYPEPTIDASE T REATMENT)
Use the normalization procedure. (See Chromatography <621>.)
Standard solution: USP Rituximab RS in an appropriate diluent
Sample solution: Rituximab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. Resolution solution: USP Rituximab RS in an appropriate diluent without carboxypeptidase treatment
Analytical system: Use a procedure validated as described in MC general chapter Assessing Validation Parameters for Reference and Acceptable Procedures <10>.
System performance requirements
Specificity
Peak profile: The chromatogram obtained from the Resolution solution shows presence of lysine (K1) variant when compared to the Standard solution.
Resolution: NLT 1.5 between the main peak (K0) and lysine variant peak (K1)
[N OTE—The following criteria are with respect to the main peak (K0).]
Precision
Repeatability: NMT 2.0% RSD
Intermediate precision: NMT 4.0% RSD
Accuracy: Probability NLT 0.95 at 95.0%–105.0%
Linearity: R2 NLT 0.99
Range: 80.0%–120.0% of the Acceptance criteria
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of acidic and basic variants in the Sample solution.
竹直锥大象虫Acceptance criteria
Acidic variants: NMT 20.0%
Basic variants: NMT 20.0%
IMPURITIES
• CE-SDS (UNDER R EDUCING C ONDITION)
Analyze Rituximab using an electrophoretic method capable of giving separation in the range 10–250 kDa followed by UV detection by normalization procedure.
Standard solution: USP Rituximab RS in an appropriate diluent.
Sample solution: Rituximab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. Non-glycosylated heavy chain (NGHC) solution: Combine a portion of the Standard solution with PNGase enzyme under suitable conditions to achieve at least 95% deglycosylation.
Resolution solution: 2.0% NGHC spiked in Standard solution
Analytical system: Use a validated procedure carried out under reducing conditions. (See Biotechnology-Derived Articles—Capillary Electrophoresis <1053>.)
Use a procedure validated as described in MC general chapter Assessing Validation Parameters for Reference and Acceptable Procedures <10>. (Although general chapter <10> is directed to chromatographic methods, concepts in the guideline are general.) System performance requirement
s
Specificity: The electropherogram obtained from the Standard solution includes a peak corresponding to light chain and heavy chain.
Resolution: NLT 1.5 between the NGHC and heavy chain peaks from the Resolution solution
[N OTE—The following criteria are with respect to the NGHC peak in the Standard solution.]
Precision
Repeatability: NMT 2.0% RSD
Intermediate precision: NMT 4.0% RSD
Accuracy: Probability NLT 0.95 at 90.0%–110.0%
Linearity: R2 NLT 0.99
Range: 50.0%–200.0% of the Acceptance criteria
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of NGHC in the Sample solution.
Acceptance criteria
NGHC impurity: NMT 2.0%
• CE-SDS (UNDER N ON-R EDUCING C ONDITION)
Analyze Rituximab using an electrophoretic method capable of giving separation in the range 10–250 kDa followed by UV detection by normalization procedure.
Standard solution: USP Rituximab RS in an appropriate diluent
Sample solution: Rituximab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. NGHC solution: Combine a portion of the Standard solution with PNGase enzyme under suitable conditions to achieve at least 95% deglycosylation.
Resolution solution: 2.0% NGHC spiked in S tandard solution under reducing condition
Analytical system: Use a validated procedure carried out under non-reducing conditions. (See Biotechnology-Derived Articles—Capillary Electrophoresis <1053>.)
Use a procedure validated as described in MC general chapter Assessing Validation Parameters for Reference and Acceptable Procedures <10>. (Although MC general chapter <10> is directed to chromatographic methods, concepts in the guideline are general.)
System performance requirements
Specificity: The Standard solution contains a principal peak corresponding to Rituximab.
Resolution: NLT 1.5 between the NGHC and heavy chain peaks from the Resolution solution
中国行为医学科学[N OTE—The following criteria is with respect to the light chain peak.] Precision
Repeatability: NMT 2.0% RSD
Intermediate precision: NMT 4.0% RSD
Accuracy: Probability NLT 0.95 at 90.0%–110.0%
Linearity: R2 NLT 0.99
Range: 50.0%–200.0% of the Acceptance criteria
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of impurities in the Sample solution.
Acceptance criteria
Sum of low molecular weight impurities: NMT 8.0%
• S IZE E XCLUSION C HROMATOGRAPHY
Use the normalization procedure. (See Chromatography <621>.)
Standard solution: USP Rituximab RS in an appropriate diluent
Sample solution: Rituximab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. Resolution solution: Dilute USP Rituximab RS in water to obtain a 1.0 mg/mL solution, incubate under UV light (at 254 nm) for 2 h. Analytical system: Use a procedure validated as described in MC general chapter Assessing Validation Parameters for Reference and Acceptable Procedures <10>.
System performance requirements
Specificity
Peak profile: The chromatogram obtained from the Resolution solution shows a high molecular weight (HMW) peak eluting before the main peak.
Resolution: NLT 1.5 between the HMW peak and the main peak from the Resolution solution
[N OTE—The following criteria is with respect to the main peak.]
Precision
Repeatability: NMT 4.0% RSD
Intermediate precision: NMT 4.0% RSD
Accuracy: Probability NLT 0.95 for 90.0%–110.0%
Linearity: R2 NLT 0.99
Range: 50.0%–200.0% of the Acceptance criteria
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of HMW impurities in the Sample solution.
Acceptance criteria
Total impurities: NMT 2.0%
• P ROCESS R ELATED I MPURITIES
Protein A: NMT 10 ppm
Host cell proteins: NMT 100 ppm
Host cell DNA: NMT 10 ng/dose
SPECIFIC TESTS
• M ICROBIAL E NUMERATION T ESTS <61> and T ESTS FOR S PECIFIED M ICROORGANISMS <62>: Total aerobic count does not exceed 1 cfu/mL.
• B ACTERIAL E NDOTOXINS T EST <85>: NMT 0.1 EU/mg of Rituximab
ADDITIONAL REQUIREMENTS
• S TORAGE: Store at 2°–8° C in an airtight container.
• L ABELING: The label states the name, content in mg/mL, and potency of the drug substance.
• R EFERENCE S TANDARDS <11>
USP Rituximab RS
REFERENCE PROCEDURES
(This section provides detailed descriptions of procedures that may be used for the evaluation of the material under test. These procedures have been fully validated, and the data is available on the MC website.)
IDENTIFICATION
• A. P EPTIDE M APPING
Solution A: 0.1% Trifluoroacetic acid in water
Solution B: 0.1% Trifluoroacetic acid in acetonitrile
Solution C: 0.5 M dithiothreitol in water
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