USP Medicines Compendium - Trastuzumab - 2014-02-

Trastuzumab

Final Authorized Version 1.0

ANTI-HER2 LIGHT CHAIN 1

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ

HYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

ANTI-HER2 HEAVY CHAIN 1

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED

TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

C 70H 10012N 1726O 2013S 42 Molecular weight, approx. 1,45,423 Da

Immunoglobulin G1 (human-mouse monoclonal rhuMAb HER2γ1-chain antihuman p185c-erbB2 receptor), disulfide with human-mouse monoclonal rhuMAb HER2 light chain, dimer [180288-69-1].

Trastuzumab is a recombinant DNA-derived, humanized monoclonal antibody (IgG1 kappa) that contains human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody selectively binds with high affinity in a cell-based assay (Kd = 5 nM) to the extracellular domain of the human epidermal growth factor receptor protein.The recombinant protein is produced in CHO cell culture.

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

Trastuzumab contains the recombinant DNA-derived humanized monoclonal IgG1 kappa antibody having a measured potency of NLT 80.0% and NMT 125.0% of the stated potency in a low bioburden solution.

IDENTIFICATION

• A. B IOASSAY

Standard solution: USP Trastuzumab RS in an appropriate diluent

Sample solution: Trastuzumab 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: The dose response of the Sample solution should be significant with defined lower asymptote and upper

asymptote and similar to that of the Standard solution . Ratio of the highest response and lowest response of the dose response curve should be ≥1.3.

• B. P EPTIDE M APPING

Use a chromatographic procedure. (See Biotechnology-Derived Articles—Peptide Mapping <1055>.)

Analyze the material to be tested by a suitable technique capable of resolving peptides generated from a Trypsin digest. The digest is conducted under reducing conditions and provides NLT 80% digestion. The analytical method used provides a minimum of 90%coverage of the sequence.

Standard solution: Digest and dilute a quantity of USP Trastuzumab RS in an appropriate diluent.

Sample solution: Digest and dilute a quantity of Trastuzumab in an appropriate diluent to obtain a nominal concentration of trastuzumab similar to that of the Standard solution .

Control solution: Digest and dilute a quantity of an appropriate negative control (non-trastuzumab monoclonal antibody) in an appropriate diluent to obtain a nominal concentration 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.]

System performance requirements

Specificity: In the profile of the Standard solution, CDR regions 1, 2, and 3 should be identified using a suitable procedure. The peptide profile of the Control solution is distinctly different from that of the Standard solution.

Analysis

Samples:Standard solution and Sample solution

The peptide profiles of the Standard solution are visually compared to the Sample solution.

Acceptance criteria: The profile of the Sample solution corresponds to the profile of the Standard solution. The relative retention time of the peaks corresponding to the CDR regions (with respect to a reference peak) in the Sample solution should differ from the Standard solution by NMT ± 0.03.

• C. I SOELECTRIC F OCUSING

Analyze Trastuzumab using an isoelectrophoretic focusing procedure using a broad range ampholyte (isoelectric point (pI) range of 3.0–10.0). (See Capillary Electrophoresis <1053>).

Standard solution: USP Trastuzumab RS in an appropriate diluent

Sample solution: Dilute a quantity of Trastuzumab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution.

Control solution: Dilute a quantity of an appropriate negative control (non-trastuzumab monoclonal antibody with known pI range between 7.5 and 10.0) in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. [N OTE—The pI of negative control should not fall between 8.5 and 9.0.]

Analytical system: Use a validated procedure. Refer to 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 requirements

Specificity: The pI of the Standard solution is between 8.5 and 9.0. The pI of the Control solution should be different from the pI of the Standard solution.

Analysis

Samples:Standard solution and Sample solution

Compare the pI from the Sample solution and the Standard solution.

Acceptance criteria: The pI of the main peak of the Sample solution differs by NMT ± 0.2 pI units from the pI obtained with the corresponding peak of the Standard solution.

ASSAY

• P OTENCY [A NTI-PROLIFERATION A SSAY]

Determine the potency using a suitable HER2 receptor expressing cell line (similar to BT-474) in an anti-proliferation 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 Trastuzumab RS in an appropriate diluent

Sample solution: Trastuzumab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. Control solution: Dilute an appropriate negative control (non-trastuzumab monoclonal antibody) in the same diluent as used for the Standard solution to obtain a nominal concentration similar to that of the Standard solution.

Analytical system: Use a procedure validated as described in Biological Assay Validation <1033>.

System performance requirements

Specificity: The Standard solution provides a dose response and the Control solution shows no response.

Precision

Repeatability: NMT 15% GCV

Intermediate precision: NMT 15% GCV

Linearity: Plot the measured potency versus expected potency. The R2 is NLT 0.95. [N OTE—The slope should be 0.80–1.20.] Accuracy: Spike recovery 85%–115%

Range: Should encompass 80%–125%, and satisfy the above acceptance criteria for linearity, precision, and accuracy.

Analysis

Samples: Standard solution and Sample solution

The potency of the Sample solution is calculated relative to the Standard solution using a suitable parallel line method or parallel logistic (full curve) method. (Proceed as directed in Analysis of Biological Assays <1034>.)

Calculate the 95% confidence limits for each independent determination of the measured potency.

Acceptance criteria

95% Confidence limits for independent relative potency determination: 74.0%–136.0%

Mean measured relative potency: 80.0%–125.0% of the Stated potency obtained as a geometric mean of a minimum of threeindependent determinations of measured potency.

95% Confidence limits of the mean measured relative potency: 85.0%–118.0%. [N OTE—Measure as many independent replicates of the Sample solution as necessary to achieve the 95% confidence limits.]

• G LYCAN P ROFILING

Use a procedure that is capable of separating all glycans. (See Glycoprotein and Glycan Analysis—General Considerations <1084>.) Standard solution: USP Trastuzumab RS in an appropriate diluent

Sample solution: Trastuzumab in an appropriate diluent

Resolution solution: Use Standard solution.

Control solution: Use an appropriate negative control (non-glycosylated protein) in an appropriate diluent.

Analytical system: Use a validated procedure. Refer to MC general chapter Assessing Validation Parameters for Reference and Acceptable Procedures <10>.

System performance requirements. [N OTE—Identify the peaks using commercial glycan standards of G0F-GN, G0, G0F, Man5, G1, G1’, G1F-GN, G1F, G1F', Man6, G2, G2F, Man 7, G2S1F, Man 8, and Man 9, or any other suitable procedure.]

Specificity

Peak profile: The profile of the Resolution solution shows prominent peaks for G0F-GN, G0, G0F, Man5, G1, G1F, G1F', and G2F.

Resolution: NLT 1.8 between G1F and G1F' glycan peaks from the Resolution solution

Negative control: The Control solution shows no glycan peaks.

Precision (Repeatability): NMT 4.0% RSD, of each glycan variant. [N OTE—Galactosylation (G0F), afucosylation (AF), mannosylation (HM), and total afucosylation (AF+HM) to be considered.]

Accuracy: Probability NLT 0.95 for 90.0%–110.0%. [N OTE—The criteria is with respect to the G0F peak in the Standard solution.] Range: 80.0%–120.0% of the Acceptance criteria

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of oligosaccharides in the Sample solution.

Acceptance criteria

Sum of all oligosaccharides with galactose: NLT 32.0%

Sum of all oligosaccharides without fucose: 5.0%–9.0%

Sum of all oligosaccharides with mannose: NMT 3.0%

• L IMIT OF C HARGED V ARIANTS (AFTER C ARBOXYPEPTIDASE T REATMENT)

Use a suitable chromatographic procedure capable of separating acidic and basic variants. Use the normalization procedure. (See Chromatography <621>.)

Standard solution: USP Trastuzumab RS in an appropriate diluent

Sample solution: Trastuzumab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. Resolution solution: USP Trastuzumab RS treated with ammonium bicarbonate with 1% final concentration at 60° 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 of the Resolution solution shows an increase in acidic variant when compared to the Standard solution.

Resolution: NLT 1.3 between the main peak (K0) and the pre peak

[N OTE—The following criteria are with respect to the main peak (K0).]

Precision

Repeatability: NMT 2.0% RSD

Ruggedness: NMT 4.0% RSD

Accuracy: Probability NLT 0.95 at 90.0%–110.0%

Range: 50.0%–200.0% of the Acceptance criteria

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of acidic and basic variants in the Sample solution.

Acceptance criteria

Acidic variants: NMT 35.0%

Basic variants: NMT 15.0%

IMPURITIES

• L IMIT OF N ON-G LYCOSYLATED H EAVY C HAIN (NGHC) I MPURITIESAnalyze Trastuzumab using an electrophoretic method capable of giving separation in the range 10–225 kDa followed by UV detection by normalization procedure under reducing condition. (See Capillary Electrophoresis <1053>.)

Standard solution: USP Trastuzumab RS in an appropriate diluent

Sample solution: Trastuzumab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. Resolution solution: Standard solution spiked to a final concentration of 2.0% NGHC solution. [N OTE—NGHC solution: Combine a portion of the Standard solution with PNGase F enzyme under suitable conditions to achieve at least 95% deglycosylation.] Analytical system: Use a validated procedure. Refer to 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 requirements

Specificity: The electropherogram of the Standard solution shows peaks corresponding to light chain and heavy chain.

Resolution: NLT 1.4 between the NGHC peak and the heavy chain peak, Resolution solution

[N OTE—The following criteria are with respect to the NGHC peak in the Standard solution.]

Precision

Repeatability: NMT 2.0% RSD

Ruggedness: NMT 4.0% RSD

Accuracy: Probability NLT 0.95 at 95.0%–105.0%

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%

• L IMIT OF L OW M OLECULAR W EIGHT I MPURITIES

Analyze Trastuzumab using an electrophoretic method capable of giving separation in the range 10–225 kDa followed by UV detection by normalization procedure under non-reducing condition. (See Capillary Electrophoresis <1053>.)

Standard solution: USP Trastuzumab RS in an appropriate diluent

Sample solution: Trastuzumab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. Resolution solution: Dilute USP Trastuzumab RS and incubate at 70° for 25 min.

Analytical system: Use a validated procedure. Refer to 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 requirements

Specificity: The electropherogram of the Standard solution shows a peak corresponding to trastuzumab intact peak.

Resolution: NLT 1.1 between the heavy heavy light (HHL) chain peak and the peak corresponding to intact trastuzumab,

Resolution solution

[N OTE—The following criteria are with respect to the light chain peak in the Standard solution.]

Precision

Repeatability: NMT 2.0% RSD

Ruggedness: NMT 4.0% RSD

Accuracy: Probability NLT 0.95 at 95.0%–105.0%

Range: 50.0%–200.0% of the Acceptance criteria

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of low molecular weight impurities in the Sample solution.

Acceptance criteria

Sum of low molecular weight impurities: NMT 5.0%

• L IMIT OF H IGH M OLECULAR W EIGHT I MPURITIES WITH M OLECULAR M ASS H IGHER THAN THAT OF T RASTUZUMAB

Use a suitable chromatographic procedure, like size exclusion chromatography, with the normalization procedure. (See Chromatography <621>.)

Standard solution: USP Trastuzumab RS in an appropriate diluent

Sample solution: Trastuzumab in an appropriate diluent to obtain a nominal concentration similar to that of the Standard solution. Resolution solution: Dilute USP Trastuzumab RS to obtain a 10.0 mg/mL solution, and 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 requirementsSpecificity

Peak profile: The chromatogram of the Resolution solution shows a high molecular weight peak eluting before the main peak.

Resolution: NLT 2.0 between the high molecular weight peak and the peak corresponding to intact trastuzumab, Resolution solution [N OTE—The following criteria is with respect to the main peak corresponding to intact trastuzumab.]

Precision

Repeatability: NMT 2.0% RSD

Ruggedness: NMT 4.0% RSD

Accuracy: Probability NLT 0.95 at 95.0%–105.0%

Range: 50.0%–200.0% of the Acceptance criteria

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of high molecular weight impurities in the Sample solution.

Acceptance criteria

Sum of high molecular weight 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/maximum adult dose (see Nucleic Acid-Based Techniques—Approaches for Detecting Trace Nucleic Acids (Residual DNA Testing) <1130>)

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 0.5 EU/mg

ADDITIONAL REQUIREMENTS

• R EFERENCE S TANDARDS <11>

USP Trastuzumab 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

Solution D: 0.5 M iodoacetamide in water

Solution E: 0.25 M tris buffer in water. Adjust with dilute hydrochloric acid to a pH of 7.5.

Solution F: 6 M guanidine hydrochloride and 1 mM EDTA in Solution E (denaturing buffer)

Solution G: 0.1 M tris buffer in water. Adjust with dilute hydrochloric acid to a pH of 7.8.

Solution H: 2 M urea in Solution G (digest buffer)

Solution I: 0.05 M acetic acid in water

Solution J: 1 mg/mL of trypsin in Solution I

Solution K: 21 mg/mL of USP Trastuzumab RS in water

Mobile phase: See Table 1.

Table 1

Time (min)Solution A (%)

Solution B

(%)

0982 59821205545

1250100

1300100

131982

135982

Standard stock solution 1: Mix 48 µL of Solution K, 452 µL of Solution F, and 10 µL of Solution C, and incubate at 37° for 30 min. Add 24 µL of Solution D, and incubate at room temperature for an additional 30 min in dark. Add 10 µL of Solution C and mix well. Standard stock solution 2: Wash a PD-10 Sephadex G-25 column with 20 mL of water and equilibrate with 35 mL of Solution H.

Load Standard stock solution 1 on the column, and elute using Solution H in volumes of 750 μL each. Collect 7 independent fractions. Measure the absorbance of each fraction at 280 nm. The fraction having an absorbance between 1.3 and 2.0 is used for digestion. [

N OTE—If the absorbance of the fraction is more than 2.0, dilute it using Solution H to get an absorbance of 2.0.]

Standard solution: Mix well 50 μL of Standard stock solution 2 and 2 μL of Solution J, and incubate for 18 h at 37°. Add 1 μL of trifluoroacetic acid and store the Standard solution at -20°.

Sample solution: Prepare Trastuzumab similar to that of the Standard solution. [N OTE—Prepare the Standard solution and the Sample solution at the same time, using the same stock of reagents and concentrations.]

Chromatographic system

(See Chromatography <621>.)

Mode: UPLC

Detector: UV 215 nm

Column: 2.1-mm × 100-mm; 130Å, 1.7-μm, packing L1 (similar to Waters BEH C18)

Flow rate: 0.3 mL/min

Temperatures

Column oven: 60°

Autosampler: 5°

Injection volume: 10 μL

System suitability

Sample: Standard solution

Suitability requirements

Relative retention times

Relative retention time of heavy chain CDR 1, 2, and 3 peaks relative to reference peak (retention time=about .0 min):

about 0.50 ± 0.03, 0.30 ± 0.03, and 0.81 ± 0.03, respectively.

Relative retention time of light chain CDR 1, 2, and 3 peaks relative to reference peak (retention time=about .0 min): about 0.46± 0.03, 0.79 ± 0.03, and 0.84 ± 0.03, respectively.

Analysis

Samples: Standard solution and Sample solution

The peptide profiles of the Sample solution are visually compared to those of the Standard solution.

• B. C APILLARY I SOELECTRIC F OCUSING (C IEF)

Solution A: 0.2 M phosphoric acid in water (anolyte)

Solution B: 0.3 M sodium hydroxide in water (catholyte)

Solution C: 0.35 M acetic acid in water (chemical mobilizer)

Solution D: 0.2 M iminodiacetic acid in water (anodic stabilizer)

Solution E: 0.5 M arginine in water (cathodic stabilizer)

Solution F: 4.3 M urea in water

Solution G: 3.0 M urea in cIEF gel

Solution H: 5 mg/mL of USP Trastuzumab RS in water

System suitability solution: Mix 200 µL of Solution G, 12 µL of Pharmalyte 3-10, 20 µL of Solution E, 2 µL of Solution D, and 2 µL of each of the pI markers (10.0, 9.5, 7.0, 5.5, and 4.1). Vortex the solution for 15 s and mix well. Transfer 200 µL to a micro vial for analysis.

Standard solution: Mix well 10 µL of Solution H, 200 µL of Solution G, 12 µL of Pharmalyte 3-10, 20 µL of Solution E, 2 µL of Solution D, and 2 µL of each of the pI markers (10.0, 5.5, and 4.1). Vortex the solution for 15 s and mix well. Transfer 200 µL to a micro vial for analysis.

Sample solution: Prepare Trastuzumab similar to that of the Standard solution. [N OTE—Prepare the Standard solution and the Sample solution at the same time, using the same stock reagents and concentrations.]

Capillary electrophoresis system

Mode: cIEF

Detector: UV 280 nm

Capillary: 30.2-cm × 50-μm (similar to Beckman coulter, Neutral coated)

Effective length: 20 cm

Temperatures

Capillary: 20°

Sample: 10°

Polarity: Anode at the inlet and cathode at the outlet

Injection time: 25 psi for 99.0 s

Focusing voltage: 25.0 kV for 15.0 min

Chemical mobilization: 30.0 kV for 30.0 min

System suitability

Sample: System suitability solution and Standard solution

Suitability requirements

R2: NLT 0.990, System suitability solution. [N OTE—Determine the R2 value from the calibration curve between pI and migration time.]

pI: Should be 8.5–9.0, Standard solution

Analysis

Samples: Standard solution and Sample solution

Plot a curve using the migration time and pI of the markers spiked in the Sample solution. Calculate the pI of trastuzumab main peak in the Sample solution.

ASSAY

• P OTENCY [A NTI-PROLIFERATION A SSAY]

Determination of the biological activity of trastuzumab solution based on the anti-proliferation activity in BT-474 cells (ATCC No.

HTB-20, human breast carcinoma-derived) using Resazurin sodium staining method.

Medium A: Mix 1:1 ratio of Dulbecco’s modified eagle’s medium with Ham’s nutrient mixture F-12 (DMEM/F12) with 2 mM glutamine, 1.2 g/L of sodium bicarbonate, 3.15 g/L of glucose, 15 mM HEPES, 1 mM sodium pyruvate, and 10% heat inactivated fetal bovine serum (FBS).

Medium B: Mix 1:1 ratio of Dulbecco’s modified eagle’s medium with Ham’s nutrient mixture F-12 (DMEM/F12) with 2 mM glutamine, 1.2 g/L of sodium bicarbonate, 3.15 g/L of glucose, 15 mM HEPES, 1 mM sodium pyruvate, and 2% FBS.

Cell line: BT-474, grow cells in Medium A

Cell suspension: 0.9–1.0 ×105 BT-474 cells/mL in Medium B. [N OTE—Maintain the cells in a uniform suspension during addition.] Standard solution 1: 2.5 µg/mL of USP Trastuzumab RS in Medium B

Standard solution 2: 1.25 µg/mL of USP Trastuzumab RS from Standard solution 1, in Medium B

Standard solution 3: 0.625 µg/mL of USP Trastuzumab RS from Standard solution 2, in Medium B

Standard solution 4: 0.313 µg/mL of USP Trastuzumab RS from Standard solution 3, in Medium B

Standard solution 5: 0.156 µg/mL of USP Trastuzumab RS from Standard solution 4, in Medium B

Standard solution 6: 0.078 µg/mL of USP Trastuzumab RS from Standard solution 5, in Medium B

Standard solution 7: 0.039 µg/mL of USP Trastuzumab RS from Standard solution 6, in Medium B

Standard solution 8: 0.02 µg/mL of USP Trastuzumab RS from Standard solution 7, in Medium B

Standard solution 9: 0.01 µg/mL of USP Trastuzumab RS from Standard solution 8, in Medium B

Standard solution 10: 0.005 µg/mL of USP Trastuzumab RS from Standard solution 9, in Medium B

Sample solution 1: 2.5 µg/mL of Trastuzumab in Medium B

Sample solution 2: 1.25 µg/mL of Trastuzumab from Sample solution 1, in Medium B

Sample solution 3: 0.625 µg/mL of Trastuzumab from Sample solution 2, in Medium B

Sample solution 4: 0.313 µg/mL of Trastuzumab from Sample solution 3, in Medium B

Sample solution 5: 0.156 µg/mL of Trastuzumab from Sample solution 4, in Medium B

Sample solution 6: 0.078 µg/mL of Trastuzumab from Sample solution 5, in Medium B

Sample solution 7: 0.039 µg/mL of Trastuzumab from Sample solution 6, in Medium B

Sample solution 8: 0.02 µg/mL of Trastuzumab from Sample solution 7, in Medium B

Sample solution 9: 0.01 µg/mL of Trastuzumab from Sample solution 8, in Medium B

Sample solution 10: 0.005 µg/mL of Trastuzumab from Sample solution 9, in Medium B

Procedure: To a suitable 96-well microtitre plate, transfer 100 µL of Cell suspension to each standard, sample, and cell control wells. Transfer 100 µL of Medium B to the wells designated as medium control and cell control. Mix and incubate the plate at 37° for a period of 3 h in a humidified incubator with 5% CO

. Transfer 100 µL each of Standard solutions1–10 and Sample solutions 1–10 to the wells

2designated for the standard and sample solutions.

[N OTE—To trypsinize the cells, add 2–3 mL of cold trypsin-EDTA to a 75 cm2 flask and spread uniformly across the monolayer for 45 s.

Discard excess trypsin and incubate for 2–3 min in an incubator. Neutralize the trypsin with Medium A, wash the cell pellet with Medium B, and resuspend with Medium B. Use an appropriate design to distribute the standard and sample solutions in a plate to achieve randomization (see Design and Development of Biological Assays <1032>).]

Mix and incubate the plate at 37° for a period of 90 ± 3 h in a humidified incubator with 5% CO

2

. Remove the plate from the incubator and add 30 µL of pre-warmed resazurin (sodium) to each well. Gently agitate the plate and incubate for an additional 7 ± 1

h. Remove the plates from the incubator and allow them to cool to room temperature by placing on a plate shaker for 30 min.

Determine the relative potency by measuring the fluorescence (relative fluorescence units, RFU) using a suitable 96-well plate reader using an excitation wavelength of 530 nm and emission wavelength of 590 nm.

Generate dose response curves for Standard solutions and Sample solutions using the final concentrations of trastuzumab. [N OTE —The concentrations are before the addition of alarm blue.]

System suitability

Samples:Standard solutions 1–10

Suitability requirements

Relative standard deviation: NMT 10.0% between the replicate RFU

Ratio: The ratio between the RFU of lowest concentration and highest concentration of the Standard solution should be ≥ 1.3.

Outlier: NMT 4 per curve [N OTE—Based on 10% RSD and/or technical problems during the performance of the assay, such as problems with pipetting or air bubbles.]

[N OTE—Medium control and cell control are included for assay monitoring purpose only and have no Acceptance criteria.]

Analysis

Samples: Standard solutions 1–10 and Sample solutions 1–10

Calculate the relative potency and 95% confidence intervals using a validated data analysis software. Perform 4-Parameter Logistic (4-PL) curve fitting of the raw data (RFU) for test of curve fitting and parallelism. (See Analysis of Biological Assays <1034>.) [N OTE—Measure four independent Sample solutions to achieve 95% confidence limits of the mean measured relative potency.]

• G LYCAN P ROFILING—N ORMAL P HASE C HROMATOGRAPHY

Solution A: 0.1 M ammonium formate solution in water. Adjust with formic acid to a pH of 4.5.

Solution B: Acetonitrile

Solution C: 1 M sodium hydroxide in water

Solution D: 5% Acetonitrile in water

Solution E: 50% Acetonitrile in water

Solution F: 0.1% Trifluoroacetic acid in Solution D

Solution G: 0.1% Trifluoroacetic acid in Solution E

Solution H: 10X G7 reaction buffer (0.5 M sodium phosphate at pH 7.5), Make: New England Biolabs

Solution I (2 AB glycan labeling reagent): Transfer 150 µL of acetic acid to a vial of DMSO, and mix well. Transfer 100 µL of DMSO-acetic acid mixture to a vial of tag 2-AB dye (Make: Ludger), and mix gently until dye is dissolved. Add dissolved dye to a vial of sodium cyanoborohydride (Make: Ludger), and mix gently until the solution is completely dissolved. [N OTE—For proper solubilization, heat the final labeling reagent at 65° for 30 s. Use within 60 min.]

Solution J: 30% Acetic acid in water

Solution K: 96% Acetonitrile in water

Solution L: 80% Acetonitrile in water

Solution M: 21 mg/mL of USP Trastuzumab RS in water

Mobile phase: See Table 2.

Table 2

Time (min)Flow rate

(mL/min)

Solution A (%)Solution B (%)

0.010.402872

450.403862

45.10.258020

500.258020

50.10.402872

600.402872

Standard solution: Mix 5 µL of Solution M, 5 µL of Solution H, 2 µL of PNGase F (500,000 units/mL, Make: New England Bio) andmake up to 50 µL with water. [N OTE—The unit definition for PNGase F may differ among commercial sources. It may be necessary to experimentally determine the appropriate quantity of PNGase F required for complete release of N-linked oligosaccharides in the sample.] Gently mix the solution and incubate for 18–20 h at 37°. Process the solution with Ludger Clean EB-10 Cartridges and EB-10 Cleanup procedure as described below.

Washing: Wash the cartridge with 500 µL of methanol, 500 µL of Solution C, 1 mL of water, 1 mL of Solution J, and 1 mL of water, sequentially.

Priming: Prime the active surface of the EB-10 cartridge resin with 500 µL of Solution G and 1 mL of Solution F, sequentially. Sample loading: Load the Standard solution on EB-10 cartridge and allow to pass it through resin. Rinse the above sample tubes with 200 µL of water and again load on EB-10 cartridge for complete binding. Wash again with 700 µL of water. Elute non-glycan contaminants with 700 µL of Solution F. Collect the glycan sample in 1.5-mL microcentrifuge tube by adding 200 µL of Solution G. Repeat this step twice. Vacuum dry the above cleaned glycan solution. Reconstitute the above solution with 5 µL of Solution I and mix well. Incubate the solution for 2 h 30 min at 65 ± 2°.

Prime the Ludger clean S cartridge with 1 mL of water, 2 mL of Solution J, and 1 mL of Solution B, sequentially. Load the labeled glycan sample onto S clean cartridge. [N OTE—Ensure the disc is in wet condition with acetonitrile.] Rinse the sample tube with 100 µL of Solution B. Allow the sample to adsorb onto membrane for 15 min. Wash each disc with 1 mL of Solution B followed by 1 mL of Solution K. Repeat the step with Solution K for five times. Elute the glycans with three aliquots of 0.5 mL of water in a single microcentrifuge tube. Freeze dry the eluted glycans. Reconstitute the aliquots in a total volume of 150 µL with Solution L.

Sample solution: Prepare Trastuzumab similar to that of the Standard solution. [N OTE—Prepare the Standard solution and the Sample solution at the same time, using the same reagents and concentrations.]

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: Fluorescence

Excitation wavelength: 330 nm

Emission wavelength: 420 nm

Column: 2.1-mm × 150-mm; 130 Å, 1.7-μm, packing L68 (similar to Acquity UPLC BEH, Make: Waters, Amide)

Temperatures

Column oven: 40°

Autosampler: 5°

Injection volume: 10 μL

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 1.8 between the G1F and the G1F' peaks

Analysis

Samples: Standard solution and Sample solution

Integrate peaks in the resulting chromatogram, and report relative percentage peak areas of major glycan structures in the Sample solution.

• L IMIT OF C HARGED V ARIANTS (AFTER C ARBOXYPEPTIDASE T REATMENT): C ATION E XCHANGE C HROMATOGRAPHY

Solution A: 10 mM sodium phosphate buffer solution in water. Adjust with dilute sodium hydroxide to a pH of 7.5.

Solution B: 10 mM sodium phosphate buffer solution and 100 mM sodium chloride solution in water. Adjust with dilute sodium hydroxide to a pH of 7.5.

Solution C: 5.0 mg/mL of carboxypeptidase B enzyme in water

Solution D: Dissolve 1.9 g of trehalose dihydrate, 47.14 mg of L-histidine hydrochloride, and 30.4 mg of L-histidine in 80 mL of water. Add 857 µL of Polysorbate-20 and 1.06 mL of benzyl alcohol to the solution, and make up the final volume to 100 mL with water. Solution E: 10% Ammonium bicarbonate in water

Solution F: 1 mg/mL of USP Trastuzumab RS in Solution A

Mobile phase: See Table 3.

Table 3

Time (min)Solution A

(%)

Solution B

(%)

0.011000

51000 437030 440100460100

46.011000

551000

Standard solution: Add 2 µL of Solution C to 200 µL of Solution F and incubate for 2 h at 37 ± 2°.

Sample solution: Prepare Trastuzumab similar to that of the Standard solution. [N OTE—Prepare the Standard solution and the Sample solution at the same time, using the same reagents and concentrations.]

Resolution solution: Mix 15 mg/mL of USP Trastuzumab RS with Solution E to a final concentration of 1%. Incubate the solution at 60° for 2 h. Dilute the above solution to 1 mg/mL in Solution A prior to analysis.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: PDA (280 nm)

Column: 4.6-mm × 100-mm; packing L53 (similar to Tosoh, TSKgel CM-STAT)

Flow rate: 0.8 mL/min

Temperatures

Column oven: 45°

Autosampler: 8°

Injection volume: 80 μL

System suitability

Samples: Standard solution and Resolution solution

Suitability requirements

Relative standard deviation: NMT 1.0% for the trastuzumab peak retention time in replicate injections, Standard solution Resolution: NLT 1.3 between the pre peak and the main peak corresponding to trastuzumab (K0), Resolution solution

[N OTE—The relative retention time of the pre peak relative to trastuzumab (K0) is about 0.93.]

Analysis

Samples: Standard solution and Sample solution

Integrate peaks in the resulting chromatogram, and report relative percentage peak areas of acidic variants and basic variants in the Sample solution.

IMPURITIES

• L IMIT OF NGHC I MPURITIES: CE-SDS (UNDER R EDUCING C ONDITION)

Use IgG purity/heterogeneity kit (Make: Beckman Coulter).

Solution A: 0.1 M tris HCl at pH 9.0 and 1% SDS (sample buffer) (Make: Beckman Coulter)

Solution B: 10 kDa internal marker (Make: Beckman Coulter)

Solution C: Molecular weight marker, 10–225 kDa (Make: Beckman Coulter)

Solution D: β-Mercaptoethanol

Solution E: 4 mg/mL of USP Trastuzumab RS in water

Standard solution 1: Mix 25 µL of Solution E, 70 µL of Solution A, 2 µL of Solution B, and 5 µL of Solution D. Heat the solution for 10 min at 70°. Transfer 100 µL to micro vial for analysis.

Standard solution 2: Add 85 µL of Solution A, 2 µL of Solution B, and 5 µL of Solution D to 10 µL of Solution C. Heat solution for 3 min at 96 ± 2°. Transfer 100 µL to micro vial for analysis.

Sample solution: Prepare Trastuzumab similar to that of Standard solution 1. [N OTE—Prepare the Standard solutions and the Sample solution at the same time, using the same stock of reagents and concentrations.]

NGHC solution: Mix 25 µL of Solution E, 70 µL of Solution A, and 5 µL of Solution D. Heat the solution for 10 min at 70°. Cool to room temperature and add 0.38 µL of PNGase F enzyme (Make: New England Biolabs). Incubate the solution for 15 h at 37°.

Resolution solution: NGHC solution spiked to 2% in Standard solution 1.

Capillary electrophoresis system

Mode: Capillary electrophoresis

Detector: UV 220 nm

Capillary: 30.2-cm × 50-μm (Bare fused silica coated, similar to Beckman Coulter)

Effective length: 20 cm

Temperatures

Capillary: 25°

Sample: 10°Polarity: Anode at the inlet and cathode at the outlet

Injection time: 5.0 kV for 20 s at reverse polarity

Separation voltage: 15.0 kV for 30 min at reverse polarity

System suitability

Samples: Standard solution 2 and Resolution solution

Suitability requirements

Resolution: NLT 1.4 between the NGHC and the heavy chain peaks, Resolution solution

R2: NLT 0.990, Standard solution 2. [N OTE—Determine the R2 value from the calibration curve between the log molecular weight from 20 kDa to 225 kDa and migration time.]

Analysis

Samples: Standard solution 1 and Sample solution

Calculate the percentage of NGHC impurity in the Sample solution.

• L IMIT OF L OW M OLECULAR W EIGHT I MPURITIES: CE-SDS (UNDER N ON-REDUCING C ONDITION)

Use IgG purity/heterogeneity kit (Make: Beckman Coulter).

Solution A: 0.10 M tris HCl at pH 9.0 and 1% SDS (sample buffer) (Make: Beckman Coulter)

Solution B: 10 kDa internal marker (Make: Beckman Coulter)

Solution C: Molecular weight marker, 10–225 kDa (Make: Beckman Coulter)

Solution D: 0.25 M iodoacetamide in water

Solution E: β-Mercaptoethanol

Solution F: 4 mg/mL of USP Trastuzumab RS in water

Standard solution 1: Mix 25 µL of Solution F, 70 µL of Solution A, 2 µL of Solution B, and 5 µL of Solution D. Heat the solution for 4 min at 65°. Transfer 100 µL to a micro vial for analysis.

Standard solution 2: Add 85 µL of Solution A, 2 µL of Solution B, and 5 µL of Solution E to 10 µL of Solution C. Make up the final volume to 100 µL. Heat the solution for 3 min at 96 ± 2°. Transfer 100 µL to micro vial for analysis.

Sample solution: Prepare Trastuzumab similar to that of Standard solution 1. [N OTE—Prepare the Standard solutions and the Sample solution at the same time, using the same stock reagents and concentrations.]

Resolution solution: Mix 25 µL of Solution F, 70 µL of Solution A, 2 µL of Solution B, and 5 µL of Solution D. Heat the solution for 25 min at 70°. Transfer 100 µL to micro vial for analysis.

Capillary electrophoresis system

Mode: Capillary electrophoresis

Detector: UV 220 nm

Capillary: 30.2-cm × 50-μm (Bare fused silica coated, similar to Beckman Coulter)

Effective length: 20 cm

Temperatures

Capillary: 25°

Sample: 10°

Polarity: Anode at the inlet and cathode at the outlet

Injection time: 5.0 kV for 20 s at reverse polarity

Separation voltage: 15.0 kV for 40 min at reverse polarity

System suitability

Samples: Standard solution 2 and Resolution solution

Suitability requirements

Resolution: NLT 1.1 between the heavy heavy light (HHL) chain peak and the peak corresponding to intact trastuzumab,

Resolution solution

R2: NLT 0.990, Standard solution 2. [N OTE—Determine the R2 value from the calibration curve between the log molecular weight from 20 kDa to 225 kDa and migration time.]

Analysis

Samples: Standard solution 1 and Sample solution

Calculate the percentage of low molecular weight impurities in the Sample solution.

• L IMIT OF H IGH M OLECULAR W EIGHT I MPURITIES WITH M OLECULAR M ASS H IGHER THAN THAT OF T RASTUZUMAB: S IZE E XCLUSION

C HROMATOGRAPHY

Solution A: 0.2 M potassium phosphate and 0.25 M potassium chloride solution in water. Adjust with dilute sodium hydroxide to a pH of 6.2.

Standard solution: 10 mg/mL of USP Trastuzumab RS in Solution A

Sample solution: 10 mg/mL of Trastuzumab in Solution A

Resolution solution: Incubate Standard solution under UV light at 254 nm for 2 h.Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: PDA (280 nm)

Column: 7.8-mm × 30-cm; 250 Å, 5-μm packing L59 (similar to TOSOH Biosciences G3000SWXL)

Temperatures

Column oven: 25°

Autosampler: 5°

Injection volume: 20 μL

Run time: 35 min

System suitability

Sample: Resolution solution

Suitability requirements

Resolution: NLT 2.0 between the high molecular weight peak and the peak corresponding to intact trastuzumab

[N OTE—The relative retention time of the high molecular weight peak relative to the trastuzumab peak is about 0.85.]

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of high molecular weight impurities in the Sample solution.

[N OTE—

1.

Protein estimation: Protein estimation is not part of the monograph and there is no acceptance criterion. Manufacturer is left with flexibility of deciding the concentration of Trastuzumab preparation. Use a validated procedure to estimate the protein concentration. 2.

Antibody Dependent Cellular Cytotoxicity (ADCC) assay: ADCC assay is not a compendial test as per the monograph. However, the Trastuzumab preparation must comply with the ADCC activity through analysis of representative batches using a validated procedure or a surrogate test for ADCC assay. Acceptance criteria: NLT 80.0% of USP Trastuzumab RS.]

Source URL (modified on 2014/02/13 - 12:53am): https://mc.usp.org/monographs/trastuzumab-1-0