JPMA 06, Dec., Case Studies of Established Conditions in Analytical Procedures. Biopharmaceutical Committee Technology Working Committee

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1 CMC Strategy Forum Japan 2016 Case Studies of Established Conditions in Analytical Procedures Biopharmaceutical Committee Technology Working Committee JPMA 06, Dec., 2016 Akira Nonoyama Santen Pharmaceutical Co., Ltd. 1

2 Objectives Provide an example of Established Condition (EC) determination for biologics-specific analytical procedures; a cell-based bioassay Propose preferred descriptions of the analytical procedure as ECs Give you tips for a further discussion on ECs for analytical procedures 2

3 Contents Discussion Process (Background) Model SOP for potency assay of G-CSF Example for potency assay in Japan module 1.2 Case study of Established Conditions Optimized description of method Performance-based description Summary 3

4 Discussion Process (Background) Potency assay of G-CSF was selected considering following points. Unique to Biologics Complexity of an analytical procedure Monograph of G-CSF is admitted in USP, Ph.Eur and JP A model SOP for potency assay was generated based on the monographs for G-CSF products in Japanese pharmacopoeia. The following assumption was set to be generalized. 4-parameter logistic curve fitting(4-pl) as a calculation method A fluorescence measurement Based on the SOP, three types of descriptions were prepared. 1. Japan Module 1.2(Approved matters) 2. Optimized description of method 3. Performance-based description 4

5 List of model SOP for potency assay of G-CSF 1. Preparation of medium 2. Preparation of assay cells 3. Preparation of sample solution 4. Preparation of standard solution 5. Addition of each solution to the plate 6. Preparation of cell suspension 7. Addition of cell suspension to the plate 8. Cell culture 9. Addition of cell counting reagent and incubation 10. Measurement of fluorescence intensity Calculation of potency System suitability (Criteria for standard solution) Sample suitability(criteria for sample solution) 12. Reagents and Solutions SOP 5

6 A model SOP for potency assay of G-CSF (1/5) <Operating procedure> 1. Preparation of medium Assay medium: Iscove s modified Dulbecco s medium 445 ml is combined with inactivated fetal bovine serum 50 ml, a 5 ml solution containing benzyl penicillin potassium ( U/mL) and streptomycin sulfate (0.01 g potency/ml), and 25 μl of 2-mercaptoethanol solution (9 in125), followed by filtration sterilization using a 0.22 μm membrane filter. Storage conditions: 2-8 C Shelf life: months Culture medium: 0.2 mg of G-CSF is dissolved with 20 ml of phosphate-buffered sodium chloride TS, followed by filtration sterilization using 0.22 μm membrane filter. An aliquot (0.1 ml) of this solution is combined with 100 ml of the assay medium. Storage conditions: 2-8 C Shelf life: months 2. Preparation of assay cells (not described in JP but described in USP) NFS-60 cells stored frozen are thawed in a water bath (37 o C), suspended in the culture medium (10 ml) and transferred into a centrifuge tube for centrifugation (200 g, 4 C, 5 min). Cell pellets are harvested and re-suspended in the culture medium (10 ml). Viable cells are counted and the culture medium is added to make the cell density equal to cells/ml. The cell suspension (15 ml) is transferred into a 75-cm 2 culture flask, followed by incubation for 3-4 days until the culture becomes 80% confluent. By similar procedure, the cells are passaged for 2 or more generations before using in the assay. <Culture conditions> Culture temperature: C CO 2 concentration of incubator: 4-6% <Number of generations allowed for passage> Can be used in the range of X-Y generations. 6

7 A model SOP for potency assay of G-CSF (Enlarged view) <Operating procedure> 1. Preparation of medium Assay medium: Iscove s modified Dulbecco s medium 445 ml is combined with inactivated fetal bovine serum 50 ml, a 5 ml solution containing benzyl penicillin potassium ( U/mL) and streptomycin sulfate (0.01 g potency/ml), and 25 μl of 2- mercaptoethanol solution (9 in125), followed by filtration sterilization using a 0.22 μm membrane filter. Storage conditions: 2-8 C Shelf life: months 7

8 A model SOP for potency assay of G-CSF (2/5) <Operating procedure> 3. Preparation of sample solution This product (1 ml) is combined with A ml of the assay medium (G-CSF concentration x ng/ml) to make an sample solution. 4. Preparation of standard solution Reference standard material (1 ml) is combined with A ml of the assay medium (G-CSF concentration x ng/ml) to make a standard solution. 5. Addition of each solution to the plate Add 50 μl of assay medium to each well of the microplate (8 rows x 12 columns, 96 wells). Then, add 50 μl of the standard solution to the wells in Row 2-4 of Column 12 and 50 μl of the sample solution to the well in Row 5-7 of the same column. Then, take 50 μl from each well of Column 12 and place it to the well of corresponding row of Column 1. Then, take 50 μl from each well of Column 1 and place it to the well of corresponding row of Column 2. Repeat the same procedure until Column 10, to prepare 2-fold serially diluted wells. No operation is made on Column 11 (Column 11 serves as the negative control). Four plates are prepared in the same way. 6. Preparation of cell suspension Transfer the culture containing NFS-60 cells into a 15 ml centrifuge tube, and centrifuge (200 g, 4 C, 5 min). Remove the supernatant by aspiration. Resuspend the harvested cells with 10 ml of the assay medium. Repeat this procedure twice more. Count viable cells and suspend in the assay medium to yield a cell suspension ( cells/ml). This suspension is prepared in a volume of 20 ml or more. 8

9 A model SOP for potency assay of G-CSF (Enlarged view) <Operating procedure> 3. Preparation of sample solution This product (1 ml) is combined with A ml of the assay medium (G-CSF concentration x ng/ml) to make an sample solution. 4. Preparation of standard solution Reference standard material (1 ml) is combined with A ml of the assay medium (G-CSF concentration x ng/ml) to make a standard solution. 9

10 A model SOP for potency assay of G-CSF (3/5) 7. Addition of cell suspension to the plate Add 50 μl of cell suspension to each well of the microplate (the wells in row 1 and 8 are not used for the assay). 8. Cell culture CO 2 incubator is used for incubation. <Culture conditions> Culture temperature: C CO 2 concentration of incubator: 4-6% Incubation period: hours 9. Addition of cell counting reagent and incubation Add 10 μl of resazurin solution to each well of the culture plate and mix homogeneously. CO 2 incubator is used for incubation. <Culture conditions> Culture temperature: C CO 2 concentration of incubator: 4-6% Incubation period: 4-6 hours 10. Measurement of fluorescence intensity. After incubation, measure the fluorescence intensity at excitation wavelength nm and measuring wavelength 590 nm (fluorescence of resorufin) using a microplate reader. Method of measurement (fluorescence): SOP-Y1 10

11 A model SOP for potency assay of G-CSF (Enlarged view) 9. Addition of cell counting reagent and incubation Add 10 μl of resazurin solution to each well of the culture plate and mix homogeneously. CO 2 incubator is used for incubation. <Culture conditions> Culture temperature: C CO 2 concentration of incubator: 4-6% Incubation period: 4-6 hours 11

12 A model SOP for potency assay of G-CSF (4/5) Calculation of potency Data from measurement are imported in accordance with SOP-Z, using the analytical software ( corporation, XX Program or equivalent), and are then analyzed. Construct 4-parameter logistic (4-PL) fit curves for sample solution and for standard solution from the fluorescence intensities at respective concentrations, and estimate the 4-PL parameters, as follows; X: Concentrations of solutions y: Fluorescence intensities A: Lower asymptote B: Slope of the curve at EC 50 C: Effective concentration 50% (EC 50 ) D: Upper asymptote The ratio of potency of sample solution to that of standard solution (Pr) is determined as follows: P r = C value of standard solution / C value of sample solution Calculate geometric mean (GM) of the Pr values from three or more plates which met system suitability criteria and sample suitability criteria, subsequently, the specific activity (U/mg) per mg protein of this product using the equation given below. Specific activity of this product (U/mg) = S GM of P r D T /D S /C S: Potency of the reference standard material (U/mL) D T : Dilution factor for sample solution D S : Dilution factor for standard solution C: Protein concentration of this product (mg/ml) 12

13 A model SOP for potency assay of G-CSF (Enlarged view) Calculation of potency Construct 4-parameter logistic (4-PL) fit curves for sample solution and for standard solution from the fluorescence intensities at respective concentrations, and estimate the 4- PL parameters, as follows; X: Concentrations of solutions y: Fluorescence intensities A: Lower asymptote B: Slope of the curve at EC 50 C: Effective concentration 50% (EC 50 ) D: Upper asymptote Specific activity of this product (U/mg) = S GM of P r D T /D S /C S: Potency of the reference standard material (U/mL) P r = C value of standard solution / C value of sample solution D T : Dilution factor for sample solution D S : Dilution factor for standard solution C: Protein concentration of this product (mg/ml) 13

14 A model SOP for potency assay of G-CSF (5/5) System suitability Curve shape of standard solution: S-shape curve with a linear part between the asymptote R 2 of 4-PL regression curve of standard solution: 0.95 The fluorescence intensity of the negative control (wells in Column 11 of the plate) should not exceed xx A value of standard solution: zz1 < A < zz2 D value of standard solution: zz3 < D < zz Sample suitability A value of sample solution / A value of standard solution: D value of sample solution / D value of standard solution: B value of sample solution / B value of standard solution: Percent geometric CV (%GCV): 25% %GCV = 100 (e SDln -1) SD ln : the standard deviation of the natural log-transformed replicate relative potency (P r ) results for each sample. (Details given in SOP-Z) 12. Reagents and Solutions Reference standard material: The in-house reference standard material is used as the reference standard material. Iscove s modified Dulbecco s medium: Iscove s modified Dulbecco s medium manufactured by corporation, Catalog number: XXXXX Fetal bovine serum: Fetal bovine serum manufactured by corporation, Catalog number: XXXXX NFS-60 cells: ATCC CRL-1838 was adapted in the culture medium and divided into multiple portions for storage frozen at -150 C or lower. Resazurin solution: Resazurin solution manufactured by corporation, Catalog number: XXXXX 14

15 A model SOP for potency assay of G-CSF (Enlarged view) System suitability Curve shape of standard solution: S-shape curve with a linear part between the asymptote R 2 of 4-PL regression curve of standard solution: 0.95 The fluorescence intensity of the negative control (wells in Column 11 of the plate) should not exceed xx A value of standard solution: zz1 < A < zz2 D value of standard solution: zz3 < D < zz Sample suitability A value of sample solution / A value of standard solution: D value of sample solution / D value of standard solution: B value of sample solution / B value of standard solution: Percent geometric CV (%GCV): 25% %GCV = 100 (e SDln -1) SD ln : the standard deviation of the natural log-transformed replicate relative potency (P r ) results for each sample. (Details given in SOP-Z) 15

16 Discussion Process (Background) Potency assay of G-CSF was selected considering following points. Unique to Biologics Complexity of an analytical procedure Monograph of G-CSF is admitted in USP, Ph.Eur and JP A model SOP for potency assay was generated based on the monographs for G-CSF products in Japanese pharmacopoeia. The following assumption was set to be generalized. 4-parameter logistic curve fitting(4-pl) as a calculation method A fluorescence measurement Based on the SOP, three types of descriptions were prepared. 1. Japan Module 1.2(Approved matters) 2. Optimized description of method 3. Performance-based description 16

17 Peculiar features of Japan M1.2 description Descriptions are based on procedures with details, which can easily be reproduced by laboratory technicians, rather than conditions: Solutions used for the assay is described not by concentrations but by procedure for preparation As a result, detailed information, which are not described in Module 3 of CTD, is required (e.g., dilute 1 ml of XX with 20 ml of distilled water) Most of procedures are described with narratives and include acceptable ranges for the specification in the narratives 17

18 Example of Description on Japan Module 1.2 (1/3) Dilution method rather than concentrations <Procedure> Add 1 ml of this product to A ml of the assay medium to make a sample solution. Add 1 ml of the reference standard to A ml of the assay medium to make a standard solution. Using three or more microplates (each plate, 8 rows 12 columns, 96 wells), add the assay medium (50 μl) to each well of the microplate. Then, add 50 μl of the standard solution to the wells in Row 2-4 of Column 12 and 50 μl of the sample solution to the well in Row 5-7 of the same column. Then, take 50 μl from each well of Column 12 and place it to the well of corresponding row of Column 1. Then, take 50 μl from each well of Column 1 and place it to the well of corresponding row of Column 2. Repeat the same procedure until Column 10, to prepare 2-fold serially diluted wells. No operation is made on Column 11. Culture NFS-60 cells with the culture medium, centrifuge the culture, and remove the supernatant liquid by aspiration, wash the cells with the assay medium. Repeat the washing procedure twice more. Then, suspend the cells in the assay medium to yield a cell suspension containing cells. Add 50 μl of the cell suspension to each well of the microplate (the wells in Row 1 and 8 are not used for the test). Incubate the plate under the atmosphere of 5 vol% carbon dioxide at 37 C for hours. After incubation, add to the all well 10 μl each of the resazurin solution, and incubate under 5 vol% CO 2 for 4-6 hours at 37 C. After incubation, measure the fluorescence intensity at excitation wavelength nm and measuring wavelength 590 nm. Detailed narrative of procedures Construct 4-parameter logistic (4-PL) fit curves for sample solution and for standard solution from the fluorescence intensities at respective concentrations, and calculate the ratio of potency of sample solution to that of standard solution (Pr), as follows; X: Concentrations of solutions y: Fluorescence intensities A: Lower asymptote B: Slope of the curve at EC 50 C: Effective concentration 50% (EC 50 ) D: Upper asymptote 18

19 Example of Description on Japan Module 1.2 (2/3) <Procedure(Continue)> P r = C value of standard solution / C value of sample solution Calculate geometric mean (GM) of the Pr values from three or more plates which met system suitability criteria and sample suitability criteria. When, subsequently, the specific activity (U/mg) per mg protein of this product is calculated according to the following equation, the specific activity must be not less than xx 10 8 U/mg and not more than yy 10 8 U/mg. Specific activity of this product (U/mg) = S GM of P r D T /D S /C S: Potency of the reference standard material (U/mL) D T : Dilution factor for sample solution D S : Dilution factor for standard solution C: Protein concentration of this product (mg/ml) <System suitability> Curve shape of standard solution must be S-shape curve with a linear part between the asymptote. R 2 of 4-PL regression curve of standard solution must be not less than The fluorescence intensity of the negative control (wells in Column 11 of the plate) should not exceed xx. A value of standard solution must be zz1 - zz2. D value of standard solution must be zz3 - zz4. Acceptance criteria here <Sample suitability> Ratio of A value of sample solution to A value of standard solution must be Ratio of D value of sample solution to D value of standard solution must be Ratio of B value of sample solution to B value of standard solution must be Percent geometric CV (%GCV) calculated according to the following equation must be not more than 25%. %GCV = 100 (esdln-1) SDln: the standard deviation of the natural log-transformed replicate relative potency (Pr) results for each sample. 19

20 Example of Description on Japan Module 1.2 (3/3) <Reagents and Solutions> Reference standard material: The in-house reference standard material is used as the reference standard material. Iscove s modified Dulbecco s medium: Iscove s modified Dulbecco s medium manufactured by corporation, Catalog number: XXXXX or equivalent is used. Assay medium: Iscove s modified Dulbecco s medium (90 ml) is combined with 10 ml of fetal bovine serum (heating at 56 C for 30 minutes), 1 ml of benzyl penicillin potassium ( U/mL) and streptomycin sulfate (0.1 g potency) dissolved in 10 ml physiological saline and 2-mercaptoethanol solution (9 in 125) 5μL, followed by filtration sterilization. Culture medium: Weigh an amount corresponding to 0.20 mg of G-CSF and dissolve with 20 ml of phosphate-buffered sodium chloride TS. Add 0.1 ml of this solution to 100 ml of the assay medium. NFS-60 cells: ATCC CRL-1838 was adapted in the culture medium and divided into multiple portions for storage frozen at -150 C or lower. Resazurin solution: Resazurin solution manufactured by corporation, Catalog number: XXXXX or equivalent is used. 20

21 Possible concern from M1.2 description The detailed descriptions and parameters are included in M1.2. Therefore, even a minor change in the assay procedure may require prior regulatory approval. Steep path 21

22 What should ECs include? Acceptance criteria? Yes Fundamental information of the assay? Yes Detailed Procedure? No Non-Key conditions? No Detailed procedure including Non-Key conditions can be defined in SOP under GMP Key conditions? Yes or No; two cases are proposed Case Operational Flexibility Clarification of Critical conditions Yes Low High No High Low Optimized description Performance-based description Required assay performance should be defined for appropriate quality control 22

23 Key concept for Optimized description of method (1/2) Switch from detailed procedure to assay conditions Information of Non-key conditions such as culture plate/wells are completely removed Mixing procedures defined with solution volumes are replaced with target concentrations Ex) Add 50 µl of solution to Row X for 2-fold serial dilutions Concentration range: x y ng/ml. Number of concentration levels: not less than Eliminate minor parameters which unlikely affect assay performance GMP control CO 2 concentration in incubator and culture duration Acceptance criteria are described in an independent column 23

24 Key concept for Optimized description of method (2/2) Focus on criteria derived from assay performance regarding system and sample suitability System and sample suitability criteria includes two types of criteria: 1. Criteria derived from assay performance Repeatability of Pr value between plates 4-PL curve shape and goodness of fit 2. Criteria derived assay conditions, such as reagent lots and plate reader Levels of asymptotes, slope and counts from negative controls 24

25 Optimized description of method (1/3) <Acceptance criteria> Acceptance criteria are separated Not less than xx 10 8 U/mg and not more than yy 10 8 U/mg <Procedure> The specific activity of the sample solution is measured by cell proliferation assay using the NFS-60 cells (ATCC CRL-1838). Standard and sample solutions are added to NFS-60 cells, followed by incubation and staining of viable cells. From fluorescence intensities of the standard and sample solutions, the ratio of potency of sample solution to that of standard solution (Pr) is determined and, subsequently, specific activity per mg protein of this product (U/mg) is determined according to the following conditions: Brief narrative of procedures Testing conditions: Standard solutions: The reference standard is serially diluted at a constant dilution factor. Concentration range: x y ng/ml. Number of concentration levels: not less than Sample solutions: This product is serially diluted at a constant dilution factor. Concentration range: x y ng/ml. Number of concentration levels: not less than List of test conditions 25

26 Optimized description of method (2/3) Testing conditions(continue): Cell suspension of NFS-60 cells: Suspend cells with the assay medium to contain cells per 1 ml Volume added to culture plates Standard and sample solutions: 50 µl/well NFS-60 cell suspension: 50 µl/well Culture condition: 37 C (CO 2 incubator) Viable cell staining: Add 10 µl/well of the resazurin solution and culture at 37 C Fluorescence measurement: Excitation wavelength nm Measuring wavelength 590 nm Number of plates: Not less than three plates Regression model: 4 parameter logistic regression (4-PL) Ratio of potency (Pr): Pr = C value of standard solution / C value of sample solution Specific activity: Specific activity of this product (U/mg) = S GM DT/DS/C List of test conditions S: Potency of the reference standard material (U/mL) GM: Geometric mean of Pr DT: Dilution ratio for sample solution (1) DS: Dilution ratio for standard solution (1) C: Protein concentration of this product (mg/ml) 26

27 Optimized description of method (3/3) <System and sample suitability> Curve shape of standard solution: S-shape curve with a linear part between the asymptote. R 2 with the regression curve of standard solution must be not less than0.95. Percent geometric CV (%GCV) calculated according to the following equation must be not more than 25%. %GCV = 100 (esdln-1) SDln: the standard deviation of the natural log-transformed replicate relative potency (Pr) results for each sample. Criteria for assay performance <Reagents and Solutions> Iscove s modified Dulbecco s medium: Iscove s modified Dulbecco s medium manufactured by corporation, Catalog number: XXXXX or equivalent is used. NFS-60 cells: ATCC CRL-1838 is adapted with appropriate medium and freeze preservation. Assay medium: Add 10 vol% of fetal bovine serum, 1 vol% of a solution containing benzyl penicillin potassium ( U/mL) and streptomycin sulfate (0.01 g potency/ml), and vol% of 2-mercaptoethanol solution (9 in 125) to Iscove s modified Dulbecco s medium, and sterilize the medium by filtration. Resazurin solution: Resazurin solution manufactured by corporation, Catalog number: XXXXX or 27 equivalent is used.

28 Key Concept for Performance-based description Performance-based description consists of: 1. Fundamental information of the assay without any details of procedure e.g. Cell proliferation assay with NFS-60 cells 2. Performance required for the assay Test method can be changed under GMP control as long as the assay fundamental is same and the method satisfies the required performance Key is to secure assay performance required for a control of the quality attribute tested (The assay performance is regarded as ECs). 28

29 Performance-based description(1/2) <Acceptance criteria> Not less than xx 10 8 U/mg and not more than yy 10 8 U/mg <Principle of Testing Method> The specific activity of the sample solution is measured by cell proliferation assay using the NFS-60 cells. Standard and sample solutions are added to NFS-60 cells, followed by incubation and staining of viable cells. Ratio of potency of sample solution to that of standard solution is determined and, subsequently, specific activity per mg protein of this product is determined. <Performance criteria>* 1 Specificity: No specific activity by the addition of the other growth factors. Precision: Repeatability: Relative standard deviation should be less than 15% Intermediate precision: Relative standard deviation should be less than 20% *1:The method should satisfy the following performance criteria (Should be verified by analytical method validation in accordance with ICH-Q2) 29

30 Performance-based description(2/2) <Performance criteria(continue)> Linearity: R 2 with the regression line/curve should be 0.95 or higher Accuracy: Recovery of spiked samples should be within % Range: The above-mentioned performance criteria should be satisfied in the % range of the labeled potency. <System and sample suitability > Curve shape of standard solution: S-shape curve with a linear part between the asymptote. R 2 with the regression curve of standard solution must be not less than0.95. Percent geometric CV (%GCV) calculated according to the following equation must be not more than 25%. %GCV = 100 (esdln-1) SDln: the standard deviation of the natural log-transformed replicate relative potency (Pr) results for each sample. 30

31 Comparison table for 3 descriptions Description Pros Cons Japan M1.2 Optimized Performancebased Easiness to reproduce testing for regulators Easiness to follow analytical procedure and reproduce testing for regulators Clear understanding for key parameter & process Easiness for improvement of analytical procedure due to assay performance-driven Operational flexibility given Difficulty to understand what is key parameter & process Resource demanded for change control Hurdle for improvement of analytical procedure due to prior regulatory approval required Resource demanded for change control compared with performance-based Difficulty to understand actual analytical procedure for regulators 31

32 Summary The example of Japan M1.2 for cell-based bioassay on G-CSF was provided based on model SOP. Two descriptions of Established Conditions, optimized description and performance-based description, are proposed. Further discussion on ECs for analytical procedures are being held at JPMA and a collaboration with regulators will accelerate mutual understanding and directional harmony to ECs. 32

33 Thank you for your attention. 33

34 BACKUP 34

35 Analytical procedure steps described in each option Step of SOP Japan M1.2 Optimized Performancebased 1. Preparation of medium No No No 2. Preparation of assay cells No No No 3. Preparation of sample solution Yes Yes (concentration range) 4. Preparation of standard solution Yes Yes (concentration range) 5. Addition of each solution to the plate Yes Yes No 6. Preparation of cell suspension Yes Yes No 7. Addition of cell suspension to the plate Yes Yes No 8. Cell culture Yes Yes (minus CO 2 level) No 9. Addition of cell counting reagent and incubation Yes Yes (minus CO 2 level and culture duration) 10. Measurement of fluorescence intensity Yes Yes No No No No 35

36 Analytical procedure steps described in each option Step of SOP Japan M1.2 Optimized Performancebased Calculation of potency Yes Yes No System suitability Yes Yes/No* Yes/No* Sample suitability Yes Yes/No* Yes/No* 12. Reagents and Solution Yes Yes No Performance of the method Analytical target profile/ validation criteria No No Yes * List criteria which are based on assay performance (e.g., fitness of the curve). System/sample suitability criteria which possibly change when testing conditions (reagents and equipment) change (e.g., counts from negative control, levels of asymptotes) are not indicated. 36

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