Potency Bioassays: Cell-based assay

Transcription

1 Potency Bioassays: Cell-based assay Nongyao Somdach Institution of Biological Product Department of Medical Sciences Ministry of Public Health 1

2 Outline Potency-What?-Why? Vaccine-What?- Why? Potency assay Challenges Attributes Approaches Past-Present-Future Cell-based assay 2

3 Potency assay 3

4 Potency assay (contd.) 4

5 Potency assay-why? Demonstrate product quality. To be efficacious & safe Stable, acceptable for use within expiry date Demonstrate manufacturing consistency & comparability 5

6 Potency assay-challenges Variety : licensed vaccine types Vary degrees : purity Differing complexities: mono/multimeric Chemical heterogeneity: active moieties Valency: single/ >1 type/serotypes 6

7 Potency assay-attributes Specific to product Quantitative, accurate & linear in the rage of interest Precise, reproducible & transferable Validatable for intended use in a compliant environment Stability indicating, providing information on shelf-life/expiry date claims Linked to biological functionality clinical functionality 7

8 Potency assay-rational Approach For potency assay selection & development, a critical linkage is clinical efficacy But results of clinical efficacy are not available during early phase product/potency assay development Understanding of critical structural attribute(s) of the product, its role in mechanism of action, and expected biological response (clinical Biomarker) should be developed during research/preclinical phase 8

9 Potency assay-rational Approach (contd.) For example: Prevention/ Reduction in culture confirmed illness/ symptoms Neutralizing Ab level as Protective titer (Surrogate) Measurable close PFUs, CFUs, Ag mass (Surrogate of Surrogate) 9

10 Potency assay-in the Past ( ) Direct measure; biological activity-unique & specific In vivo: Early 1900s Lethal challenge in animal module or clinical data Refinement s Measurement of reduction in clinical symptoms Pertussis, Rabies, Diphtheria, Tetanus 10

11 Potency assay-present In Vivo + Vitro Immunogenicity Potency assay (Hybrid) In Vitro Biological replication of virus /bacteria In Vitro Immunochemical- Antigenicity potency assay In Vitro Physico-chemical- Mass potency assay 11

12 Potency assay-present In Vivo + Vitro Immunogenicity Potency assay (Hybrid) In Vitro measurement of In Vivo biological response in relevant animal model Measure biomarker functional response (neutralization assays) Measure biomarker total response (ELISA) e.g. Pertussis, Rabies, Diphtheria, Tetanus 12

13 Potency assay-present In Vitro Biological replication of virus /bacteria Virus replication: End point measurement: cytopathic effect/ plaque formation Early measurement : qpcr/ viral Ag expression Bacterial replication: End point measurement: Visible colony formation Early measurement : ATP bioluminescence / Ag expression 13

14 Potency assay-present In Vitro Immunochemical- Antigenicity potency assay Nephelometry measuring formation light scattering complexes SRID ELISA IVRP measuring precipitating complexes measuring Ag-Ab binding from std curve measuring Ag-Ab binding from parallel line analysis Competitive measuring binding competition using 4 parameter fit Except: live vaccine 14

15 Potency assay-present In Vitro Physico-chemical- Mass potency assay HPLC/ HPSEC assay polysaccharide content Chemical assay protein & specific Ag measurement. 15

16 Potency assay-future New vaccines AIDS, Tuberculosis, Tetravalent Influenza Cancer, Diabetes, Autoimmune, Alzheimer s Malaria, West Nile, Dengue DNA vaccines 16

17 Potency Bioassays: Cell-based assay In Vitro Biological replication of virus /bacteria End point measurement 17

18 Potency Bioassays: Cell-based assay End point measurement Colony Forming Unit: CFU Plaque assay Plaque Reduction Neutralization Test: PRNT Cell Culture Infective Dose 50%: CCID 50 18

19 Colony Forming Unit: CFU Bacterial replication on selective media Visible colony formation March 11, 2016 By Nongyao Somdach IBP,DMSc 19

20 Terminology and abbreviation Colony Forming Units (CFU) is a colony of bacteria forming from single or a cluster of bacteria cell. 20

21 BCG Vaccine Potency Test: CFU 21

22 Principle Freeze dried BCG Vaccine is a dried preparation of live bacteria. Determination of potency can be carried out by culturing vaccine suspension on a selective media and find out the number of culturable particles (CFU) of BCG. 22

23 Equipment and Supplies 23

24 Reagent and Medium Sauton solution L-asparagine monohydrate Citric acid trisodium salt dihydrate K2HPO4 anhydrate MgSO4.7H20 Feric ammonium citrate Glycerin Lowenstein Jensen Medium Lowenstein Medium Base Glycerol Fresh egg 24

25 Sauton solution 25

26 Lowenstein Jensen Medium Glycerol 26

27 Procedure Sample Medium Potency test : kept at 4 C Warm medium at 37 C, 2-3 hrs Stability test : kept at 37 C for 4 weeks Give a random number to a tube by Bioassay random 27

28 Procedure vaccine WRP (Sauton solution) (Sauton solution) 1 ml./vial 1 ml./vial Pool (vaccine) 28 WRP

29 Procedure Pool vaccine 1 ml. 1 ml. 1 ml. 2 ml. 2 ml. (Sauton solution) 99 ml. 99 ml. 2 ml. 6 ml. 7 ml. A B C D E :2x10 4 1:4x10 4 1:8x

30 Procedure Inoculate 0.1 ml of each dilution to medium Potency test Stability test WRP Used dilution C, D, E (Medium 5,5,10 tubes) Used dilution B, C, D (Medium 5,5,10 tubes) Used dilution C, D, E (Medium 5,5,10 tubes) 30

31 Procedure Tighten screw caps of each tube and reciprocate every 5 min until the inoculums is completely absorbed by the medium. Wrap all tubes with aluminium foil. Incubate at 37 C for 4 weeks. Count the Colony Forming Unit 31

32 Calculation An appropriate formula for calculation of CFU / ml is selected depending on a condition as follows If 2ω > X 1 +X 2 +2X 3, CFU/ml = d 1 /v. 1/2. (X 1 +X 2 +2X 3 ) If X 1 +X 2 +2X 3 > 2ω > X 2 +2X 3, CFU/ml = d 2 /v. ωx 1 / 2ω+ X 1 - (X 2 + 2X 3 ) 3. If X 2 +2X 3 > 2ω > 2X 3, CFU/ml = d 3 /v. ωx 2 / 2ω+ X 2-2X 3 4. If 2X 3 > 2ω, CFU/ml = d 3 /v. X 3 32

33 Quality Control 1. Working reference vaccine should be tested along with the sample. 2. Working reference should be consistency within +3SD of the titer calibrated by the Biological Standardization Section. 3. The working reference vaccine should be re-calibrate at least once a year or when its testing result is out of the range of + 3SD of the titer calibrated previously. 33

34 Quality Control Error within dilution (X 2 ) of dilution should be following the table I. and difference between dilution should be small than 2 Table I : X 2 ( n = number of inoculated tube, n-1 = degree of freedom) n n X

35 Reference 1. WHO, Requirement for Dried BCG Vaccine, WHO TRS no.771,1988, p WHO/TB/Technical Guide/77.9, WHO,BLG/UNDP/82.1 Rev.1,1982, p Minimum Requirement for Biological Products Volume I,1998, Thailand, p

36 Plaque assay JE Vero cell Varicella MRC-5 Yellow fever Vero cell Dengue C6/36, BHK-21, LLCMK2 March 11, 2016 By Nongyao Somdach IBP,DMSc 36

37 Potency Test Varicella Vaccine by Plaque assay 37

38 Potency Test Varicella Vaccine by Plaque assay Terminology and abbreviation Plaque Forming Units (PFU) areas of cell lysis (CPE) in monolayer cell culture, under overlay conditions, initiated by infection with a single virus particle 38

39 SPGC solution for VZV 0.5% sucrose 10 gm 0.1% Sodium glutamate 0.2 ml PBS ml Total 200 ml Adjust the ph to 7 and then add PBS up to 200 ml. Sterile by membrane filtration with 0.22 µm 39

40 Procedures MRC-5 cell preparation Warm growth media, PBS - and Trypsin - EDTA solution at water bath 37 o C for 15 min. Observe monolayer MRC-5 cells under microscope to check microorganism contaminations and morphology of cells. 40

41 MRC-5 cell 41

42 Discharge culture medium from the flask and wash monolayer cells with PBS - Add 0.025% Trypsin-EDTA solution and incubated at 37 o C, 5% CO 2 for 2 min. Tap the flask gently for cell detachment. 42

43 Add 5 ml of culture medium, then pipette cell several time until becoming a single cell. Count the number of cells. Normally use the ratio 1: 5 dilution. Adjust the cell number to 0.8 x 10 5 cells/ ml. 43

44 Add 3 ml of cell suspension (2.4 x 10 5 cells/well) into each well of 6- well plates. Incubate at 37 o C, 5% CO 2 for 2 days. 44

45 Vaccine virus infection Thaw freeze-dried varicella vaccine at water bath, 37 o C and then keep on ice. Dilute the vaccine sample at 0.5 log dilution (1x10-1, 1x10-1.5, 1x10-2, 1x and 1x10-3 ) with SPGC solution. 45

46 Varicella vaccine Virus dilulent SPGC solution Keep on ice all the time. 46

47 Discharge culture medium from 6 -well plate by using 10 ml serological pipette Gently add 3 ml of PBS - from the edge of the well for cell washing and then rocking gently by hand. Take PBS - out from the plate with serological pipette. 47

48 Add 100 µl of SPGC solution in control well by using micropipette tip. Add 100 µl of diluted vaccine into each well. Start from the highest dilution 1x10-3 to 1x10-1 respectively. Incubate at 37 o C, 5% CO 2 for 60 min rock every min. 48

49 Pipette 3 ml of maintenance medium into each well. Start with cell control first and then with highest dilution, respectively. Incubate at 37 o C, 5% CO 2 for 5 days. Observe infected cells every day after infection. 49

50 MRC-5 cell VZV infected, 4 days Uninfected cells VZV infected, 7 days 50

51 Cell staining After incubation, fix the cells with 2-3 ml of 5% Formaldehyde for min. Discharge formaldehyde and wash with pipe water 2-3 times. 51

52 Add 0.3 ml of 0.02% crystal violet for 1 min. Wash again several times and keep it dry at RT 52

53 Figure 1. Plaques of varicella zoster virus Infected MRC-5 cell (a) infected cells (b) uninfected cells 53

54 Calculations PFU/ml (Av. Plaques/ well) x Dilution factor Vol. inoculated/well 54

55 Plaque Reduction Neutralization Test: PRNT 55

56 Potency assay: inact. JE by PRNT Terminology and abbreviation "a serological test which utilizes the ability of specific antibody to neutralize virus and thereby prevent it from causing the formation of plaques in a cell monolayer. 56

57 Inact. JE vaccine Potency test : Plaque Reduction Neutralization Test JE vaccine (1:8,1:16, 1:32, 1:64) 1ml+ 3 ml PBS 1 st immunize mice 7 days 2 nd immunize Bleeding (1:10,1:40, 1:160, 1:640) 14 days Collect serum Ab Standard JE virus Virus- serum Ab mixture Inoculate in cell culture Incubation time : 6 days Plaque observation 57

58 Plaques 58

59 Calculations Calculate % of reduction of plaques (PRNT) in the sample vaccine and reference vaccine % PR= 100-((Mean no. of plaques in each dilution)x100) Mean no. of virus in virus control NT antibody titer (log10 Neutralizing antibody titer 50%) using Spearman and Kabur method. The parallel line programme: Japan and WHO 59

60 Calculation of log 50% PRNT where d m = largest log dilution = log(10) in this example d = difference between log dilutions = log(10)-log(40) = log(0.25) in this example p i = proportion of plaque reduction at serum dilution i. March 11, 2016 By Nongyao Somdach IBP,DMSc 60

61 Validity criteria Mean value of plaques shown in the virus control should be plaque-forming units (PFUs)/0.2 ml. The cells control are free from any plaques and abnormality. The PRNT should be calculated from the dilution used showing 50% reduction of neutralizing antibody titer. The requirements for the assay to give the statistically valid fit to the parallel. 61

62 Tissue/Cell culture infective dose 50% : TCID 50 / CCID 50 62

63 Cells susceptible to virus Vero cell Vero cell RK-13 cell MA 104 cell Hep-2 cell 63

64 Potency Test MMR Vaccine by CCID 50 assay 64

65 Identification : MMR vaccine Method: Seroneutralization Specific antibody neutralization Measles component : Anti-mumps + MMR vaccine Mumps component : Anti-measles + MMR vaccine Rubella component : Anti-mumps + MMR vaccine 65

66 Potency: MMR vaccine Method: microtitration assay on cell culture Measles component : on Vero cell Mumps component : on Vero cell Rubella component : on RK-13 66

67 Procedure Dilution preparation Media Virus Cell preparation Add: virus dilution & cells suspension Read the results & Calculation 67

68 Dilution preparation WRP/ Vaccine MM (ml) Dilution

69 Cell Counting using a haemocytometer March 11, 2016 Average number of cells in one large square x dilution factor* x 10 4 By Nongyao Somdach IBP,DMSc 69

70 Calculation of Cell Viability: No. of Viable Cells Counted x 100 = % viable cells Total Cells Counted (viable + dead) Ex. (( )/4) x 5 x 10 4 = 250 x 10 4 cells /ml = 2.5 x 10 5 cells /ml *dilution factor is usually 5 (1:4 dilution with trypan blue), but may need to further dilute (or concentrate) cell suspensions = conversion factor to convert 10-4 ml to 1 ml (refer to figure 3 to view a diagram of the arrangement and dimensions) 70

71 Adding virus suspension & cells suspension WPR : -Measles & Rubella Mumps sample : March 11, 2016 Measles : 2.0 x 10 5 cells/ml Mumps : 2.0 x 10 5 cells/ml Rubella : 2.5 x 10 5 cells/ml By Nongyao Somdach IBP,DMSc 71

72 Incubation Measles : 7-9 days at CO 2 incubator 37 C Mumps : 9-12 days at CO 2 incubator 37 C Rubella : 7-9 days at incubator 32 C March 11, 2016 By Nongyao Somdach IBP,DMSc 72

73 Vero Cytophatic effect Vero RK-13 March 11, 2016 Measles Mumps By Nongyao Somdach IBP,DMSc Rubella 73

74 Spearman & Karber formula logccid 50 = -log of lowest dilution used +{[( Σ of CPE wells /No. of well inoculated per dil n )- 0.5] x log dilution factor} Ex. Potency titer -Positive CPE 30 wells/plate -inoculated vaccine 0.05 ml/well ( 10 wells/dilution) March 11, 2016 Calculation: logccid 50 = - (-2.0) + [ (30/10) ] 0.5 logccid 50 = 3.25 CCID 50 = By Nongyao Somdach IBP,DMSc 74

75 Calculation: CCID 50 /dose/0.5ml CCID 50 per 0.05 = CCID 50 per dose = = log CCID 50 per dose = log log10 = = 4.25 CCID 50 per dose = or 4.25 logccid 50 /dose March 11, 2016 By Nongyao Somdach IBP,DMSc 75

76 The dilution range selected should cover dilutions that will infect between 0% and 100% of the culture inoculated. The distribution of CPE should be uniform The control cell should not demonstrate any CPE The titer of WRP should be within the range, which is obtained from the recent standardization (Mean ± 3SD) March 11, 2016 By Nongyao Somdach IBP,DMSc 76

77 Potency Test: OPV by CCID 50 assay 77

78 Potency Test: OPV CCID 50 assay 78

79 Potency Test: OPV CCID 50 assay 79

80 Potency Test: OPV CCID 50 assay 80

81 Potency Test: OPV CCID 50 assay 81

82 Potency Test: OPV CCID 50 assay 82

83 Potency Test: OPV CCID 50 assay 83

84 Potency Test: OPV CCID 50 assay 84

85 Potency Test: OPV CCID 50 assay 85

86 Potency Test: OPV CCID 50 assay -3.0 PV1 PV CC CC 86

87 Example: Calculation 87

88 Potency Test: OPV CCID 50 assay 88

89 Potency Test: OPV CCID 50 assay 89

90 Thank you March 11, 2016 By Nongyao Somdach IBP,DMSc 90