Federal University of Santa Maria

SÉRGIO LUIZ DALMORA, PhD

CASSS – Brasília – 2014

BIOASSAYS AND ALTERNATIVES FOR THE

QUALITY ASSESSMENT OF BIOTECHNOLOGY -

DERIVED MEDICINES

BIOASSAYS

• BIOLOGICAL POTENCY ASSESSMENT

• Design

• Precision

• Validation

• CHALLENGES

• Variability

• Three Rs

• QUALITY CONTROL OF BIOTHERAPEUTICS. BIOSIMILAR STUDIES

• ALTERNATIVES

2

• QUALITY

• EFFICACY

• SAFETY

ANALYTICAL TECHNOLOGIES

IDENTITY

PURITY

STABILITY

BIOLOGICAL POTENCY/CONTENT

3

Combination of biological, physicochemical and immunological methods

(rhEPO)

The complex structure of the sialoglycoprotein hormone

consists of a 165 aminoacids polypeptide chain, heavily

glycosilated at three N-linked and one O-linked

glycosilation sites, about 40%, and sialic acids. Molecular

mass of 30.4 kDa.

Isoforms

RECOMBINANT HUMAN

ERYTHROPOIETIN

4

BIOLOGICAL POTENCY EVALUATION OF

RECOMBINANT HUMAN ERYTHROPOIETIN

- BIOASSAYS -

Strains: CF1, BALB/c, B6D2F1

8 weeks-old females :

weight according to the strain: ± 2 g

Random distribution of animals per dose

• POLYCYTHAEMIC MICE

NORMOCYTHAEMIC MICE

5

8 mice per treatment group

Bioassay: 3 x 3 doses-1st day 10, 20, 40 5, 15, 45 IU/0.5 ml/mouse

- SINGLE INJECTION PROTOCOL

- MULTIPLE INJECTION PROTOCOL

6 mice per treatment group

Bioassay: 3 x 3 doses: Four days: 0.8 ; 2.4; 7.2 IU/0.2 l/injection/mouse

Blood sampling 5th day

RETICULOCYTES COUNTING BY FLOW CYTOMETRY

STATISTICAL ANALYSIS

-COMBSTATS (EDQM- EP.)

6

Linearity of dose

response curve

Limit of quantitation

Specificity

Precision

Accuracy

Robustness

Validation

LABORATORY ANIMALS CERTIFICATION

7Guidelines

VariablesPotency found

IU/ml

Confidence intervals

(P= 0.95)

Optimized

conditions

Animal strain

CF1 3.972 3.240 – 5.040

-BALB/c 4.036 3.560 – 4.840

B6D2F1 4.048 3.320 – 5.040

Animal age (weeks)

7 4.156 3.320 – 5.880

88 4.056 3.160 – 5.400

9 3.936 2.760 – 5.960

Buffer (pH)

7.0 4.132 3.360 – 5.040

7.27.2 4.044 3.320 – 5.000

7.4 3.868 3.200 – 4.840

Injection (ml)

0.1 4.112 2.760 – 5.880

0.20.2 3.984 3.040 – 5.200

0.3 3.932 2.960 – 5.360

Solutions stability, preparedDaily 4.032 3.200 – 5.160

WeeklyWeekly 3.988 3.160 – 5.000

Injection protocolSingle 4.088 2.360 – 6.400

MultipleMultiple 3.972 3.040 – 5.560

Animals per group

4 3.936 2.440 – 6.360

66 4.092 2.840 – 5.800

8 4.024 3.080 – 5.480

Validation parameter of the Normocythaemic Mice Bioassay

a

ROBUSTNESS TESTING

8

Cell culture assays

Physicochemical methods

ALTERNATIVES AND ADVANCES

Cell lines: AS-E2, TF-1

- LIQUID CHROMATOGRAPHY METHODS

- CAPILLARY ELECTROPHORESIS METHOD

9

A

A = Ph. Eur. BRP for rhEPO 1.7 µg/50µl = 200 IU Retention time: 53.45

B = Sample 1.7 µg/50µl = 200 IU

B

CORRELATION BETWEEN REVERSED-PHASE LIQUID

CHROMATOGRAPHY METHOD AND BIOASSAY FOR rhEPO

Content/Potency: peak 3

Bioassay: 106.70%

RP-LC: 99.53%Area: 112.855

Area: 113.388

3

3

3

10

SIZE EXCLUSION- SE-LC - chromatograms of rhEPO. Peaks 1: aggregates, 2: dimer, 3:

monomer and 4: excipients:

(A) Ph. Eur. BRP for Erythropoietin

(B) Biopharmaceutical formulation

(C) Formulation: stress conditions by temperature

11

12

(A) N-acetilneuraminic acid reference substance

(B) Sample of rhEPO

Peaks 1 e 2: sialic acids

DETERMINATION OF SIALIC ACIDS

- rhEPO -

Cell line TF-1 (ATCC CRL-2003): 1.0 x 105 cells/ml (3 x 104 cells/well)

Dilutions series: 0.10-1.60 IU/ml of rhEPO

Tetrazolium sodium salt (XTT) (1mg/ml)

Absorbance at 450 nm

BIOACTIVITY AND SIALILATION

BIOACTIVITIES

Dimer/aggregates 10% ± 2.76 (n=3)

Deamidated/sulphoxides 9% ± 3.49 (n=3)

In vitro TF-1 cell culture assay

13

Sample

Theoretical

amount

IU/mL

Mice bioassay a TF-1 proliferation bioassay a

Sialic acids

(ng/mg rhEPO)Potency

(%)

Confidence

intervals

(P = 0.95)

Potency

(%)

Confidence

intervals

(P = 0.95)

1 10.000 96.00 69.30–138.68 94.20 69.30–138.68 140.61

2 4.000 110.00 75.65–141.42 108.40 75.65–141.42 171.54

3 10.000 93.30 67.56–136.76 90.50 67.56–136.76 108.74

4 10.000 96.50 69.97–134.01 93.10 69.97–134.01 115.38

5 4.000 96.40 70.83–133.88 93.00 70.83–133.88 122.67

6 4.000 102.90 74.21–131.53 99.30 74.21–131.53 145.46

7 10.000 98.00 71.24–129.32 96.30 71.24–129.32 144.90

8 10.000 105.30 75.10–138.26 102.50 75.10–138.26 150.42

9 10.000 123.50 82.59–155.43 120.60 82.59–155.43 188.15

Mean a – 102.43 – 99.76 – 143.09

SD b – 9.52 – 9.59 – 20.21

a Mean of three replicates. b = SD, standard deviation.

Comparative potency assessment of rhEPO in biopharmaceutical formulations by

in vivo and in vitro bioassay, and determination of sialic acids by the RP-LC

method

14

Sample

Theoretical

amount

IU/mL

RP-LCa SE-LCa

Deamidated/

sulphoxides

forms (%)

Main

peak

(%)

HMM

(%)

Dimer

(%)

Monomer

(%)

1 10.000 0,19 94.12 0.23 0.10 95.34

2 4.000 0.28 107.78 0.31 0.17 107.98

3 10.000 0.22 91.98 0.21 0.08 92.76

4 10.000 2.71 93.42 1.22 3.12 95.54

5 4.000 0.45 93.91 0.35 0.15 94.36

6 4.000 0.35 100.92 0.29 0.09 101.99

7 10.000 0.31 97.40 0.32 0.05 97.56

8 10.000 0.42 101.55 0.11 0.12 102.97

9 10.000 0.68 121.86 0.49 0.20 122.49

Mean a - 0.36 100.32 0.28 0.11 101.22

SD b - 0.15 9.53 0.10 0.05 8.82

a Mean of three replicates. b SD = standard deviation.

Comparative content/potency evaluation of rhEPO in biopharmaceutical

formulations by RP-LC and SE-LC methods

15

FILGRASTIM

Non-glycosilated Human Granulocytes

Colony-stimulating Factor - (rhG-CSF)

Molecular structure consists of a 175 aminoacids

polypeptide chain containing an extra methionine at its N-

terminus. Molecular mass 19 kDa.

16

NEUTROPENIA MICE BIOASSAY IN VIVO

-- 8 week-old female Balb/c mice

-- Ifosfamide: 4.3 mg/mouse

- - 5th day anaesthesia blood sampling

- - Neutrophils counting

BIOASSAYS

IN VITRO CELL CULTURE

- Cell line MNFS-60: 7 x 105 cells/ml

- Dilutions series: 12.5 - 200 IU/ml

- MTT and absorbance at 595 nm

17

Peaks: 1 - Deamidated 2 - Sulphoxides 3 - Non-altered form

REVERSED-PHASE LIQUID CHROMATOGRAPHY

- FILGRASTIM -

0

5

10

Minutes

0 5 10 15 20 25 30 35 40 45

0

10

20

mA

Um

AU

B

A

(2)

(3)

(4)

(1)

(1)

0

5

10

Minutes

0 5 10 15 20 25 30 35 40 45

0

10

20

mA

Um

AU

B

A

(2)

(3)

(4)

(1)

(1)

1

2

3

Linearity: 10 – 300 µg/ml

LOQ: 10 µg/ml

18

Peaks: 1 - Aggregates 2 - Dimers 3 - Monomer 4 - Excipients

mA

U

Minutes

0 5 10 15 20 25 30

0

100

200

25

.02

18

.11

16

.94

13

.41

3

mA

U

Minutes

0 5 10 15 20 25 30

0

100

200

25

.02

18

.11

16

.94

13

.41

1

2 4

SIZE EXCLUSION LIQUID CHROMATOGRAPHY

- FILGRASTIM -

Linearity: 100 - 300µg/ml

LOQ: 45 µg/ml19

BIOACTIVITY OF HIGHER MOLECULAR MASS AND

RELATED PROTEINS

Bioassay using MNFS-60 cell line

Neutropenia mouse bioassay

Desamidated and sulphoxides: 15.67 ± 5.73% (n=3)

Dimers and aggregates: 14.60 ± 3.81% (n=3)

20

min5 10 15 20 25 30

mAU

0

5

10

15

20

25

DAD1 C, Sig=195,4 Ref =of f (FILGR\08051608.D)

2

1

Peak:

(1) Internal Standard – Leuprorelin acetate

(2) Filgrastim

Eletrophoretic conditions : Electrolitic solution of 50 mM di-sodium tetraborate decahydrated, pH 9.0.

Capillary of fused-silica, thermostatized at 15 ºC. Hydrodynamic injection for 6 s at 50 mbar and a constant

voltage of 15 kV. Detection at 195 nm

ELECTROPHEROGRAM OF FILGRASTIM

21

Sample

CZEa SE-LCa RP-LCa In vitro bioassaya

(%)

Monomer

(%)

HMMb

(%)

Main peak

(%)

Deamidated/

sulphoxides (%)

Potency

(%)

Confidence intervals

(P = 0.95)

1 100.23 100.02 0.12 100.05 2.11 103.54 98.71 – 108.44

2 89.25 86.37 0.31 90.58 1.00 91.72 86.43 – 97.30

3 76.76 77.91 0.61 80.33 1.45 82.41 78.35 – 86.67

4 91.84 87.74 4.25 89.90 0.71 92.02 88.61 – 95.59

5 95.68 95.53 0.16 97.79 1.56 100.53 96.90 – 104.23

6 101.47 100.92 0.09 102.35 0.82 104.91 98.32 – 112.01

7 103.20 102.15 0.21 104.13 0.35 105.21 100.10 – 110.62

Mean 94.06 92.95 0.82 95.02 1.14 97.19 –

SDc 9.19 9.14 1.52 8.48 0.60 8.67 –

aMean of three replicatesbHMM = higher molecular mass

substancescSD = standard deviation

Comparative content/potency of filgrastim in biopharmaceutical formulations

22

CELL CULTURE ASSAY IN VITRO

• Daudi cells anti-proliferative assay

23

RECOMBINANT INTERFERON-α2a

LIQUID CHROMATOGRAPHY METHODS

RP-LC Chromatogram of rhIFN-α2a (6 MIU/ml): SBR-rhIFN-α2a after

degradation by oxidative conditions:

peak 1 – deamidates

peak 2 – sulphoxides

peak 3 – rhIFN-α2a

24

SE-LC Chromatogram after degradation by glutaraldehyde:

peaks 1,5 and 7 – excipients

peak 2 = aggregates

peak 3 = dimmers

peak 4 = rhIFN-α2a

peak 6 = glutaraldehyde25

Comparative content/potency evaluation of Interferon-α2a in

biopharmaceutical formulations by LC methods and bioassay

Sample

SE-LCa,b RP-LCa,b Bioassaya

Monomer HMW Main peakDeamidate/sulphoxides

Potency Confidence intervals

(%) (%) (%) (%) (%) (P = 0.95)

1 105.17 n.d.d 98.75 n.d.d 102.80 94.80–109.98

2 103.17 0.23 97.04 0.38 101.70 92.55–107.81

3 97.50 0.41 92.77 0.45 99.96 93.86–108.96

4 101.17 n.d.d 95.18 0.51 98.80 91.78–106.23

5 102.44 0.79 100.13 0.26 98.95 92.03–108.85

6 95.67 0.18 94.87 0.19 91.10 82.81–99.39

7 107.90 0.54 105.84 n.d.d 106.42 98.79–114.93

8 96.22 0.15 93.01 0.89 97.50 88.72–104.32

Mean 101.15 0.28 97.20 0.33 99.65 –

SDc 4.39 0.27 4.35 0.29 4.46 –

aNon-significant difference (p> 0.05).

bMean of three replicates.

cSD = relative standard deviation.

dn.d = non detected.26

BIOASSAY

6 female rats wistar (19 – 22 days) per dose

Random distribution standard and sample

Subcutaneous administration: three days - 0.5 ml

4th day: Remove and weight the ovaries

1.5 - 3 - 6 IU/rat

Statistical analysis- Parallel line method (3x3)

RP-LC CORRELATION

FOLLICLE-STIMULATING HORMONE – rhFSH-

27

Cell line UMR-106 (ATCC CRL-1661): 6.0 x 105 cells/ml

Dilutions series: 25 - 100 µg/ml of rhPTH

MTT solution and microplate reader absorbances at 595 nm

UMR-106 cell proliferation bioassay In vitro

Chicken hypercalcemia bioassay

Male chickens COBB (120-140 g)

Standard and test samples dilutions concentrations of 10, 25 and 62.5 µg/ml

Absorbances at 620 nm. Assessment of serum calcium concentration

RECOMBINANT PARATHYROID HORMONE

TERIPARATIDE – rhPTH (1-34)

28

Comparative content/potency evaluation of rhPTH in biopharmaceutical

formulations by LC methods, in vivo and in vitro bioassays

Sample

RP-LCa SE-LCa In vivo bioassay In vitro bioassay

SulphoxidesMain peak

Agregates/dimer

Monomer PotencyConfidence

intervalsPotency

Confidenceintervals

(%) (%) (%) (%) (%) (P = 0.95) (%) (P = 0.95)

1 0.45 99.31 0.08 99.84 100.93 95.63–106.52 98.43 92.74–104.52

2 0.22 101.35 0.46 101.13 103.56 95.68–112.07 97.72 91.37–104.58

3 0.53 99.29 1.32 98.87 99.84 85.54–116.51 101.57 96.53–106.71

4 1.84 98.42 0.19 99.05 100.29 94.27–106.62 97.29 92.12–102.65

5 0.31 107.56 0.14 107.18 108.05 99.81–117.03 106.94 101.76–112.43

6 4.24 93.12 3.15 91.43 91.47 82.65–101.18 91.18 84.12–98.34

7 0.39 100.74 0.13 100.45 102.81 96.32–109.89 99.65 93.04–106.65

8 0.28 102.89 0.72 99.30 100.83 94.28–107.81 102.06 96.75–107.78

Mean 1.03 100.33 0.77 99.66 100.97 – 99.35 –

SDb 1.40 4.10 1.05 4.28 4.66 – 4.54 –

a Mean of three replicates. b SD = relative standard deviation.29

INTACT BIOMOLECULE IC50= 69.43 µg/ml

SULPHOXIDES IC50= 46.90 µg/ml

DIMERS AND AGGREGATES IC50= 49.64 µg/ml

TERIPARATIDE – rhPTH (1-34)

Cell line: NCTC 929 - 2 x 105 cells/ml

CYTOTOXICITY TEST

Neutral red uptake

30

Cell line TF-1 (ATCC CRL-2003): 4.0 x 105 cells/ml

Dilution series: starting with 65 IU/ml (250 μg/ml)

MTT solution and absorbance measured at 595 nm

TF-1 Cell Proliferation Bioassay In vitro

Thrombocytopenia Bioassay In vivo

Female Balb/c mice (18-23 g)

Standard and test concentrations between 350 and 700 µg/ml

Mytomycin C: dose of 40 µg

Results as platelets count and neutrophils number.

RECOMBINANT HUMAN INTERLEUKIN-11

(rhIL-11)

31

Comparative content/potency evaluation of rhIL-11 in biopharmaceutical formulations

by RP-LC method and TF-1 proliferation bioassay

Sample

RP-LC a In vitro Bioassay a*

Main peak (%) Deamidated/

Sulphoxides (%)Potency (%) Confidence Intervals (P=0.95)

1 97.69 0.25 95.04 88.15 – 105.64

2 101.17 1.16 97.89 94.11 – 108.27

3 98.09 0.88 100.14 95.66 – 107.46

4 97.60 0.56 93.23 85.74 – 106.61

5 102.40 1.63 103.30 96.14 – 97.93

6 101.36 1.57 96.45 92.15 – 108.95

7 96.95 0.79 93.26 85.16 – 100.33

8 103.83 0.34 98.92 93.67 – 106.84

Mean 99.88 0.77 97.28 –

SD b 2.60 0.43 4.10 –

a Mean of three replicates. b SD = relative standard deviation.

*In vivo bioassay showed potency 3.82% lower than in vitro assay.

32