Development of Analytical Control Strategies for L-DOS47,

a Novel Antibody-Enzyme Conjugate

IBC Life Sciences March 1, 2012

L-DOS47 is a novel therapeutic candidate for lung adenocarcinoma • L-DOS47 is a conjugate of jack bean urease and the

single domain AFAIKL2 antibody fragment • AFAIKL2 localizes L-DOS47 to lung adenocarcinoma

cells through its binding to the carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6)

• Urease provides L-DOS47 with enzymatic activity that generates a cytotoxic microenvironment that can be targeted to cancer cells

Overview of L-DOS47 as a Therapeutic Drug

From the Helix BioPharma website – used with permission

Overview of L-DOS47 as a Therapeutic Drug

From the Helix BioPharma website – used with permission

Contract Development at KBI Biopharma

• Transfer/optimize or develop analytical methods for formulation development, API characterization, and release/stability testing

• Develop a robust lyophilized formulation for the L-DOS47 drug product

• Produce a non-cGMP lot of drug product for preclinical studies

• Perform release and stability testing of clinical (cGMP) material

A lyophilized formulation for L-DOS47 The target formulation for L-DOS47 was a lyophilized product with a 0.9 mL fill volume and 1.8 mg/mL API. Buffer and excipient selections were driven, in part, by the desire to produce an elegant cake with a good stability profile.

Methods have been developed for release and stability of the L-DOS47 drug product

and drug substance

• Conjugation ratio using the Bio-Rad Experion automated electrophoresis station

• Binding potency by ELISA • Enzymatic activity using a colorimetric assay • SEC-HPLC is the main Purity method

Conjugation Ratio using the BioRad Experion Automated Electrophoresis System

Electropherogram of L-DOS47

Urease subunit with 0 antibodies Urease subunit with 1 antibody Urease subunit with 2 antibodies Urease subunit with 3 antibodies Urease subunit with 4 antibodies

Samples with varying conjugation ratios displayed in a traditional gel format

Virtual Gel Lane Sample Conjugation

Ratio

1 Urease 0

2 L-DOS47 1.7

3 L-DOS47 3.0

4 L-DOS47 4.8

5 L-DOS47 5.8

6 L-DOS47 (reference material)

9.9

Relative Binding Potency by ELISA

0

0.5

1

1.5

2

2.5

3

3.5

4

0.001 0.01 0.1 1

Standard Curve

Conjugation Ratio=1.7

Conjugation Ratio=3.0

Conjugation Ratio=4.8

Conjugation Ratio=5.8

Conjugation Ratio=9.9

Concentration of L-DOS47 (μM)

Increasing the conjugation ratio (AFAIKL2:Urease) increases the relative binding to the CEACAM6-A antigen. A standard curve is generated from the L-DOS47 reference standard, which is used to determine a relative binding potency of test samples.

Enzymatic Activity by Colorimetric Assay

The enzymatic assay is performed in a 96-deepwell plate. Aliquots from the reaction wells are transferred to a 96-well plate for development (image above on left). The absorbance of indophenol blue is proportional to the amount of ammonia that was generated. A standard curve (above on right) is generated using an ammonia standard, which is used to quantify the ammonia generated from the test samples. This assay is used for the L-DOS47 drug substance and drug product, as well as the drug intermediate (urease).

Standard Curve

Test Samples

The SEC-HPLC method can resolve the L-DOS47 Drug Substance from the Drug Intermediates

m in10 20 30 40 50

m AU

0

200

400

600

800

1000

34.

314

36.

626

m in10 20 30 40 50

m AU

0

10

20

30

40

50 27.

142

m in10 20 30 40 50

m AU

0

10

20

30

40

50

60

70 24.

807

AFAIKL2 Antibody Fragment (Drug Intermediate) 14 kDa

Urease (Drug Intermediate) 545 kDa

L-DOS47 (Drug Substance) 680 kDa

SEC-HPLC of Urease and L-DOS47

m in10 15 20 25 30 35

m AU

0

10

20

30

40

50

60

70

80

90

27.

142

24.

807

Urease

L-DOS47

Aggregation of L-DOS47 and the solution pH

• Aggregation is strongly pH dependent • pH was a critical factor in formulation

development and analytical method development

• SEC-HPLC was used as the primary method for monitoring L-DOS47 aggregation

Investigation of aggregation • The pH of the DS Lot 1 drug substance

was adjusted and the samples were measured by SEC-HPLC

• One set of samples was placed at 2-8°C and a second set was placed at 25°C/60% RH. Both samples were held for two weeks. The samples were monitored by SEC-HPLC after 1 week and 2 weeks.

Overlay of 1 week at 25°C

m in10 15 20 25 30 35

m AU

0

20

40

60

80

100

120

140

160pH 6.0 pH 6.2 pH 6.4 pH 6.6 pH 6.8 pH 7.0

2 weeks at 25°C and pH 6.0

m in10 15 20 25 30 35

m AU

0

25

50

75

100

125

150

175

DAD1 A, Sig=280,4 Ref=off (D:\BEANST~1\SECRAW~1\T_0~1\052109P8.D) DAD1 A, Sig=280,4 Ref=off (D:\BEANST~1\SECRAW~1\T_2W5C~1\60509008.D) DAD1 A, Sig=280,4 Ref=off (D:\BEANST~1\SECRAW~1\T_2W25~1\60509021.D)

T=0 T= 1 week T=2 weeks

2 weeks at 25°C and pH 7.0

m in10 15 20 25 30 35

m AU

0

20

40

60

80

DAD1 A, Sig=280,4 Ref=off (D:\BEANST~1\SECRAW~1\T_0~1\011-1102.D) DAD1 A, Sig=280,4 Ref=off (D:\BEANST~1\SECRAW~1\T_2W5C~1\60509018.D) DAD1 A, Sig=280,4 Ref=off (D:\BEANST~1\SECRAW~1\T_2W25~1\60509031.D)

T=0 T= 1 week T=2 weeks

SEC-HPLC of Two Lots of L-DOS47 Drug Substance

Both lots show out of specification results at the 25°C/60% RH conditions at 1 and 3 months. The frozen (-70°C) and 2-8°C conditions remain within specification throughout the study (6M or 18M).

SEC-HPLC of Three Lots of L-DOS47 Drug Product The frozen (-20°C) and 2-8°C conditions remain within specification throughout the studies (21M or 24M). Out of specification results were reported for the 25°C/60% RH and 40°C/75% RH conditions.

SEC-HPLC of an Out of Specification Drug Substance Sample

Blue = L-DOS47 ref std Red = DS Lot 2, 3 Months @ 40°C/75% RH

A shoulder peak appeared at an apparent lower molecular weight and a large aggregate peak appeared at the excluded volume.

SEC-HPLC of Out of Specification Drug Product Samples

In the drug product, the main impurity is a shoulder peak at an apparent lower molecular weight.

DP Lot 2 6 months @ 40°C/75%RH

DP Lot 3 21 months @ 25°C/60%RH

Enzymatic Activity of Two Lots of L-DOS47 Drug Substance Both lots show a major loss of activity at the 25°C/60% RH conditions at 1 and 3 months. Enzymatic activity was preserved in the frozen conditions and a small loss of activity appears to occur after 6 months in the refrigerated samples.

Enzymatic Activity of Three Lots of L-DOS47 Drug Product

Enzymatic activity remained stable in the frozen, refrigerated, and 25°C/60% RH conditions. A loss of activity was seen at 40°C/75% RH over a 6 month period.

Binding Activity of Two Lots of L-DOS47 Drug Substance Both lots show a major loss of binding activity at the 25°C/60% RH conditions at 3 months. Binding activity was preserved in the frozen conditions and refrigerated samples.

Binding Activity of Three Lots of L-DOS47 Drug Product

Binding activity remained stable in the lyophilized drug product at all stability conditions.

Conjugation Ratio of Two Lots of L-DOS47 Drug Substance Conjugation ratio remained stable for both lots of drug substance at all stability conditions.

Conjugation Ratio of Three Lots of L-DOS47 Drug Product

Conjugation ratio remained stable in the lyophilized drug product at all stability conditions.

Summary and Conclusions • Analytical methods have been developed to monitor

the key attributes of L-DOS47 necessary for its therapeutic function.

• Specifications have been defined that are compatible with the sponsor’s ability to manufacture the drug substance and drug product and reflect the target quality of the drug substance and drug product.

Summary and Conclusions • The methods are able to detect impurities in the drug

substance and drug product at accelerated conditions and correlate the loss of purity with loss of enzymatic activity and binding activity.

• Real time stability studies have shown that the frozen drug substance is stable for > 1.5 years and the drug product is stable for > 2 years at 2-8°C (or an alternate storage condition of -20°C).

Acknowledgements

Juan Davagnino, PhD Steven Cottle, MS Pawel Pirog Crystal Gordon Vivian Ring Amy Venturini

Heman Chao, PhD Kim Gaspar, PhD Carl DeLuca, PhD Baomin Tian, PhD Wah Wong, PhD