Gene Therapy and AAV: Advancing Analytical Characterization to Improve Product Understanding, Control and Comparability Exercises

CMC Strategy Forum17 July 2017

Herb Runnels, PhD

Pfizer, BioTx Pharmaceutical Sciences, Analytical R&D

Pfizer Proprietary

Practical Challenges:• Attribute criticality – field still in early understanding• Sample retain limitations due to small batch sizes• Limitations of standard assays – poor sensitivity, high

variability, technology availability

Comparability

Advancement of assays can improve attribute and product understanding, thus improving

comparability exercises

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• Introduction of New Assays: Mass Spec, RP-HPLC, Peptide Mapping and SEC-HPLC

• Case Study of Comparability Finding due to New Analytical Capabilities

• Importance and Challenge of Strong Potency Assay

• Residual Host Cell DNA Characterization: Qualitative PCR

• Improved Characterization (Full / Empty Ratio): EM and AUC

Presentation Outline

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Characterization and Comparability of AAV Capsid Proteins

• Intact Capsid Protein Analysis– Denature capsid and separate by RP chromatography prior to MS (RP-

HPLC/MS)

• Proteolytic Digest of Capsid Proteins (Peptide Mapping)– Denature capsid and digest with proteolytic enzyme prior to RP-HPLC-MS/MS

Capabilities of Mass Spec-Based Methods• Confirm amino acid sequence• Monitor clips/truncations• Determine cysteine oxidation state• Identify capsid protein modifications• Characterize unknowns/impurities

Mass Spectrometry

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AU

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes36.00 36.20 36.40 36.60 36.80 37.00 37.20 37.40 37.60 37.80 38.00 38.20 38.40 38.60 38.80 39.00 39.20 39.40 39.60 39.80 40.00 40.20 40.40 40.60 40.80 41.00 41.20 41.40 41.60 41.80 42.00 42.20 42.40 42.60 42.80 43.00

AU

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

Minutes34.20 34.40 34.60 34.80 35.00 35.20 35.40 35.60 35.80 36.00 36.20 36.40 36.60 36.80 37.00 37.20 37.40 37.60 37.80 38.00 38.20 38.40 38.60 38.80 39.00 39.20 39.40 39.60 39.80 40.00 40.20 40.40 40.60 40.80 41.00 41.20 41.40

VP2

VP3

VP1VP1VP2VP3

Traditional: SDS-PAGE Purity Advanced: RP-HPLC Purity

AAV Serotype “A”

VP2 VP1

VP3AAV Serotype “B”

More quantitative Greater sensitivity

Less resource intensive Amenable to characterization by on-line mass spectrometry

Advancement of Traditional Purity Assay

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AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

0.38

Minutes10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00 150.00 160.00 170.00 180.00

Traditional: Western Blot Advanced: Peptide Mapping

Less resource intensive Platform assay for different serotypes

Amenable to characterization by mass spectrometry Increased ability to distinguish highly similar sequences

Vector Capsid Identity:

6

VP1

VP2

VP3

AAV Serotype “B”

Advancement of Traditional Assays

Sequence Coverage (98.6%)

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Advancement of Traditional Assays

Traditional: ELISA

Advanced: SEC-HPLC

Less variability Less resource intensive

Platform assay for different serotypes

Vector Particle Titer:

7

1.00E+13

5.10E+14

1.01E+15

1.51E+15

2.01E+15

2.51E+15

3.01E+15

0 2000000 4000000 6000000 8000000 10000000 12000000 14000000

Linear from 1.0E+13 – 2.5E+15 vp/mL

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

AAV MonomerParticle Aggregates

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Case Study

Denaturation

38.5

39

38.8

28

39.1

61

AU

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

Minutes35.20 35.40 35.60 35.80 36.00 36.20 36.40 36.60 36.80 37.00 37.20 37.40 37.60 37.80 38.00 38.20 38.40 38.60 38.80 39.00 39.20 39.40 39.60 39.80 40.00 40.20 40.40 40.60 40.80 41.00 41.20 41.40 41.60 41.80 42.00 42.20 42.40 42.60 42.80 43.00 43.20 43.40

VP2 VP1 VP360 capsid protein subunits (VP1,VP2,VP3) assembled into icosahedron at a ratio of 1:1:10

The RP-HPLC assay is a denaturing assay

RP-HPLC Method:

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Case Study

41.6

06

43.2

83

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes36.00 37.00 38.00 39.00 40.00 41.00 42.00 43.00 44.00 45.00 46.00 47.00 48.00

37.4

39

39.0

71

AU

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

Minutes31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 39.50 40.00 40.50 41.00 41.50 42.00 42.50 43.00 43.50 44.00

39.1

61

AU

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

Minutes35.20 35.40 35.60 35.80 36.00 36.20 36.40 36.60 36.80 37.00 37.20 37.40 37.60 37.80 38.00 38.20 38.40 38.60 38.80 39.00 39.20 39.40 39.60 39.80 40.00 40.20 40.40 40.60 40.80 41.00 41.20 41.40 41.60 41.80 42.00 42.20 42.40 42.60 42.80 43.00 43.20 43.40

38.5

34

AU

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

Minutes34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 39.50 40.00 40.50 41.00 41.50 42.00

Impurity Impurity

Process 1 - 1 Process 1 - 2

Process 2 - 1 Process 2 - 2

Identity of the impurity determined by MS with Heightened Characterization

New impurity was detected by RP-HPLC following process change

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Case Study

The new impurity observed by RP-HPLC was NOT detected in SDS-PAGE

MWM Process 2 Process 1

100 kDa75 kDa

50 kDa

37 kDa

VP1

VP3VP2

SDS-PAGE Method:

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Case Study

RP-HPLC coupled to MS determines the identity of the impurity Knowledge is Power in understanding how to control the process

Intact Protein

RP-HPLC/MS

Process 1

Process 2

RP-HPLC/MS Method:

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Case StudyA

U

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

--- Batch 1--- Batch 3

The SEC-HPLC is a non-denaturing assay No difference in particle aggregates was observed between Process 1 and 2

Impurity is not inter-particle aggregates

Inter-particle multimers

Process 1Process 2

SEC-HPLC Method:

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Case Study

Small-scale DOE identifies causative step

AU

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Minutes34.00 35.00 36.00 37.00 38.00 39.00 40.00 41.00 42.00 43.00 44.00 45.00 46.00 47.00 48.00 49.00 50.00 51.00 52.00

Process Condition 1

Process Condition 2

Process Condition 1b

RP-HPLC Method:

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Case Study

RT: 39.7 - 48.7

40 41 42 43 44 45 46 47 48Time (min)

0

20

40

60

80

100 NL: 6.10E6m/z= 232.2903-232.2927+278.5470-278.5498+347.9320-347.9354+463.5735-463.5781+694.8566-694.8636+1388.7059-1388.7197 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20170315TWP03NL: 6.10E6m/z= 232.4543-232.4567+278.7438-278.7466+348.1780-348.1814+463.9015-463.9061+695.3486-695.3556+1389.6900-1389.7038 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20170315TWP03

Process 2-2

RT: 39.7 - 48.7

40 41 42 43 44 45 46 47 48Time (min)

0

20

40

60

80

100 NL: 1.70E7m/z= 232.2903-232.2927+278.5470-278.5498+347.9320-347.9354+463.5735-463.5781+694.8566-694.8636+1388.7059-1388.7197 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20160209TWP02NL: 1.70E7m/z= 232.4543-232.4567+278.7438-278.7466+348.1780-348.1814+463.9015-463.9061+695.3486-695.3556+1389.6900-1389.7038 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20160209TWP02

Process 1-1

RT: 39.7 - 48.7

40 41 42 43 44 45 46 47 48Time (min)

0

20

40

60

80

100 NL: 1.40E7m/z= 232.2903-232.2927+278.5470-278.5498+347.9320-347.9354+463.5735-463.5781+694.8566-694.8636+1388.7059-1388.7197 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20160209TWP04NL: 1.40E7m/z= 232.4543-232.4567+278.7438-278.7466+348.1780-348.1814+463.9015-463.9061+695.3486-695.3556+1389.6900-1389.7038 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20160209TWP04

Process 2-1

23.0% Deamidation

42.6% Deamidation

46.4% Deamidation

Peptide map-MS also revealed higher deamidation levels in Process 2

Peptide Mapping

RP-HPLC-MS/MSBesides the impurity, an additional change was discovered:

RP-HPLC-MS/MS Method:

Deamidation:

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Case Study: Summary

Process was changed to include new step and unit operation Newly implemented purity method (RP-HPLC) showed a new impurity after

this change Traditional purity method (SDS-PAGE) did not detect this new impurity RP-HPLC/MS determined the identity of the impurity SEC-HPLC confirmed the impurity was not inter-particle aggregates Additionally, RP-HPLC-MS/MS (peptide mapping) identified increased

deamidation levels after the process change

Follow-up experiments confirmed/mapped the impurity was caused by a specific change in the process

Process change was removed because of greater analytical understanding of the product

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1. AAV Infection

Tissue Selective Infectivity of AAVSerotypes

Find a cell line that can be infected

Readout: Usually qPCR

2. Protein Expression

Tissue Specific Promoters

Find a cell line that results in promoter activity and protein expression

Readout: ELISA, western blot

3. Protein Activity

Activity Assays require successful infectivity and protein expression

Required for pivotal trial material

Readout: Depends on the protein (e.g., enzymatic activity)

4. Relative Potency

Activity relative to a reference material enables • lot to lot consistency• controls for assay

variability

Reference Material choice is critical

Readout: Relative Potency (e.g., 103%)

Robust potency assay(s) are critical for comparability

Multi-Component Potency Assays

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Optimal target cell line

Infect with virus encoding Collect conditioned medium

from infected cells Assess Activity

Single , All-inclusive Potency Assay Encompasses: Infectivity Expression of Gene Functional Activity

210% 55%

Cell-Based Assay: Relative Potency

100% Vs 50% mock potency100% Vs 200% mock potency

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DOEs: Optimizing Potency Assays

Cell Line Screeningo Target cell line with maximum promoter

activity driving Gene-of-Interesto Cell line with high infectivity rate

Viral Infectiono Cell density at harvesto Cell density for infectiono Cell passageo Medium conditions to maximize infection and

downstream expression of Gene-of-Interest

Activity measurement

o Time of harvest of conditioned mediumo Choice of assay for activity measurement

Good potency assay robustness and precision are critical for gaining meaningful data to assess comparability.

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Process-Related Impurity: Host Cell DNA

• Residual host cell DNA levels in AAV products across industry tend to exceed regulatory guidelines (10 ng/dose)– Necessary to have process understanding to control as much as possible

and support comparability

• Advanced characterization is key for process and product understanding– Why can’t DNA be purified beyond a certain level?

• qPCR is used to quantitate DNA levels during AAV downstream processing

• When AAV samples are treated with benzonase or DNase I prior to qPCR, we find that a percentage of the DNA is protected, potentially inside the AAV particles

– What is the size of the residual DNA?• Qualitative assessment can be performed by PCR and agarose gel

electrophoresis

• Approaches such as qPCR with nested primers, electrophoresis and other techniques are under evaluation for quantitation of DNA of different sizes

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Host Cell DNA Size Characterization

Human 18S rRNA gene (1870 bp)

Fixed Forward Primer

• Forward primer(s) & different reverse primers were designed to attempt to amplify different lengths of a host cell gene sequence in AAV samples.

• PCR is performed and PCR products are visualized on an agarose gel to determine if DNA of varying sizes is present.

• This qualitative assessment tells us that we have host cell DNA >200 bp.

• This DNA appears to be protected from DNase by the particle.

Different Reverse Primers

PCR products of different sizes visualized on a gel

Fixed Forward Primer

~150 bp ~300 bp ~630 bp

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Sedimentation Coefficient (S20,w)20 40 60 80 100 120 140

c(s

20,w

)

0.00

0.05

0.10

0.15

0.20

Advanced: Analytical Ultracentrifugation

Direct quantitation Less susceptible to impurity interferences

Correlates well to electron microscopy images Ability to distinguish full, partially full, and empty particles

However, difficult to make a QC release assay

Empty

FullPartial

Traditional: Spectrophotometry

Full : Empty Ratio

Improved Characterization: Full/Empty Ratio

Vector Particle Content:

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11,000x30,000x

10% Empty

69% “Fully Full”

Sedimentation Coefficient (S20,w)50 100 150 200

c(s 20

,w)

0.00

0.02

0.04

0.06

0.08

0.10

Empty Capsids

FilledCapsids

Transmission Electron Microscopy (TEM)

Analytical Ultracentrifugation (AUC)

Sample enriched for full particles

Improved Characterization: Full/Empty Ratio

• TEM– Size and shape consistent with expectations– Empty & full capsids distinguished

• AUC– Separation of empty & full capsids

• TEM consistent with AUCPfizer Proprietary

Sedimentation Coefficient (S20,w)50 100 150 200

c(s 20

,w)

0.00

0.05

0.10

0.15

0.20

15,000x 11,000x

Empty Capsids

Transmission Electron Microscopy (TEM)

Analytical Ultracentrifugation (AUC)• TEM– High % empty AAV capsids observed

• AUC– High % empty AAV capsids confirmed

• TEM consistent with AUC

Sample enriched for empty particles

Improved Characterization: Full/Empty Ratio

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• Introduction of assays with advanced capabilities improves comparability assessments and product understanding

• Robust release assays are necessary for process and product understanding

• Orthogonal assays advance product characterization and support validity and understanding of release assays

• Advanced product understanding could facilitate better critical quality attribute definition

Presentation Conclusion

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Acknowledgements: For These Data

Bioassay & Impurity

• Phoebe Baldus

• Herb Runnels

• Stephanie Botos

• Emilia Byrne

• Suzanne DeMarco

• Savita Sankar

• Kun Zhang

Mass Spec & Biophysical

• Tom Lerch

• Thomas Powers

• Caitlin Wappelhorst

Project Progression Line

• Amanda Werle

• Tatiana Shapkina

• John Amery

• Missy Anderson

• Svetlana Averianova

• Jasmina Fazlic

• Halyna Narepekha

• Rathna Nostra

Quality Control

• Tom Schomogy

• Amy St. Charles

Special Thanks As Sponsors of These GTx Projects

• Pfizer’s Rare Disease Research Unit and Bamboo Therapeutics, Inc.

– Jude Samulski

– Josh Grieger

– Michael Linden

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