2017 Well Characterized Biological Products

Gene Therapy Treatments for Hemophilia A & BProcess Validation Considerations in the Manufacturing of Gene Therapy Products

Robert Baffi, Ph.D., MBAExecutive Vice President, Technical OperationsBioMarin Pharmaceutical Inc.

“You measure the size of the accomplishment by the obstacles you had to overcome to reach your goals." Booker T. Washington (American educator, author, orator, and advisor to presidents of the United States)

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Gene Therapy & Hemophilia AUnderstanding the potential of BMN-270

http://www.nibsc.org/science_and_research/biotherapeutics/haemostasis/haemophilia.aspxhttp://www.the-scientist.com/?articles.view/articleNo/32141/title/Targeting-DNA/

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Some Facts About AAV5 Capsid Structure• AAV5 capsid is composed of 60 viral protein subunits VP1, VP2, and VP3, in approximately

the ratio 1:1:10.

•

Approximate molecular weights:

VP1: 80.42 kDa

VP2: 65.38 kDa

VP3: 59.55 kDa

Average molecular weight of empty vector ~ 3706.5 kD.

D = 25nm

AAV: Why is this virus a vector?

• Non-pathogenic in humans

• ITRs only required elements for packaging

• Many non-human serotypes

• Capsid-based tissue tropism

• Stable long term gene expression

• Little, if any, risk of insertional mutagenesis

• rAAV can be grown to high titers

Integrating Very rarely

Delivery Method In vivo

Advantages Low risk of insertion site mutagenesisNon-pathogenicNot very immunogenicRobust particleWorks on cells that aren't dividing

Disadvantages Small genome capacityEpisome gets diluted from dividing cellsExposure to AAV may limit addressable populationEffect may not be as durable

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Gene Therapy Products in

Clinical Development for Hemophilia

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Structure of Human Factor VIII

Gordon A. Vehar*, Bruce Keyt*, Dan Eaton*, Henry Rodriguez*,

Donogh P. O'Brien†, Frances Rotblat†, Herman Oppermann‡, Rodney Keck*, William I. Wood‡,

Richard N. Harkins*, Edward G. D. Tuddenham†, Richard M. Lawn‡ &

Daniel J. Capon‡

Nature 312, 337-342 (22 November 1984)

Treatment of Coagulation Disease by Administration of Recombinant VWF

•Abstract: Treating coagulation disease, including hemophilia and von Willebrand disease by administering recombinant von Willebrand Factor alone or in combination with Factor VIII.•Filed: June 11, 2012 Patent number: 2012/0316,116•Inventors: Friedrich Scheiflinger, Peter Turecek , Bruce Ewenstein, Wing Yen Wong, Tobias Suiter.

Gordon Wing Barrie

A Human Parvovirus, Adeno-Associated Virus, as a Eucaryotic Vector: Transient

Expression and Encapsidation of the Procarotic Gene for Chloramphenicol

Acetyltransferase

Jon-Duri Tratschin, Michael H.P. West, Tracey Sandbank, and

Barrie J. Carter*

Molecular and Cellular BiologyVol 4 No. 10 p. 2072-2081

(October 1984)

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BMN 270 Most Recent Data:

Data from high-dose patients

• FVIII levels stabilized

• Mean Annualized Bleed Rate declined 91% for patients

previously on prophylactic Factor VIII

• All patients off steroids

• ALT levels (liver function) in or around the normal range

Weeks

N=

NormalFVIII

Range:50%-150%

BMN 270: FVIII Levels Stabilized and Maintained out to 50 weeks

Baseline FVIII activity for all subjects at study start was <1% of normal level

BMN 270 Effectiveness Maintained out to 50 Weeks

High-doseSubject #

FVIII level (%)at last update

July 6

Most recent week of

observation

FVIII level (%) at most recent

observation*

1 89 50 121

2 219 42 133

3 271 40 222

4 12 41 16

5 133 40 175

6 69 38 77

7 79 34 62

*Data as of Dec. 9

Baseline FVIII activity for all subjects in BMN 270 study was <1% of normal level

Prior to Treatment with BMN 270 Annualized Bleeding Rate (ABR) and Annualized Number of FVIII Infusions are Significant

Mean ABR for 6 prophylactic subjects in high dose cohort

Mean Annualized Number of FVIII Infusions for 6 prophylactic subjects in high dose cohort

Before BMN 270* Before BMN 270*

16.3 136.7

Mea

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* Obtained from medical records

Annualized Bleeding Rate (ABR) and Annualized Number of FVIII Infusions Declines Dramatically with BMN 270 Treatment

After BMN 270** After BMN 270**

91% Reduction 98% Reduction

136.7

Mean ABR for 6 prophylactic subjects in high dose cohort

Mean Annualized Number of FVIII Infusions for 6 prophylactic subjects in high dose cohort

1.5 2.9

5 of 6 had no bleeds requiring FVIII use after week 2

5 of 6 had no FVIII infusions after week 2

* Obtained from medical records **Rates after BMN 270 were based on data after week 2 through last follow-up visit

16.3

Mea

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Before BMN 270* Before BMN 270*

Liver Function Tests (ALT) in or Around Normal Range

High-doseSubject#

ALT (U/L); (ULN = 43 (U/L))

Peak ALT level Last ALT level*Last ALT Level

Status

1 60 15 Normal

2 95 16 Normal

3 82 42 Normal

4 87 33 Normal

5 43 38 Normal

6 81 45 <1.1 ULN

7 66 27 Normal

Since WFH all ALT results have improved and all patients are off steroids

*Data as of Dec. 9

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ValidationCategories

rDNA Protein Production Vector Production Ex-Vivo Transduction

Facility cGMP design requirements well understood

Air pressure positive in the core

Air Pressure differential meant to protect the process

Conflicts between Biosafety and cGMP’s requirements

Air pressure negative in the core

Air Pressure differential meant to provide operator safety

Often BL-2 or BL-2+ requirement

Facility extends into hospital centers with additional / different regulatory oversite

Equipment Focused on closed systems with fairly standard equipment

More likely to share equipment

Reactors and storage vessels more likely to be stainless steel

Fill Finish – Can use RABS

Atypical bioprocess equipment not routinely used for cGMP production

Less likely to share equipment

Reactors and storage vessels more likely to be single-use technology

Fill Finish - Isolators for operator safety

Equipment not necessarily developed for cGMP operations creating cGMP and validation difficulities

Clinical equipment being adapted to cGMP use

Validation Considerations: rDNA vs Vector vs Transduction

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ValidationCategories

rDNA Protein Production Vector Production Ex-Vivo Transduction

Process Characterization of proteins to demonstrate comparability well understood

Industry settled on a few well understood cell lines

PK and PD of proteins better understood for comparability evaluations

Characterization of capsids, packaged materials, or nanoparticles for delivery more challenging than proteins

Ongoing development with multiple different production systems

PK and PD of Vectors containing RNA / DNA less well understood for comparability evaluations

Additional challenges for validating processes which may include transduction of primary cells of various homogeneity from different patients with varying results

Raw Materials

75% of products produced in CHO or E.coli. Readily chacracterizied and banked

Primarily serum and animal free soucres

Primarily defined media

Readily sampled and tested

A developing mix of baculovirus, insect and human cell lines. Insect cell lines generally easier to characterize and bank than human cell lines

Human cell lines much more likely to require serum or animal sourced raw materials

Human cell lines likely to use undefined media. Testing of serum or animal components more challenging

Exclusively reliant on aseptic control as filtration of human cells not possible

Many pseudo GMP reagents required including growth factors, HSA and serum

Limited sampling and testing options. Human cells susceptible to viral exposure and contamination

Validation Considerations: rDNA vs Vector vs Transduction

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ValidationCategories

rDNA Protein Production Vector Production Ex-Vivo Transduction

Viral Removal and Inactivation

Well understood models and experience

Orthogonal use of detergents, pH, heat, chromatography and filtration routinely used for nearly all products providing high levels of putative adventitious virus removal or inactivation

Less well understood models and experience

Nano filtration cannot be used for any of these products.

Detergents cannot be used for any enveloped vectors such as lenti

pH and heat will have limited utility and will need to be evaluated on a case by case basis

Limited options beyond aseptic technique

Cleaning Generally readily measured – TOC

Sensitivity of TOC in ppb range adequate to provide safety assurance for carryover

Single-use technology gaining favor

Generally readily measured – TOC

Sensitivity of TOC in ppb range may not be adequate to provide safety assurance for carryover. PCRmethods may be overly sensitive and susceptible to false positives

Dedicated single-use technology simplifies cleaning requirement

Challenges for autologoustreatment of large number of patients and potential for cross-contamination

Dedicated single-use technology a likely requirement

Validation Considerations: rDNA vs Vector vs Transduction

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