2017 well characterized biological products...2017 well characterized biological products gene...
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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
n A
nn
ual
ized
nu
mb
er o
f FV
IIIIn
fusi
on
s (n
o. o
f FV
III in
fusi
on
s/ye
ar)
Mea
n A
BR
(n
o. o
f ep
iso
des
/yea
r)
* 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
n A
nn
ual
ized
nu
mb
er o
f FV
IIIIn
fusi
on
s (n
o. o
f FV
III in
fusi
on
s/ye
ar)
Mea
n A
BR
(n
o. o
f ep
iso
des
/yea
r)
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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