Presentation in World Vaccine Congress, Singapore, 17-20 June 2013.

1. Overcoming challenges of host cell DNA
removal in vaccine manufacturing
Dr. Priyabrata Pattnaik
Director – Asia Vaccine Initiative

2. Agenda
DNA removal needs and regulatory position1
2 Nucleic acid removal by adsorptive depth filter
Nuclease treatment3
4 Methods for removal and detection of residual nuclease
Chromatography based removal of nucleic acid5
6 Tangential flow filtration for DNA removal
Summary7

3. Vaccine and DNA
• Viral vaccines and biological products contain contaminating residual DNA
from cell substrate
• WHO Expert Committee on Biological Standardization says.................
“DNA considered as cellular contaminant rather than risk factor which
requires removal to extremely low levels”
• The amount of residual cell-substrate DNA in a vaccine will depend on the
vaccine and the manufacturing process
• DNA makes downstream processing difficult (viscosity, complex formation)
3

4. Regulatory requirement on Purity and Safety
- Residual DNA content
10 ng/dose
WHO 1998
100 pg/dose
WHO 1987
Vero* and MDCK*
Based Viral Vaccine
40 pg/dose
Per.C6
Adeno-HIV
10 pg/dose
HepB (CHO)
EU Pharmaco
* Non tumerigenic at the passage of production.
* DNA <10 ng/dose commonly accepted
EMEA position on tumerigenic cells of human origin
DNA as low as possible with risk assessment study
FDA: Case by case
Reduce size (<200bp)
and amount (<10ng/dose)
4

5. How much nucleic acid removal is “good
enough”?
 Adenovirus-specific regulatory guidance: 10 ng would only be
acceptable provided that the DNA was digested to less than 100-200
base pairs in length[1]
 Adenoviruses are typically produced at about 104-105 viral particles
(vp)/cell[2]
 Mammalian cells have a genome of about 10 pg/cell[3]
 Assuming 2x106- 8x108 cell/ml; 20µg - 8 mg DNA /ml at virus harvest
 Minimum 7 logs of DNA clearance would be required in order to attain
levels below 100 pg/dose for a high (1012 vp) dose of adenovirus.
[1] Bauer et al., Testing of Adenoviral Vector Gene Transfer Products: FDA Expectations. In Adenoviral Vectors for Gene Therapy; Curiel, D. T., Douglas, J. T., Eds.; Academic
Press: New York, 2002; pp 615-654.
[2] Nadeau and Kamen. Production of adenovirus vector for gene therapy. Biotechnol. Adv. 2003, 20 (7-8), 475-89.
[3] Kraiselbuld et al., Presence of aherpes simplex virus DNA fragment in a L cell clone obtained after infection with irradiated herpes simplex virus 1. J. Mol. Biol. 1975, 97, 533-
542.0
5

6. How to remove residual DNA
 Precipitation (Acid/base treatment, organic solvent)
- Ex. Conjugated polysaccharide vaccine
 Treatment by β-propiolactone (BPL)
- Ex. Killed viral vaccine
 Adsorptive Depth Filters
- Inactivated Polio
Chromatographic methods
- Bind and elute (chromatography media)
- Flow Through (membrane absorber)
 Nuclease treatment
- HepA, LAIV, Rabies, HPV
 Tangential Flow Filtration (TFF)
- Several vaccines
6

7. Nucleic Acid Removal by
Adsorptive Depth Filtration

8. DNA removal by depth filtration (Millistak+®)
mostly by adsorption-based retention mechanism
– Attraction forces between
particles and filter material
– DNA is adsorbed by a
combination of electrostatic
and hydrophobic interaction
– Not size-dependent
– Adsorptive capacity is limited
and “breakthrough” eventually
occurs
– DNA adsorption depends on
solution composition. pH and
conductivity plays a major role
– DNA reduction: 1 - 2 log
8

9. Cell based flu clarification by Millistak+®D0HC
Performance on HA yield, DNA removal rate and Sterile filter capacity
0
10
20
30
40
50
60
70
80
90
100
HAYield(%)
0
150
300
450
600
750
900
1050
1200
1350
SterileFilter
Capacity(L/m2)
0
10
20
30
40
50
60
70
80
90
100
DNAremoval
(%)
200 400 600 800 1000
D0HC Flux [LMH]
9
Millistak+® D0HC can show 50-90% removal of DNA

10. Nuclease Treatment

11. FDA briefing document on cell line derived
vaccines
http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/VaccinesandRelatedBiological
ProductsAdvisoryCommittee/UCM319573.pdf
……..Benzonase® digestion for live vaccines can reduce the
infectivity of DNA by more than 100,000 fold …….
11

12. Genetically engineered endonuclease that cleaves all forms of DNA and RNA.
Origin: Serratia marcescens
Expression: E.coli K -12 mutant
Molecular mass: ca. 30 kD (subunit, exist as dimer)
Isoelectric point (pI): 6.85
Functional in pH range: 6–10
Temperature: 0 - 42ºC
Presence of Mg2+ (1-2 mM) is required for enzyme activity.
One unit of Benzonase® degrades approximately 37µg DNA in 30 min to as low
as 3-8 base pairs (<6 kDa).
Benzonase® Endonuclease
12

13. Mixture of pLAI DNA and uninfected Jurkat DNA in equal amounts was digested with Benzonase® at 30 °C.
DNA was purified, analyzed by 1.8% agarose-gel electrophoresis
1.5 μg of each time point was transfected into 293T cells followed by co-culture with Jurkat cells.
Virus prodn was detected by RT activity and virus prodn from each sample.
SOURCE: Sheng-Fowler et al. (2009) Biologicals, 37(4): 259-269. Division of Viral Products, CBER, FDA
Benzonase® is effective within 4 min
Effect of Benzonase® digestion on the infectivity of DNA
13

14. Benzonase® is widely recognized…….
...........For Vero cell–produced
vaccine, nucleic acid can be
reduced in size by treatment with
Benzonase® then removed by
ultrafiltration using a 50,000 MW
membrane or removed by ion-
exchange chromatography. It is
not necessary to incorporate
steps to remove nucleic acid
from vaccine produced on diploid
cells.......
(Page 21)
14

15. Benzonase® can prevent Virus-DNA complex
formation during purification
Fractogel® SO3¯
SOURCE: Chahal et al., Journal of Virological Methods 139 (2007) 61–70.
Adeno-assocuated virus (AAV) and
DNA can form aggregates, since there
is a net positive charge on AAV at pH
7.5 and negative on DNA
Digesting cellular DNA in by adding
Benzonase® in lysis buffer prevents
binding of DNA to AAV during and
after the cell rupture step
 Lysis Buffer: 50mM Tris, 2mM MgCl2
and 5U of Benzonase®/million cells at
pH 7.5
15

16. Benzonase® activity optimization
Temperature Magnesium Ions pH
Monovalent Cations Detergents
SDS
UreaPhosphate Ions
16

17. Removal and Detection of Residual Benzonase®
Benzonase® is not an API or Excipient.
Like any other process additives, Benzonase®
need to be removed from the vaccine process.

18. Removal of Benzonase®
 Flow Through TMAE or DMAE Fractogel® pH 7- 8 , 50 – 200 Mm Nacl ,
50 mM Tris
 Benzonase® is not or only weakly bound to anion exchange resins under
a variety of conditions; pH 7.0 – 9.0 at 50 mM NaCl; different Fractogel®
anion exchangers
 Benzonase® elutes from cation exchange resins below 200 mM NaCl at
pH 6.0 and is not bound to weak cation exchange resin at pH 6.0
 Ultrafiltration 500 kDa Biomax® membrane
 Retains Viral Particle
 Diafilter out Benzonase® and small nucleic acid base pairs
Reference: Yi Lu et al, Development of Economic Production Platform for Live Attenuated Influenza Vaccine. IMVAC Aug 2009.
18

19. Benzonase® clearance by AIEX and GF
Process
step
Total
Benzonase®
input (ng)
Total
Benzonase®
output (ng)
Benzonase®
clearance
Anion
Exchange
3 0.006 98%
Gel
Filtration
3.9 0.12 97%
Samples were assayed using the Benzonase® ELISA Kit II
• Solid line shows UV at 280 nm
• Solid columns represent the Benzonase®
concentration
• Arrows indicate the target Ad5-GFP peaks
SOURCE: Eglon et al., Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography. J Gene Med 2009; 11: 978–989.
19

20. Benzonase® ELISA Kit II
Description: Immunological
detection of Benzonase®
Sensitivity: ca. 0.2 ng/ml
Benzonase® (0.2 ng/ml
(correspond to < 1ppm in the
presence of other proteins at
conc. > 0.5mg/ml.)
Validation: Test method is
validated
20

21. Nucleic Acid Removal by Chromatography
1. Bind and elute based removal
(packed bed chromatography)
2. Flow-through chromatography
(membrane absorber)

22. 22
Bind & elute based process
Example: Adenovirus purification
Kamen and Henry, Development and optimization of an adenovirus production process, J Gene Med 6, S184–S192, 2004
Adenovirus production
Harvest
Liquid
Cell lysis
Benzonase®
treatment/
Centrifugation
Anion Exchange
Chromatography on
Fractogel® DEAE
media
Solid
Filtration
Ultrafiltration/
Concentration
Retentate
(adenovirus)
Size Exclusion
Chromatography
Purified
Adenovirus

23. Performance level of different Fractogel®
media for DNA removal from rabies vaccine
SOURCE: Method for Purifying the rabies virus, Patent-US2010/0260798A1, Date: Oct 14. 2010 (Sanofi Pasteur)
®
®
®
23

24. 24
Flow-through based chromatography for
DNA removal using ChromaSorbTM
membrane adsorber
0.08 mL
50 mL
Polyethelyene (0.65µm)
Positively Charged gel (PAA)
500 mL
8 layers of membrane
Membrane bed volume = 0.0798mL

25. Virus-DNA separation using ChromaSorbTM
membrane adsorber in flow-through mode
Feed
MDCK cell culture Influenza A/WS
(10KHAU/ml), DNA (1-2µg/ml) in Buffer
Pure Virus
Most DNA
bound
100% DNA removal corresponds to <10ng of whole DNA
0
20
40
60
80
100
120
50mM Tris 50mM Phos +
0.3 M NaCl
50mM Tris+
50mM
Phos+0.3 M
NaCl
50mM Tris+
50mM
Citrate+0.3 M
NaCl
0
20
40
60
80
100
120
50mM Tris 50mM Phos
+ 0.3 M
NaCl
50mM Tris+
50mM
Phos+0.3 M
NaCl
50mM Tris+
50mM
Citrate+0.3
M NaCl
0
20
40
60
80
100
120
50mM Tris 50mM Phos +
0.3 M NaCl
50mM Tris+
50mM
Phos+0.3 M
NaCl
50mM Tris+
50mM
Citrate+0.3 M
NaCl
%VirusRecovery
%DNARemoval
% Virus Recovery % DNA removal
• Complete flow-through of virus in presence of multivalent ions
• High capacity for DNA and high throughput (~187CV)
25

26. Nucleic Acid Removal by
Tangential Flow Filtration (TFF)

27. Clearance of Benzonase® digested DNA across TFF
(Pellicon® 2, Biomax® 500kDa)
27
1 2 3 4 5 6 7 8 9
Various UF samples
 Lane 1 – Marker (100 BP)
 Lane 2 – Undigested DNA in
Feed
 Lane 3 – After Benzonase®
digestion
 Lane 4 – Post Recirc
retentate
 Lanes 5, 6, 7, 8 – Retentate
samples after 1, 3, 5, 8 DV
 Lane 9 – Permeate at 5DV

28. Diafiltration of Residual Benzonase®
28
99.5% clearance at 5 diavolumes and > 99.9% (3 log) clearance
after 8 diavolumes across the UF/DF step

29. Summary
 There are multiple methods for DNA removal from vaccine processes
 Adsorptive depth filter (Millistak+ ®) can also remove nucleic acid from
vaccine process
 Benzonase® is the proven endonuclease for digestion of nucleic acid in
vaccine processes
 Optimization of reaction conditions using Benzonase® is critical for
success of DNA digestion
 Combination of Chromatography (Fractogel®) and TFF (Pellicon® 2) is
good enough for removal of residual DNA and residual Benzonase®
 Multiple analytical methods (Benzonase® ELISA Kit II) are available for
quantization of residual Benzonase® in final product
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30. 3030
Thank you