Overcoming challenges of host cell DNA removal in vaccine manufacturing

Dr. Priyabrata Pattnaik Director – Asia Vaccine Initiative

Agenda

DNA removal needs and regulatory position 1

2 Nucleic acid removal by adsorptive depth filter

Nuclease treatment 3

4 Methods for removal and detection of residual nuclease

Chromatography based removal of nucleic acid 5

6 Tangential flow filtration for DNA removal

Summary 7

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)

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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)

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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

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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

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Nucleic Acid Removal by Adsorptive Depth Filtration

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

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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

HA

Yiel

d (%

)

0 150 300 450 600 750 900

1050 1200 1350

Ste

rile

Filte

r

Cap

acity

(L/m

2 )

0 10 20 30 40 50 60 70 80 90

100

DN

A re

mov

al

(%)

200 400 600 800 1000 D0HC Flux [LMH]

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Millistak+® D0HC can show 50-90% removal of DNA

Nuclease Treatment

FDA briefing document on cell line derived vaccines

http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/VaccinesandRelatedBiologicalProductsAdvisoryCommittee/UCM319573.pdf

……..Benzonase® digestion for live vaccines can reduce the infectivity of DNA by more than 100,000 fold …….

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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

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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

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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)

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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

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Benzonase® activity optimization

Temperature Magnesium Ions pH

Monovalent Cations Detergents

SDS

Urea Phosphate Ions

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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.

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

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

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

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Nucleic Acid Removal by Chromatography

1. Bind and elute based removal (packed bed chromatography) 2. Flow-through chromatography (membrane absorber)

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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

Ret

enta

te

(ade

novi

rus)

Size Exclusion Chromatography

Purified Adenovirus

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)

®

®

®

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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

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 MNaCl

50mM Tris+50mM

Citrate+0.3 MNaCl

0

20

40

60

80

100

120

50mM Tris 50mM Phos+ 0.3 MNaCl

50mM Tris+50mM

Phos+0.3 MNaCl

50mM Tris+50mM

Citrate+0.3M NaCl

0

20

40

60

80

100

120

50mM Tris 50mM Phos +0.3 M NaCl

50mM Tris+50mM

Phos+0.3 MNaCl

50mM Tris+50mM

Citrate+0.3 MNaCl

% V

irus

Rec

over

y

% D

NA

Rem

oval

% Virus Recovery % DNA removal

• Complete flow-through of virus in presence of multivalent ions

• High capacity for DNA and high throughput (~187CV) 25

Nucleic Acid Removal by Tangential Flow Filtration (TFF)

Clearance of Benzonase® digested DNA across TFF (Pellicon® 2, Biomax® 500kDa)

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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

Diafiltration of Residual Benzonase®

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99.5% clearance at 5 diavolumes and > 99.9% (3 log) clearance after 8 diavolumes across the UF/DF step

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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Thank you