Best practices in nucleic acid removal from vaccine processes

Frank Appel, Senior Manager

Europe Vaccines Segments I Process Solutions I Merck Millipore

Agenda

DNA removal needs and regulatory position1

2 Density gradient centrifugation and depth filtration methods

Nuclease treatment method3 Nuclease treatment method3

4 Methods for removal and detection of residual nuclease

Chromatography based removal method of nucleic acid5

6 Tangential flow filtration method for DNA removal

Summary7

2

At a Glance

“... Manufacturing vaccine is a complex,

time-consuming, capital intensive, and

highly regulated process requiring an

efficient supply chain and supportingefficient supply chain and supporting

infrastructure of highly qualified staff, and

reliable and continuous supplies of utilities...”

“The Vaccine Industry: Does it Need a Shot in the Arm?” National Health Policy

Forum Background Paper. 25 January 2006. A publication of GlaxoSmithKline

Government Affairs, Public Policy and Patient Advocacy

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"Got a few problems going from lab

scale to full-scale manufacturing!"

Vaccine and DNA

� Viral vaccines and biological products contain contaminating residual

DNA from cell substrate

� Primary and diploid cell based vaccines is not of concern, but continuous

cell line is a concern due to potential oncogenicity

� 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

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Interference caused by nucleic acids

Increase of viscosity

� impedes liquid handling

� extended risk of proteolytic attack

� reduced efficiency of separation methods

"Got a few problems going from lab

scale to full-scale manufacturing!"

− Filtration

− Centrifugation,

− Chromatography

Formation of protein/nucleic acid complexes

� changes in virus / protein characteristics

� unpredictable purification (shift in pI, retention times etc.)

� low recovery, purity, no easy scale-up

5

Effect of position of nuclease treatment onDNA concentrations (HepA, Merck & Co.)

(1) Lysate was treated with Benzonase®

endonuclease (30 µl/L), then concentrated on a capture column and PEG precipitated.

(2) Lysate was concentrated on a capture column, then Benzonase®column, then Benzonase®

endonuclease treated (1.2 mL/L, to keep the enzyme/Hep A ratio same as in 1) and PEG precipitated.

(3) Lysate was concentrated on a capture column, and PEG precipitated without nuclease digestion.

SOURCE: Hagen et al., Biotechnol. Prog. 1996, 12, 406-406

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Regulatory requirement on Purity and Safety Residual DNA content

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)

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.

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Agenda

DNA removal needs and regulatory position1

2 Density gradient centrifugation and depth filtration methods

Nuclease treatment method3 Nuclease treatment method3

4 Methods for removal and detection of residual nuclease

Chromatography based removal method of nucleic acid5

6 Tangential flow filtration method for DNA removal

Summary7

8

Traditional method for nucleic acid removalDensity Gradient Centrifugation

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Steps% Overall Flu

Recovery% HCP removal

% DNA removal

Step -1:Gradient pelleting

60 99.9% 67-75%

Step-2: Gradient fractionation

30 Below detection limitBelow

detection limit

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

� Used primarily for colloid removal and clarification

� Reduction of DNA by 1-2 log and HCP by 0.5 – 1 log

� Useful for cell culture based vaccine to take load off of downstream steps

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Agenda

DNA removal needs and regulatory position1

2 Density gradient centrifugation and depth filtration methods

Nuclease treatment method3 Nuclease treatment method3

4 Methods for removal and detection of residual nuclease

Chromatography based removal method of nucleic acid5

6 Tangential flow filtration method for DNA removal

Summary7

11

FDA briefing document on cell line derived Vaccines

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

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

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Benzonase® endonuclease 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 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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� 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

Benzonase® Endonuclease

� Temperature: 0 - 42ºC

� Presence of Mg2+ (1-2 mM) is required for enzyme activity.

� Free of detectable proteolytic activity

� Manufactured under cGMP conditions

� Supported by Emprove® Bio dossier and a DMF type II file (FDA Reg. No.

BBMF 5403; current version 2013, available as eCTD format)

�One unit of Benzonase® endonuclease degrades approximately 37µg DNA in 30 min to as low as 3-8 base pairs (<6 kDa).

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Agenda

DNA removal needs and regulatory position1

2 Density gradient centrifugation and depth filtration methods

Nuclease treatment method3 Nuclease treatment method3

4 Methods for removal and detection of residual nuclease

Chromatography based removal method of nucleic acid5

6 Tangential flow filtration method for DNA removal

Summary7

15

Removal of Benzonase® endonuclease

� Chromatography Flow Through

− Fractogel® TMAE or DMAE resin pH 7- 8 , 50 – 200 Mm Nacl , 50 mM Tris

� Benzonase® endonuclease is not or only weakly bound to anion exchange resins under a variety of conditions; pH 7.0 – 9.0 at 50 mM NaClresins under a variety of conditions; pH 7.0 – 9.0 at 50 mM NaCl

� Benzonase® endonulcease 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 300 kDa Biomax® membrane

− Retains Viral Particle

− Diafilter out Benzonase® endonuclease and small nucleic acid base pairs

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Detection systems for Benzonase® endonuclease

� Semi-quantitative assay for the detection of residual activity of nucleases (digestion of salmon-sperm, photometric measure of cleavage products, LLOQ ~ 1 U/mL, Merck Millipore monograph)

� Electrophoretic test (based on a publication by Nycomed)

� Fluorescence test (see publication by MedImmune, R. Strouse et. al., BioPharm April 2000; LLOQ ~ 0.001 U/mL)

� Benzonase® standard activity assay (volume activity measuring Benzonase®

activity of the active Benzonase®)

� ELISA II by Merck Millipore (is measuring Benzonase® protein concentration, this does not correspond to activity) Sensitivity: Approx. 0.2 ng/ml Benzonase®

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Agenda

DNA removal needs and regulatory position1

2 Density gradient centrifugation and depth filtration methods

Nuclease treatment method3 Nuclease treatment method3

4 Methods for removal and detection of residual nuclease

Chromatography based removal method of nucleic acid5

6 Tangential flow filtration method for DNA removal

Summary7

18

Performance level of different Fractogel®

resins 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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Agenda

DNA removal needs and regulatory position1

2 Density gradient centrifugation and depth filtration methods

Nuclease treatment method3 Nuclease treatment method3

4 Methods for removal and detection of residual nuclease

Chromatography based removal method of nucleic acid5

6 Tangential flow filtration method for DNA removal

Summary7

20

Cumulative DNA removal through different unit operations

SOURCE: Method for Purifying the rabies virus, Patent-US2010/0260798A1, Date: Oct 14. 2010 (Sanofi Pasteur)

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Diafiltration of Residual Benzonase®

endonucelase

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99.5% clearance at 5 diavolumes and > 99.9% (3 log) clearance after 8 diavolumes across the UF/DF step with Pellicon® 2, Biomax® 300kDa

Agenda

DNA removal needs and regulatory position1

2 Density gradient centrifugation and depth filtration methods

Nuclease treatment method3 Nuclease treatment method3

4 Methods for removal and detection of residual nuclease

Chromatography based removal method of nucleic acid5

6 Tangential flow filtration method for DNA removal

Summary7

23

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 � Optimization of reaction conditions using Benzonase® endonucelase is critical for success of DNA digestion

� Combination of Chromatography (Fractogel®

resin) and TFF (Pellicon® 2 cassettes) is good enough for removal of residual DNA and residual Benzonase® endonuclease

� Multiple analytical methods (Benzonase® ELISA Kit II) are available for quantization of residual Benzonase® endonuclease in final product

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Thank You For Your Attendance And Attention

Question & Answer

Forthcoming event

Frank Appel

Frank.Appel@merckgroup.com

TFF Experts Community Meeting

December 4-5, 2013 Strasbourg , France

http://www.millipore.com/downstream/flx4/tff_user_group