Life Sciences

Case Study - Orthogonal

Membrane Technologies
for Viral and DNA Clearance
Jerold Martin* and E.J. Brandreth**

*Pall Life Sciences, East Hills, NY

**BioMarin Pharmaceuticals, Novato, CA
(Present address: Genitope)
 Overview
ƒ Two membrane technology steps
ƒ Virus filtration – size exclusion
ƒ Chromatography – cationic adsorption
ƒ Enhanced Viral safety
ƒ Addition of clearance factors
ƒ Regulatory views
ƒ Enhanced DNA safety and economics
ƒ Added clearance factor
ƒ Implications for DNA QC lot release testing
© Pall Corporation 2005
 Introduction
Viral Clearance
ƒ Viral Clearance
ƒ “…it is expected that the safety of these
products with regard to viral contamination
can be reasonably assured only by the
application of a virus testing program and
assessment of virus removal and inactivation
achieved by the manufacturing process…”

FDA/ICH Guideline on Viral

© Pall Corporation 2005
Safety… (1997)
 R-Protein Purification Process
Low pH
Clarified UF
Chrom Chrom elute Chrom
Harvest and
I II III DF
Fluid hold

Flexible
Container

© Pall Corporation 2005

 Viruses for Clearance Evaluation
Virus XMuLV Reo 3 PRV MMV*
Family Retroviridae Reoviridae Herpesviridae Parvoviridae

Genome RNA RNA DNA DNA

Enveloped Yes No Yes No
Phys/Chem Low High Medium High
Resistance
Size (nm) 80-110 60-80 150-250 20-25

© Pall Corporation 2005

* a.k.a. MVM
 Process Viral Clearance*
Process XMuLV Reo 3 PRV MMV
Step
Chrom II 2.82 ≥ 4.57 ** 1.33
+ 0.14 + 0.32 + 0.54

Low pH ≥ 5.02 <1 ≥ 4.73 <1

+ 0.31 + 0.25

Chrom III 3.29 ≥ 3.24 <1 2.14

+ 0.28 + 0.00 + 0.24

Total ≥11.83 ≥ 7.81 ≥ 4.73 3.47

* Viral clearance was qualified according to the ICH
Retroviral ~8 Q5A Guideline for Viral Safety. Viral challenge studies
included a pre-filtration step to ensure that aggregation
burden of viral particles was not occurring

Margin ~4 ** Reduction through the column removal process

could not be separated from Low pH inactivation,
© Pall Corporation 2005 therefore only the Low pH claim was made for PRV
 Virus Membrane Filtration

© Pall Corporation 2005 Pall Ultipor® VF DV50 membrane and filters

Virus and membrane SEMs not to scale
 Virus Membrane Filtration
ƒ Orthogonal mechanism
ƒ Size-based exclusion
ƒ Non-interfering
ƒ High product recovery
ƒ Non-additive
ƒ Correlated integrity test
ƒ Easily added to process

© Pall Corporation 2005

 Virus Membrane Filtration*
VIRUS CARRIER FLUID LTR REFERENCE
(SIZE,NM )
HSV-1 TCM 1 + 5% serum > 5.8 Roberts, 1997
(120-300) PBS + 5% HSA > 6.4 Anazawa et al.,1997
HIV-1 TCM + 10% > 4.0 Anazawa et al.,1997
(80-100) Serum
PPSB > 5.0 Personal comm.
IvIg > 5.0 Personal comm.
M uLV M onoclonal IgG > 7.6 Aranha, et al., 1998
(80-120)
Sindbis TCM 1 + 5% serum 6.3 Roberts, 1997
(40-70) PBS + 0.5% HSA > 8.9 Roberts, 1997
PPSB > 6.9 Personal comm.
IvIg > 7.5 Personal comm.
SV40 (40-55) TCM +10% serum > 6.9 Oshima et al., 1998
rProtein > 4.1 Shi et al. 1999
BVDV (40-70) TCM +10% serum > 5.3 Unpublished data
HBV (42-47) TCM +10% serum 5.4 2 Oshima et al., 1998
HCV (30-65) TCM +10% serum 3.2 2 Oshima et al., 1998
*Pall Ultipor® VF DV50 membranes 1: TCM: Tissue Culture Medium;
© Pall Corporation 2005 2: PCR based assays; all others based on infectivity assays
 Membrane
Filtration
Scale-up

© Pall Corporation 2005

 Viral Clearance by
Membrane Filtration*
Virus XMuLV Reo 3 PRV MMV
Size (nm) 80-110 60-80 150-250 20-25

Enveloped Yes No Yes No

Log10 TR ≥ 5.22 3.64 ≥ 5.14 1.41
+ 0.37 + 0.56 + 0.36 + 0.61

*Pall Ultipor® VF DV50 membranes

© Pall Corporation 2005
 Combined Viral Clearances
Process XMuLV Reo 3 PRV MMV
Chrom II 2.82 ≥ 4.57 - 1.33
+ 0.14 + 0.32 + 0.54
Low pH ≥ 5.02 - ≥ 4.73 -
+ 0.31 + 0.25
Chrom III 3.29 ≥ 3.24 - 2.14
+ 0.28 + 0.00 + 0.24
Membrane ≥ 5.22 3.64 ≥ 5.14 1.41
Filtration + 0.37 + 0.56 + 0.36 + 0.61

Total ≥17.05 ≥ 11.45 ≥ 9.87 4.88

Retroviral ~8
burden
Margin ~9
© Pall Corporation 2005
 Virus Filter Placement
“Appropriate precautions should be taken
to prevent potential viral contamination from
pre-viral to post-viral removal/inactivation steps.
Therefore, open processing should be performed
in areas that are separate from other processing
activities and have separate air handling units.”

ICH Q7A Good Manufacturing Practice for

Active Pharmaceutical Ingredients
18.5 Viral Removal/Inactivation Steps

© Pall Corporation 2005

CPMP/ICH/4106/00 (2000)
 R-Protein Purification Process
Low pH
Clarified UF
Chrom Chrom elute Chrom
Harvest and
I II III DF
Fluid hold

Barrier

Virus
Flexible Membrane
Container Filtration

© Pall Corporation 2005

 DNA Clearance
“It is necessary to guarantee that
such impurities are reduced to
an acceptable level…
For that, two approaches can be envisaged:”
ƒ “Validation approach”
ƒ Predict and guarantee residual level
ƒ “Routine approach”
ƒ Monitor levels and set fixed limits
CPMP Position Statement on DNA and Host Cell
Proteins (HCP) Impurities. Routine Testing versus
© Pall Corporation 2004
2005 Validation Studies CPMP/BWP/382/97 (1997)
 FDA Comments on DNA Removal
“… It is suggested that, whenever
possible, the final product contain no
more than 100 pg cellular DNA per dose.
It is suggested that a method with a
sensitivity of 10 pg be used to determine
DNA levels”
FDA ‘Points to Consider in the Manufacture of
Monoclonal Antibodies,’ CBER (1996)

© Pall Corporation 2005

 “Acceptable Levels” - DNA Limits
ƒ WHO, EU - 100 pg/dose
ƒ FDA - 100 pg/dose*
ƒ For 1-100 ml dose = <1-100 pg/ml
ƒ Harvest fluid DNA not detectable
ƒ <2 pg/ml by Threshold** assay

*Up to 10 ng/dose may be accepted

for large dosages, e.g MAb
© Pall Corporation 2004
2005 ** Trademark of Molecular Devices, Inc.
 DNA Clearance Lot Testing

“Lot-to-lot testing for DNA

content, ….is recommended
as a way to monitor
purification efficiency and
reproducibility”

FDA “Points to Consider in the

Manufacture of Monoclonal
© Pall Corporation 2005
Antibodies,” CBER (1996)
 DNA Clearance Lot Testing

ƒ Implications – potential costs

ƒ Lot release testing costs - $
ƒ Risk of test failure - $$
ƒ Risk of batch failure - $$$$
ƒ Consider Validation Approach to
eliminate DNA lot QC testing.

© Pall Corporation 2005

 Membrane Chromatography

© Pall Corporation 2005 Pall Mustang™ Q Capsules and Cartridges

 Membrane Chromatography Benefits
Cartridge/Capsule format No packing

Autoclavable Clean process

No cleaning /
No cleaning validation
Single use
Reduce chemicals needs

High capacity Reduce buffer costs

High flow rates Fast processing time

Easy scale up Speed development time

© Pall Corporation 2005
 Ion Exchange Membranes*
ƒ Polyethersulphone (PES)
membrane substrate
ƒ Cross-linked polymers
containing functional
groups – Q, S, other
ƒ Nodular surface -
high surface area
ƒ High voids -
0.8 µm pores
ƒ Multiple layers - *Mustang™ Q membranes
controlled bed depth
© Pall Corporation 2005
 Beaded vs. Membrane Chrom
Membrane

© Pall Corporation 2005

 Beaded vs. Membrane
Chromatography
Membrane

Q
Q
Q
Q

© Pall Corporation 2005

Q - Virus
Q Membrane DNA Capacity Vs. Flow
35 mg/ml
30
25
20
15
10
5
0
10 CV/min 40 CV/min
Mustang™ Q membrane

© Pall Corporation 2005

 Q Membrane
Chromatography
Scale-up

© Pall Corporation 2005

Mustang™ Q membranes and capsules
 Q Membrane Scaleability

NP6
(260 mL)

© Pall Corporation 2005

Q Membrane Capsule Qualification
ƒ Flow rate
ƒ Capacity
ƒ Product recovery Q

ƒ Extractables
ƒ Sizes (membrane volume/units)
ƒ DNA clearance – small scale
ƒ DNA clearance – process scale
© Pall Corporation 2005
 Q Membrane Unit* Flow Rates
20

18

16

14
10 ml Q Capsule
Flow Rate (L/min)

12
60 ml Q Capsule
10
300 ml Q Cartridge
8

0
0 10 20 30 40 50 60 70
© Pall Corporation 2005
Pressure (psi) *Mustang™ Q capsules
 DNA Clearance - Q Membrane*
Product Run Influent Effluent Log
DNA (ng) DNA (ng) Reduction
1 1,000,000 0.4 >6.4

2 1,100,000 2.0 >5.7

3 1,200,000 2.0 >5.8

Average 1,100,000 1.5 >5.9

Production Scale (extrapolation) >8.1

Qualification Lots (3 US, 5 EU) >5.7

© Pall Corporation 2005
*Mustang™ Q membranes
 Virus Membrane Chromatography

Pall Mustang™ Q Capsules and Cartridges

© Pall Corporation 2005
Virus and membrane SEMs not to scale
 Viral Clearance - Q Membrane
Model Study Design
OD 280 nm Injection Elution Cleaning
Equilibration
(100 ml) Washing 0.7 M NaCl 1.5 M NaCl
(10 ml) 10 ml 10 ml Discard
(20 ml)

• Test unit: Q Membrane «coin » unit (0.35 ml bed volume)

• Flow rate: 3.5 ml/min (10 CV/min)
• Model protein sol’n: Lysozyme; 2.0 mg/ml in 10mM phosphate buffer (pH7)
• Test viruses: PRV, MuLV, BVDV, HAV, PPV
• Washing Buffer: 10 mM phosphate buffer, pH 7.0
• Elution Buffer: 0.7 M NaCl-10 mM phosphate buffer, pH 7.0
• Cleaning Buffer: 1.5 M NaCl-10 mM phosphate buffer, pH 7.0

© Pall Corporation 2005

 Viral Clearance - Q Membrane*
Model Study Results
Virus Size Envel- Removal of Recovery of
(nm) oped virus from flow- input viruses
through (log10) (%)
PRV 130 Yes >4.13 18.5
MuLV 80-110 Yes >4.72 0.1
BVDV 25-40 Yes 0.40 42.5
HAV 22-30 No >3.84 37.5
PPV 18-26 No >6.98 102.5

*Pall Mustang® Q membrane testing conducted by

© Pall Corporation 2005 Analysis Biomedizinische Test GmbH, Köln, Germany
 Viral Clearance - Q Membrane
Model Study Summary
ƒ >4 log removal of MuLV, PRV, PPV and HAV
ƒ No removal of BVDV observed
ƒ 18-100 % recovery of PRV, BVDV, PPV and HAV
ƒ No significant losses
ƒ Low recovery of MuLV
ƒ Full recovery of PRV (130 nm), HAV (22-30 nm)
and PPV (18-26 nm) after near complete removal
ƒ Indicates adsorptive mechanism with no size exclusion
effect.

© Pall Corporation 2005

 Viral Clearance - Q Membrane*
Qualification Results
Virus XMuLV Reo PRV MMV
Enveloped Yes No Yes No
Size (nm) 80-110 60-80 150-250 20-25
Membrane 2.48 <1 ≥ 5.49 <1
Chrom + 0.55 + 0.43

*Mustang™ Q membrane.
Conditions for optimal virus clearances were not determined.

© Pall Corporation 2005

 Viral Clearance - Q Membrane
Qualification Summary
ƒ CHO cells are known to contain noninfectious retroviral
particles.
ƒ Retroviral particle load of the Harvested Production Cell
Culture Fluid was ~ 5 logs per mL.
ƒ Complete clearance, with an additional safety factor, was
required of the purification train.
ƒ Viral clearance claims for each orthogonal step in the
process – 2 chromatography steps, low pH inactivation, size
exclusion filtration and Q-membrane chromatography were
combined
ƒ Results which indicated a log reduction of one log or less
were not included, and were assigned a 0 value.
© Pall Corporation 2005
 ICH Guidance on Viral Safety Evaluation
of Biotechnology Products
Derived From Cell Lines of
Human or Animal Origin

“An overall reduction factor is generally expressed

as the sum of the individual factors.
However, reduction in virus titer of the order of
1 log10 or less would be considered negligible
and would be ignored unless justified.”

CPMP/ICH/4106/00 (2000)
© Pall Corporation 2005
 Combined Viral Clearances
Process Step XMuLV Reo PRV MMV
Chrom II 2.82 ≥ 4.57 - 1.33
+ 0.14 + 0.32 + 0.54
Low pH ≥ 5.02 - ≥ 4.73 -
+ 0.31 + 0.25
Chrom III 3.29 ≥ 3.24 - 2.14
+ 0.28 + 0.00 + 0.24
Membrane ≥ 5.22 3.64 ≥ 5.14 1.41
Filtration + 0.37 + 0.56 + 0.36 + 0.61

Membrane 2.48 - ≥ 5.49 -

Chrom + 0.55 + 0.43

Total ≥18.83 ≥ 11.45 ≥ 15.36 4.88

Margin ~11
© Pall Corporation 2005
 R-Protein Purification Process
Low pH
Clarified UF
Chrom Chrom elute Chrom
Harvest and
I II III DF
Fluid hold

Barrier

Virus DNA + Virus

Flexible Membrane Membrane
Container Filtration Chrom

© Pall Corporation 2005

 Summary and Conclusions (1)

Viral Clearance
ƒ Calculation of viral particles per dose (per ICH)
ƒ Retroviral particles per mL of the HCCF = 105.63
ƒ mL of HCCF per dose = 103.61
ƒ Accumulated logarithmic clearance value = ≥1018.83

(105.63 virus units/ml) (103.61 mL/dose) = 109.24 virus units/dose = ≤10-9.59

≥1018.83

ƒ ≤10-9.59 indicates one retroviral particle per

3.9 billion doses expected.
ƒ Other viruses also cleared by significant amounts:
ƒ MVM, a small non-enveloped virus (most challenging)
~ 5 logs were achieved – high safety margin
© Pall Corporation 2005
 Summary and Conclusions (1)

DNA Clearance
ƒ DNA derived from CHO cell lysis - undetectable in
Chrom III eluate (<40 µg/ml IDU)

ƒ Manufacturing scale Q membrane provides

additional 8.1 logs DNA removal.

ƒ DNA in formulated bulk substance > 8.1 logs lower

than limit (0.2 ng/mg) = < 0.2 x10-8 ng/mg

ƒ Significant DNA removal capability obviates the

need for lot-to-lot DNA testing.
© Pall Corporation 2005
 Regulatory Views
ƒ Process approved by FDA and EMEA
ƒ Viral Clearance
ƒ Orthogonal mechanisms recognized
ƒ Size + Q Membrane TR’s additive
ƒ 1-2 log reductions per step accepted
ƒ DNA Clearance
ƒ Lot release without DNA QC accepted
ƒ Validated Q membrane performance
© Pall Corporation 2005
 Acknowledgements
ƒ Min Min Qin – BioMarin Pharmaceuticals
ƒ Dan Wendt – BioMarin Pharmaceuticals
ƒ Andreas Immelt – Analysis Laboratories
ƒ Mohammed Haque – Pall Corp

© Pall Corporation 2005