Heather Bethea Horne, Ph.D.

October 15, 2015

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• Program overview

• Background

• Scope of Work

• Virus Filters for Evaluation

• Model Molecules

• Experimental Plan & Results

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• Our current preferred viral filter is the Asahi Kasei Planova® 15N filter as it consistently provides excellent log reduction value (LRV) of small viruses at ~ 20 nm

• Limitations include lower batch throughput for feeds with higher product concentrations, the need for a gold particle test for integrity testing, and high operational cost

Molecule Type Viral Filter Manufacturing Target

Loading (L/m2) XMuLV LRV (80-110 nm)

MMV LRV (18-24 nm)

IgG4 Planova® 15N ≤1152 6.42 3.94

IgG4 Planova® 15N ≤351 ≥ 6.08 3.80

gp120 Planova® 15N ≤501 4.72 4.99

Recombinant Enzyme Planova® 15N ≤1002 5.34 4.08

IgG1 Planova® 15N ≤1002 3.93 4.48

1 Established as true limits during PD development; 2 Limit based on material availability for the PD development study

XMuLV = Xenotropic Murine Leukemia Virus; MMV = Mouse Minute Virus

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• As a CMO focusing on clinical development of > 12 IND stage molecules per year, we are looking for a viral filter that offers consistent and reliable performance from product to product

• The filter must meet the following requirements: • High capacity and scalable

• Disposable flow path at manufacturing scale

• Easy installation, utilization, and integrity test

• Consistent and robust clearance of parvovirus

• There are several established virus filters that are currently used in industry including the Asahi Kasei Planova®, EMD Millipore Viresolve®, and Sartorius Stedim Virosart® • Sartorius Stedim recently launched a hollow fiber Virosart® filter in March 2015

• EMD Millipore has a new Shield H filter that will be launched this year (beta version tested in this study)

• EMD Millipore and Sartorius Stedim virus filters can be integrity tested by diffusional flow rate of air through the water wetted membrane

• The air/water diffusion integrity test is currently standard practice for other filters in our manufacturing facility

• With the exception of Planova® BioEX, the Asahi Kasei filters require the use of the gold particle test for integrity testing

• The GPT could be eliminated if the Planova® BioEX, EMD Millipore or Sartorius Stedim viral filters were integrated into the platform

• The elimination of the gold particle integrity test would help simplify and reduce the number of operations in manufacturing

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• Identify a platform filter that offers increased batch throughput, consistent performance from product to product, and allows for easy manufacturing operations

• The filter could become part of our platform approach to monoclonal antibody (mAb) purification

• Rely on the vendor for viral clearance data to demonstrate acceptable LRV for each filter type being evaluated

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• Rely on LRV data provided by vendors

• All filters demonstrate similar clearance of small viruses (18-24 nm)

Pre-Filter Viral Filter Vendor Viral Filter Composition MMV LRV PPV LRV

0.1 µm PES (VPF, Shield H

beta evaluated)

Viresolve® Pro (Vpro)

EMD Millipore PES Asymmetric Membrane

5.8

N/A 4.8

5.7

0.1 µm PES Planova® 15N Asahi Kasei Bioprocess

Regenerated Cellulose Hollow Fiber

N/A > 4.6

0.1 µm PES Planova® 20N Asahi Kasei Bioprocess

Regenerated Cellulose Hollow Fiber

6.9

> 5.2

> 5.1

> 4.2

0.1 µm PES Planova® BioEX Asahi Kasei Bioprocess

PVDF Hollow Fiber > 4.8

> 5.3 > 6.1

0.1 µm PES Virosart® HF Sartorius Stedim PES Hollow Fiber 6.2 N/A

EMD Millipore Viresolve® Pre-filter (VPF) = diatomaceous earth, positive charge resin, cellulose esters EMD Millipore Shield H beta Pre-filter = Mixed mode PES PPV = Porcine Parvovirus

1 LRV data provided by vendor

1 1

• Model molecules selected for testing in the various viral filters:

• All material was stored at -80°C so a single freeze-thaw cycle was required; a 0.1 µm PES filtration was performed just prior to viral filtration operation to remove potential aggregates generated due to freeze-thaw

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Molecule mAb Type

Product Concentration

(mg/mL) Buffer Matrix

Feed Conductivity

(mS/cm) Feed pH

HMW Levels by SEC

mAb1 IgG1 3.9 20 mM phosphate, 10 mM citric acid, 300 mM NaCl, pH 5.0 29-31 5.0 2.3%

mAb2 IgG1 5.4 50mM Tris, 240mM NaCl, pH 8.0 25 8.0 3.3%

mAb3 IgG4 11.6 10 mM phosphate, 140 mM NaCl, pH 6.1 13-16 6.1 5.8%

mAb4a IgG4 10.5 10 mM phosphate, 140 mM NaCl, pH 6.1 13-16 6.1 3.1%

mAb4b IgG4 3.3 20 mM phosphate, 0.6M AmSO4, pH 6.1 83-105 6.1 2.3%

• Evaluate model molecules in selected virus filter options, which includes new and established virus filters

• The naked virus filters are evaluated for a direct comparison

• The Viresolve® Prefilter and new Shield-H are evaluated to assess the performance impact with the inclusion of pre-filtration with the EMD Millipore virus filter

• Monitor filter performance with the following parameters for each model molecule:

• Flow decay at a fixed volumetric loading

• Throughput at a fixed volumetric loading

• Operational cost at 25% flow decay for 100L batch volume

» 25% flow decay criteria based on Parenteral Drug Association (PDA) committee recommendation corresponding to ~ 4 LRV

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Lute, S., Riordan, W., and Pease, L. et al. (2008) PDA J Pharm Sci and Tech 62, 318-333

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• The Planova® 20N and BioEX filters demonstrated the lowest flow decay among the virus filters evaluated

• The Viresolve® Pro and Planova® 15N filters possessed the highest flow decay

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N/A

Note: mAb4a was not evaluated in BioEX due to limited material availability

• Throughput calculated based on processing time required to achieve a fixed volumetric loading of 50 L/m2

• The Viresolve® Pro and Virosart® HF filters were the top performers

• The Planova® filters demonstrated the poorest throughput

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N/A

*

*

* 50 L/m2 loading was not achieved due to significant flux decay therefore throughput per time could not be calculated Note: mAb4a was not evaluated in BioEX due to limited material availability

• Cost calculated based on surface area required to process 100-liter batch up to 25% flow decay

• The Planova® 20N and BioEX filters were the most cost effective among the filter options for the molecules tested

• The Viresolve® Pro and Virosart® HF filters possessed the highest operational cost among the various filters

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N/A

<

<

< < <

1, 2 1, 2 2 2 2

1 Cost includes the gold particle test 2 Cost includes a 0.1µm PES filter

Note: < was included for runs that did not reach 25% flux decay therefore the cost was calculated using the test throughput

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• The inclusion of the EMD Millipore pre-filters significantly reduced flow decay compared to the naked Viresolve® Pro filter

• The Viresolve® pre-filter + Pro virus filter (VPF + VPro) and Planova® 20N filter setups demonstrated the lowest flow decay

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N/A

• Throughput calculated based on processing time required to achieve a fixed volumetric loading of 50 L/m2

• With the exception of mAb2, the inclusion of the EMD Millipore pre-filters significantly enhanced throughput over time across all molecules tested in comparison to naked VPro

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N/A

*

*

* 50 L/m2 loading was not achieved due to significant flux decay therefore throughput per time could not be calculated

Note: mAb4a was not evaluated in BioEX due to limited material availability

• Cost calculated based on surface area required to process 100-liter batch up to 25% flow decay

• The Viresolve® pre-filter (VPF) significantly reduced cost in comparison to the naked VPro

• The Viresolve® pre-filter + Pro virus filter (VPF + VPro) and Planova® 20N setups were the most cost effective filters among those evaluated

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N/A

<

< <

< < <

Note: < was included for runs that did not reach 25% flux decay therefore the cost was calculated using the test throughput

1 Cost includes the gold particle test 2 Cost includes a 0.1µm PES filter

1, 2 1, 2 2 2 2

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• The Planova® filters can be classified as low flux filters with low fouling incidence however the Planova® 15N fouling incidence is highly dependent on the feed material

• The Viresolve® Pro and Virosart® HF virus filters can be classified as high flux filters with the Viresolve® Pro filter demonstrating the highest fouling incidence among all virus filters evaluated

• The use of the EMD Millipore pre-filters facilitates achievement of the range of desirability (i.e. high flux and low fouling incidence)

Note: Data points are average values for all molecules tested

• A viral filtration performance study was completed for four monoclonal antibodies possessing varied product concentrations and buffer matrices

• The Planova® 20N and BioEX filters presented the lowest flow decay, lowest throughput and lowest cost in comparison to the Viresolve® Pro and Virosart® HF

• The inclusion of a pre-filter significantly improved the performance and reduced operational costs of the Viresolve® Pro filter

• The Viresolve® pre-filter + Pro virus filter (VPF + VPro) and Planova® 20N filter setups demonstrated the lowest flow decay and operational costs for all molecules in comparison to the other filter setups

• This initial screening study has identified the Millipore Viresolve® pre-filter + Pro virus filter and the Asahi Kasei Planova® 20N filter setups as top contenders for integration into the KBI mAb platform

• Additional model molecules will be evaluated with the top performers to make a final filter section for the platform

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KBI Biopharma, Inc.

Process Development:

• Carnley Norman, Ph.D

• Abhinav Shukla, Ph.D

• Sigma Mostafa, Ph.D

• Leslie Wolfe, Ph.D.

• Aishwarya Sivakumar

Analytical Development:

• Jimmy Smedley, Ph.D.

• Helena Gaweska

• Mike Pollock

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EMD Millipore

• Mireille Deschamps

• Juan Costano

• Kenneth Scott

• Jaime Desouza

Asahi Kasei Bioprocess, Inc.

• Todd Nixon

• Hirotomi Naokatsu

Sartorius Stedim Biotech

• Carl Breuning

• Sherri Dolan

Viresolve is a registered trademark of EMD Millipore Planova is a registered trademark of Asahi Kasei Bioprocess, Inc. Virosart is a registered trademark of Sartorius Stedim Biotech

• The Viresolve® pre-filter + Pro virus filter and Planova® 20N filter setups demonstrated the lowest flow decay among the molecules evaluated

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N/A

• The Viresolve® pre-filter + Pro virus filter setup demonstrated the best overall performance for all molecules evaluated

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* Vmax could not be calculated due to 0% flux decay

N/A *

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N/A

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N/A

9/21/2016

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9/21/2016

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