Successful Sterile Filtration of a Squalane Emulsion - Pall€¦ · Filtration Occurs at Many Points in a Biopharmaceutical Process Sources of Contaminants (particulate or microbial)

Filtration Occurs at Many Points in a BiopharmaceuticalProcess

Sources of Contaminants (particulate or microbial)

Contamination from external sourcesProcess fluids like water, solvents or buffers, etc.Raw materialsAir/personnel/premises

Contamination generated within the process

Wear from moving components like pumps or valvesUndesired components as by-products of a chemical reactionor fermentation process (possible bacterial growth)Oxidation and chemical decomposition of fluid componentsover time or temperature changes

Contamination generated during maintenanceDebris from cleaning towelsGrease and lubricantsManufacturing debris from newly installed components

Relative Sizes of Small Contaminants

Relative Sizes of Some Microorganisms

Key Particle Removal Mechanisms

Surface View of 0.2 µm Rated Membrane Challenged With Bacteria1

Edge View of 0.2 µm Rated Membrane Challenged With Bacteria2

How a Sterilizing Grade Filter is Defined (Rated)

Industry standards require that sterilizing filters are challenged withthe microorganism Brevundimunasdiminuta at a minimum concentrationof 107 CFU /cm2 of effective filterarea. Regulatory and industry expectations are that filters challenged according to this method provide a sterile effluent.

Filter Challenge Tests

Filter efficiencies of sterilizing-grade filters can be determinedvery effectively using bacterial challenge tests.Suspensions of the test organisms are prepared and pumpedor transferred by a pressure vessel through the filter to betested.Any bacteria that might have penetrated the filter tested can be detected on a downstream-analysis membrane.This is achieved by placing the analysis membrane to an agarplate, which supports growth of the challenge bacteria and allows them to grow to a bacterial colony.

Schematic Setup for Bacterial Challenge Tests

Titer Reduction (TR)

Process Specific Bacterial Retention Summary

This test validates that the filter produces sterile filtrate under simulated worst-case process conditionsIncludes three test filters from three different lots – One lot number is representative of minimum

specification filter membrane

Typically uses Brevundimunas diminuta (ATCC 19146)– Controlled culture conditions (ASTM F838-15)– Minimal size (B. diminuta 0.3 x 0.8 µm)

– Monodispersed – Penetration of 0.45 µm rated control filter assures

appropriate size of bacterial cells – Total challenge ≥ 1 x 107 CFU/cm2

Analyzes total effluent for sterility

Most filter validation are successful, however, emulsionsand similar fluids can lead to increased risk of bacterialpenetration (N=267)

Category of filtered fluids which resulted in reduced bacterial retention (%)

Conducting bacterial retention tests prior to finalization ofthe manufacturing process may be beneficial for final-fillapplications involving sterile filtration of emulsions andsimilar fluids

Area Benefit

High Risk Fluid • Determines likelihood of obtaining sterile product prior to conducting Filter Validation Studies

• Minimizes laboratory re-work due to failed bacterial retention

Process Optimization • Helps evaluate likelihood of success for bacterial (incl. High Risk Process) retention when developing a new process or

changing process parameters• Risk may be based on process parameters, filter

type, fluid, or all three

Example of an pre-screening bacterial challenge test withan emulsion fluid (squalane based (minus a drug product)

0.75% Tween 800.75% Span 855.0% Squalane

All filters showed complete retention at 10, 30 and 60 psidThere was no change in emulsion particle (the drug deliveryvesicle) size distribution post-filtration (data available for the 30and 60 psid tests only)Provides an example of the benefits of early screening to pro-vide a technical solution for complex applications

Results of a Bacterial Challenge of a 0.2 µm rated Fluorodyne EX filter with a Squalane Emulsion at a test pressure of 60 psid*

Total Total Bacterial Bacterial Challenge Recovery

Flux Challenge Level (CFU/Filter Filter (mL/min/cm2) (CFU/Filter) (CFU/cm2) Effluent)

1 14.0 6.8 x 108 4.9 x 107 0

2 10.7 6.8 x 108 4.9 x 107 0

3 14.7 6.8 x 108 4.9 x 107 0*Penetration through a 0.45 rated control filter was detected. All test filters passedpre- and post Bubble Point Integrity Tests




1 4.8 5.8 x 108 4.2 x 107 0

2 4.8 5.8 x 108 4.2 x 107 0





1 2.4 7.0x 108 5.1 x 107 0

2 2.1 7.0 x 108 5.1 x 107 0


Results of Particle Size Analysis Pre and Post-filtrationThrough a 0.2 µm-rated Fluorodyne EX Filter

Successful Sterile Filtration of a Squalane EmulsionMartha Folmsbee, Ph.D, Scientific and Laboratory Services, Pall Corporation, Port Washington, NY, USA

Ross Turmell, Senior Technical Specialist, Pall Corporation, Covina, CA, USA

BACKGROUND

y p

Media

MediaFiltration

Chromatography Steps (capture & purification)

UF/DF Virus FiltrationDNA/HCPRemovalFinal Bulk Filtration

Vent

Bioreactor

Cell harvesting

Clarification

Formulation and Filling

UF/DF

© 2015, Pall Corporation. Pall, , Fluorodyne, and Supor are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 11/15, GN15.6417Contact: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • E-mail: [email protected] • Web: www.pall.com/biopharm

Pencil point (40 m)

Red blood cell (7 m)

Silica particle (20 m)

Bacteria ( > 0.3 m)

Yeast (3 m)

Brevundimonas diminuta

Acholeplasma laidlawii

laidlawiiAcholeplasma

laidlawiiAcholeplasma

Three Removal mechanisms work to get the particles to interact with the

filter matrix.

Two Retention mechanisms work to ensure that particles stay in place.

Direct interception

Diffusional interception

Inertial impaction

Mechanical retention Adsorption interception

Direct

fusional intercept fffusional interception Di

interception impaction Inertial

fusional interception

retention Mechanical

Adsorption Adsorption interception impaction Adsorption Adsorption

Brevundimonas diminutabacteria (~0.3 – 0.4 µm

by ~0.6 – 1.0 µm)

Many pore openings atmembrane surface

are > 0.2 µm

Brevundimonas diminutabacteria

• Cells may penetratesome distance into membrane depth.

• Typical membrane thickness ~40–150 µm.

Cutting LineMembraneSurface

Dep

th o

f M

embr

ane

Bacterial Retention TestTest Solution Pre-Filter:Supor® Grade EKV (PartNumber KA3EKVP1G)Test Filter: 0.2 micron ratedFluorodyne EX Grade EDF(Part Number FTKEDF)Test Solution: 5% Squalanein water emulsion

Test Pressure: 10, 30 and 60 psid

Particle SizingLaser Diffraction technologyfrom Horiba (Horiba LA 950).The refractive index was1.470-0.010i and the diluent was Deionized water (17.1 mega ohms)

ACKNOWLEDGEMENTSAngel Lorenzo, Kevin Marino, Daniel Eshete, Julie Grace at Pall Corporation. Special thanks to Dr. Yang Su, Kyle Jandrasitz,Yuxian Zhang, and Steven Mesite at Microfluidics InternationalCorporation for formulating and producing the emulsion and forall particle size measurements.

Challengefilter

AnalysisMembrane filter

Drain

Pressure vesselwith bacterialsuspension

Regulated air inlet

Bacteria colonies onan analysis disc afterincubation on an agarplate

For microbial filters, filter efficiencies

are expressed as

TR is measured in bacterial challenge tests

as described in Pall validation guides

Titer reduction or TR

For sterilizing grade filters, the downstream count has to be zero;

therefore the titer reduction claim is expressed as > the total challenge

count.

2 2 6

21

31

38

Salts/chelator

Uncategorized

Blood products (and related)

Lipid and Lipid-like

Surfactant containing

Liposomal

0

20

40

60

80

100

120

140

160

180

d10 d50 d90

Part

icle

Cou

nt (N

=3)

Particle Size Range (nm)

Pre-filtration

30 psid

60 psid

RESULTS AND SUMMARY

MATERIALS AND METHODS

~10 µm

Documents

Successful Sterile Filtration of a Squalane Emulsion - Pall€¦ · Filtration Occurs at Many Points in a Biopharmaceutical Process Sources of Contaminants (particulate or microbial)