1 Evaluation of Prion Reduction Filters with a Highly Sensitive Cell Culture-Based Infectivity Assay Evaluation of Prion Reduction Filters with a Highly Sensitive Cell Culture-Based Infectivity Assay Presented By Samuel Coker, PhD Senior Technical Director Pall LifeScience R&D FDA-TSEAC Meeting on October 28-29, 2010

FDA Meeting, October 28-29, Background  The clearance of prion infectivity from biologic fluids with prion removal devices is usually quantified by:  The use surrogate marker of infection in in vitro assays such as:  Enzyme-linked Immunosorbent assay (ELISA).  SDS-PAGE Western blot.  Conformational dependent immunoassay (CDI).  Bioassays based on intracerebral inoculation of hamsters or mice.

FDA Meeting, October 28-29, Disadvantages of current methods  Prion infectivity may accumulate in the absence of detectable levels of PrP Sc ;  Levels of PrP Sc do not necessarily correlate with infectivity;  Current bioassays using mice and hamsters are slow, cumbersome, involve the use of hundreds of hamsters for example, and are extremely expensive with a typical endogenous infectivity study costing as much as $250,000 to $500,000 with a single study with a duration of 500 to 600 days.  Therefore, the development of a reliable and highly sensitive cell culture-based infectivity assay may greatly accelerate the evaluation of new prion removal devices.

FDA Meeting, October 28-29, Objective of study  The main objective of this present study was to evaluate the use of a a highly sensitive cell culture based infectivity assay to evaluate the effectiveness of the following prototypes of leukocyte and prion reduction filters in removing prion infectivity from 300 mL of red cell concentrates (RCC):  Leukotrap affinity prion reduction filter (10 layer variant-PRM3)  Leukocyte and prion reduction filter (22 layer variant-PRM3)  Leukocyte and prion reduction filter (22 layer variant-PRM6)  Leukocyte and prion reduction filter (22 layer variant-PRM7)  Leukocyte-reduction filter (BPF4)

FDA Meeting, October 28-29, Filter design and configuration  All the filters contained essentially the same prion binding chemistry on polyester fibrous media.  All the prion reduction filters are also capable of removing leukocytes.  PRM3, PRM6 and PRM7 have the same base polyester media but with different hydrophilic and hydrophobic properties which may or may not enhance prion removal.  BPF4 contained polyester fibers with surface chemistry for binding leukocytes.

FDA Meeting, October 28-29, Leukocyte-reduction component of the prion reduction filter  Mechanical trapping or sieving based on the structure of the fibers (fiber diameter, pore size, or distribution etc.)  Activation of leukocytes to enhance binding to the polyester fibers,  Indirectly through interaction with platelets resulting in the formation of cell aggregates that are then removed through sieving.

FDA Meeting, October 28-29, Materials and Methods Preparation of 10%(wt/vol) Mouse Brain Homogenate Mice brain homogenate from mice infected with the Rocky Mountain Laboratory (RML) scrapie strain were prepared according to the standard protocol of Professor Weissmann's laboratory at SCRIPPS, FL, USA. Briefly, mice were first inoculated intracranially with high titer brain homogenate from mice infected with RMLscrapie strain. The animals were sacrificed after about 145 days at an advanced stage of disease, and the brains were removed to prepare 10% suspensions in phosphate buffered saline (PBS), pH 7.4. When this method is used with scrapie infected mouse brain homogenate (MBH), the titer is of the order of about of LD 50 units per mL.

FDA Meeting, October 28-29, Materials and Methods  Five units of 1-2 day-old ABO compatible nonleukocyte-reduced RCC were purchased directly from an AABB accredited blood bank. All 5 units were transferred into a 2-liter blood bag to create a homogenous pool.  Approximately 10.5mL of infectious MBH-RML were added to about 1570mL of pooled RCC such that the final dilution of the MBH-RM with RCC was 1:150.  The infectious prions were mixed with the pooled RCC. The pooled RCC was then divided into 300 mL aliquots. Preparation of Red Cell Concentrates

FDA Meeting, October 28-29, Experimental design BPF4 Pr-Filter #1 #2 #3 #4#5 Pool of 5 units of RCC (1570mL). 11mL of 10%(wt/vol) mice brain homogenate RML scrapie strain (MBH-RML). 1:150 Dilution Prion & Leukocyte Reduced RCC Leukocyte-Reduced RCC 300mL RCC Containing MBH A Unit ( mL) of RCC

FDA Meeting, October 28-29, MATERIALS AND METHODS Standard Scrapie Cell Assay (SSCA)  The SSCA is:  Based on the isolation of a cell line (Cath-a differentiated cells, CAD5; Scripps, FL) that is highly susceptible to RML scrapie strain;  A method for identifying and quantifying prion- infected cells.

FDA Meeting, October 28-29, Procedure for SSCA CAD5 cells in reduced serum medium (Opti-MEM) were dispensed into 96 well tissue culture plates; 2.The cells were allowed to attach to the plates over night in humidified CO 2 incubator; 3.The attached cells were exposed to either serial dilutions of MBH-RML (1:5, 1:10, and 1:30) or the test samples (Samples 1-5) and then incubated for 4 days in a humidified CO 2 incubator and allowed to grow to confluence. 4.After 4 days, the cells were split 1:10 and then seeded again onto tissue culture plates. 5.After the third split, 20,000 cells of each sample were filtered onto membranes of a 96-well plate (AcroRead, Pall LifeScience); 6.The cells were lysed and treated with proteinase K to eliminate normal PrP C ; 7.PrP Sc infected cells were identified by an Enzymed-Linked Immunosorbent Assay (ELISA) using monoclonal antibody (D18) and alkaline phosphatase – linked anti-IgG antiserum 8.The infected cells (PrP Sc positive cells) were counted using an automated imaging system. The settings of the imaging system was optimized to give maximal ratio of positive cells relative to negative cells 9.The data are expressed as the number of infected cells per 20,000 CAD5 cells.

FDA Meeting, October 28-29, Flow diagram of SSCA procedure Copyright 2008 TSRI Step 1: Expose susceptible CAD5 cells to brain homogenate or red cell suspensions containing infectious prions Step 2: Serially propagate cells and seed ELISPOT Step 4: Colorimetric Detection Step5: Analysis – Zeiss KS Elispot Automated Imaging Step 3: Digest & Denature

FDA Meeting, October 28-29, Representative well of an SSCA plate as imaged with the Zeiss KS Elispot system: Infected spots on 20,000 CAD5 cells.

FDA Meeting, October 28-29, Spiking study to determine the inhibitory effects of test samples  To confirm that any observed reductions in infectivity were not due to components in the test samples that were inhibitory to the cell line, aliquots of postfiltration samples at different dilutions (1:5,1:10,1:30 and 1:90) were mixed with a predetermined amount of MBH-RML.  1mL of test sample was added to 10µL of LD 50 / mL MBH-RML, and 0.145mL of the suspension was added to 5000 CAD5 cells  The proportion of infected cells at the different dilutions of the test samples was determined as previously described.

FDA Meeting, October 28-29, Copyright 2008 TSRI Figure 1A Standard curve of serial dilutions of MBH-RML in the presence and absence of Inhibitor (Pentosan Polysulfate) of infection

FDA Meeting, October 28-29, Standard calibration curve of SSCA

FDA Meeting, October 28-29, Determination of inhibitory effects of RCC on SSCA

FDA Meeting, October 28-29, Effect of leukoreduction step on prion infectivity

FDA Meeting, October 28-29, Prion infectivity in red cell concentrates before and after filtration with different prion reduction filters

FDA Meeting, October 28-29, Endogenous infectivity studies with different prototypes of prion-reduction filters  Units ( mL) of whole blood were collected from scrapie infected hamsters ( a unit of blood was obtained from 500 hamsters) into CPD anticoagulant.  Units of whole scrapie infected blood were centrifuged at 5000g for 30 minutes.  The supernatants were removed and the red cells were resuspended in SAGM additive solution to produce a unit ( mL) of RCC.  Each unit of RCC was filtered with either 10 or 22 layer variant of prion reduction filters.  50µL of pre and postfiltration RCC were injected intracranially into healthy normal hamsters. The animals were monitored and maintained for days. Those that developed clinical symptoms of scrapie were killed and the brain tested for the presence of PrP Sc by Western blot assay using 3F4 monoclonal antibody.

FDA Meeting, October 28-29, Summary of results of SSCA  All the 22-layer prion reduction filters independent of the initial base chemistry on the polyester fibers (PRM3 vs. PRM7) removed prion infectivity below the limit of detection of the SSCA. Therefore, the important component is the number of layers of the fibers with the prion removal chemistry.  The maximum reduction observed in the present study with the SSCA was ≥ 2.0 log 10 LD 50 / mL  The 10 layer variant of the prion reduction filter showed some residual infectivity which is significantly higher than the baseline value obtained with uninfected CAD5 cells

FDA Meeting, October 28-29, Flow diagram of endogenous infectivity study :scrapie infected RCC were filtered with 10 and 22 layer prion reduction filters Scrapie Infected Hamsters Normal Hamsters Intracerebral Injection Hard spin centrifugation Remove supernatant plasma Add red cell additive solution Prion reduction filter BRAIN 10% brain homogenate BRAIN 10% brain homogenate 10% Scrapie Infected Hamster Brain Homogenate

FDA Meeting, October 28-29, Endogenous infectivity study with layer prion reduction filters

FDA Meeting, October 28-29, Endogenous infectivity study with 10 layer prion reduction filter

FDA Meeting, October 28-29, Endogenous infectivity study with 22 layer prion reduction filter

FDA Meeting, October 28-29, Summary of endogenous infectivity data and their relationships to the results of the SSCA  All the 22-layer prion reduction filters significantly prevented the transmission of scrapie infection into hamsters that received filtered RCC over the lifespan ( days) of the hamsters. In contrast, significant number of hamsters that received unfiltered RCC developed scrapie infection (Figures and 7).  In the study with the 10 layer prion reduction filter, 3/413 (0.74%) of the hamsters that received filtered RCC developed scrapie with a median onset of scrapie infection at 130 days post-treatment compared to 6/187 (3.74%) and median onset of 230 days in the control hamsters that received unfiltered RCC (Figure 6A).  These endogenous infectivity data are in agreement with the in vitro infectivity assay, the SSCA which showed residual prion infectivity with the 10 layer prion filter and none (below limit of detection) with the 22 layer.

FDA Meeting, October 28-29, Conclusions  These results demonstrate the utility of the highly sensitive cell culture-based infectivity assay for screening reduction filters.  The use of this type of in vitro infectivity assay will substantially help expedite the screening and discovery of devices aimed at reducing the risk of vCJD disease transmission through blood transfusion.  The use of this infectivity assay will also significantly reduce the cost for developing and evaluating devices for prion clearance.  It is very important that methods for screening potential prion removal chemistries or ligands include an infectivity assay at a very early stage of the screening process to complement other in vitro assays.  Although for the final release of any prion reduction device, it may still be necessary to conduct a limited endogenous infectivity bioassay, the use of SSCA should help improve and greatly expedite the process of screening and developing new devices for prion clearance from biological fluids.

FDA Meeting, October 28-29, Acknowledgments  Professor Charles Weissmann (Scripps, FL)  Dr. Christopher Baker (Scripps, FL)  Dr. Cheryl Demczyk (Scripps, FL)  Ms. Fabiola Andrade (Pall Medical Research Lab, NY)  Professor Maurizio Pocchiari (Istituto Superiore di Sanita, Rome, Italy)  Dr. Franco Cardone (Istituto Superiore di Sanita, Rome, Italy)  Dr. Richard Carp (NY Institute for Basic Research, NY)  Dr. Richard Kascsak (NY Institute for Basic Research, NY)  Ms. Regina Kascsak (NY Institute for Basic Research,NY)  Mr. Clifford Meeker ( NY Institute for Basic Research, NY)  Dr. Joseph Cervia (Pall Medical, NY)  Mr. Allan Ross (Pall Medical, NY)  Dr. Stein Holme (Pall Medical, NY)  Members of Pall QIRP internal review process (Pall Medical, NY)