TSE Agent Clearance Issues

TSE Advisory Committee

February 20, 2003

Dorothy Scott, M.D.

DH/OBRR/CBER/FDA

Paradigm: Validation of Virus Removal/inactivation Includes:

• Scaling down process steps• Spiking appropriate steps with high titer

of infectious agent (actual or model)• Determination reduction factors for each

step• Summing reduction factors [from

orthogonal processes] to give a total log10 reduction value

Scale-Down of Purification Steps

• Usually 1/10 to 1/100 scale; no set guidelines

• Must keep buffers, pH, protein concentration, and product the same as full scale manufacturing

• Must keep operation parameters as close to full scale as possible (e.g., bed height, flow rate)

• Must show product is identical to production scale

Criteria for Acceptable Pathogen Detection Assays

• Accuracy

• Assay repeatability and reproducibility

• Linearity

• The limit of detection (LOD)

• The limit of quantitation (LOQ)

• Assay robustness and reproducibility

TSE Spike Plasma

Cryoprecipitation Cryoprecipitate(FVIII)

Cryopoor Plasma Supernatant

Albumin, IGIV, 1PI, ATIII, etc.

TSE Clearance Evaluation: Example

ANTITHROMBIN III

ANION EXCHANGE CHROMATOGRAPHYHEPARIN AFFINITY CHROMATOGRAPHY

cohnfrac.org by DJFrazier 12/96

Pasteurization

(Diafiltration)(Chromatography)(Viral inactivation)(Limited proteolysis)(Lyophilization)

The Cohn-Oncley Blood Plasma Fractionation Process

Fraction IV-1 Supernate

Cryoprecipitate Supernate

Fraction I Supernate

Fraction II+III Supernate

Fraction II+IIIw Supernate

Fraction II Supernate

Fraction III Supernate

Fraction V Supernate

Fraction IV-4 Supernate

freeze, thaw

IMMUNE GLOBULINS

pH 7.425% ETOH-5 oC

pH 5.217% ETOH-6 oC

pH 7.620% ETOH

-5 oC

ALBUMIN

pH 4.610% ETOH

-2 oCFilterpH 5.1540% ETOH

-5 oCResuspend

pH 4.840% ETOH-5 oC

pH 5.840% ETOH

-5 oC

pH 5.318% ETOH

-5 oC

pH 6.8520% ETOH-5 oC

pH 7.28% ETOH-2oC

FACTOR IX COMPLEXANTIHEMOPHILIC FACTOR

Plasma

Published TSE Clearance Studies for Plasma Fractionation

1. Brown, P et al, Transfusion 1998 38:810-62. Brown, P et al, Transfusion 1999 39: 1169-783. Lee, DC et al, J. Virol. Meth. 2000 84: 77-89 4. Foster, PR et al, Transfusion Science 2000 22:53-565. Foster, PR et al, Vox Sanguinis 2000 78:86-95 6. Lee, DC et al, Transfusion 2001 41: 449-557. Cai, K et al, Biochem Biophys. Acta 2002 1597: 28-358. Stenland, JS et al, Transfusion 2002 42:1497-15009. Vey, M et al, Biologicals 2002 30:187-9610. Reichl, HE et al, Vox Sanguinis 2002 83:137-45

Challenges in Studies of Clearance of TSE Agents

• What source of infectivity to use– Brains preparations from experimentally infected animals most easily

available• Hamsters (scrapie)• Mice (GSS, BSE)

– BSL-3 facility needed to study vCJD, BSE– PrpSc partitioning similar when source is human (CJD, vCJD), or

animal TSE’s (Stenland et al, Transfusion 42: 1497-1500, 2002; single study)

• What “form” of infectious agent most relevant to blood? – Brain homogenate– Subcellular membrane fractions– Membrane-free infectious material

Challenges in Studies of Clearance of TSE Agents

• The lower limits of assay sensitivity (2-3 logs), and upper limits of titers available for spiking– Range of infectivity removal detectable 4-5 logs– “Throughput” experiments to assess additiveness of clearance

steps therefore have limitations

• What assays are best to measure outcomes– In vivo infectivity (time, expense)– In vitro surrogates – measurements of PrpSc

– Bridging in vivo to in vitro results (Transfusion 2001 41: 449-55)

• Mass balance – retention TSE agents by columns; loss of mass balance

Challenges in Evaluating Clearance of TSE Agents

• How much reduction is “enough? (risk assessment)

• How many disparate clearance steps should there be?

• What steps can be summed, which cannot?– Summed reduction factors for similar steps, e.g.

EtOH precipitation

TSE Clearance depends upon specific characteristics of starting material and process conditions:

Examples• Partitioning of infectivity depends upon pH,

ionic strength, and alcohol concentration

• Cryoprecipitation methods may influence degree of clearance

• Depth filtration effectiveness depends upon filter used and/or properties of starting material

Example (1) PrpSc Partitioning is condition-dependent

Cai, A. et. al. Biochem. Biophys. Acta 597: 28-35, 2002 • Scrapie brain homogenate spiked into

buffers with varied:– EtOH concentrations– Salt concentrations– pH

• Incubation• Centrifugation• Measurement PrpSc in supernatant

Parameters Influencing Prpsc Partitioning

Precipitation best at:a. Mildly acidic pHb. With EtOHc. At higher pH, with salt and EtOH

Cai, K. et. al. Biochem Biophys Acta 1597(1): 28-35, 2002

Example (2) Cryoprecipitation: variable clearance among studies

with different conditions

1. FVIII partitions with cryoprecipitate2. Clearance of PrpSc in cryoprecipitation

- 1 log clearance in effluent (Lee et al., Transfusion 41: 449-55, 2001)

- 1 log clearance in effluent (Brown et al., Transfusion 38: 810-16, 1998)

- <1 – 1.7 logs clearance in precipitate (Foster et al., Vox Sang 78:86- 95, 2000)

Example (3) Clearance PrPsc (microsomal spike) by Depth Filtration – Influence of Starting Materials and Filter

Starting Material Depth Filter Reduction Factor (log10)

Fr V (albumin) Seitz KS80 > 4.9

Fr V (albumin) CUNO Delipid 1 2.3

S I + III (IGIV) Millipore AP20 < 1

Fr II (IGIV) Seitz K200 > 2.8

Foster et. al., Vox Sang 78: 86-95, 2000

Fr I supernatant (IGIV, albumin) Supra P80 < 1

Fr V supernatant (albumin) Supra P80 > 1.1

Fr V supernatant (albumin) – Prp-sc spike Supra P80 > 2.4

Vey et al, Biologicals 30:187-96, 2002

TSE Clearance and the Manufacturing Process

• Manufacturing processes are highly individual – Cohn-Oncley process variations– Other fractionation methods– Variations in downstream processing/purification of

products (e.g. column chromatography)

• Rigorous demonstrations of TSE clearance therefore need to be based upon the specific manufacturing process

• Published studies may prove useful to identify steps with potential for TSE clearance

Evaluation of TSE clearance studies from industry, to support labeling claims

of lowering possible TSE risk

• Characterization of spiking agent • Accurately scaled-down processes• Robust and reproducible experiments • Well-characterized assay for TSE infectivity

– Bridging binding assays to bioassays

• Estimated logs clearance of TSE by processing steps (reduction factor and clearance factor)

• Demonstration of mass balance• Demonstration, where relevant, that non- orthogonal

(similar) clearance steps are/are not additive

Evaluation of Submissions to Support Labeling Claims

• Clearance “beltline” to support labeling– At least 2 orthogonal steps with > 4 logs clearance (total 8

logs)– At least 2 steps demonstrated to be additive with > 4 logs

clearance/step (total 8 logs) – ? At least 2 steps (orthogonal or demonstrated to be additive)

with > 3 logs/step (total 6 logs)– Is a single clearance step of > 4 logs sufficient if robust and

reproducible?– Are clearance steps of > 2 logs reliable if they are robust and

reproducible?

• Cumulative clearance/risk analysis

Labeling for TSE Risk• Current proposal: “Because this product is made from

human plasma, it carries a risk of transmitting infectious agents, e.g. viruses, and, theoretically the vCJD agent. It has been demonstrated that [the manufacturer’s] manufacturing process provides substantial clearance of agents similar to those causing CJD and vCJD. Thus the theoretical risk of transmission of CJD or vCJD is extremely remote.”

• Future improvements in risk assessment, understanding of plasma infectivity, and study methods could provide a basis for additional labeling content