# Presentation: 776003

## 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 log****10**** 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 Clearance Evaluation:  Example

***Cryoprecipitation*****	****	****	****	****	****Cryo**_**precipitate**_
**	****	****	****	****	****	****	****(FVIII)**
**	****	****	****	****	****	****	**
**	****	****	****Cryopoor Plasma **_**Supernatant**_**	****	**
**	****	****	**
**	****	****	****Albumin, IGIV, **
**	****	****	********1PI, ATIII, etc.**

_**TSE Clearance Evaluation:  Example**_

## Slide 6

## Published TSE Clearance Studies for Plasma Fractionation

- Brown, P et al, Transfusion **1998** 38:810-6
- Brown, P et al, Transfusion **1999** 39: 1169-78
- Lee, DC et al, J. Virol. Meth. **2000**  84: 77-89
- Foster, PR et al, Transfusion Science **2000**  22:53-56
- Foster, PR et al, Vox Sanguinis **2000** 78:86-95
- Lee, DC et al, Transfusion **2001** 41: 449-55
- Cai, K et al, Biochem Biophys. Acta **2002**  1597: 28-35
- Stenland, JS et al, Transfusion **2002** 42:1497-1500
- Vey, M et al, Biologicals **2002**  30:187-96
- 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**
**Prp****Sc **** 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:
- Mildly acidic pH
- With EtOH
- 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

**FVIII partitions with cryoprecipitate**
**2. Clearance of Prp****Sc**** 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 (log**__**10**__**)**_
**	****	****	****	****	****	**
**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