# Presentation: 920638

**Implications of Batch Processing for Manufacture of **
**Human Cells, Tissues and Cellular and Tissue-Based **
**Products**
***Experience of a Commercial Manufacturer***
***of Pituitary-derived Growth Hormone***
**Eshkol, Ph.D., **
**Vice President for Scientific Affaires, **
**Serono International SA, Geneva**

- A. Eshkol, TSEAC, June 26-27, 2002

## Background

**	****Human Growth Hormone had been extracted from Pituitary Glands from the 1950’s.**
**	****Until the 70’s acetone dried glands were used and basically 2 methods – Raben and Wilhelmi - were employed. The products contained aggregates and dimers and were not a purified hGH product.**
**	****From the 70’s onwards extraction and purification was done on frozen glands.  The methods were milder (no strong acid or alkali) but included chromatographic exclusion of dimers, aggregates and other impurities.**

- A. Eshkol, TSEAC, June 26-27, 2002

## Safety Depends on:

**Raw Material ****	****- Biological Source**
**Process ****	****	****- Validation**
**	****	****	****	****- Decontamination**
**	****	****	****	****- Cross Contamination**
**	****	****	****	****- Batch Size**
**Testing ****	****	****- QC**

- A. Eshkol, TSEAC, June 26-27, 2002

## Source – hGH from Human Hypophyses

**Pituitary glands collected Post Mortem**
**Majority of Donors Older People**
**Donors with Nervous System Disease or Mental **
**    ****Illness Excluded**
**Mostly no Brain Histology available**
***Conclusion : No certainty of CJD-free Donors***

- A. Eshkol, TSEAC, June 26-27, 2002

## hGH and CJD

**In March 1985 the NIH learned that a  22 year old man died of CJD. He received hGH from 1966-1976.**
**In April 1985 two additional deaths due to CJD were revealed.**
**Such a risk was considered by some investigators, and a validation study determining the clearance potential of the purification process was initiated in 1979 in the UK and published in August 1985 – Taylor et al., The Lancet, Aug. 3, 1985, 260-262.  This study demonstrated that the specific procedure employed - the Lumley-Jones method (Lumley-Jones et al. J Endocrinol 82:77-86) – had a high capacity for the Scrapie agent clearance.  **

- A. Eshkol, TSEAC, June 26-27, 2002

## Process for hGH Preparation

**Multiple steps including:**
**Aqueous phase protein extraction**
**Selective Isoelectric Precipitation – pH 4.9**
**Solubilization and concentration**
**Chromatography on Sephacryl SF200**
**Collection of monomeric hGH**
**Millipore ultrafiltration – 100,000 dalton cut-off ***
**6M Urea Treatment ***
*** Introduced in 1985**

- A. Eshkol, TSEAC, June 26-27, 2002

## Inactivation Experiments with the 263K Strain of Scrapie

**Effect of 2 filtration procedures alone and followed by 6M Urea treatment**
- a Infectivity titres are expressed as – log 10 LD50 i.c. units/g of brain, measured by endpoint titration
- B Measurement by incubation time (mean  SEM)
- *From: Pocchiari et al. Arch. Virol. 1988; 98: 131-135

| Experimental
Procedures - sample | Infectivity a
titres (log) | Reduction of Infectivity
titres (log) |
| --- | --- | --- |
| Control - A | 9.0 | -- |
| Filtration (100,000 MW) – B | 6.8 | 2.2 |
| Filtration (25nm) – C | 7.0 | 2.0 |
| Filtration - B + Urea | 3.4  0.2 b | 5.6  0.2 |
| Filtration - C + Urea | 4.6  0.2 b | 4.4  0.2 |

- A. Eshkol, TSEAC, June 26-27, 2002

## Background and Decontamination Checking

**Trial Run / Background contamination**
**	****hGH purification procedure without scrapie challenge was run till after the Sephacryl chromatography step.**
**	****The monomeric hGH was concentrated and the whole product inoculated i.c. into 66 hamsters.**
**	*****None developed scrapie.***

- A. Eshkol, TSEAC, June 26-27, 2002

## Process – ValidationPerformed as three independent runs at 1:10 scale of actual process (1)

| Sample Tested | Total infectivity, log 10 LD 50 |  |  |  |
| --- | --- | --- | --- | --- |
|  | Run 1 | Run 2 | Run 3 | mean |
| Starting material | 9.7 | 9.7 | 9.7 | 9.7 |
| hGH chromatography 
purification – Sephacryl | 5.4 | 5.4 | 5.4 | 5.4 (-4.3) |
| hGH chromatography purification with 
added ultrafiltration and 6 M urea | 0.7 | 1.0 | 0.4 | 0.7 (-9.0) (2) |

**Performed as three independent runs at a 1:10 scale of actual process (1)**

- Numbers in parentheses represent change in titer from starting material. Each batch of starting
- material was contaminated with the same lot of a 10% clarified suspension of scrapie-infected hamster
- brain (strain 263K).
- From: Pocchiari et al. Horm. Res. 1991; 35: 161-166
- (2) 	Only 7/381 animals developed scrapie after a mean incubation of 232 days. Therefore infectivity was extrapolated based on % of animals developing scrapie.
- (2) Only 7/381 animals developed scrapie after a mean incubation of 232 days. Therefore infectivity was extrapolated based on % of animals developing scrapie.

- A. Eshkol, TSEAC, June 26-27, 2002

## Background and Decontamination Checking

**2.**** ****Control Run / Decontamination**
**	****At the end of the validation/clearance experiment, laboratory equipment (glassware, columns, ultrafiltration equipment) was decontaminated either by steam autoclaving at 134°C  (2 bars) for 1 h or, if not feasible, by overnight soaking in a 1 N NaOH solution containing 1% sodium dodecyl sulfate.**
**	****Then the hGH purification was performed using the decontaminated equipment.**
**	****Aliquots of 0.05 ml of monomeric hGH (post Sephacryl) were inoculated into 12 hamsters. **
**	****Following ultrafiltration and Urea treatment, the entire product was inoculated into 135 hamsters. *****None showed clinical signs of disease nor histological lesions in the brain.***

- A. Eshkol, TSEAC, June 26-27, 2002

## Avoidance of Cross Contamination

**Entire Process Steps were Divided into 4 Risk Categories**
** **
**	****	****	****	**

| Flow(a) | Step | Risk | Traffic(b) |
| --- | --- | --- | --- |
|  | Gland handling & first extraction | High |  |
|  | Crude Extract | Medium |  |
|  | Semi-purified protein | Low |  |
|  | Purified hGH | No |  |

- Flow of extraction and purification process
- Traffic of personnel, equipment, reagents, etc.

- A. Eshkol, TSEAC, June 26-27, 2002

## Batch Contamination / Batch Size

**Assumption : CJD bearing donors - 1/10,000 ****(1)**
**(1) Brown, Gajdusek, Gibbs & Asher ; NEJM, 1985; 313:728-731**
**(2) Of 4 IU of hGH**
**(3) Serono Procedure**

| Batch
size | Contaminated batches | Max. No. of vials (2) per batch |
| --- | --- | --- |
| 5,000 | 1/2 | 25,000 |
| 2,000 | 1/5 | 10,000 |
| 500-600 (3) | 1/10 – 1/20 | 2,500 |

- A. Eshkol, TSEAC, June 26-27, 2002

## Calculated Level (Worst Case) of Contamination of hGH following Production Procedures With and Without Added Ultrafiltration and Urea Treatment Steps

**(1) From: Pocchiari et al., 1991**

|  | hGH 
purification | hGH purification
with filtration/urea |
| --- | --- | --- |
| Infectivity (logLD50) in pituitary batch | 4.0 | 4.0 |
| Infectivity (logLD50) per hGH batch | -0.3 | -5.0 |
| Infectivity (logLD50) per hGH vial | -4.7 | -9.4 |
| Probability that a single vial is contaminated | 1 in 50,000 | 1 in 2.5 billion |
| Probability that 1 patient receives a contaminated vial during course of treatment | 1 in 64 | 1 in 3.2 million |

- A. Eshkol, TSEAC, June 26-27, 2002

## Incubation Periods in Pituitary Gland-derived GH Recipients

| Country | Age/
Gender | Treatment Period | Onset of illness/
incubation  | Reference |
| --- | --- | --- | --- | --- |
| USA | 41 M | 1956-1966 | 1991/ 27-37y. | Brown, Preece &
Will, 1992 |
| Netherlands | 39 F | 1963-1969 | 1989/ 20-26y. | Quoted by Croes
et al., 2002 |
| Netherlands | 47 M | 1963 (5 days) | 2001/ 38y. | Croes et al., 2002 |
| USA | 4 cases |  1977 | 1998-1999/ > 21y. | Brown et al., 2000 |
|  | 139 cases |  | 5 – 30 years
(median 12 y.) | Brown et al., 2000 |
| Australia
(Gonadotropins) | 4 |  | 12 – 16 years
(median 13 y.) | Brown et al., 2000 |

- A. Eshkol, TSEAC, June 26-27, 2002

## Conclusions:

**1. A protein like hGH (single chain, non-glycosilated) can be purified with a large safety margin provided that:**
**In the procedure, steps are included which can significantly reduce and inactivate CJD/TSE agents.**
**The process is carried out under strict conditions and includes effective decontamination procedures.**
**Care is taken to avoid cross-contamination.**

- A. Eshkol, TSEAC, June 26-27, 2002

**2. Can***** total elimination***** of potential infectivity be guaranteed ?**
**Serono has had no CJD cases reported, but in view of some very long incubation periods reported, we remain vigilant. If there was residual infectivity in some batches, it must have been extremely low.**
***Total absence (?)***** of risk would also need assurance of the safety of the raw material, which is particularly important in the case of older age group donors.**
**Currently available test methods do not have the sensitivity which would make this feasible.**

- A. Eshkol, TSEAC, June 26-27, 2002

## References

**P. Brown, M. Preece, J.-P. Brandel, T. Sato, L. McShane, I. Zerr, A. Fletcher, M. Pocchiari, N.R. Cashman, J.H. Aignaux, L. Cerven****áková, J. Fradkin, L.B. Schonberger, S.J. Collins.  Neurology 2002;55:1075-1081.**
**P. Brown, M.A. Preece, R.G. Will.  Lancet 1992;340:24-27.**
**P. Brown, D.C. Gajdusek, C.J. Gibbs Jr, D.M. Asher.  NEJM 1985;313:728-731.**
**E.A. Croes, G. Roks, G.H. Jansen, P.C.G. Nijssen, C.M. van Duijn.  J Neurol Neurosurg Psychiatry 2002;72:792-793.**
**R. Lumley-Jones, G. Benker, P.R. Salacinski, T.J. Lloyd, P.J. Lowry.  J Endocrinol 1979;82:77-86.**
**M. Pocchiari, S. Peano, A. Conz, A. Eshkol, F. Maillard, P. Brown, C.J. Gibbs Jr, Y. Geng Xl, E. Tenham-Fisher, G. Macchi.  Horm Res 1991;35:161-166.**
**M. Pocchiari, G. Macchi, S. Peano, A. Conz.  Arch Virol 1988: 131-135.**
**D.M. Taylor, H. Fraser, P.R. Salacinski, A.G. Dickinson, P.A. Robertson, P.J. Lowry.  Lancet 1985: 260-262.**

- A. Eshkol, TSEAC, June 26-27, 2002