Chapter 9
Virus Removal and Inactivation A Decade of Validation Studies: Critical Evaluation of the Data Set Joachim K. Walter, Franz Nothelfer, and William Werz Downloaded by CORNELL UNIV on June 11, 2017 | http://pubs.acs.org Publication Date: July 23, 1998 | doi: 10.1021/bk-1998-0698.ch009
Boehringer Ingelheim Pharma Deutschland KG, D-88397 Biberach, Germany
Recombinant DNA technology as well as hybridoma technology provide an outstanding opportunity for the preparation of complex human proteins with significant impact on replacement therapy. Acting as living production facilities, the microorganisms and animal cells used for the manufacturing of proteins are subject to biological deviations and are influenced by the environment. With respect to mammalian cell culture, the cell's susceptibility for potential viral infections is of concern regarding the drug safety. Measures for the removal of potential viral contaminants during the downstream processing would be more reliable, if only cell cultures essentiallyfreeof endogenous virus are employed for the expression of the desired protein product; however, potential viral contamination may be induced adventitiously by application of biological raw materials to the production process. In addition, a failure in proper containment e.g. in venting and aeration of the fermentation culture may definitely lead to a viral infection. Especially in the case of viruses of unknown species, it is theoretically and practically impossible to monitor the inactivation or removal of such species. As they are expected to have unknown but potentially harmful biological effects, specific assays can not be devised to monitor their presence and/or behavior during the manufacture of a pharmaceutical protein. Hence, a number of "next best" preventative measures have to be established around the manufacturing process. All have the common goal to reduce the probability of a potential viral contamination. These measures include tests for a great number of specific viruses potentially present in the designated Master Cell Bank (MCB) and define specific tests to detect potential adventitious virus infections during the production process. Nevertheless, the capability and the capacity to reduce potential viral contaminants has to be demonstrated for individual unit operations of the downstream processing. To this end however, model viruses must be selected which represent the whole range of virus species. Table I lists the model viruses typically used in validation studies. Criteria for the selection of such viruses are:
114
©1998 American Chemical Society
Kelley and Ramelmeier; Validation of Biopharmaceutical Manufacturing Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
Kelley and Ramelmeier; Validation of Biopharmaceutical Manufacturing Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
Vesicular stomatitis virus Parainfluenza virus Human immunodeficiency virus Murine leukaemia virus (MuLV) Poliovirus Sabin type I Encephalomyocarditis (EMC) Reovirus 3 SV 40 Parvoviruses (canine, porcine)
Virus
Low
100-200 nm Pleo/Spherical 80- 100 nm Spherical Spherical Icosahedral Icosahedral Spherical Icosahedral Icosahedral
80- llOnm 25 - 30 nm 25 - 30 nm 60 - 80 nm 40 - 50 nm 18 -24nm
+ +
-
RNA RNA RNA DNA DNA
Man Mouse Various Monkey Canine Porcine
Picorna
Picorna
Reo Papova Parvo -
+
RNA
Mouse
Retro
Medium Very High Very High
Medium
Medium
Low Low
70 x 175 nm Bullet shape
Shape
+
RNA RNA RNA
Rhabdo
Size
Resistance to Physicochemical Treatment Low
Envelope
Genome
Paramyxo Retro
Natural Host
Equine Bovine Various Man
Familiy
Table I: Model Viruses for Validation Studies (adapted from [7, 8])
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116 • • • • •
size of the viral particle enveloped / non-enveloped virus genome structure: DNA / RNA strandedness of the virus genome resistance to inactivation methods
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With regard to the feasibility of validation work on virus inactivation / removal, criteria also include: • • • •
relevance of the virus species for the producer cells achievable high virus titer high sensitivity of detection ease of detection
Virus clearance during downstream processing is performed by methodologies for virus inactivation and virus removal. Regarding virus removal, two principal unit operations of the downstream process may contribute: filtration and chromatography (1-4). This paper presents datafromnumerous validation studies on the inactivation and removal of virus during the downstream processing. This set of data has been collected, compared and evaluated to rank such unit operations regarding their impact, importance and reliability for the drug safety of mammalian cell culture derived pharmaceutical proteins. Validation of Virus Removal and Inactivation The capability and capacity of individual unit operations in the downstream processing regarding virus removal and inactivation needs to be validated according to the very same concepts and criteria established for process validation in general (5-8). With rare exceptions such as microwave-induced High Temperature Short Time (HTST)-Heat Treatment the scale of the respective validation equipment might be different from the manufacturing scale: typically the validation needs to be performed using the equipment at least of identical type and scale as intended for the production; validation of viral clearance however has found acceptance as an exception to this rule: the contamination of equipment for the production with infectious virus as a spike would impair such equipment to an irresponsible degree. In addition, the availability of the required amount of respective virus at high titer for a production scale of several thousand liters is not feasible. Accordingly, the design of the downstream processing should consider some basic requirements for the validation of viral clearance: • • •
Every process step should be transferable to the required scale. All process equipment that is not disposable has to be at least sanitizable. The design of all process components must permit the necessary validation procedures.
Kelley and Ramelmeier; Validation of Biopharmaceutical Manufacturing Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
117 Unit Operations for Viral Clearance Data for viral clearance have been obtained from a number of downstream processes for various unit operations and modes of operation:
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•
Chromatography - Affinity - Hydrophobic Interaction - Cation Exchange - Anion Exchange - Gel permeation • Filtration - Nanofiltration - Ultrafiltration • Inactivation - Acid - Urea - Convective Heat - Microwave Heat Chromatography Data Any chromatographic separation which is claimed for viral clearance must be validated first with respect to the down-scaled chromatographic process, before it is applied to validation experiments: • •
Proof of product quality before and after the chromatographic separation indicates the process performance. Validity and reliability of the chromatographic process is demonstrated by the reproducibility and comparability of process data derived from production scale and down-scale. Typical process parameters to be checked are: - yield - linear flow rate - resolution - pH-value - selectivity - conductivity - capacity - system pressure - profiles - sample load respective matrix volume
Conclusions Chromatography The results in Table II, which were obtained from a number of different chromatographic processes, indicate that - even comparable modes of chromatography contribute to virus removal to a different and in inconsistent extent. Despite the fact that an individual chromatographic separation is highly reproducible, the data clearly show that chromatography works as it is designed for: it separates proteins by
Kelley and Ramelmeier; Validation of Biopharmaceutical Manufacturing Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
118 Table II: Viral Clearance in Chromatographic Separations T v
e
Downloaded by CORNELL UNIV on June 11, 2017 | http://pubs.acs.org Publication Date: July 23, 1998 | doi: 10.1021/bk-1998-0698.ch009
l P Affinity
Protein 1 MuLV 1 PI-3 1 Reo3 1 SV-40 [
Prot A
150 Kd
4.0
3.2
6.3
6.5
1.1
4.0
Process Buffer
L:pH9.0 190mS E: pH3.0 38mS L:pH6.25 4.5mS E:pH3.4 lmS 26mS L: pH 6.0 E: pH 7.5 15mS
Hydrophobic Interaction Pyridyl
46 Kd
3.6
1.7
>6.8
5.5
Octyl
80 Kd
5.0
6.3
2.8
1.1
46 Kd
>4.0
1.2
2.8
5.9
80 Kd
>6.3
6.5
1.1
4.0
60 Kd
3.6
1.7
>6.8
>6.3
3.0
>6.7
150 Kd
L: pH 8.0 115mS E: pH 5.5 lOmS FT: pH 5.8 135mS
Cation Exchange S Sepharose FF
SP Sepharose FF
L:pH6.0 E: pH 7.8 L: pH 6.3 E:pH6.5 FT: pH5.0
6mS 20mS 6mS 9mS 5mS
5.5
L: pH 6.8 E: pH 7.5
8.5mS 40mS
5.3
3.5
5.3
6.1
>6.7
5.0
6.0
6.0
5.8
150 Kd
3.6
1.7
>6.8
5.5
60 Kd 150 Kd
5.0 >5.7
5.7
1.7 >6.7
6.9
5mS L: pH 7.5 E: pH 7.5 12mS 2mS L: pH 8.5 E: pH 7.5 15mS 4mS L: pH 8.8 E: pH 8.5 8.5mS 6mS L: pH 8.5 E: pH 8.5 16mS FT: pH 7.5 15mS FT: pH 8.0 6mS
80Kd 60 Kd
>6.4 7.0
1.5 1.3
6.1 >7.7
2.7 n.d.
46 Kd
>3.8
3.8
>3.1
>3.9
>3.3
pH 7.1
14mS
46 Kd 80 Kd
>3.8 4.2
>3.0 3.5
>3.1 5.4
>4.3 2.9
pH5.5 pH5.0
22mS 5mS
60 Kd 150 Kd
n.d. 4.0
>6.9 5.2
>7.2 2.6
>5.5 3.6 4.9 >5.2 >5.9
3.7 >6.6 >4.4 4.8 7.5 >7.1
6.0 >3.0
4.2 1.2 3.8
