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ARTICULO SOBRE TECNICAS / PAPER ON TECHNIQUE SENDAI VIRUS REMOVAL AND INACTIVATION DURING MONOCLONAL ANTIBODY PURIFICACION. Rodolfo Valdes^1, Tania Diaz^1, Aymara Nieto^2, Calixto Garcia^2, Maite Perez^1, Janet Garcia^1,Yair Quinones^3 1Monoclonal Antibody Production Department. Center for Genetic Engineering and Biotechnology.P.O.Box 6162, Havana 6, Cuba.^ 2Quality Control Department of the National Center for Laboratory Animal Production. P.O.Box 6162 Havana 6, Cuba. ^3Hepatitis B Vaccine Department. Center for Genetic Engineering and Biotechnology. P.O.Box 6162. Havana 6, Cuba
Code Number: BA95048
File Sizes:
Text: 24K
Graphics: Line Drawings (gif) 28K
Recibido en agosto de 1994. Aprobado en febrero de 1995.Key words: Sendai virus, monoclonal antibody, Hepatitis B, virus validation. SUMMARY A feature common to all biologicals in whose production any material of animal or human origin have been used is the risk of viral contamination. Validation of viral removal during their manufacture is one of several techniques which can be used to establish product safety. The usefulness of this validation technique in the manufacture of monoclonal antibody CB/HEP/1 is demonstrated in this paper. Several procedures were used to examine ways eliminating infectious viruses by experimenting with a scale-down manufacturing procedure, and spiking of individual step or the undivided purification process to determine the extent of removal and viral inactivation. In this case cumulative clearance factor of 16 logs was achieved for the Sendai virus used as a relevant and model virus. It can be concluded that this clearance factor provides a highly safe purification standard in the production process of monoclonal antibody from ascitic fluid. RESUMEN Un aspecto comun para todos los productos biologicos en cuya produccion se involucra el uso de algun material de origen humano o animal, es el riesgo de contaminacion viral. La validacion de la eliminacion de agentes virales durante la fabricacion de un producto es una de las tecnicas que se emplean para establecer la seguridad del mismo. La utilidad de las tecnicas de validacion puede ser ilustrada en la manufactura del anticuerpo monoclonal CB/HEP/1 dirigido contra el antigeno de superficie del virus de la Hepatitis B. Varios procedimientos se basan en el principio de la eliminacion de virus infecciosos mediante experimentos que involucran un desescalado del proceso de purificacion, desafio del proceso o de cada paso individualmente, para determinar el poder de eliminacion y de inactivacion de virus. En este caso, el factor total de eliminacion encontrado para el virus Sendai fue de 16 logs, por lo que se concluye que este proceso de purificacion brinda un elevado margen de seguridad para anticuerpos obtenidos a partir de fluido ascitico. INTRODUCTION Recent biotechnological developments have brought new quality demands into the pharmaceutical manufactures for human use. Many of this products are based on monoclonal antibodies or recombinant DNA technologies and most of them are purified from cell tissues, organs, blood or other biological fluids. The potential for contamination by infectious viruses comes from many sources. A major problem is the fact that cell line particular those of rodent or human origin commonly used in the production of biopharmaceuticals often contain endogenous or latent retroviruses (1 and 2). In the past, a number of biologicals administered to humans was contaminated with viruses, and in several instances the contaminant was identified only many years after the product's introduction into the market. Yellow fever vaccine was contaminated with Avian Leukosis virus by virtue of its production in infected hen eggs and also by Hepatitis B virus contained in human sera added to the vaccine as a stabilizer. Other example is Sendai virus 40 contamination of polio virus and adenovirus vaccines prepared in the 1950s on primary cultures of kidney cells obtained from rhesus monkeys which naturally harboured an imperceptible clinical infection with Sendai virus 40, and HIV has also contaminated blood products (2). Three main approaches can be adopted to control potential virus contamination of biologicals: selecting and testing source materials for the absence of the viruses, testing the capacity of the production processes to remove or inactivate viruses and testing the product for contaminating viruses at appropriate stages of production (2).
This will be achieved by the deliberate addition (spiking) of significant amounts of a virus to the row material to be purified and to different fractions obtained during the various purification stages. A major aim in performing a validation is the determination of which viruses should be used. These fall in two categories; relevant virus and model virus. relevant viruses are viruses which are known or likely to contaminate the source material or other materials used in the production process. Model viruses: if the use of relevant viruses does not encompass viruses with a wide range of physico-chemical properties, then validation should be performed with model viruses. Validation of Ab CB/HEP/1 purification process was performed on a research scale (figure 1) and this monoclonal antibody purified from BALB/c ascitic fluid. The viral agent used in this study was an RNA virus, family Paramyxoviridae, genus Paramyxovirus, species parainfluenza 1[Sendai virus] (table 1). The virus particles are spherical, 150-200 nm in diameter, have a helical nucleocapsid, a continuous single RNA genome and low resistance to physical and chemical agents such as UV light, temperatures above 37^oC, lipid solvents and extremes pHs (3). This virus provides a representative panel of possible mouse viral contaminant such as Retrovirus, Influenza or other small enveloped viruses (4). MATERIALS AND METHODS Purification of Monoclonal Antibodies from Ascitic Fluid Samples of ascitic fluid were obtained from routine large scale ascitic fluid production derived from hybridome cell line CB/HEP/1, which secretes a murine monoclonal antibody (IgG2b) specific for the rHBsAg(5). The viral removal experiments were performed with ascitic fluid and inactivation experiments were performed with purified monoclonal antibody. To design the virus study validation, a scaling down on the purification process was done. For our experiments; the scale-down was brought to the 1% of the real production scale and the level of purification of the product mimiked the production process and the products generated were similar in terms of purity, specific activity, mouse DNA and yield. (table 2).
Elisa PVC plates were coated with rHBsAg (100 uL, 10 ug/mL in NaHCO3 100 mM, pH 9.6, 20 min at 56^oC). The plates were washed with PBS/ 0.05% Tween-20. Several dilutions of the samples were added and incubated during two hours at RT. The plates were washed again and incubated at 37^oC for one hour with HRPO-labeled, affinity purified goat anti mouse-IgG diluted 1/1000 in PBS/Tween-20/BSA. The plates were washed and subsequently incubated 20 min with substrate solution (5 uL H2O2 and 5 mg OPD diluted in 10 mL of Citrate 50 mM, pH 5.5). The reaction was stopped with 250 mM SO4H2 (50 uL/well) and the absorbency was measured at 492 nm in an ELISA reader. The average value curve of CB/HEP/1 was used to quantify Ab activity. Electrophoresis
Protein purity was analyzed by electrophoresis in 12.5%
polyacrylamide gel containing 0.1% SDS at pH 8.6 as described by
Laemmli (6). Prior to electrophoretic run, eluates were treated
with 1% SDS at 50
Protein Concentration
The protein concentration was determined according to Lowry
Murine DNA
The murine DNA in purified monoclonal antibody preparations was
assayed by dot-blot hybridization using a mouse genomic DNA
probe. Standard mouse DNA was used to determine the DNA content
in the same experiment.
Table 1. Specification of the virus strain and read-out
systems used for measuring the reduction and inactivaton factors
of Sendai virus
Spike Experiments
Viral Removal
To determine the virus reduction factor in the down stream
purification process of ascitic fluid, a preparation of
infectious Sendai virus was added to the initial fluid in order
to measure the total reduction factor of the scale-down
purification process. We worked with only one virus strain to
reduce the interference from other viruses in the measurement of
the infectivity titers. Before and after each purification, 2 mL
of the samples were taken from each test and immediately frozen
at-70^oC (2).
Viral inactivation
Citrate low pH buffer was used to elute the Ab CB/HEP/1 from the
Protein A-Sepharose CL4B column. In this study, we spiked the
citrate buffer with Sendai virus in order to test its ability to
inactivate this virus. We conducted antibody inactivating studies
at 4^oC, 37^oC and 60^oC for 10 h to determine the condition in
which the antibody does not lose its recognition capacity of the
antigen and subsequent measure of the virus stability (table 3).
Table 2. Sendai virus reduction factor of CB/HEP/1 purification
method
Hemagglutination Test
An HA test was carried out to determine the infective titers of
Sendai virus. For this purpose two log serial dilutions were made
in PBS 7.4 and human erytrocyte sedimentation was observed after
30-45 min at room temperature (22^oC). The titers were expressed
as the reciprocal of the dilution still showing
hemagglutination.
Embryonated Egg Infectivity Doses
The Reed-Muech method was applied to determine the infectivity
titers. Several dilutions of the samples were made in PBS and 0.1
mL of these dilutions were inoculated in the allaontoic cavity
of embryonated chicken eggs (8).
Clearance/Inactivation Factor
The virus reduction factor of a purification or inactivation step
is defined as the log10 of the ratio between the virus load in
the preparation material and the virus load in the
post-purification material which is ready for the next step of
the process. The following abbreviations are used ( 9):
Starting material: vol v'; titer 10a'
Viral load: v'*10a'
Final material: vol v"; titer 10a" Viral load: v"*10a"
The individual reduction (RF) or inactivation factor (IF) is
calculated according to 10rf = v'*10a'/ v"*10a"
RESULTS AND DISCUSSION
Sendai virus inactivation
A low pH is frequently used to elute proteins from affinity
chromatography. In our purification process we used 0.1 M, pH 3
citrate buffer to elute CB/HEP/1 from Protein A Sepharose CL-4B
column (Pharmacia) (5). Heat treatment however, has a mayor
disadvantage: not all products tolerate heat and this could have
an adverse effect on the efficacy, stability or biochemistry of
the product (10). Kinetic studies for inactivation of CB/HEP/1
have been important in demonstrating at which condition this
monoclonal antibody was more resistant. For example, CB/HEP/1 Ab
were completely inactivated after one hour at 60^oC in citrate
buffer pH 3 while after 10 h at 37^oC the activity was 23.4%. For
this reason, we selected citrate pH 3 at 4^oC as the best
experimental condition as in which, after seven h the activity
was more than 80% of the initial activity (table 3).
This buffer was spiked with Sendai virus. We conducted the
inactivation studies at 4^oC and 7 h for periods from one hour
to 7 h. The reduction in Sendai virus titers was 4.2 logs (first
experiment) and 8.4 logs (second experiment) (tables 4 and 5).
Inactivation of this virus occured rapidly, and was dependent on
buffer formulation and not on temperature because the reduction
in virus titer used as control (PBS, pH 7.4 at 4^oC) was not
observed (table 4).
Table 3. Ability of citric acid pH3 treatment at different
temperatures to inactivate a monoclonal antibody CB/HEP/1
preparation
FC final concentration measurement by ELISA (mg/ml)
IC initial concentration measurement by ELISA (mg/ml)
Sendai viral removal
Validation was performed on a research scale that represented 1%
of the real scale of the purification process (figure 1) (11).
The Ab purity, especific activity and yield provided by the
protocol are equivalent to real production scale, 50 mL of
ascitic fluid were spiked with Sendai virus. This virus is
representative of a panel of possible contaminating agents with
similar physico-chemical structures: a murine retrovirus and
other viruses of RNA, envelope, size between 100-250 nm,
spherical shape and little resistance to physico-chemical
agents.
The removal of the viral agent in this standard purification
system worked only on protein A affinity chromatography. There
is a reduction factor of up to 8.4 logs (table 2). The overall
clearance factor (inactivating and removal factor, 16.8 logs) was
substantially greater than the potential virus titer in the
source material; concentration of this group of contaminating
viruses in a cell culture rarely exceeds 10^6 (11) and ascitic
fluid 10^9 (12). Now we are making spiking experiments of
the purification process with other viral particles that
represent a different viral system in order to complete the panel
representing a model virus group in terms of physico-chemical
properties of all murine viruses.
Table 4. Ability of citric acid pH 3 at 4 C treatment to
inactivate the Sendai virus.
Table 5. Ability of citric acid pH 3 at 4 C treatment to
inactivate the Sendai virus
A cumulative removal of more than 16 logs of removal/inactivation
can be estimated if it is assumed that the steps are independent
events. The degree of removal shown by these logs would be more
reliable if the actual amount of virus contaminating the ascitis
bulk were known.
We are trying at present to measure the virus level in ascitic
fluid risk in CB/HEP/1 in order to determine the real
concentrations of the different viruses that are included in this
group, and as well as we are using a MAP Hageman test on the
master cell bank, ascitic fluid and final product as an
additional test to confirm the safety of the manufacturing
process. In all cases, the results were negative for this kind
of virus, suggesting that there is a substantial margin of safety
in the process and for Hepatitis B vaccine since this monoclonal
antibody is used to immunopurify the recombinat Hepatitis B
surface antigen used in this biological preparation.
ACKNOWLEDGMENTS
Our thanks to Lic. Beatriz Gago, Dr. Jorge Gavilon-do, Lic. Suany
Ojeda and Ing. Alberto Agrass for critical review of this
manuscript and Dr. Luis Herrera for his scientific
contributions.
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