WO1998017786A1

WO1998017786A1 - Heat inactivation of viruses in antibody preparations

Info

Publication number: WO1998017786A1
Application number: PCT/US1997/018851
Authority: WO
Inventors: Ilene Sussman; Gopalan Soman; Samuel K. Ackerman
Original assignee: Oravax, Inc.
Priority date: 1996-10-23
Filing date: 1997-10-23
Publication date: 1998-04-30
Also published as: AU5082698A

Abstract

The invention provides methods for inactivating viruses in antibody preparations, such as monoclonal antibody preparations, by gentle heating.

Description

HEAT INACTIVATION OF VIRUSES IN ANTIBODY PREPARATIONS Statement as to Federally Sponsored Research Background of the Invention

This invention relates to inactivation of viruses in antibody preparations. Monoclonal antibodies have been applied clinically in the diagnosis and therapy of an array of human disorders, including cancer and infectious diseases, and have been used for the modulation of immune responses. A problem inherent in the use of biological products, such as antibodies, in therapy is that there is a possibility that these products are contaminated with pathogens, such as viruses.

Summary of the Invention We have shown that gentle heating of antibody preparations inactivates model viruses, while maintaining antibody integrity.

Accordingly, the invention features methods of inactivating a virus in a sample containing an antibody that binds to an antigen. In these methods, the sample is heated under conditions in which (1) the virus is inactivated, and (2) the antibody retains its capacity to bind the antigen. The virus inactivated by the methods of the invention is no longer able to replicate.

Preferably, after treating a sample using the methods of the invention, there is at least a 50% reduction in replicable virus in the sample. For example, there can be at least a 60%, 75%, 90%, 95%, or 100% reduction. The level of inactivation can also be measured by determining the log reduction of active virus (see below). Preferably, there is at least a 1 log reduction in virus titer, for example, at least a 2, 3, 4, 5, or 6 log reduction.

The antibody in the sample treated using the methods of the invention can be a monoclonal or a polyclonal antibody. For example, the antibody can be an IgA (e.g., the antibody produced by hybridoma cell line HNK20 (American Type Culture Collection Accession No. HB 11514)), IgG, IgM, IgE, or IgD antibody. Alternatively, other protein preparations can be treated using the methods of the invention. The heating in the methods of the invention can be carried out at 50-57 °C for up to around 60 minutes. For example, the heating can be carried out at 54 ± 1 °C for 0.1-10 minutes, 1-5 minutes, or about 3 minutes.

The invention also includes antibodies (e.g., monoclonal or polyclonal antibodies, such as IgA (e.g., the antibody produced by hybridoma cell line FTNK20 (American Type

Culture Collection Accession No. HB 11514)), IgG, IgM, IgE, or IgD antibodies) treated according to the methods of the invention.

The invention provides several advantages. For example, the invention enables the production of therapeutic antibody preparations that, when administered to patients, such as human patients, have decreased risks of causing the patients to become infected with a contaminating virus. Additional advantages of the methods of the invention include, e.g., that they do not require the use of compounds such as stabilizers, and that they are rapid and can readily be incorporated into large-scale production processes.

Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.

Brief Description of the Drawings Fig. 1 is a schematic illustration of a laboratory-scale purification scheme for monoclonal antibody FINK20.

Fig. 2 is a schematic illustration of an apparatus for heat inactivation of an antibody sample.

Fig. 3 is a schematic illustration of a large-scale purification scheme for monoclonal antibody HNK20.

Detailed Description The invention provides methods for inactivating viruses in antibody (e.g., monoclonal antibody) preparations by gentle heating, without significantly affecting antibody purity, integrity, effectiveness, and stability.

Antibodies present in the samples treated using the methods of the invention include, e.g., monoclonal antibodies, such as monoclonal antibodies of the IgA, IgG, IgM, IgE, and IgD classes, and subclasses of these antibody classes. In addition to monoclonal antibodies, polyclonal antibodies and other protein preparations can be treated using the methods of the invention. The antibodies can be contained in, e.g., a physiologically balanced buffer, such as phosphate buffered saline, at a pH of, e.g., about 7.2. The antibody concentration in the preparation can range from, e.g., less than 0.1 mg/mL-20 mg/mL. For example, the antibody concentration can be 2 ± 0.2 mg/mL. Viruses that can be inactivated by the methods of the invention include, e.g., enveloped and non-enveloped viruses such as poliovirus, pseudorabies virus, xenotropic murine leukemia virus (MuLV), vesicular stomatitis virus, parainfluenza virus, human immunodeficiency virus, sindbis virus, bovine viral diarrheal virus, encephalomyocarditis virus, reovirus, hepatitis viruses, such as hepatitis A virus, and simian virus 40. The temperatures and heating times that can be used to inactivate specific viruses in samples containing specific types of antibodies, while maintaining antibody stability, can readily be determined by one skilled in the art. Generally, the antibody-containing sample is heated at 50-57 °C (e.g., at 54 ± 1 °C) for up to one hour (e.g., for 15 or 30 minutes). For example, in the case of a sample containing an IgA antibody, such as HNK20 (see below), antibody integrity is maintained when the sample is heated at about 53 °C for up to about

60 minutes, followed by cooling at room temperature or 4°C. Alternatively, the sample can be heated to about 55 °C for up to around 30 minutes and then cooled at 4°C.

Viruses such as poliovirus can be inactivated in antibody samples by heating at about 54 ± 1 °C for less than one minute, for example, for 0.1 minute (times longer than one minute can also be used). Partial inactivation of poliovirus is achieved by heating at about 50 °C for about 30-60 minutes. Viruses such as pseudorabies virus can be inactivated by heating at about 54 ± 1 °C for about 30 minutes; partial inactivation is achieved by heating at this temperature for about 15 minutes. Viruses such as MuLV are inactivated by heating at 54 ± 1 °C for less than one minute, for example, for 0.1 minute (times longer than one minute can also be used).

Heating can be carried out in any appropriate vessel as can readily be selected by one skilled in the art. For example, heating can be carried out in a flask (see Examples I and II, below), a tube, such as a 1.5 mL microcentrifuge tube (Example III), or a 15 mL conical tube (Example III). Vessels used for inactivation can be made of any material determined to be appropriate by one skilled in the art, e.g., plastic, glass, or steel. For large scale preparations, the sample can be heated in an integrated heating system, such as that described in Example IV.

Maintenance of antibody integrity after gentle heating can be measured by, e.g., determining the ability of the heated antibody to bind to its respective antigen (e.g., by ELISA analysis, see below), or by measuring the physical properties of the heated antibody

(e.g., by SDS-PAGE or size exclusion chromatography (SEC)-HPLC, see below). As will be understood in the art, loss of some antibody binding activity or structural integrity, in the interest of achieving more complete virus inactivation, is an acceptable consequence of the methods of the invention. Antibodies are preferably purified before heating. Standard antibody purification methods can be used and can readily be selected by one skilled in the art depending on, e.g., the antibody isotype. For example, Fig. 3 shows a purification and heating scheme that can be used with IgA antibody HNK20. This scheme can readily be adapted by one skilled in the art for use with other antibodies. After heating and purification, the antibody can be prepared for administration or can be stored using standard methods. Appropriate antibody concentrations and storage buffers, which vary depending on, e.g., selected storage conditions or the intended route of administration, can readily be determined by one skilled in the art.

The following examples are intended to illustrate and not to limit the scope of the invention.

Example I

Clearance of model viruses is an important part of the characterization of processes used to manufacture monoclonal antibodies. The following study was undertaken to characterize the inactivation of model viruses achieved by a gentle heating step included in the manufacturing process for the antibody produced by hybridoma cell line HNK20. (The

HNK20 hybridoma line is available from the American Type Culture Collection in Rockville, Maryland, and is designated with ATCC Accession No. HB 11514.) Following consultation with the FDA, three model viruses were chosen for this study, poliovirus, pseudorabies virus, and xenotropic murine leukemia virus (MuLV). The heating step was designed to inactivate poliovirus, which is traditionally resistant to many forms of biochemical agents and physical processes included in protein manufacturing schemes. Heat inactivation was chosen to be performed on the final purified bulk HNK20, since this would not affect the purification of the product. Pilot data indicated that heating to 50-57 °C under defined conditions for short periods of time (under 60 minutes) was effective in inactivating poliovirus. Careful preliminary study showed that heating to 53 °C for such periods had no detectable effect on the structural or functional characteristics of HNK20. Thus, this temperature was chosen for further study using all three model viruses.

The experiments described below show the effect of gentle heating on the three model viruses under conditions applicable to the manufacture of the HNK20 monoclonal antibody. Poliovirus, pseudorabies virus, and xenotropic murine leukemia virus (MuLV) were each separately spiked into purified bulk HNK20. The spiked products were heated to 53 °C and maintained at 53 °C for 45 minutes. Samples were taken at the time the products first reached 53 °C ("Time 0") and at 15, 30, and 45 minutes later. Vims titers were measured from samples taken at each time point to determine the kinetics of inactivation. Two separate heating runs were performed for each virus. The study was carried out under GLP conditions.

The results of these experiments show that poliovirus and MuLV were fully inactivated at the time the product first reached 53 °C. Duplicate runs showed good reproducibility. Poliovirus was inactivated at least 6.3 logs and MuLV at least 5.8 logs. Pseudorabies virus was more resistant to heat, and thus was inactivated more slowly, showing one log of inactivation at Time 0, approximately 4.1 logs at 15 minutes, and 5.4 logs at 30 and 45 minutes (complete inactivation). The virus titers and log reductions in these experiments are shown in Tables 1 and 2, respectively.

These results show that a short heating step after the last column chromatography purification step is highly effective in inactivating viruses for which MuLV or poliovirus are good models. Inactivation of viruses comparable to pseudorabies is also possible, but requires longer periods of heating.

The following methods and materials were used to carry out the experiments described above and illustrated in Tables 1 and 2. Preparation of HNK20 Purified bulk HNK20 was purified by methods comparable to those used for manufacture of clinical product. Briefly, harvested antibody was pH inactivated, and passed through a Q-column and a DEAE column. Purified HNK20 obtained from S-300 sizing chromatography, the final chromatographic step in the manufacturing process, was adjusted to 2 mg/mL ± 20% in phosphate buffered saline, pH 7.2. Experimental protocol A baseline sample of purified bulk HNK20 was taken for later analysis and 26 mL of the remaining product were spiked with 2.6 mL of concentrated stock poliovirus, pseudorabies virus, or murine leukemia virus. Additional samples were taken to be used as baseline and 45 minute room temperature controls. The remainder of the product was pumped into a flask set in a waterbath calibrated to raise and maintain the temperature of the sample at 53 °C. When the temperature of the sample reached 53 °C (about 3 minutes), a sample was taken for determination of virus titer ("Time 0"). Fifteen, 30, and 45 minutes later, additional samples were drawn for quantitation of viable virus. Two separate heating runs were performed for each virus. Quantitation of virus Poliovirus and Pseudorabies virus

Monkey kidney (Vero) cells were inoculated into six well tissue culture plates and grown to confluence. Cells were inoculated with 0.1 mL of test sample, placed at and overlaid with agarose containing fresh medium. When positive control plates demonstrated plaque formation, a second overlay containing neutral red was added and plaques were counted in all plates. Pseudorabies virus was assayed comparably to poliovirus, as described above. Xenotropic murine leukemia virus

The mink cell (S+L-) focus assay was used to quantitate MuLV. S+L- cells were inoculated with 0.1 mL of test sample and placed at 37°C under 5% CO₂ for 60 minutes. Wells were then overlaid with complete RPMI- 1640 growth media containing 1 μg/mL polybrene. On Day 3 post inoculation, growth medium was replaced with complete RPMI- 1640. On Day 7 post-inoculation, the wells were read for focus formation. Heating Apparatus

The heating apparatus used in these experiments is schematically illustrated in Fig. 3. In this apparatus, the antibody is heated in a glass vacuum flask set in a waterbath containing water heated to the appropriate temperature. The contents of the vacuum flask are stirred by a stir bar, in order to keep the temperature of the flask contents uniform. The antibody is introduced into the vacuum flask by being pumped through a glass condenser that is heated with water from the waterbath, by the use of a submersible pump. The heating process is monitored by temperature probes placed in the waterbath and in the vacuum flask.

Table 1 - Virus titers

* Time when 53 °C is first attained "pseudo" = pseudorabies virus

Table 2 - Log Reductions

* Time when 53 °C is first attained "pseudo" = pseudorabies virus

Example II

As is described above, gentle heating of HNK20 IgA is an effective method for inactivation of model viruses. The experiments described below were carried out to determine whether heating conditions that are effective at inactivating polio virus cause changes in the structural or functional characteristics of HNK20 that are immediately measurable or that might appear during product storage.

Aliquots of two HNK20 IgA lots, purified bulk lot 4E002 and laboratory lot DR057A (purified from pooled harvest lot 4H001; see, e.g., Fig. 1), were heated for short intervals (30 minutes) at temperatures shown to inactivate poliovirus (53 °C and 55 °C). The heated aliquots were put on accelerated stability (37 °C) with changes to structure and function evaluated weekly vs. unheated/real time (2-8 °C) and unheated/accelerated (37 °C) controls using SDS-PAGE, SEC-HPLC, and ELISA analysis. Briefly, the test results showed no significant differences between heated samples and controls throughout the 4-week study interval, indicating that HNK20 IgA can be effectively heat-inactivated without affecting its long-term stability. These results are described in more detail, as follows.

SDS-PAGE: At each of the 4-week timepoints, the band patterns of the room temperature controls, the accelerated temperature controls, and the 53° C and 55 °C heated samples were essentially equivalent. SEC-HPLC: Although the chromatograms showed no indication of differences in product profile between controls and heated samples, results of the analyses of weeks 1-3 are not included in this report because of poor resolution between polymer, dimer, and monomer peaks. The chromatograms for the 4-week timepoint have acceptable resolution between peaks, and the results are tabulated below. The data show that no significant change in monomer/dimer/polymer ratio or in total IgA level occurred in either lot as a result of heating for 30 minutes at 53 °C, or in 4E002 following heating for 30 minutes at 55 °C. DR057A at 55 °C showed a slight decrease in level of dimer with an apparent corresponding increase in peaks of RT>11.7 minutes (low molecular weight); this variability is probably within the precision of the method for single injections. The total IgA percent for DR057A at 55 °C still meets release specifications of > 90% total peak area. The results of these experiments are shown in Table 3. Table 3 - Results of SEC-HPLC of HNK20 IgA Accelerated

Stability Timepoints

Peak Area Percent

RSV Binding ELISA: As is shown in Table 4, below, within the precision of the method, no significant changes in RSV binding occurred in either lot as a result of 30 minute heating intervals at 53 °C and 55°C.

Table 4 - Results of RSV Binding ELISA of HNK20 IgA Accelerated Stability Timepoints

This accelerated stability study showed that heating HNK20 IgA, at approximately 2 mg/mL in PBS, for short intervals at temperatures effective at viral inactivation (53-55 °C) does not significantly change its structural or functional characteristics, as measured by SDS-PAGE, SEC-HPLC, and ELISA analysis, or adversely impact its long-term stability.

The following methods and materials were used to carry out the experiments described above and illustrated in Tables 3 and 4. Preparation of HNK20 Materials

Two lots of HNK20 were included in the study. Lot DR057A was purified at laboratory scale from pooled harvest lot 4H001. Lot 4E002, a purified bulk lot manufactured, tested and subsequently released for clinical studies as final dosage form Lot 94G02, was also evaluated. The two HNK20 lots were diluted to approximately 2 mg/mL (based on initial protein concentrations) in lx PBS. The diluted test samples were sterile filtered, and 500 μL aliquots were transferred to sterile tubes. Experimental protocol

Four test aliquots were treated in test tubes and stored according to each set of conditions shown in Table 5.

Table 5 - Design of Heating Time Course/ Accelerated Stability Study

Test procedures

Individual aliquots were removed from storage at 1 week intervals and evaluated for structural and functional changes by the test procedures described below.

SDS-PAGE: Test samples were combined 1:1 with non-reducing 2x sample buffer without boiling, and 10 μL aliquots of the resulting solutions and a molecular weight marker solution were loaded into individual wells of a 3-15% non-reducing SDS- PAGE gel. Following electrophoresis, the gel was stained with Coomassie blue and dried.

SEC-HPLC: Test sample aliquots (100 μL) were chromatographed on a 30 cm x 7.8 mm ID Progel-TSK G4000SWXL column equipped with a 4.0 cm x 6.0 mm ID SWXL guard column in a 100 mM PO₄, 100 mM NaCl, pH 7.0, elution buffer at a flow rate of 1.0 mL/minute. Eluted sample peaks were detected by UV absorbance at 280 nm, and their relative areas were determined by peak area integration.

RSV Binding ELISA: Test samples were diluted to 125, 63, and 31 ng/mL with antibody diluent (PBS/Tween/2.5% NFDM). A series of reference standard dilutions were also prepared in antibody diluent over a range of 8-1000 ng/mL. Test sample and reference standard dilutions (100 μL) were transferred in triplicate to ELISA plates that had been coated overnight (2-8 °C) with a 1 :2000 dilution of RSV Cell Lysate Antigen

(lot 7) in carbonate/bicarbonate buffer, then blocked with lx PBS/Tween 2.5% NFDM (1 hour at approximately 28 °C). The plates were incubated for 1 hour at approximately 28 °C. Following incubation, the test and reference dilutions were removed, then the plates were washed with lx PBS/Tween and blotted dry. One hundred μL of a 1 :500 dilution of rabbit anti-mouse IgG/HRP in antibody diluent was added to each well, and the plates were again incubated for 1 hour at approximately 28 °C. Following removal of the secondary antibody solution and washing with lx PBS/Tween, 100 μL of substrate solution (ABTS/H₂O₂ in citrate/phosphate buffer) was added to each well. Plates were incubated for 40 minutes at approximately 28 °C, then read in a microtiter plate reader at 405/490 nm using a dual endpoint program. Mean absorbances of the reference standard dilutions (y-axis) were plotted vs. concentration (x-axis) using a 4-parameter curve fit program. Concentrations of the test sample dilutions falling on the linear portion of the standard curve were averaged and reported.

Example III Using many of the methods described above, another IgA was tested for stability and viral inactivation upon gentle heating. The IgA used in these studies was a rat myeloma IgA purchased from Zymed Laboratories, Catalog # 02-9400.

In initial studies, heating of the antibody (lot #60530375) was carried out in a 1.5 mL microcentrifuge tube in a waterbath heated to 53 °C. Stability was measured by SEC- HPLC, as described above. The antibody was about 100% stable 15 minutes after the tube had been placed in the waterbath, and there was a small amount of instability at 30 minutes after incubation.

In additional studies, the rat myeloma IgA (a 50/50 mixture of lot #60530375 and lot #60832163) was spiked with polio virus, as described above, and gently heated in a 15 mL conical tube in a waterbath heated to 53 °C. At 15 minutes after the tube reached

53 °C, the estimated loss of polio virus was about 3 logs. There was instability of the IgA, as measured by SEC-HPLC, at this time point.

Example IV A thermal inactivation system designed for large-scale heat inactivation of monoclonal IgA HNK20 is described as follows. The system can be used as part of the

HNK20 purification scheme shown in Fig. 3.

The thermal inactivation system was designed to produce at least a 4 log reduction of virus concentration in a monoclonal antibody product solution by heat inactivation. The experimental procedures developed that achieve this goal are as follows:

1. The product solution is heated uniformly to a temperature of 53 °C from some initial temperature (typically around 2° C).

2. The product solution is held at the elevated temperature for a minimum of 3 minutes. 3. The product is then cooled to some final temperature that is less than 52 °C (typically 15 °C)

The goal of this system is to be able to continuously process 10 to 100 liter batches of product solution in an aseptic manner and to achieve the same reduction in virus concentration as produced in smaller scale laboratory experiments. Further operational design constraints on the system are as follows:

4. The product solution is heated from an initial temperature of approximately 2°C to a temperature of 53.5 ± 0.5 °C.

5. The product solution is held at the design inactivation temperature of 53.5 ± 0.5°C for a minimum of 3 minutes and a maximum of 10 minutes. The system temperature profile is further defined in such a way that 99.9% of the total volume of product solution processed is held at the design temperature for a minimum of 3 minutes, and that no more than 1% of the total volume of product solution processed is held at the design temperature for longer than 10 minutes. 6. The temperature of the product solution at no time exceeds 54°C.

7. Buffer is supplied to the system at approximately 2°C ahead of the product solution in order to equilibrate the system and to allow system temperatures to stabilize.

8. Buffer is supplied to the system after the product solution to chase the solution from the system.

9. The system is designed in such a way so that it is supplied from both a product solution feed tank and a buffer solution feed tank, and it delivers the product solution and buffer to a common recovery tank.

10. The system is designed in such a way so that it minimizes product damage due to shear forces.

11. The system is designed to be cleaned in place via connection to a separate CIP system.

12. The system is designed to be steamed in place by direct injection of a 30 lb clean steam; alternatively, chemical sanitization can be carried out. 13. The system has as available running utilities chilled water at 1 °C and

220v single phase power. The philosophy behind item (5) above is that, as a system priority, a sufficient amount of the product solution must reach and be maintained at the design temperature for the minimum time required in order to insure the desired reduction in live viral organisms. Product degradation can be expected to occur if the product solution is held at design temperature for longer than 10 minutes. However, some degradation of product is preferred to an insufficient amount of virus reduction, as the latter would make the product unusable, while the former would just reduce the total usable product yield. The thermal inactivation system is a portable, self-contained, skid mounted system. It includes inlet and outlet valves, a positive displacement product feed pump, a heating sub-system, a heat retention tube, a cooling heat exchanger, and several RTD temperature probes linked to a chart recorder to measure product solution temperature at critical points in the system. The skid has an enclosed control panel that contains displays for the RTD probes, the chart recorder, pump controls, On/Off switches, control switches for the valves, and alarm lights and switches. There are quick disconnect-style connections for the chilled water supply and return to the skid, as well as a low-point condensate drain valve for the removal of clean steam condensate from the process side of the system. The individual components and sub-systems, as well as their intended operation and performance, are described in the following sections. Product Feed Pump and Flow Control Valves The product is moved through the process side of the system with a PulsaFeeder brand positive displacement pump (PO25). This pump has a vertical flow path with sanitary end connections. It utilizes a flexible PFA tube element surrounded by a product compatible hydraulic fluid (i.e., WFI), in conjunction with inlet and outlet check valves to pump the product solution. The pump motor has a remote controlled (via the skid mounted control panel) variable stroke controller to allow varying of the flow rate. This style of pump was chosen because the flow rate changes insignificantly with fluctuating pressures, allowing for precise control of flow rate in the system. A design flow rate of about 600 mL/minute has been selected to provide good product mixing characteristics in the heating coil and heat retention coil, without creating undue turbulence in the flow stream, which could contribute to product degradation due to shear forces. The inlet and outlet valves on the process side of the system are 1/2" ITT sanitary diaphragm valves. There are two inlet valves mounted upstream of the feed pump, one for a product solution inlet (VO05) and one for a buffer solution inlet (VOIO). These valves have sanitary tri-clamp inlet connections, and are welded together at their outlets via a sanitary tee fitting, which feeds directly to the inlet of the feed pump. The inlet valves are pneumatically operated, air-to-open, spring-to-close, and are activated from the control panel. The outlet valves are mounted in-line at the outlet of the process side of the system. The last valve in-line (VO40) is pneumatically operated, air-to-open, spring- to-close, with a sanitary tri-clamp outlet, and is activated from the control panel. The other outlet valve (Vxxx) upstream of the pneumatic outlet valve is manually operated, and is used to set the back pressure on the process side of the system. This is necessary because the check valves on the feed pump require some back pressure to operate properly. During normal operation, the pneumatic outlet valve will be in the open position, and connected to the product solution recovery tank, and either the product solution inlet valve (VO05) or the buffer solution inlet valve (VOIO) will be in the open position. The back pressure outlet valve will be in a preset position to provide adequate system back pressure (determined during system testing and validation).

Heating Sub-System Several methods for heating the product solution from an initial temperature to the design temperature were investigated, and a recirculating hot waterbath was selected because of the stable temperatures that could be achieved and simple controls associated with such a system. The heating subsystem is made up of the following components: ^■ An insulated 50 Liter hot water storage tank (Water Heater 200) with a solid state controlled immersion heater to provide recirculating hot water at a constant temperature.

^• A small centrifugal pump (P210) for recirculating the hot water.

^• A calibrated balancing valve (CV225) for accurately controlling the flow rate of the recirculating hot water. - A sanitary, specially designed shell- in- tube style heat exchanger (Product

Heater 030). The water heater stores hot water to recirculate to the shell side of the product heater. The water is supplied to the shell side product heater at a preset temperature of 55 ± 0.5 °C. This temperature ensures that the product solution on the process side of the product heater will not exceed 54°C. However, this temperature setting can be adjusted if necessary during system testing and validation.

The recirculation pump and balancing valve work in tandem to provide the correct flow rate of hot water to the shell side of the product heater. The pump is run at wide open speed, and the balancing valve applies back pressure to the outlet of the pump, which affects the flow rate. (For centrifugal pumps, flow rate decreases with increasing back pressure according to a flow/pressure curve that is unique for a given pump at a given speed). The balancing valve is calibrated to allow a specific flow rate for a specific pressure drop across the valve. By measuring the pressure on the inlet and outlet of the valve, the exact recirculation rate can be found. More importantly, however, is the ability to control precisely the flow rate to the shell side of the product heater. During system testing and validation, the hot water recirculating flow rate is slowly decreased until the outlet temperature of the test solution on the process side of the product heater drops just below the required set point of 53.5 °C. At this point, the flow rate is increased just enough to ensure that the product solution leaves the product heater at the design set point temperature. The product heater is based on the Spiratherm® design, as manufactured by

Cherry-Burrell, and is mounted as close as possible to the outlet of the product feed pump (PO25). The design consists of a spiral product tube, or coil, set in an insulated, closed annulus shell. The coil is constructed of 3/8" OD, 16 Ga, 316L SS, with 1/2" tri-clamp fittings at the inlet and outlet. The product heater is oriented on the skid so that the coil axis is vertical, and the product solution will move up through the coil from bottom to top. The hot water will recirculate in a counter flow direction through the annulus on the outside surface of the coil, providing the highest heat transfer coefficient possible for this configuration. The length of the coil is determined from several factors, including the inlet temperature of both the product solution (2°C) and hot water (55 °C), the desired outlet temperature of the product solution (53.5 °C), the flow rate of the product solution

(about 600 mL/minute), and the recirculation flow rate of the hot water. The required coil length was calculated using these values, and a factor of safety was employed to ensure that the product heater would be capable of the process specified within the flow rate range of the recirculation pump. Heat Retention Tube The heat retention tube (Hold Tube 060) is essentially identical to the product heater in design, with a few exceptions. Instead of recirculating a fluid on the shell side, the space between the coil and the inside of the annulus is packed with insulation to prevent heat loss. The length of the tube is calculated based on the cross sectional area of the process side of the tube, the flow rate of the product solution through the tube (about 600 mL/minute), and the required time the product solution must be held at temperature

(3 minutes). An immersion RTD probe is mounted at the inlet and outlet of the hold tube to monitor the temperature of the product solution. Cooling Heat Exchanger

The cooling heat exchanger (Cooler 075) is a sanitary U-tube design as provided by Allegheny Bradford. The tube side is constructed of 316L SS and has 1/2" tri-clamp connections. The heat exchanger is designed to cool the product solution from 53 °C to approximately 15 °C using chilled water on the shell side supplied at 1 °C. The outlet temperature of the product solution can be varied within a certain range by adjusting the chilled water outlet control valve (CV330), which can accommodate user preferences for a give process. An immersion RTD probe is mounted at the outlet of the heat exchanger to monitor the exit temperature of the product solution. System Sanitization

The system can be sanitized by standard chemical treatment or by the use of steam-in-place components, as are described as follows. Steam-In-Place Components

Due to the simplicity of the system, there are very few components required for effective sterilization of the process side of the system with clean steam. The system is designed to be completely drainable, with one exception, to a common low point where a manual diaphragm valve (VO15) is connected to a thermostatic steam trap (ST010). The exception is the product feed pump, which has two check valves, one at the inlet and one at the outlet. These check valves prevent flow, and hence draining, in the downward direction through the pump. For this reason, clean steam will have to be introduced to the process side of the system through the inlet diaphragm valves (VO05 & VOIO). The steam vapor rising up through the pump lifts the check valves off their respective seats, allowing condensate to flow downward past them to the system low point. To ensure that the steam moves adequately up through the system and reaches all product contact surfaces on the process side of the system, a 1/2" SS tube is connected to the outlet diaphragm valve (VO90) with a sanitary tri-clamp swing elbow. This tube is also routed to the steam trap (SM1O), and has a check valve in-line to prevent contaminated steam or condensate from entering the outlet of the system from the wrong direction. Since the size of the system, as well as the process tubing, is small (less than 100 lbs of stainless steel), the clean steam load required to completely sterilize the system should be low (approximately 10 lbs/hour of 30 psig saturated clean steam). The setup for steaming the system should include isolation valves for introducing both clean steam and either pharmaceutical compressed air or dry nitrogen as a purge and hold after steaming. In addition, the shell side of both the product heater and cooler should be drained before steaming to ensure complete sterilization (no cold spots) and to decrease the SIP cycle time. Alarms and Controls

The thermal inactivation system is designed to induce a 4 log reduction in live viral organisms in the product solution by using heat. Therefore, it is important to monitor process temperatures at critical points in the system. These points include the product heater outlet/hold tube inlet (CM035), the hold tube outlet/cooler inlet (CM65), and the cooler outlet (TE080). The temperature elements at these points are all connected to a common chart recorder, and each element is connected to a dedicated display that has alarm capabilities. These alarms will be set to act when the system drops below or goes above the set point temperatures for their corresponding points in the system.

Due to the design of the system and the continuous nature of the process, it is imperative that the process not be interrupted once it is started, unless something has gone wrong with the system. For this reason, the alarms will light a red alarm display to notify the operator, who will have to acknowledge the alarm and decide on an action. Due to the sensitivity of the electronic equipment in the system, it is possible that momentary fluctuations in temperature readings can occur and cause an alarm. The operator has the option of checking the chart recorder and displays to determine if system shut down and process termination is warranted. If it is determined that the alarm warrants system shut down, the product feed pump is turned off, and the inlet and outlet pneumatic diaphragm valves are closed. In this way, no potentially contaminated or unusable product still within the system is allowed to proceed to the recovery tank containing product solution that has been successfully processed. The system can be disconnected from the feed and recovery tanks, the product solution can be flushed and deactivated, and the cause of the alarm can then be found. The specific operating procedures, as well as the temperature set points and alarms, can be determined during system testing and validation.

General System Operation

An outline of the general operating procedures is listed below: 1. Start up

1.1. The skid is connected to the chilled water supply and return lines from the plant, and the associated valves on the skid are opened.

1.2. The water heater is filled with chilled water if it is not already full.

1.3. The hot water sub-system is turned on, which includes supplying power to the immersion heating element and turning on the recirculation pump.

2. System Stabilization

2.1. The product feed tank and buffer tank are connected in an aseptic fashion to the corresponding inlet diaphragm valves on the skid.

2.2. The product solution recovery tank is connected in an aseptic fashion to the outlet diaphragm on the skid. 2.3. The buffer inlet diaphragm valve and the outlet diaphragm valve on the skid are opened via the hand switches on the control panel.

2.4. The product feed pump is started via the hand switch on the control panel, and buffer is pumped through the system until the heat retention tube has reached operating temperature as indicated by the temperature probe at the outlet of the retention tube. 3. Product Solution Processing

3.1. The hand switch that controls the inlet diaphragm valves is switched from the buffer tank to the product solution tank, allowing the system to start processing product solution. 3.2. The system is monitored for alarms and temperature indications during the duration of the process.

3.3. When all of the product solution has been processed (as determined by time, weight, or other method), the system is switched back to the buffer tank via the inlet valve control switch on the control panel. 3.4. Buffer is pumped through the system until the product solution has been sufficiently flushed from the system.

4. System Shut Down

4.1. The product feed pump, hot water recirculation pump, and immersion heater are turned off.

4.2. The chilled water supply and return valves are closed.

4.3. The inlet and outlet diaphragm valves are closed via the hand switch on the control panel.

4.4. The system is disconnected from the product feed tank and the buffer tank and readied for CIP.

It should be understood that this general operating procedure can be supplemented with additional steps developed during system testing and validation.

The system described above was used to heat monoclonal antibody HNK20 at 53.5 ± 0.5 °C for 3 minutes. SEC-HPLC analysis of the heated antibody revealed that the structure of the antibody did not change significantly during this process.

Other Embodiments It is to be understood that the foregoing description is intended to illustrate and not to limit the scope of the invention. Other aspects, advantages, and modifications of the invention are within the scope of the following claims.

What is claimed is:

Claims

1. A method of inactivating a virus in a sample comprising an antibody that binds to an antigen, said method comprising heating said sample under conditions wherein (1) said virus is inactivated, and (2) said antibody retains its capacity to bind said antigen.

2. The method of claim 1, wherein said antibody is a monoclonal antibody.

3. The method of claim 1, wherein said antibody is an IgA antibody.

4. The method of claim 1, wherein said heating is at 54 ± 1 °C for 0.1-10 minutes.

5. The method of claim 4, wherein said heating is for 1-5 minutes.

6. The method of claim 5, wherein said heating is for about 3 minutes.

7. The method of claim 1, wherein said virus is selected from the group consisting of poliovirus, pseudorabies virus, and xenotropic murine leukemia virus (MuLV).

8. The method of claim 3, wherein said IgA antibody is the antibody produced by hybridoma cell line HNK20 (American Type Culture Collection Accession

No. HB 11514).

9. An antibody treated according to the method of claim 1.

10. The antibody of claim 9, wherein said antibody is a monoclonal antibody.

11. The antibody of claim 9, wherein said antibody is an IgA antibody.

12. The antibody of claim 11, wherein said antibody is produced by hybridoma cell line HNK20 (American Type Culture Collection Accession No. HB 11514).

13. The antibody of claim 9, wherein said antibody was heated at 54 ± 1 °C for 0.1-10 minutes.

14. The antibody of claim 13, wherein said antibody was heated for 1-5 minutes.

15. The antibody of claim 14, wherein said antibody was heated for about 3 minutes.