Field of the Invention

This invention relates to filtration. Background of the Invention

Viruses can be used to deliver genes, e.g. in gene therapy. Such viruses include retroviruses, adenovirus, adeno-associated virus and herpes simplex virus. For example, an adenovirus that delivers functional thymidine kinase, for use in therapy relating to the treatment of brain tumours and the prevention of their recurrence, is disclosed in WO00/28059. This technology is known as Cerepro®. A preferred buffer system (HEPES-glycerol) is described in WO2009/022174.

It will be appreciated that, for therapeutic use, various regulatory requirements must be met. Stringent controls are placed on the production of viruses for gene therapy, and the stability and potency of viral formulations are critical considerations. GMP (Good Manufacturing Practice) standards must be met.

Bovine serum albumin (BSA) is a major constituent of bovine serum. Fetal bovine serum (FBS) containing BSA is used in cell culture media in the production of Cerepro®. BSA should not be more than the limit approved for the particular preparation, e.g. as determined by a suitable immunochemical method.

During manufacture, a virus preparation must be filtered, in order to provide a clean product. In order to avoid damage to the virus, many filter changes are required.

The molecular weight of adenovirus is 175,000 kDa and the molecular weight of BSA sub-unit is 66 kDa. In general, a five-fold size difference between components is sufficient to achieve separation in TFF ultrafiltration. Therefore, in optimal conditions, these components should be readily separated by tangential flow filtration (TFF). However, BSA has a natural tendency to aggregation. BSA is also known to interact with adenovirus. Therefore, in addition to selection of optimum nominal molecular weight cut-off (NMWCO), it may be desirable to minimise BSA aggregation and interactions with other molecules present in the material. Summary of ...the I nyention

The present invention is based on the discovery that, by reversing flow during filtration, recovery is improved and damage to the virus can be reduced or avoided. In addition, throughput can be increased.

According to the present invention, a filtration process comprises one or more optional gradient ultrafiltration steps followed by TFF incorporating forward flow and/or reverse recirculation, one or more times. A peristaltic pump may be used with TFF.

In particular, the invention is a suspension process based on single use systems (SUS) for manufacturing commercial batches of adenoviral gene-based medicines ready for fill. SUS is based on disposable wavebag and membrane based purification processing technology, and utilises a reversing flow system.

The new process has resulted in a very high quality finished product with improved yields of up to 100 times those achieved by more conventional adherence cell or non-disposable bioreactor suspension cell culture and chromatographic purification processes. An object of this invention was to achieve BSA clearance to low levels, of < 50 ng/ml, which is the limit for vaccines in WHO guidelines. Another aim was to maintain a residual CsCI level of < 25 ppm and virus particle concentration of > 0.7E+12 vp/ml by OD ₂₆ o-SDS.

The SUS system occupies a quarter of the space previously needed for conventional production and, being disposable, results in considerable cost and time savings with reduced facility and equipment sterilisation 'down time'. The extensive use of contained process elements opens up hitherto product-specific production theatres for potential manufacture of more than one product line. Description of the Invention

In general terms, a method according to the invention may comprise components as or of the type described in the prior art to which reference is made above; the content of each of these publications is incorporated herein by reference. More specific embodiments and illustrative examples relating to the invention are described below. It will of course be understood by the skilled person that forward and reverse flow in TFF may each be conducted one or a larger number of times, as necessary or desired.

The production of virus, e.g. adenovirus for use in Cerepro®, preferably comprises one or two caesium chloride (CsCI) gradient ultrafiltration steps prior to TFF. A CsCI wash procedure has been found to improve the BSA clearance of TFF.

The following Examples illustrate the invention. Example 1

A 100 kDa TFF cassette (Pall) was used. Two CsCI washing steps were conducted using 5mM HEPES and, optionally 20%, glycerol as buffer.

An adenovirus band is formed at 1.35 g/ml density in CsCI gradient ultracentrifugation. This density equals to 478 mg/ml CsCI concentration. An average of 32 ml is collected from second ultracentrifugation followed by dilution to 200 ml by using 5 mM HEPES. This dilution results to 77 mg/ml CsCI concentration in the product before TFF. This 77 mg/ml CsCI start concentration was maintained over 5 diafiltration factors during TFF diafiltration. This was followed by normal de-salting (10 x DF against 5 mM HEPES) and final formulation (5 x DF against 5 mM HEPES-20% glycerol) steps.

Tests were conducted, using forward flow recirculation of retentate at the end to release the virus from the membrane. Reverse recirculation of retentate was also tested.

Permeate was closed during final concentration at beaker volume ~70 ml. The retentate was first re-circulated by forward flow, sample was taken and analysed as 3.75E+11/p/ml by AEX-HPLC. After 5 min, the retentate flow was reversed, re-circulated for 5 min, sampled and analysed as 4.76E+1 1 vp.ml. Reversing the retentate flow showed significant 27% increase in the adenovirus concentration. Main 47% and overall 71 % recoveries were achieved. A further run was carried out by performing the retentate reverse re-circulation for main recovery and for each flush. Adenovirus recovery of 77% was now achieved (main 56%, 1st flush 7%, 2nd flush 11%, 3rd flush 2%).

The effect of reverse recirculation on CsCI and BSA impurity levels was tested. A BSA concentration of 68.4 ng.ml was detected before the reverse recirculation and 66.5 ng/ml was detected after the reverse recirculation. The result confirms that there is no BSA build-up in the retentate which could be released during the reverse recirculation. A CsCI concentration of 0.1 ppm was detected before the reverse recirculation and 0.6 ppm was detected after the reverse recirculation. The result shows an increase in CsCI levels by the reverse recirculation. However, CsCl levels were still clearly within specification of < 25 ppm. Example 2

1500 ml (=DF x 7,5) of 25 mg/ml CsCI was used followed by 4000 ml (=DF x 20) 5 mM HEPES-20% glycerol and a 300 kDa TFF cassette (Pall). There was no detectable BSA in the purified sub-batch (PSB), confirming that a CsCI wash is required to achieve the BSA target. The retentate was sampled throughout and analysed by inductively coupled plasma mass spectrometry, in order to confirm the volume of 5mM HEPES-20% glycerol needed and to check the effect of reverse recirculation on residual CsCI levels.

Results showed that 3000 ml (DF x 15) of 5 mM HEPES-20% glycerol is sufficient to clear out the CsCI. Total CsCI level increased 104 μm from a concentration to recovered PSB (final PSB concentration 2.8 ppm). 33 μm of the increase was associated with the first retentate reverse recirculation performed for the main recovery.

Reversing the retentate flow at the end showed significant (27%) increase in the adenovirus concentration. This indicated build-up of adenovirus somewhere in the system. The increase in the CsCI level could be due to ionic interactions between adenoviruses and caesium ions.