Asahi Kasei Biopocess Europe Monthly report August 2011 / EK

Visit Roche, Germany with Stefan As reported Roche has interest to evaluation BioEX for one mab project, which is in phase I early development, but quite promising The official letter that Asahi decided establishes a new idea of integrity test was already handed over to them in June. We performed at Roche the scalability study using BioEX. The results were very satisfied for them. Also we showed them the Pre-and post use Leakage test. For more details please refer to our report and filtration results:

2011 08 10 Roche visit report BioEX.docx

Roche BioEX Filtration Report Aug 2011.xls

In September they do the first technical run under GMP. We need the official SOP end of August which we received on Aug. 31th. Thank you all very much for your support.

SIP will be checked on August 16, 2011. 3 SIP cycles will be tested. QSD presentation has been handed over to Dr. von Hirschheydt, who showed an interest. Audit: For Audit of BioEx and new plant in Oita two dates have been proposed: 1. option 13.12.2011 - 15.12.2011 2. option 20.12.2011 - 22.12.2011. Option 1 is fine for Roche and confirmed. Charles River

CR was invited by us to show Virus removal date between small scale filter and process filters at our next workshop. The idea came to them to do such study because they and also I heard the rumor that Millipore filters at least NFP has a different wider pore structure in process filter then in their small capsule filters, which means that filtration capacity, is better with process filters, but virus might go through. This rumor I heard from Sartorius. We are discussing now to perform following further experiments as you see on next page. Small scale experiments under point 4 and 5 has been done already, except P15N. Depending on the schedule we might add P15N as well The next size from Sartorius is 0.7 m. This surface is too large for handing at CR.

Asahi Kasei Biopocess Europe Monthly report August 2011 / EK

For P20N, BioEX Vpro we are now checking the right equipment to handle the needed large volumes and big devices for 0.1 m prefiltration. We reduced the filtration volume from 300 ml to 150 m/m It is a big challenge for CR and we offered out support and equipment.

Filtration conditions:
1) Filter Planova 20N 0,12 fiber Planova BioEX 0, 1 Fiber Pall Novasip DV20 0,07 cartridge Millipore VPro Sartorius Virosart CPV Planova N 15N 0,12 fiber ?

2)

Filter area m2

0,22 plate

Single

3)

Total filtration volume [L] Comparison with smallest surface areas m Total filtration volume [ml] Protein solution conc.

18

15

10,5

33

18

Single

4)

0.001 fiber

0.001 fiber

Disc 0.0011

Capsule 0,00035

Capsule 0.001 fiber Dublicate 0.0005 75-150 150-300 Dublicate

5)

150-300 150-300 BSA BSA 5mg/ml 5mg/ml 0.1 % 0.1 %

165 -330 50,25-105 BSA 5mg/ml 0.1 %

6)

BSA BSA BSA 5 mg/ml 5 mg/ml 5mg/ml 0.1 % 0.1% 0.1 %

7) Test virus PPV Titer Dead-end with constant pressure [bar] Temperature

8)

0.8

3.0

2.0

2.0

2.0

0.8

9)

RT

RT

RT

RT

RT

RT

I hope we can realize this study. The purpose of this study is to show if a linear scalability is possible regarding filters shapes (i.e. capsule to cartridge) and virus retention.

Asahi Kasei Biopocess Europe Monthly report August 2011 / EK Presentation from Charles River at pre-conference virus removal by filtration sin Barcelona
Marcel Asper.pdf

We got following information from him afterwards and we placed some questions which I listen up: 1. Which filter were used: Filter 1 is BioEX, Filter 3 is Planova 20N, Vpro without shield filter showed no passive virus leaking during pressure release for 5 min. Due to confidential reasons CR cannot give us more information regarding the filters. 2. What were the pressures applied for each filters? As recommended by the filter supplier 3. How long did you release the pressure, 30 min, 1h or more? 5 min 4. What is the initial virus titer (log10) in the feed? Something around 8? Yes 5. When measuring the virus titer for each permeate fractions, was the limit of detection almost the same for each filter? Something around 1 log? Less than 1 log 6. Was the quantity of proteins/m2 applied to the filters the same (for both cases with BSA and MAb)? For example the sample 6 of one filter can correspond to a higher loading/m2 than the sample 6 of another filter? He did not take care of the loading /m2, means unfortunately you cannot really compare 7. In the case with MAb, you only showed the results for filters 2 and 6. Does it mean that the other filters did not show any breakthrough after the pressure release? He did not test the other filters under these conditions. 8. In the slide 21, was it the case with BSA or MAb? BSA 9. Are you continuing this big study, in particular to check the impact of the MAb concentration, duration of the pause, etc? It is not decided yet 10.We (Asahi) tried to give an explanation (presentation from my colleague Dr Hongo). Did the other filter suppliers give any tentative explanations to the breakthrough phenomenon? No reaction from the other filter suppliers His presentation made a first big wave . BioReliance and I think also LFB contacted us about our opinion of passive virus leaking during post wash (pressure release between filtration and post wash). I send Bio Reliance our data and they are fine. PEI Germany: Circo virus study PCV-1

Asahi Kasei Biopocess Europe Monthly report August 2011 / EK As reported before PEI is investigating the clearance of PCV1 by the Planova 15 N and the BioEX filters. So far they have been doing this by a PCR-based approach. They have an purified stock of PCV1, it is nuclease treated before application to the filters and then samples of the fractions are nuclease treated before extraction/amplification. All samples are collected and they have to decide if they want to look at the infectivity as well. They have used the Planova 20N as a pre-filter for both the Planova 15N and the BioEx filters simply done to make sure any possible aggregates are removed. What they have found is that with the Planova 15N there is reduction of PCV1 in the order of 2-3 logs. When they washed the filter then they saw some breakthrough. They would not expect this to be due to free DNA since they have a purified virus stock and are nuclease treating twice prior to extraction. This result was repeatable. In the case of the BioEx, they see an approximately 0.5-1.5 log reduction. This would need repeating, but one certainly would not call this effective reduction. They have a different virus stock and we want to repeat the experiments with this. The German authorities asked for further 6 samples of P20 and 2 more BioEX Filters, which we have sent .

Also in this case they found a breakthrough during pressure release (before post wash). In September we get more new internal data during out EU meeting from Tsuboi san.

ViruSure, Austria Circo virus study PCV-2 As reported ViruSure is also performing the circo virus study. We did some preliminary runs with the Mab from BI and it went well no flux decay We have seen no significant flow decay using following different concentrations of mab and spike:

Test Filter : Concentration (mg/mL) : Concentration (Circo) : Buffer : Total filtration time (min) : Total filtration volume (mL) :

Test 1 P20N 10 1%

Test 2 P15N 3 0.5 %

Test 3

Test 4

pH 7, PBS buffer 86 90 74 50

Asahi Kasei Biopocess Europe Monthly report August 2011 / EK Average flux (L/h/m2) : Pressure (kPa) : Vmax (L/m2) : 47,1 80,0 >1000 35,9 80,0 130 L

They have finished the interference study and send us the final study plan very soon.

Image of the main study: Filter Concentration Mab Filtration volume Planova 15N Planova 20N Planova BioEX Adsorber QSD 0.5 % plus 0.5 % virus 50 ml

Duplicate Yes Yes Yes Yes

1.0 % plus 1 % virus 200 ml 1.0 % plus 1 % virus 200 ml 1.0 % plus 1 % virus 200 ml

On September 5 we will visit them again. Contract has been signed.

Baxter, Austria Baxter received a sample of QSD on their request. It was a delivery quite on short notice because they have done a comparison study with different adsorber membranes at that time. They did an internal judgment which candidates of adsorber membranes will be selected for further evaluation study. Their target was to do trials with their IgG solution. They want to bind protein impurities of high MW while recovering the IgG in the flow through mode. We asked for a meeting on September 6 and it is confirmed. Baxter (QC) Austria: As reported before Baxter has to rearrange their own in-house specification on the basis of our new announced COA. Baxter could understand why we are not using the current updated latest references.

Asahi Kasei Biopocess Europe Monthly report August 2011 / EK It was good that Baxter received an official document as follows, which is acceptable to Baxter.
Baxter Description on Safety Test of COA 2011.7.19.pdf

Also we explained in an official document that the current reference from 2005 Approval Standard or dialyzer, 1983 does not focus anymore on Hollow fiber made of Cellulose, therefore fore Asahi needs to refer to the document from 1983.
Baxter Approval_Standards_for_Dialyzer_Type.pdf

I hope now we can close this topic and Baxter is satisfied.

Novartis, Switzerland As reported Novartis is doing a virus validation study at Charles River in the second or third week of August. Mathithas made the AGP Test for them and he found out that the product is an mab sourced from camels for cancer treatment (more details, please refere to Mathithas report) .Due to the good news that we change the integrity test for BioEX, Novartis will internally discuss and re-consider again which position BioEX will have in the future within Novartis end of October 2011. A meeting was offered in September, but they refused for unknown reason for me, it was maybe too early for them to have a further meeting or as Bixente reported they have a delay as usual.

Anyhow for one project which has been postponed to next year, definitely BioEX will be used. CSL Bern Switzerland
As reported last time Cytogam Cytomegalovirus Immune Globulin Intravenous (Human) will be revalidated in Switzerland. They are testing all different filters including BioEX. Due to good results of BioEX, which they have experienced, BioEX has a good chance to get the filter of choice. I informed them unofficially that we have change or Integrity test to a simple Leakage test. They expressed their high interest and agreed to a meeting with us on 20. September after summer holidays.

Asahi Kasei Biopocess Europe Monthly report August 2011 / EK Octapharma / Germany and Austria
QSD will be presented at Octapharma in Vienna on September 6. We will find out as well the current status of fibrinogen and IvIgG project.

Boehringer Ingelheim During our last training in CTC Mrs. Dilger, who was joining the first open training course said to us that she would like to coordinate a big meeting with BI Employees from different divisions like small scale, process development and production. We have sent to BI a proposal of an agenda which includes all our products and engineering. We will make two blocks Focus more on practical points for the handling for Planova technology, also new strategy for BioEX Lecture on QSD, MF-SL, IBD etc. Mrs. Dilger will make also a proposal from their side and she will suggest a meeting date in October 2011. We wait for her response. I think it is a good opportunity to meet many people within BI.

Sanofi Aventis Germany: They contacted us for getting an offer for a dynamic and static synthesis column of 60 cm diameter. I have forwarded this request to Jacques.

Others: 14th Planova workshop preparation November 9/10th, 2011

Final program for WS has been finished together with Masao and will be send out send out beginning of September with the reviewed registration sheet included which will be ready on Sept 5

DRAFT Planova Workshop 2011 3rd announcem ent.pdf

Annual Leave:

Asahi Kasei Biopocess Europe Monthly report August 2011 / EK July 27-August 4 2011 and August 22-26, 2011