Needs of antisera production centres. Prof. Sumana Khomvilai

Transcription

1 Needs of antisera production centres Prof. Sumana Khomvilai

2 Rabies and snakebites are serious public health problems in the developing countries. Reported cases of rabies and snakebites from the government sector in Thailand showed that animal-derived sera for rabies and envenoming are still needed.

3 Reported cases of Snakebites and Rabies in Thailand Cases Deaths Cases Deaths Cases Deaths Cases Deaths Snake bite Rabies (PEP) 390, , , PEP = Post exposure prophylaxis Reported quantities are only from government sector Sources : CDC, ministry of public health

4 To increase the quality production of anti sera production of animal derive sera to meet the public health needs:. Increase the production capacity of existing production plant (Production centres) who Prequllification Ivestment.Technology transfer.expert Meeting

5 Potential for increasing the production quantities For production centres - Increase the number of healthy horses - Improve the horses immunization process - Improve the purification process (To increase the yield, efficacy, purity and stability of products) - Validation laboratory testing method, improve consistency of test results

6 Increase the number of healthy horses

7 Improve the horses immunization process Several animal species are used for immunization but horses are most commonly used. Healthy horses prior to be immunized should be as follows : - Ages : 3½ - 10 years old - Serology control of bacterial and viral diseases eg. Equine infectious anemia (EIA) - Vaccination - Routine clinical chemistry and haematology - Antiparastic treatment - Free from antibiotics, penicillins

8 Immunization schedules for Snake Antivenoms - Immunization the appropriate national reference lyophilized snake venoms into horses, for each species, to raise the antivenoms. - Depend on many factors related to the particular venom and the horses being immunized, no single schedule for all venoms can be established - In some venoms, the total amount of venoms given could be small amount of 2-20 mg (maximum 40 mg) and short (about 2 months) but with more complex venoms, the immunization course may longer than 6-7 months

9 Immunization Schedule for Rabies Immunoglobulins Veterinary rabies vaccines (Rabisin ) or concentrated human rabies vaccines (supplied by Chiron Behring ) and Freund s Complete Adjuvant (FCA) containing Mycobacterium tuberculosis are being used for horses immunization in production of rabies antibody plasma. Immunization schedules are similar to snake antivenoms

10 Improve horses immunization process

11 Bleeding is done by Plasmapheresis

12 Bleeding is done by Plasmapheresis

13 Improve the purification process (To increase the yield,efficacy, purity and stability) Animal derived plasma containing antibody is purified by pepsin digestion and thermocoagulation, It is hydrolysed into F(ab )2 fragment and Fc fragment. Caprylic acid is using for further precipitation of non-igg proteins and aggregate proteins. All precipitations are removed by filter press filtration and the residue caprylic acid is eliminated with ultrafiltration technique. A purified immunoglobulin [F(ab )2] with high efficacy is obtained.

14 Filter through filter press for precipitation of unwanted protein

15 Purification and concentration by Ultrafiltration, then sterile filtration

16 Production/Purification 1. Pepsin Hydrolysis Aninal derived Plasma containing antibody Pepsin Hydrolysis/Digestion F(ab ) 2 +Fc, non specific Ig and lipoprotein 2. Ammonium Sulphate precipitation (Salting out ) and thermocoagulation + Caprylic acid precipetate Solution Fc, non specific Ig and lipoprotein F (ab ) 2 are filtered by filter press Ultrafiltration Concentrated F(ab ) 2 and removed caprylic acid AdjustpH& Osmolarity Sterile filtration Purified F (ab ) 2 Filling

17 Laboratory testing 1. Control of crude plasma Appearance Potency test Sterile and free from contaminating micro-organisms. 2. Control of other raw materials

18 3. In-process control and final bulk control Appearance ph Phenol content : < 0.25% Protein content : < 17.0% Ammonium sulphate : < 0.1% Sterility test : passes Pyrogen test : passes Abnormal toxicity : conforms Potency test : conforms

19 4. Finished Products Control Specifications of Snake Antivenoms 1. Appearance : Freeze-dried cake 2. Identification : Identify specific antibodies against snake venoms 3. Sterility test : Sterile 4. Abnormal toxicity : None of the animals dies or shows signs of illness 5. Potency : 1 ml of antivenin can neutralize venoms not less than the labelled amount

20 6. Pyrogen : Passed 7. Protein content : Not more than 17% w/v 8. ph : Phenol content : Not more than 0.25% w/v 10. Osmolarity : mosmol/kg 11. Determination of F(ab ) 2 : Not less than 75%

21 Specifications of TRCS ERIG TRCS Antirabies Serum) 1. Appearance : Clear or pale-yellow to light-brown solution 2. Identification : Identify specific antibodies against rabies virus 3. Sterility test : Sterile 4. Abnormal toxicity : None of the animals dies or shows signs of illness 5. Potency : 1 ml of contains not less than 200 IU 6. Pyrogen test : Passed 7. Protein content : Not more than 17% w/v 8. ph : Phenol content : Not more than 0.25% w/v 10. Moisture content : Not more than 3.0% 11. Determination of F(ab ) 2 : Not less than 75%

22 Laboratory testing - Manufacturer s laboratory has to perform the chemical and biological testing every batch according to WHO and Pharmacopoeia requirements - NCL (National control laboratory) will confirming the manufacturer s test results and issue certificate of lot release every batch before sales.

23 Content of F(ab )2 and foreign proteins The contents of F(ab )2 and foreign proteins in production batches produced by new technique are compared to the old methods and commercial products by using SDS Page and HPLC technique and found that products produced by new technology contain high content of F(ab )2 and high purity (foreign protein content is too small quantity and cannot be detected).

24 Lane Description / Source Protein (mg/ml) Volume ( mcl ) F(ab )2 1 TRCS ERIG TRCS Antirabies Serum (dialysis bag with caprylic acid), old method 3 TRCS Antirabies Serum (dialysis bag without caprylic acid), old method Horse F(ab) 2 (Reference) Commercial ERIG A (whole IgG) Commercial ERIG B (split IgG chromatograph) Crude plasma (Horse)

25 TRCS ERIG UF+CA Bag+CA (Old) Bag+CA Bag no CA Bag no CA (Old) F(ab) 2 (Reference) Com A Gel picture Com B Crude

26 Protein bands in Thai venomous snake venoms 1 : 60 KD 2 : NTX 7 KD 3 : T. albolabris (Green Pit Viper) 4 : C. rhodostoma (Malayan Pit Viper) 5 : D. russelii siamensis (Russell s Viper) 6 : O. hannah (King Cobra) 7 : B. fasciatus (Branded Krait) 8 : N. kaouthia (Cobra) 9 : B. candidus (Malayan Krait)

27 Safety and Immunogenicity Study QSMI never received the serum sickness reports after QSMI had distributed more than 100,000 vials of QSMI s snake antivenoms products produced by this improved technology to worldwide since A statistical kinetic profile of TRCS ERIG has performed in year 2001 in QSMI s clinic with Good Clinical Practices and with local regulatory requirements. The protocols were approved by QSMI s Ethic committee and Ministry of Public Health s Ethic committee.

28 I. ERIG (old process without UF) II. ERIG (new process with UF) batch no. 10/44 (year 2001) 11/44 (year 2001) 10/45 (year 2002) 4620 (year 2003) year 2004 year 2005 year 2006 (non-volunteer subjects) Safety Study for TRCS ERIG Subject : WHO Category III Dose : 40 IU/Kg body weight Products Total number of patients Local reactions No. of patients % General reaction No. of patients The results showed that TRCS ERIG is safe with no serious adverse events and no anaphylactic reactions or severe serum sickness were reported %

29 Pharmacokinetic analysis of TRCS ERIG Determine the content of F(ab )2 in the collected blood from subjects administered with TRCS ERIG (Q Group), and split IgG chromatographed (P Group) by using ELISA technique. The contents of F(ab )2 was found immediately after administration and further increased up to day 3.

30 Comparison of Q-group and P-group Amount of F(ab')2 (ug/ml) P-group Q-group Time (hour) The results showed that the content of F(ab )2 in the collected blood of subjects administered by TRCS ERIG and split IgG chromatographed are equal.

31 Efficacy of RIG determined by challenging with CVS-strain in Hamster RIG Without vaccine %Efficacy QSMI Whole IgG HRIG 20 IU/ml HRIG 40 IU/ml % Efficacy Whole IgG (commercial) TRCS ERIG Split F(ab )2, chromatograph (commercial) QSMI Whole IgG HRIG 20 IU HRIG 40 IU Whole IgG (commercial) TRCS ERIG Split F(ab )2, chromatograph Q SM I Whole I gg HRI G 20 I U HRI G 40 I U Whole I gg ( comm er cial) TRCS ERI G Split F( ab ) 2, chr om at ogr aph Cont r ol Control

32 Production Quantity Manufacturer Products Quantity (vials) Current maximum Monovalent Snake Antivenoms(10ml) 60, ,000 QSMI TRCS ERIG (5 ml of 200IU/ml lyophilized product) 100, ,000

33 Unit Cost Details % Plasma 50.0 Raw Materials 5.0 Packing Materials 5.0 QA Cost 10.0 Other Expenses / Depreciation 25.0 Solvent Cost 5.0 Total Cost 100.0

34 Acknowledgement Pakamatz Khowplod Lalida Skolpap Wachiragporn Hemmala Wipaporn Jaijaroensab Suraseha Ouisuwan Maneerat Benjawongkulchai Narumol Pakmanee Duangporn Pornmuttakun Kornvika Limsuwun Surasak Kesowan Artikaya Sawangyaree Thanphet Tantawichien

35 Thank you