1
FORM 2
THE PATENTS ACT 1970
[39 OF 1970]
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See Section 10; rule 13]
“Isolation of Highly Pure, Monomeric Tetanus Toxoid Using Gel-Filtration Chromatography”
Serum Institute of India Pvt. Limited, 212/2, Soli Poonawalla road, Hadapsar, Pune-411028, India (Nationality: Indian)
The following specification particularly describes the invention and the manner in which it is to be performed.
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FIELD OF THE INVENTION
[0001] The present invention is in the field of Biotechnology applicable in vaccine manufacturing biopharmaceutical industry. It involves use of biotechnology tool; gel-filtration chromatography for the isolation of monomeric tetanus toxoid mainly suitable for use as carrier protein (adjuvant) for conjugation vaccines additionally, it can also be used as an antigen for monovalent or multivalent vaccine preparations involving tetanus toxoid.
BACKGROUND OF THE INVENTION
[0002] Tetanus is an acute, often fatal disease caused by the action of highly potent neurotoxin tetanospasmin which is produced during the growth of an anaerobic bacterium Clostridium tetani. Due to effective immunization strategies by WHO and UNICEF, although tetanus morbidity and mortality has been decreased in developed countries, it remains a major public health problem in most of the developing countries. Hence, Tetanus and combination vaccines involving tetanus toxoid still remains part of the national immunization schedule in almost all countries world-wide. Additionally, Tetanus toxoid is not only effectively used as one of the component in new generation multivalent vaccines like Pentavalent and Hexavalent vaccines, but also it is used as a carrier protein to boost immunogenicity of bacterial polysaccharides in case of conjugation vaccines like Haemophilus influenzae type b (Hib) vaccine, Meningococcal vaccine (Men-A) and pneumococcal conjugate vaccine.
[0003] Tetanus toxoid used for vaccine preparation should be purified in order to eliminate constituents of growth medium and metabolites which tend to provoke undesirable reactions (Gupta R.K., et. al., 1991; Relyveld E.H., et. al., 1962; Henocq E., et. al., 1962; Fanet G., et. al., 1972; Relyveld E.H., et. al., 1979; Relyveld E.H., et. al., 1984; Bizzini B. et. al., 1984; Pappenheimer A.M., et. al., 1984). Currently, commercial purification of tetanus toxoid for vaccine preparation is carried out by fractional precipitation using ammonium sulphate (WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1.). Tetanus toxoid
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purified by this method very well achieves WHO requirement of purity which is 1000 Lf/mg of protein nitrogen. But this method is not designed to remove aggregates as well as criteria of purity determination doesn’t consider presence or absence of low molecular weight impurities (Doshi J.B. et. al. 2003).
[0004] Another important aspect is heterogenecity in purified tetanus toxoid which has been reported long back (Latham W. C.; et.al., 1965, Raynaud M; et. al. 1971, Runova V. F.; et. al 1994) and it was correlated with process of chemical detoxification of tetanus toxin using formalin (Rappuoli R., 1997; Schwendeman S.P., et al., 1995). Formalin treatments lead to inter and intra molecular covalent cross-linking involving additional amino groups of Lysine and histidine creating heterogeneous detoxified tetanus toxoid; additionally having aggregates and dimmers. This heterogenecity was not matter of much concern until recently it was discovered that aggregated and dimeric tetanus toxoid are responsible for inconsistency, poor stability and potency in conjugation vaccine (Michael B. R; et. al. 2016) wherein, tetanus toxoid is used as a carrier protein. It was also suggested that immunogenicity of the polysaccharide component is improved by reducing the protein content of the conjugate antigen (International Patent no. CA 2221511 C) and one of way to achieve this is by use of highly pure monomeric tetaus toxoid. Recent research in conjugation vaccine further suggested use of tetanus > 80% monomeric tetanus toxoid so as to produce more stable and homogeneous conjugation vaccine (Ali Saber Abdelhameed et. al, 2012).
[0005] Though not reported in case of tetanus toxoid, it has been revealed recently that aggregation in therapeutic proteins can lead to elicitation of anti-drug antibodies (ADA) against biotherapeutics which can have detrimental effects on drug safety, efficacy, and pharmacokinetics (Kirsty D. R; et. al. 2013). Use of highly pure antigen for vaccine manufacturing has been one of the basic and essential requirements as it gives advantage of less reactogenicity and better potency.
[0006] Considering above aspects inventors assessed that there is need to develop method for the purification of tetanus toxoid with main objective to achieve highly pure Monomeric tetanus toxoid.
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[0007] In case of purification of tetanus toxoid one of the major challenge is the separation of minute amount of the active compound from the large volumes of culture media. Ideal toxoid purification system should be the one that has following features (Latham et. al., 1965):
1. Separate the specific component from the other bacterial proteins and constituents of the culture medium.
2. Should not significantly affect the yield or the biological activity of the toxoid.
3. It should be reproducible procedure, which lends itself to large scale production.
[0008] Various methods have been well documented for the purification of tetanus toxin and toxoid in the past, mostly based on principles of Salt Precipitation, precipitation using Organic Solvents and Acid Precipitation. Most of them had some or other constrains for implementation at large scale like lower recovery, lower purity, non-economical, residual toxicity of reagents used for purification, non-feasibility for operating at bulk stage and denaturation of toxoid. And most importantly none of the method had specific objective of achieving monomeric tetanus toxoid.
Chromatographic Techniques
[0009] It has been known that Gel-filtration is a good chromatographic technique for obtaining molecularly homogeneous and highly pure bacterial antigens (GoLshmid V.K. et.al.; 1974).
[0010] Latham W. C. and his group (1965) first evaluated use of chromatographic technique for the purification of tetanus toxoid. The technique was further modified by the same team for the large scale purification of toxoid in 1967. They used sephadex G-100 as a gel-filtration media. Although they could get high purity toxoid, recovery was just 50% also toxoid obtained was not monomeric in nature. Immuno-purification of tetanus toxoid using affinity chromatography was tried by M. Hughes et. al., in 1974. Sheppard and his group employed similar technique for the purification of tetanus toxin in 1987. In both the cases purity
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achieved was very high as well as recovery was also good, but this method could not get acceptance probably due to its limitations for use at large scale as well as aspect of high production cost. In 1997 a very novel and economical approach was tried by Vancetto M.D.C. and his group (Vancetto M.D.C.;et. al., 1997) wherein, first toxin was ultrafiltered followed by formalin treatment, diafiltration and lastly gel filtration using Sephadex G-50. Though method had achieved about 92% recovery the final average purity was not substantially high as compare to that of reported earlier by Latham W.C., 1965. In 1999 and 2001 Prado S. M. and his group reported sephacryl-HR-100 and HR-200 as a better media for the chromatographic purification of the toxoid (Prado S. M. et.al; 1999 and 2001). They employed this technique only at small scale alongwith ultrafiltration by tangential flow filtration method but reported gel-filtration chromatograph shows extremely poor separation. Secondly, they did not consider aspect of aggregate free toxoid with more than 80% monomeric toxoid.
[0011] Very recently in 2011 hydrophobic chromatography and immobilized metal affinity chromatography has been tried for the purification of tetanus toxoid (Stojiæeviæ et al., 2011). Results of hydrophobic chromatography were similar to that of ammonium sulphate precipitation method while results of immobilized metal affinity chromatography were very poor.
Need for the Present Invention
[0012] 1) For almost all the purification techniques developed in the past as explained in above section, main objective was to get high toxoid purity only in generalized terms i.e. high anitigenicity against lower protein content but none of them had specific objective to remove aggregates and dimmers of toxoid and obtain highly pure and aggregate free monomeric toxoid. In view of toxoid purity following two aspects of tetanus toxoid has got lot of importance due to recent research findings:
A) Aggregates and dimmers
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[0013] Recently researchers have expressed their concern over presence of aggregation in bio-therapeutics due to formation of anti-drug antibodies (ADA) and other related issues (RatanjiKirsty et.al; 2013). With reference to tetanus toxoid used for the conjugation vaccine it has been suggested to have minimum aggregates/dimmers (Ali Saber Abdelhameed et. al, 2012, International Patent no. CA 2221511 C). Even WHO has suggested to have minimum aggregates/dimmers and impurities for the tetanus toxoid used for conjugation vaccine and hence, purity limits for such toxoid has been raised from 1000 Lf/mg of PN to 1500 Lf/mg of PN.
B) Low molecular weight impurities
[0014] Though low molecular weight proteins and peptides may not be immunogenic, their removal is essential as they may be reactogenic in nature (Relyveld E. H., 1998; Gupta R. K., 1991; Relyveld E. H., 1979; Levine M.M., 1997). Such accessory proteins and peptides can compete for the adjuvant-binding site and can lead to reduction in antigen adsorption ultimately affecting vaccine potency (Ellis R. W., 1999).The present era of combination vaccine also demands for the highly purified toxoid (Doshi J. B., et. al., 2003).
[0015] 2) Most of the toxoid purification studies done in the past were at lab scale level only. Many of them were not implemented commercially due to scale up issues with reference to quality and cost.
[0016] In view of above aspects, inventors assessed that there is need to develop alternate commercially feasible and scalable process for the tetanus toxoid purification technique mainly with reference to manufacturing of tetanus toxoid antigen of homogeneous nature (monomeric) with high degree of purity specially for its use in conjugation vaccines as well as other new generation vaccines (viz. Tdap, Penta-valent, Hexa-valent etc).
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Why Gel-filtration technique
[0017] To achieve above objective Gel-filtration chromatography technique seems to be having more advantage over other methods (Prado S. M. et.al; 1999 and 2001, Golshmid V. K; 1974) because of following reasons:
1) As formalin detoxification process generates cross-linked toxoid molecules, aggregates and presence of low molecular weight impurities makes toxoid having proteins of wide molecular weight range. Under such conditions, purification based on molecular size is more logical and safe method to achieve monomeric tetanus toxoid of 158 kD.
2) It is much safe technique as it doesn’t involve any chemical interaction or modifications, helpful in retaining conformational integrity of toxoid antigen during processing.
3) Formalin detoxification of crude or partially purified toxin results in inter and intra molecular covalent cross-linking (Rappuoli R., 1997; Schwendeman S.P., et al., 1995). This process being uncontrolled and nonspecific cellular and media protein also gets cross-linked to toxin, generating heterogeneous pool of toxoid. Across vaccine industry different culture medium is used for the growth of C. Tetani as well as there are variations in some of the critical process steps as well as parameters like formalin concentration and duration of incubation for detoxification etc. Under such circumstances best purification method for achieving monomeric toxoid which can be applied universally is gel-filtration.
4) Large scale manufacturing possible.
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Table no. 1 Comparison with other closely related research works.
Inventor
Principle of purification
Monomeric TT achieved (%)
Monomeric, % of other components
Scale
Toxicity studies
Potency
studies
Remark
Latham W.C.; et.al., 1967
Gel-filtration
No
NA
Small scale
No
No
Poor recovery
M. Hughes et. al., in 1974
Affinity Purifcation
No
NA
Small scale
No
No
Scale up and Economical feasibility issues
Vancetto M.D.C.;et. al., 1997
Mmbrane Filtration and gel-filtration
No
NA
Large Scale
No
No
Purity similar to conventional method.
Prado S. M. 1999 and 2001
Gel-filtration
No
NA
Small scale
Yes
No
Poor resolution and not monomeric
Stojicevic et. al., 2011
Immobilized metal affinity and hydrophobic chromatography
No
NA
Small scale
No
No
Same as conventional method.
Douglas J. C, 1996. (Patent no. WO1996025425 A1)
Affinity chromatography
65%
(± 5.4)
26.9
(± 7.1)
Small Scale
Yes
Yes
Involves chemical interactions, After chromatography monomer content is poor. Final product has aggregates, dimmers and low molecular weight moieties as well
Present Invention
Gel-filtration chromatography
87.45
(± 0. 74)
12.5
(± 0. 71)
Large scale
Yes
Yes
Being more than 80% monomeric suitable for conjugation vaccine purpose. Largest scale i.e. processing of 10 Million doses per batch.
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Final product is free from aggregates and has low amount of dimmers and low molecular weight moieties.
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SUMMARY OF THE INVENTION
[0018] The present invention relates to isolation of monomeric tetanus toxoid by gel-filtration chromatography, mainly suitable for use as carrier protein (adjuvant) for conjugation vaccines additionally, it can also be used as an antigen for monovalent or multivalent vaccine preparations involving tetanus toxoid.
[0019] In an aspect of the present discloser, there is provided a scalable method for the purification of immunogenic tetanus toxoid to achieve highly pure, monomeric (molecular weight close to 158 kD) tetanus toxoid using gel-filtration chromatography. The inventors have studied that tetanus toxoid purified by WHO suggested method of tetanus toxoid purification (WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1.) provides toxoid of multimeric nature hence, further purification by gel-filtration chromatography provides excellent basis for achieving task to manufacture monomeric tetanus toxoid. This embodiment of the present invention relates to a large scale method for purification of immunogenic and monomeric tetanus toxoid using gel-filtration chromatography, said method comprising the steps of:
a. Obtaining an immunogenic multimeric tetanus toxoid (monomer being 60%) containing material prepared from fermentation of Clostridium tetani; and further manufactured by chemical detoxification using formalin followed by ultrafiltration, fractional ammonium sulphate precipitation and diafiltration;
b. Further carrying out gel-filtration chromatographic purification by loading toxoid on pre-equilibrated large scale gel-filtration multi-column setup packed with acrylamide based gel-filtration media;
wherein a substantially purified preparation of tetanus toxoid is obtained which is highly monomeric and having monomer content between 80-90%.
[0020] In another aspect tetanus toxoid being formalin detoxified product it contains molecules of wide molecular weight range involving tetanus antigen, aggregates, dimmers and low molecular weight components. Inter and intra molecular covalent cross-linking due to formalin detoxification generates antigen molecules of wide
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range of molecular weights also molecules with minor difference in surface charge. Considering these aspects inventors used gel-filtration chromatography using Sephacryl-HR gel filtration media of wide fractionation range to achieve better separation and isolation of monomeric toxoid. For best method development, different critical process parameters were varied like flow rate, sample volume and sample protein content to achieve best separation of multimeric toxoid molecules. In preferred embodiments, the method involves use of chromatographic technique which is based on principle of separation only the basis of difference in molecular weight. As there is no chemical interaction between separation media and tetanus antigen method can be applied universally even though there may be differences in fermentation media or process of detoxification.
[0021] In preferred embodiments the method includes first step as purification of crude tetanus toxoid by using WHO suggested purification method (WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1) which involved steps 1) material obtained from fermentation is further detoxified and subjected to 80-90 times concentration by ultrafiltration using tangential flow filtration system through 30 kD cutoff membrane b) Two step fractional ammonium sulphate precipitation c) Diafiltration for the removal of ammonium sulphate. Toxoid obtained at this stage is multimeric in nature due to formation of inter and intra molecular covalent cross-linking during formalin detoxification step (Schwendeman S. P.; et.al. 1995; Levine M. M.; et.al 1997).
[0022] In preferred embodiments the method further includes 1) loading of the multimeric tetanus toxoid over gel-filtration media column 2) Separation/elution of multimers on the basis of difference in molecular weight 3) Isolation of monomeric toxoid fraction which gets separated from other multimers (aggregates, dimmers and low molecular weight proteins).
[0023] In preferred embodiments the method involves use of Sephacryl-HR 300 gel filtration media (GE health care) to get monomeric tetanus toxoid. Sephacryl-HR gel filtration media has been preferred as it is being advantageous with respect to performance, stability and suitable to be used at large scale purification.
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[0024] In another aspect inventors conceived that Sephacryl-HR 300 gel filtration media (GE health care) wide fractionation range is suitable to obtain highest peak resolution /separation for multimeric tetanus toxoid as compare to other Sephacryl HR media’s like HR-100 and HR-200.
[0025] In preferred embodiments the method involves use of best elution flow rate (10-25 cm/hr) which provides best peak separation helping in isolation of highly monomeric tetanus toxoid.
[0026] In preferred embodiments the method further includes separation of multimers on the basis of their molecular weights leading to elution in the following sequence 1) Aggregates 2) Dimer 3) Monomeric toxoid 4) Low molecular weight proteins.
[0027] In preferred embodiments the small scale method includes elution step which is sufficiently gentle that a biological activity (Antigenicity) of the monomeric toxoid is preserved e.g. salt solution of 0.14 M solution.
[0028] In preferred embodiments elution and monomer fraction collection results in loss less than 20%, 30% and 40% of the biological activity which is mainly due to loss or removal of multimeric forms of tetanus toxoid other than monomeric.
[0029] In preferred embodiments, the invention at small scale features, a substantially purified preparation of tetanus toxoid which is highly monomeric. Monomer content found to be > 80% (between 80-90%) on the basis of HPLC analysis.
[0030] In preferred embodiments, inventors further successfully scaled up process by extrapolation of parameters optimized on small scale. Method was scaled up from purification of 50 ml of tetanus toxoid to 5 Litres i.e. from 0.025 million doses to 2.5 million doses.
[0031] In preferred embodiments large scale method includes use of dual column technology, which provides unique advantage of achieving desired bed height with minimum back pressure. This technology provides better support to the separation media especially at the centre of the column which is more prone to collapse under high pressure.
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[0032] In a preferred embodiment the present method comprises multi-column (each column of size 50 to 450 mm (width) x 500 mm to 1000 mm height) large scale setup packed with gel-filtration media having fractionation range between 1 × 104– 1.5 × 10⁶ being used for the purification of (multimeric) tetanus toxoid to manufacture highly pure monomeric tetanus toxoid.
[0033] In preferred embodiment of the present invention, the said method comprises total bed height for gel-filtration media being used more than 50 cm i.e. between 50-90 cm.
[0034] In preferred embodiments the large scale method also involves use of elution flow rate 10-25 cm/hr more preferably 16 cm/hr which provides best peak separation helping in isolation of highly monomeric tetanus toxoid.
[0035] In preferred embodiments the large scale method also includes elution step which is sufficiently gentle that a biological activity (Antigenicity) of the monomeric toxoid is preserved e.g. salt solution of 0.14 M solution. In preferred embodiments elution and monomer fraction collection results in loss less than 20%, 30% and 40% of the biological activity which is mainly due to loss or removal of multimeric forms of tetanus toxoid other than monomeric.
[0036] In preferred embodiments, invention at large scale features, a substantially purified preparation of tetanus toxoid which is highly monomeric with monomer content found to be between 80-90% on the basis of HPLC analysis.
[0037] Monomeric toxoid produced was tested as per WHO guidelines involving tests; Sterility, Specific toxicity, Irreversibility and antigenic purity. It passed for all the quality control tests while antigenic purity was found to be higher than conventional ammonium sulphate based purification.
[0038] Additionally, Monomeric toxoid was analysed for SDS-PAGE analysis (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) which revealed that new method of toxoid purification is producing toxoid of highly monomeric in nature with high degree of batch to batch consistency.
[0039] Finally inventors formulated monovalent tetanus toxoid vaccine using AlPO4 as an adjuvant for toxoid before gel-filtration as well as for toxoid after gel-filtration
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(monomeric tetanus toxoid). Both the formulation were tested for animal potency as per WHO suggested challenge assay (WHO TRS 980; http://www.who.int/biologicals/WHO_TRS_980_WEB.pdf) Potency results were satisfactory for both type of formulations values being 1) Conventional toxoid (before gel-filtration) vaccine formulation potency : 96 IU/dose 2) Monomeric toxoid (after gel-filtraion) vaccine potency was : 109 IU/dose. These results clearly indicated tetanus toxoid purified by gel-filtration chromatography has retained its conformation, preserving its immunogenicity and hence, potency.
[0040] In a preferred embodiment of the present invention, the large scale manufacturing method is capable of processing 1 to 10 L of toxoid ( i.e. about 0.5 million doses to 5 million doses, 15 to 150 gms) within one day while 20 L toxoid (about 10 million doses, 300 gms) requires two days for processing.
In a preferred embodiment of the present invention, the monomeric purity of the tetanus toxoid improved by > 40% than conventional one.
[0041] One embodiment of the present invention relates to a substantially purified preparation of tetanus toxoid made by the present method.
[0042] In a preferred embodiment, a substantially purified preparation of tetanus toxoid is monomeric tetanus toxoid preparation and is free from higher order aggregates.
[0043] In another preferred embodiment, a substantially purified preparation of tetanus toxoid wherein the tetanus toxoid preparation is less than 6% dimeric.
[0044] There are other features and advantages of the invention which will be apparent from following description and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0045] Figure 1 : HPLC analysis ; tetanus toxoid purified by conventional method of purification showing presence of aggregates and low molecular weight components along with monomeric tetanus toxoid at the centre.
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[0046] Figure 2 : SDS-PAGE analysis (reducing gradient gel) ; 1) Molecular weight marker 2) Tetanus toxoid 10 μg 3) Tetanus toxoid 20 μg. Band 1 : 300 kD Dimer, Band 2 : 150 kD monomer, Band 3 and 4 are about 100 and 50 kD subunits of tetanus toxoid. Aggregated tetanus toxoid being very high molecular weight did not enter in resolving gel. Each band identification was performed by protein extraction from each band and further analysis by mass spectrophotometry.
[0047] Figure 3: Gel-filtration chromatography : separation of tetanus toxoid using different gel-filtration media- (A) Spehacryl HR-100 (B) Sepahcryl HR -200 (C) Sephacryl HR-300 ; best separation has been achieved using sephacryl HR-300.
Figure 4: HPLC analysis; tetanus toxoid purified by gel-filtration chromatography showing presence of highly pure monomeric tetanus toxoid free from high molecular weight aggregates.
[0048] Figure 5: Reducing SDS-PAGE analysis: samples were purposefully over loaded to 50-60 ug in each well, Lane 1: toxoid before gel-filtration, Lane 2 to 5: different fractions collected during gel chromatographic purification of multimeric tetanus toxoid. Lane no. 2: fraction 1 (aggregated toxoid, being very high molecular weight remained in spacer gel), Lane no.3: fraction 2 (aggregated toxoid and dimmer), Lane no. 4: fraction 3 (monomeric tetanus toxoid), Lane no.5: fraction 4 (low molecular weight tetanus toxoid).
[0049] Figure 6: HPLC analysis: large scale gel chromatographic purification of tetanus toxoid batch no. 878L7001, A- before gel-filtration, B-after gel-filtration.
[0050] Figure 7: HPLC analysis: large scale gel chromatographic purification of tetanus toxoid batch no. 878L7002, A- before gel-filtration, B-after gel-filtration.
[0051] Figure 8: HPLC analysis: large scale gel chromatographic purification of tetanus toxoid batch no. 878L7003, A- before gel-filtration, B-after gel-filtration.
[0052] Figure 9: Reducing SDS-PAGE analysis: samples were purposefully over loaded to 50-60 ug in each well, Lane no. 1: molecular weight marker. Lane 2: Toxoid before gel-filtration, Lane 3 to 5: Different batches of monomerc tetanus toxoid manufactured at large scale i.e. Lane no.3: Monomeric tetanus toxoid batch
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no. 878L7001, Lane no. 4: Monomeric tetanus toxoid batch no. 878L7002, Lane no.5: Monomeric tetanus toxoid batch no. 878L7003.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The present invention relates to isolation of monomeric tetanus toxoid by gel-filtration chromatography, mainly suitable for use as carrier protein (adjuvant) for conjugation vaccines additionally, it can also be used as an antigen for monovalent or multivalent vaccine preparations involving tetanus toxoid.
[0054] In an aspect of the present discloser, there is provided a scalable method for the purification of immunogenic tetanus toxoid to achieve highly pure, monomeric (molecular weight close to 158 kD) tetanus toxoid using gel-filtration chromatography. The inventors have studied that tetanus toxoid purified by WHO suggested method of tetanus toxoid purification (WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1.) provides toxoid of multimeric nature hence, further purification by gel-filtration chromatography provides excellent basis for achieving task to manufacture monomeric tetanus toxoid.
[0055] In another aspect tetanus toxoid being formalin detoxified product it contains molecules of wide molecular weight range involving tetanus antigen, aggregates, dimmers and low molecular weight components. Inter and intra molecular covalent cross-linking due to formalin detoxification generates antigen molecules of wide range of molecular weights also molecules with minor difference in surface charge. Considering these aspects inventors used gel-filtration chromatography using Sephacryl-HR gel filtration media of wide fractionation range to achieve better separation and isolation of monomeric toxoid. For best method development, different critical process parameters were varied like flow rate, sample volume and sample protein content to achieve best separation of multimeric toxoid molecules.
[0060] In preferred embodiments, the method involves use of chromatographic technique which is based on principle of separation only the basis of difference in molecular weight. As there is no chemical interaction between separation media and
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tetanus antigen method can be applied universally even though there may be differences in fermentation media or process of detoxification.
[0061] In preferred embodiments the method includes first step as purification of crude tetanus toxoid by using WHO suggested purification method (WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1)which involved steps 1) material obtained from fermentation is further detoxified and subjected to 80-90 times concentration by ultrafiltration using tangential flow filtration system through 30 kD cutoff membrane b) Two step fractional ammonium sulphate precipitation c) Diafiltration for the removal of ammonium sulphate. Toxoid obtained at this stage is multimeric in nature due to formation of inter and intra molecular covalent cross-linking during formalin detoxification step (Schwendeman S. P.; et.al. 1995; Levine M. M.; et.al 1997).
[0062] In preferred embodiments the method further includes 1) loading of the multimeric tetanus toxoid over gel-filtration media column 2) Separation/elution of multimers on the basis of difference in molecular weight 3) Isolation of monomeric toxoid fraction which gets separated from other multimers (aggregates, dimmers and low molecular weight proteins).
[0063] In preferred embodiments the method involves use of sephacryl-HR 300 gel filtration media (GE health care) to get monomeric tetanus toxoid. Sephacryl-HR gel filtration media has been preferred as it is being advantageous with respect to performance, stability and suitable to be used at large scale purification.
[0064] In another aspect inventors conceived that sephacryl-HR 300 gel filtration media (GE health care) wide fractionation range is suitable to obtain highest peak resolution / separation for multimeric tetanus toxoid as compare to other sephacryl HR media’s like HR-100 and HR-200.
[0065] In preferred embodiments the method involves use of best elution flow rate (10-25 cm/hr) which provides best peak separation helping in isolation of highly monomeric tetanus toxoid.
[0066] In preferred embodiments the method further includes separation of multimers on the basis of their molecular weights leading to elution in the following
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sequence 1) Aggregates 2) Dimer 3) Monomeric toxoid 4) Low molecular weight proteins.
[0067] In preferred embodiments the method includes elution step which is sufficiently gentle that a biological activity (Antigenicity) of the monomeric toxoid is preserved e.g. salt solution of 0.14 M solution. In preferred embodiments elution and monomer fraction collection results in loss less than 20%, 30% and 40% of the biological activity which is mainly due to loss or removal of multimeric forms of tetanus toxoid other than monomeric.
[0068] In preferred embodiments, the invention features, a substantially purified preparation of tetanus toxoid which is highly monomeric. Monomer content found to be > 80% (between 80-90%) on the basis of HPLC analysis.
[0069] In preferred embodiments, inventors further successfully scaled up process by extrapolation of parameters optimized on small scale. Method was scaled up from purification of 50 ml of tetanus toxoid to 5 Litres i.e. from 0.025 million doses to 2.5 million doses.
[0070] In preferred embodiments large scale method includes use of dual column technology, which provides unique advantage of achieving desired bed height with minimum back pressure. This technology provides better support to the separation media especially at the centre of the column which is more prone to collapse under high pressure.
[0071] In a preferred embodiment the present method comprises multi-column (each column of size 50 to 450 mm (width) x 500 mm to 1000 mm height) large scale setup packed with gel-filtration media having fractionation range between 1 × 104 – 1.5 × 10⁶ being used for the purification of (multimeric) tetanus toxoid to manufacture highly pure monomeric tetanus toxoid.
[0072] In preferred embodiments the large scale method also involves use of elution flow rate 10-25 cm/hr more preferably 16 cm/hr which provides best peak separation helping in isolation of highly monomeric tetanus toxoid.
[0073] Finally inventors formulated monovalent tetanus toxoid vaccine using AlPO4 as an adjuvant for toxoid before gel-filtration as well as for toxoid after gel-filtration
19
(monomeric tetanus toxoid). Both the formulation were tested for animal potency as per WHO suggested challenge assay (WHO TRS 980; http://www.who.int/biologicals/WHO_TRS_980_WEB.pdf) Potency results were satisfactory for both type of formulations values being 1) Conventional toxoid (before gel-filtration) vaccine formulation potency : 96 IU/dose 2) Monomeric toxoid (after gel-filtraion) vaccine potency was : 109 IU/dose. These results clearly indicated tetanus toxoid purified by gel-filtration chromatography has retained its conformation, preserving its immunogenicity and hence, potency.
Small scale manufacturing method of Monomeric Tetanus Toxoid:
[0074] In preferred embodiments formalin detoxified crude tetanus toxoid is first processed as per WHO suggested purification method (WHO Manual for the production and control of Vaccines. Tetanus toxoid BLG/UNDP/77.2 Rev1) which involves steps, 1) 80-90 times concentration of crude toxoid (batch size 500 or 1000 L) by ultrafiltration using tangential flow filtration system equipped with 30 kD cut-off membrane b) Two step ammonium sulphate precipitation; first step involves precipitation using 11% preferably 12% or 13% ammonium sulphate. After discarding precipitate isolated by centrifugation, additional ammonium sulphate is added to supernatant achieve final concentration to 22% preferably 23% or 24%. Precipitate obtained during second precipitation is retained after centrifugation and dissolved in 20 L water for injection c) Diafiltration step is performed using tangential flow filtration system equipped with 30 kD cutoff membrane for the removal of ammonium sulphate. Finally toxoid is sterile filtered using sterile grade filter under class A conditions.
[0075] In preferred embodiments even though toxoid obtained at this stage passes for all WHO requirements of safety (toxicity and irreversibility) including purity being above 1000 Lf/mg of protein nitrogen, it is multimeric in nature due to formation of inter and intra molecular covalent cross-linking during formalin detoxification step (Schwendeman S. P.; et.al. 1995; Levine M. M.; et.al 1997). This toxoid shows presence of aggregates, dimmers and low molecular weight proteins as observed in
20
HPLC analysis (Figure 1 ) making it not suitable for conjugation purpose (Ali Saber Abdelhameed et. al, 2012).
[0076] In preferred embodiments more specifically by SDS-PAGE analysis it was revealed that tetanus toxoid purified by conventional method of purification contains components of molecular weight about ~ 600 kD (probably tetramer/aggregate), ~ 300kD (probably Dimer), ~ 150 (monomer) and low molecular weight minor components of ~ 100 kD and~ 50 kD (subunits of tetanus toxoid (Figure 2). Considering wide range of molecular weight species present in tetanus toxoid, Sephacryl HR gel-filtration media was selected for the separation as it has wide molecular weight fractionation range also it has high chemical and physical strength. Sephacryl HR gel-filtration media of different fractionation range was used to develop best method using optimum elution flow rate for achieving highest possible separation of Monomeric toxoid from other components as mentioned in table no.2.
Table 2: Experimental details
Gel-filtration
Media
Fractionation Range
(Dalton)
Column
Efficiency By
Elution Flow rate (cm/hr)
Sephacryl HR-100
1 × 103–1 × 105
HETP
11
17
28
--
Sephacryl HR-200
5 × 103–2.5 × 105
HETP
23
34
45
57
Sephacryl HR-300
1 × 104–1.5 × 106
HETP
12
15
21
24
21
[0077] Column packing using gel-filtration media was done as per manufacturer’s instructions. HETP values were calculated using 8 M NaCl solution wherein it was expected to achieve > 2000 plates/meter.
[0078] For small scale experiments inventors used XK-26 and XK-50 (GE Health care) glass columns for packing gel-filtration media. While, AKTA Pure (GE Health care) chromatography system was used to perform actual chromatographic runs. Toxoid elution was online monitored by UV detection at 280 nm also other parameters like pH, conductivity pressure were also monitored online.
[0079] To achieve best possible separation between different toxoid components (Aggregates, dimmers, monomers and low molecular weight components) it is essential to have appropriate bed width and more importantly bed height combination. Higher the bed height better will be the separation but it will increase total time for the separation as well as higher bed height can be limiting factor for the large scale purification.
[0080] Inventors studied and founded that more than 50 cm height is important to achieve separation between toxoid molecules, more preferably above 70 cm bed height can provide still better separation more preferably more than 90 cm bed height can provide best toxoid separation but can create limitation with respect to large scale purification as more height increases column back pressure.
[0081] Toxoid loaded on the gel-filtration column was between 1-4% of the total bed volume while in terms of protein concentration sample with protein concentration up 10-15 mg /ml was loaded without any effect on separation efficiency.
[0082] On the basis of different experiments inventors established that best suitable gel for achieving maximum possible separation i.e. highest resolution between all the components of tetanus toxoid using optimized flow rate between 10 cm/hr to 25 cm/hr is Sephacryl HR-300 (Figure 3).
[0083] Inventors evaluated batch to batch consistency and other quality parameters by manufacturing monomeric toxoid batches using following optimized conditions:
22
1) Column : XK-50 (GE-Heath care)
2) Chromatography system : AKTA PURE
3) Gel-filtration media : Sephacryl HR-300
4) Elution buffer : 0.14 M NaCl
5) Sample Volume per run : 50-70 ml (3000-4000 Lf/ml, Protein : 10-15 mg/ml)
6) Column bed height : 70-90 cm
7) Flow rate : 10-25 cm/hr
ACCEPTANCE LIMITS:
1) Plates per meter: > 2000 plates / meter
2) Acceptance of chromatograph: 150 kD peak (main peak) should be best separated from other peaks.
3) Monomer content / Monomeric purity by HPLC : > 80%
4) Other tests: Purified toxoid should pass all safety and sterility tests.
5) Purity by WHO method: > 1500 Lf/mg of Protein nitrogen
[0084] In preferred embodiments small scale batches of monomeric tetanus toxoid were manufactured by processing 400 ml of tetanus toxoid (50-70ml for each run) using above parameters. Finally all the monomeric toxoid fractions of each run were pooled, optionally concentrated by small scale TFF system with 30 kD cutoff cassettes, sterile filtered and tested.
[0085] In preferred embodiments various quality control test results obtained for three toxoid batches purified by optimized gel-filtration chromatography method are provided in Table no.3 (before gel-filtration) and Table no. 4 (After gel-filtration). HPLC results of toxoid after gel-filtration revealed very distinct single peak appearance with excellent purity (Refer comparative HPLC analysis chromatograph of toxoid batch before and after gel-filtration in figure 1 and figure 4).
[0086] In preferred embodiments small scale studies revealed excellent quality control testing results indicating high degree of consistency in obtaining > 80% monomeric toxoid by chromatographic purification. Inventors could achieve
23
substantial increase in monomer content i.e. 50% rise in monomer content (increase from 58% to 87%) as higher order aggregates have been removed completely while dimmers and low molecular weight proteins reduced substantially (Refer table 3 and 4, figure 4).
[0087] In preferred embodiments SDS-PAGE analysis of different fractions collected during gel-filtration of tetanus toxoid revealed excellent separation of monomer from other components (figure 5).
[0088] In preferred embodiments after gel-filtration toxoid passed for all safety related testing viz specific toxicity and irreversibility tests as well as there was considerable increase in toxoid purity determined as per WHO method (Anitigenic content per mg of protein nitrogen).
[0089] In preferred embodiments though monomeric toxoid was manufactured with main objective to be used as adjuvant in conjugation vaccine still to confirm that during chromatographic purification toxoid retains its potency (confirmation/epitopes) monovalent tetanus toxoid vaccine formulation using AlPO4 adjuvant was prepared for toxoid before and after chromatography and it was further tested for potency by WHO suggested challenge assay (WHO TRS 980; http://www.who.int/biologicals/WHO_TRS_980_WEB.pdf). Potency results were satisfactory (above WHO requirement of 40 IU/dose) for both type of formulations values being; for conventional toxoid (before gel-filtration) vaccine formulation potency: 96 IU/dose and for Monomeric toxoid (after gel-filtration) vaccine potency was: 109 IU/dose. These results clearly indicated that while chromatographic purification toxoid has retained its conformation, preserving its immunogenicity and hence, potency (Table no. 3 and 4).
[0090] In preferred embodiments with reference to potency aspect inventors preferred to challenge product under worst case scenario. In general there is always synergistic effect on potency of tetanus antigen when it is in combination with pertussis vaccine in (viz. DTP vaccine) (Samore M.H; et.al.; 1996). Hence, to avoid this effect and to get real picture about monomeric tetanus toxoid antigen potency, monovalent tetanus toxoid vaccine was prepared also it was tested by
24
more realistic potency assay i.e. WHO suggested method of challenge assay (WHO TRS 980; http://www.who.int/biologicals/WHO_TRS_980_WEB.pdf) instead of indirect/partly in-vitro method of antibody induction assay. As under these conditions monomeric toxoid vaccine passed very well for potency test it provides assurance and high level of confidence that monomeric tetanus toxoid manufactured by inventors is of high quality and potency.
25
TETANUS TOXOID SMALL SCALE CHROMATOGRAPHIC PURIFICATION RESULT SUMMARY
Table no.3 RESULTS BEFORE GEL-FILTRATION
* : Analysis as per WHO guidelines
$ : Antigenicity (Lf) per mg of protein nitrogen,
Batch no.
HPLC analysis
PURITY*
(WHO method)$
POTENCY*
(TT VACCINE)
Challenge assay
% Aggregates
and Dimers
% Monomer
% Low molecular weight proteins
TT-1
15.34
60.73
23.92
1778
96 IU/Dose
(Toxoid used from batch no.TT-1)
TT-2
15.42
61.08
23.48
1741
TT-3
16.59
58.91
24.49
1886
TT-5
15.22
56.95
27.82
1812
TT-6
15.20
57.32
27.48
1812
TT-7
15.12
57.09
27.78
1996
Average (SD)
15.48
(SD ± 0. 55)
58.68
(SD ± 1. 86)
25.83
(SD ± 2. 07)
1837
(SD ± 91)
26
TETANUS TOXOID SMALL SCALE CHROMATOGRAPHIC PURIFICATION RESULT SUMMARY
Table no.4 RESULTS AFTER GEL-FILTRATION
Note : Batch no. GFTT-4 was used exclusively for analytical method development purpose, was not part of this study.
* : Analysis as per WHO guidelines.
$ : Antigenicity (Lf) per mg of protein nitrogen. @ : No aggregates present only dimmers.
Batch no.
HPLC analysis
Animal
safety studies
and sterility test *
PURITY*
(WHO method)$
POTENCY*
(TT VACCINE)
% Dimers @
% Monomer
% Low molecular weight proteins
GFTT 1
5.45
89.40
5.14
All batches
Passed test for sterility, specific toxicity and Irreversibility test performed as per
WHO guidelines
2581
109 IU/Dose
(Toxoid used from batch no.GFTT1)
GFTT 2
4.47
88.68
6.84
2381
GFTT 3
4.59
87.53
7.87
2130
GFTT 5
4.95
86.37
8.68
1979
GFTT 6
5.01
86.09
8.90
2037
GFTT 7
4.76
85.35
9.90
2126
Average (SD)
4.87
(SD ± 0. 35)
87.24
(SD ± 1. 58)
7.89
(SD ± 1. 69)
2205
(SD ± 229)
27
Large Scale Manufacturing of Monomeric Tetanus Toxoid
[0091] In preferred embodiments, inventors further successfully scaled up process by extrapolation of parameters optimized at small scale using sephacryl HR 300 gel. Method was scaled up from purification of 50 ml of toxoid to 5 Litres i.e. from 0.025 million doses to 2.5 million doses (each run).
[0092] In preferred embodiments tetanus toxoid batch of 20 L (10 million doses) was chromatographically purified in four chromatography runs each of 5 L toxoid (2.5 million doses).
[0093] In preferred embodiments inventors developed large scale method with use of multi-column (two) technology which provides unique advantage of achieving desired bed height with minimum back pressure. This arrangement i.e. limited bed height in each column provides better support to the separation media especially at the centre of the column which is more prone to collapse under high pressure.
[0094] In preferred embodiments inventors used two BPG columns of 450 cm width and 750 cm height. Total bed height of 70-90 cm was achieved using these two columns.
[0095] In preferred embodiments large scale gel-purification was performed using process scale AKTA system (GE health care). While toxoid elution was online monitored by UV detection at 280 nm also other parameters like pH, conductivity pressure were also monitored online.
[0096] In preferred embodiments inventors mmanufactured large scale monomeric tetanus toxoid batches using following process parameters:
1) Column : BPG 450 x 750 (Two column) (GE-Heath care)
2) Chromatography system : AKTA Process
3) Gel-filtration media : Sephacryl HR-300
4) Elution buffer : 0.14 M NaCl
5) Sample Volume per run : 1 - 5 L (3000-4000 Lf/ml, Protein : 10-13 mg/ml)
6) Column bed height : 70-90 cm
7) Flow rate : 10-25 cm/hr (preferably 16 cm/hr)
28
ACCEPTANCE LIMITS:
1) Plates per meter: > 2000 plates / meter
2) Acceptance of chromatograph: 150 kD peak (main peak) should be best separated from other peaks.
3) Monomer content / Monomeric purity by HPLC : > 80%
4) Other tests: Purified toxoid should pass all safety and sterility tests.
5) Purity by WHO method: > 1500 Lf/mg of Protein nitrogen
[0097] In preferred embodiments the large scale method includes elution step which is sufficiently gentle that a biological activity (Antigenicity) of the monomeric toxoid is preserved e.g. salt solution of 0.14 M solution. Method also involves use of best elution flow rate of 10-25 cm/hr preferably, 16 cm/hr which provides best peak separation helping in isolation of highly monomeric tetanus toxoid.
[0098] In preferred embodiments the large scale method also includes washing step using 0.2 N NaOH after completion of every batch.
In preferred embodiments monomeric tetanus toxoid obtained from each chromatography run was tested for monomer content by HPLC and finally toxoid from all four runs was pooled, if required concentrated by ultrafiltration using tangential flow filtration system equipped with 30 kD cut off cassettes and finally sterile filtered.
[0099] In preferred embodiments inventors further tested monomeric toxoid from each batch for all WHO suggested quality parameters including HPLC analysis for monomer content / Monomeric purity by HPLC. (Table no. 5 and 6)
In preferred embodiments elution and monomer fraction collection results in loss of less than 20%, 30% and 40% of the biological activity (monomer % recovery) which is mainly due to loss or removal of multimeric forms of tetanus toxoid other than monomeric.
[0100] In preferred embodiments, invention at large scale features, a substantially purified preparation of tetanus toxoid which is highly monomeric with monomer
29
content found to be > 80% (i.e. average 87.45%) on the basis of HPLC analysis. Inventors could achieve substantial increase in monomer content i.e. 42% rise in monomer content (increase from 61.74% to 87.45%) as higher order aggregates have been removed completely while dimmers and low molecular weight proteins reduced substantially (Table no. 5 and 6) as well as same has been observed in case of SDS-PAGE analysis (Figure 9)
[0101] Scale up of gel-filtration chromatography has various challenges like to achieve similar column packing, equivalent separation efficiency, recovery etc. same has been successfully achieved even though method was scaled up from 50ml toxoid purification to 5 litres (100 times scale up).
[0102] In preferred embodiment inventors founded out that while achieving desired high bed height in large scale column of 450 mm diameter there is always chance that higher bed height can create high back pressure. There is always possibility of bed getting collapsed at the centre of the column due to high back pressure. Hence, inventors achieved desired bed height 70-90 cm preferably 75 cm using two columns instead of one column.
[0103] In preferred embodiments entire process of large scale gel-filtration as well as column washing method was validated which revealed/concluded that method designed was not only robust but also manufactured product of desired quality consistently (Table no. 6).
[0104] In preferred embodiments, invention at large scale features, chromatographic purification of minimum 1 L to 5 L of toxoid with protein concentration of 10-15 mg/ml more preferably 13 mg /ml which comprises about 0.5 to 2.5 million doses.
[0105] In preferred embodiments at large scale inventors could purify a batch of 20 L toxoid (10 million doses) in four chromatographic runs. Each run was followed by column cleaning using 0.2-0.5 M NaOH preferably 0.2 M NaoH (25 L). Monomeric fractions collected from all four runs were analyzed for monomer content and further pooled together and optionally concentrated using TFF system equipped with 30 Kd cutoff cassettes before performing final sterile filtration
30
using 0.1 micron sterilizing grade filter. Final sterile and Gel-chromatography purified monomeric toxoid was tested for sterility, antigenic purity, irreversibility, Specific toxicity, and Monomer content by HPLC (Table no.6, Figure 6, 7 and 8).
[0106] In preferred embodiments high degree of consistency has been observed for monomer content values (HPLC results) between each chromatographic run (Table no.6) average value of 16 runs being very high as 87.45% with very minimum standard deviation of just ± 0. 74. Each run being of 5 L toxoid it can be claimed that method developed is highly consistent and robust.
[0107] In preferred embodiments inventors developed largest gel-filtration set up ever reported for tetanus toxoid gel-filtration chromatography purification, having capacity to process 20 L toxoid in just two days, manufacturing about 150-200 gm of monomeric tetanus toxoid per batch.
[0108] In preferred embodiments method developed completely removed higher order aggregates while substantially reduces dimmers to just 3% (Table no. 5 and 6) which are reported to be main culprit for inconsistency and instability of conjugation vaccine (Ali Saber Abdelhameed et. al, 2012).
[0109] In preferred embodiments inventors could successfully extrapolate small scale gel-filtration chromatography parameters (50 ml sample) to develop large scale gel-filtration chromatography (5L sample) without any compromises in the quality of toxoid.
31
TETANUS TOXOID LARGE SCALE CHROMATOGRAPHIC PURIFICATION RESULT SUMMARY*
Table no. 5 RESULTS BEFORE GEL-FILTRATION
# : HPLC analysis was done in duplicate and average values are reported herewith. Method was developed in-house in
consultation with NIBSE (UK) (WHO approved testing laboratory).
* : Analysis as per WHO guidelines
$ : Antigenicity (Lf) per mg of protein nitrogen
Batch no.
HPLC analysis#
PURITY*
(WHO method)$
% Aggregates and Dimers
% Monomer
% Low molecular weight proteins
288L6028
18.89
61.39
19.73
1742
288L6029
15.70
62.37
21.94
1810
288L6030
17.02
61.46
21.52
1764
Average (SD)
17.20
(SD ± 1.60 )
61.74
(SD ± 0. 55)
21.06
(SD ± 1. 17)
1772
(SD ± 35)
32
TETANUS TOXOID LARGE SCALE CHROMATOGRAPHIC PURIFICATION RESULT SUMMARY*
Table no. no. 6 RESULTS AFTER GEL-FILTRATION
# : HPLC analysis was done in duplicate and average values are reported herewith. Method was developed in-house in
consultation with NIBSE (UK) (WHO approved testing laboratory).
* : Analysis as per WHO guidelines, @ : No aggregates present only dimmers.
$ : Antigenicity (Lf) per mg of protein nitrogen. @ : No aggregates present only dimmers.
Batch no.
(20 L each i.e.10 million doses)
Run no.
( 5 L each)
HPLC analysis #
Final pooled batch
% Monomer
by HPLC
Animal
safety studies
and sterility test *
PURITY*
(WHO method)$
% Dimers@
% Monomer
% Low molecular weight proteins
878L7001
Run 1
5.53
86.40
8.08
86.6
All batches Passed test for sterility, specific toxicity
and Irreversibility test
performed as per WHO guidelines
2154
Run 2
4.27
87.26
8.48
Run 3
3.09
88.20
8.72
Run 4
3.43
87.41
9.21
878L7002
Run 1
3.19
88.46
8.35
86.6
2405
Run 2
2.94
88.30
8.76
Run 3
2.66
88.28
9.06
Run 4
3.77
86.87
9.37
878L7003
Run 1
3.40
86.95
9.66
85.5
2236
Run 2
3.08
86.60
10.33
Run 3
3.40
87.29
9.32
Run 4
5.53
86.40
8.08
3.52
(SD ± 0. 79)
87.45
(SD ± 0. 74)
9.03
(SD ± 0. 64)
86.23
(SD ± 0. 63)
2265
(SD ± 127)
33
EXAMPLES
[0110] The present invention will now be illustrated with working example, which is intended to illustrate the working of the invention and not intended to take restrictively to imply any limitations on the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to a person skilled in the art to which the disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is also understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
Example 1
[0111] Crude tetanus toxoid is manufactured as per WHO guideline (WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1.) Seed lot system is used for the production of Crude tetanus toxoid. This is initiated by one ampoule of lyophilized Working seed of Clostridium tetani (Harvard Strain No. 49205 of Clostridium tetani) which is removed from cold storage (2-8 oC and surface is disinfected using absolute alcohol. The ampoule is cut open and reconstituted in Fluid thioglycollate medium. For purity check, a drop of suspension is inoculated on nutrient agar and in nutrient broth tubes. The vial is then incubated at 35 ± 1˚C for 48 hours.
[0112] After incubation and confirming the purity, 2-3 ml revived culture is transferred to a tube containing Fluid thioglycollate medium for passaging and also inoculated on nutrient agar, nutrient broth for purity check. Fluid thioglycollate medium tubes are incubated at 35±1°C for 24 hours. Purity of passage culture is confirmed by observing the nutrient agar slants, nutrient broth tubes and is also confirmed under by microscopy. After completion of incubation, 10 to 15 ml of passaged culture is transferred into Seed bottle containing Fluid Thioglycollate Medium (FTG). The seed bottle is incubated at 35 ± 1 °C for 24 hours. After incubation, purity of culture is
34
confirmed microscopically and the content of seed bottles is transferred to fermenter containing Semi-synthetic (meat free) medium.
Fermentation:
[0113] Semi-synthetic (meat free) medium is prepared and sterile filtered in the presterilized fermenter. The fermenter is inoculated with 24 hrs old seed culture. Aeration and agitation are maintain and monitored during fermentation cycle. Growth is allowed to proceed for 7 days.
[0114] After 7 days, the cell mass is separated from the toxin by clarification through TFF with 0.2 μ cut-off. The sterile filtered toxin is distributed into 50 liter and 20 liter glass bottles, which are contained in the culture free section. After filtration, Crude Toxin Samples are taken and tested for Lf content, Maximal Toxin Value (MTV) and Maximum Lethal Dose (MLD) and sterility.
[0115] The sterile toxin is detoxified by the addition of 0.45 % v/v of formalin and incubated at 35 - 37C for at least 4 weeks. After 4 weeks incubation the bottles are aseptically transferred and pooled into sterile stainless steel tanks of 260 L capacity and samples taken for specific toxicity, sterility and Lf content tests. The tanks containing the detoxified crude tetanus toxoid are stored at 2 - 8C till the test is complete.
[0116] Once specific toxicity results ensured that toxin is completely detoxified in to toxoid, further this crude toxoid batch of about 1000 L is purified using WHO suggested method involving ultrafiltration, 80-90 times concentration using tangential flow filtration system followed by fractional precipitation using ammonium suplhate and diafiltration step to remove ammonium sulphate using tangential flow filtration system. Finally 20 L concentrated toxoid is sterile filtered and tested as per WHO specification for Antegenic purity (Lf /mg of protein nitrogen), Specific toxicity, Irreversibility test and sterility test. Toxoid (batch no. 288L6029 ) at this stage when analysed using HPLC to determine monomer content it is found to be having 57% of monomer. In general toxoid at this stage has 50-60% of monomer. To make it suitable for conjugation process monomer content need to be more than 80%. Hence, toxoid was further processed using dual column large scale chromatography usin sephacryl
35
HR 300 gel. Each column of size was 450 x 750 mm. Elution was performed using 0.14 M NaCl at flow rate 10-25 cm/hr more preferably 16 cm/hr. 20 L toxoid batch with 50-60% monomer having 13-15 mg /ml protein concentration, 3500-4000 Lf/ml i.e. about 10 million doses was further processed in four chromatographic run each of 5 L. Monomeric fractions collected from all four chromatographic runs were tested for monomer content, pooled together and further tested for specific toxicity test, irreversibity test, monomer content (HPLC analysis), sterility and Purity.
[0117] Chromatographically purified monomeric tetanus toxoid (batch no. 878L7002 ) passed for all WHO suggested tests while monomeric purity improved from 62% to 87% and purity by WHO suggested method improved by > 40% (Table no. 5 and 6).
INDUSTRIAL APPLICABILITY OF THE INVENTION
[0118] The present invention relates to method of purifying tetanus toxoid using gel- filtration chromatography to achieve highly monomeric form and its use has been suggested mainly in conjugation vaccines to improve quality and consistency.
[0119] Inventors have developed unique dual column large scale gel-filtration chromatography which is probably the largest set up for tetanus toxoid chromatographic purification for monomeric tetanus toxoid manufacturing.
[0120] This method being fast and high capacity. It is capable of processing of 20 L of tetanus toxoid i.e. about 300 gm (about 10 million doses) in just two days. Such large capability and fast processing makes this method suitable for the industrial application wherein monomeric tetanus toxoid is in high demand for many different conjugation vaccines viz Haemophilus influenzae type b (Hib) vaccine, Meningococcal vaccine (Men-A) and pneumococcal conjugate vaccine.
[0121] This unique method produced monomeric toxoid which is free from aggregated and substantially reduces dimmers as well as low molecular weight components.
[0122] Release of polysaccharide (PRP- polyribosyl ribitol phosphate) from conjugate vaccine has been one of the major concerns. Aggregates in tetanus toxoid has been reported to be responsible in increasing free polysaccharide as there is strong stearic hindrance in the bond between aggregated tetanus toxoid and polysaccharide. Use of
36
monomeric tetanus toxoid will provide stability to TT-Hib bonding, will minimize free PRP.
[0123] It has been reported that use of tetanus toxoid with aggregates in conjugation vaccine leads to formation of multimers of various sizes conjugates also it leads to batch to batch variations hence, use of highly pure monomeric tetanus toxoid is highly recommended.
[0124] Highly Pure and monomeric toxoid use can lead to better adsorption for tetanus toxoid as well as other antigens in penta and Hexavalent vaccines.
[0125] Improvement with respect to minimizing probable factors responsible for reactions like aggregates and dimers.
37
REFERENCES:
Ali Saber Abdelhameeda, Gordon A. Morrisa, Gary G. Adamsa, Arthur J. Rowea, Olivier Lalouxb, Louis Cernyb, Benjamin Bonnierb, Pierre Duvivierb, Karel Conrathb, Christophe Lenfantb, Stephen E. Hardinga, (2012), An asymmetric and slightly dimerized structure for the tetanus toxoid protein used in glycoconjugate vaccines. Carbohydrate Polymers, 90 (2012) 1831– 1835
Bizzini B. (1984) Tetanus. In Gennanier R. (ed), Bacterial Vaccines. Orlando, FL, Academic Press, 38-68.
Doshi J. B., Ravetkar S. D., Ghole V. S., Rehani. K. (2003) Comparative quantitation for the protein content of diphtheria and tetanus toxoids by DC protein assay and Kjeldahl method. Biologicals, 31, 187-189.
Ellis R. W. (1999) Combination vaccines, Humana Press, Totowa, NJ, 51-89.
Fanet G., Turpin A., Henocq E., Bizzini R., Relyveld E. H., Raynaud M. (1972) Asthme consecutif a l'inhalation massive de toxine tetanique Med. Maladies. Infect., 2,163.
Golshmid V.K., Gel Filtration as a Method of Obtaining Molecularly Homogeneous Bacterial Toxins and Anatoxins (A Review of the Literature). Zh Mikrobiol Epidemiol Immunobiol (12), 89-96. 12 1974.
Gupta R. K., Relyveld E. H. (1991) Adverse reactions after injection of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine are not due only to pertussis organisms or pertussis components in the vaccine. Vaccine, 9 (10), 699-702.
Henocq E., Relyveld E. H., Raynaud M. (1962) La sensibilisation aux antigenes diphteriques. Etude immunologique et clinique. Int. Arch. Allergy, 20, 262.
38
Hughes M., Thomson R. O., Knight P. (1974) Immunopurification of tetanus toxoid. J. Appl. Bact., 37, 603-621.
Kirsty D Ratanji, Jeremy P Derrick, Rebecca J Dearman , Ian Kimber (2013), Immunogenicity of Therapeutic Proteins: Influence of Aggregation. J Immunotoxicol 11 (2), 99-109. 2013 Aug 06.
Latham W. C., Jenness C. P., Timperi R. K., Michelsen C. B., Zipilivan E. M., Edsall G., Ley H. L. (1965) Purification and characterization of tetanus toxoid and toxin. The J. Immunol., 95(3), 487-493.
Latham W. C., Michelsen C. B., Edsall G. (1967) Preparative procedure for the purification of toxoids by gel-filtration. Applied Microbiology, 15(3), 616-621.
Levine M. M., Woodrow G. C., Kaper J. B., Cobon G. S. (Ed.) (1997) New Generation Vaccines, 2nd edition, Marcel Dekker Inc., 422-423.
Michael B. R., Oker F. B. and Paolo C. Factors contributing to the immunogenicity of meningococcal conjugate vaccines, HUMAN VACCINES & IMMUNOTHERAPEUTICS (2016), VOL. 12, NO. 7, 1808- 1824
Pappenheimer A. M. (1984) Jr.Diphtheria In: Bacterial Vaccine (Ed.) Germanier R., Academic Press, Orlando, 1-36.
Prado S. M., Vancetto M. D., de Oliveira J. M., Fratelli F., Higashi H. G. (2001) Use of sephacryl high resolution (HR) in tetanus anatoxin purification. Boll. Chim. Farm. May-Jun; 140 (3):160-4.
39
Prado S. M., Vancetto M. D., De Oliveira J. M., Higashi H. G. (1999) Development and validation study for the chromatographic purification process for tetanus anatoxin on Sephacryl S-200 high resolution. Boll. Chim. Farm., 138(7), 364 – 368.
Rappuoli R. (1997) New Generation Vaccines. Ed. Levine M. M., Woodrow G. C., Kaper J. B., Cobon G. S. 2nd edition, Marcel Dekker Inc., 422-423.
Raynaud M, Bizzini B, Turpin A, Ann Inst Pasteur (Paris). 1971 Jun; 120(6):801-15. [Heterogeneity of tetanus anatoxin]. [Article in French]., PMID:4997518
Relyveld E. H. (1984) Recent developments with vaccines against bacterial protein toxins. In: Bacterial Protein Toxins (Eds Alouf JE, et al). Academic Press, London. 217-226.
Relyveld E. H., Bizzini B., Gupta R. K. (1998) Rational approaches to reduce adverse reactions in man to vaccines containing tetanus and diphtheria toxoid. Vaccine, 16 (9/10), 1016 – 1023.
Relyveld E. H., Henocq E. and Bizzini B. (1979) Studies on untoward reactions to diphtheria and tetanus toxoids. Dev. Biol. Stand. 43, 33-37.
Relyveld E. H., Henocq E., Raynaud M. (1962) Etude de la reaction de Schick a l'aide d'une toxine pure. Bull. Acad. Nat. Med., 146, 101
Runova VF, Kargina TM, Selezneva, [The immunogenic and antigenic properties of the individual fractions of tetanus anatoxin]. [Article in Russian] VP Zh Mikrobiol Epidemiol Immunobiol. (1994) Nov-Dec;(6):75-6. PMID:7879554
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Samore M.H.; Siber GR.; Pertussis toxin enhanced IgG1 and IgE responses to primary tetanus immunization are mediated by interleukin-4 and persist during secondary responses to tetanus alone; Vaccine. (1996) Mar; 14(4): 290- 7.
Schwendeman S. P., Costantino H. R., Gupta R. K., Siber G. R., Klibanov A. M., Langer R. (1995) Stabilization of tetanus and diphtheria toxoids against moisture induced aggregation. Proc. Natl. Acad. Sci. USA, 92(24), 11234-11238.
Stojiæeviæ I, Dimitrijeviæ L, Dovezenski N, Živkoviæ I, Petrušiæ V, Marinkoviæ E, Iniæ-Kanada A, Stojanoviæ M. (2011) Tetanus toxoid purification: Chromatographic procedures as an alternative to ammonium-sulphate precipitation, Journal of Chromatography B., 879 (2011) 2213–2219.
Vancetto M. D., Oliveira J. M., Prado S. M., Fratelli F., Higashi H. G. (1997) Tetanus toxoid purification: a case study. Nature Biotechnology, 15, 807 –808.
WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1.
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Patents:
Douglas J. Cecchini, Patent no. WO 1996025425 A1 (Aug 22, 1996), Purified tetanus toxoid and toxin, Massachusetts Health Research Institute.
Krishna Murthy Ella, Srinivas Kannappa Vellimedu, Patent no. WO2005063794A1 (Jul 14, 2005), A process for the preparation and purification of recombinant proteins, Bharat Biotech International Limited.
Moncef Mohamed Slaoui, Pierre Hauser, Patent no. CA 2221511 C (8 Jan 2013), Vaccine comprising a polysaccharide antigen-carrier protein conjugate and free carrier protein,,Smithkline Beecham Biologicals S.A
Website:
http://www.who.int/biologicals/WHO_TRS_980_WEB.pdf
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WE CLAIM:
1. A large scale method for purification of immunogenic and monomeric tetanus toxoid using gel-filtration chromatography, said method comprising the steps of:
a. Obtaining an immunogenic multimeric tetanus toxoid (monomer being 60%) containing material prepared from fermentation of Clostridium tetani; and further manufactured by chemical detoxification using formalin followed by ultrafiltration, fractional ammonium sulphate precipitation and diafiltration;
b. Further carrying out gel-filtration chromatographic purification by loading toxoid on pre-equilibrated large scale gel-filtration multi-column setup packed with acrylamide based gel-filtration media;
wherein a substantially purified preparation of tetanus toxoid is obtained which is highly monomeric and having monomer content between 80-90%.
2. The method as claimed in claim 1, wherein the said method comprises multi-column (each column of size 50 to 450 mm (width) x 500 mm to 1000 mm height) large scale setup packed with gel-filtration media having fractionation range between 1 × 104 – 1.5 × 10⁶ being used for the purification of (multimeric) tetanus toxoid to manufacture highly pure monomeric tetanus toxoid.
3. The method as claimed in claim 2, wherein said method comprises total bed height for gel-filtration media being used more than 50 cm i.e. between 50-90 cm.
4. The method as claimed in claim 3, comprising the flow rate for elution of toxoid from gel-filtration column being between 10-25 cm/hr.
5. The method as claimed in claims 1 to 4, wherein said gel-filtration media being cross-linked insoluble polymer like acrylamide.
6. The method as claimed in claims 1 to 5, wherein said method comprises equilibration and elution buffer as 0.14 M (0.85%) NaCl solution.
7. The method as claimed in claim 1, wherein the large scale manufacturing method is capable of processing 1 to 10 L of toxoid ( i.e. about 0.5 million doses to 5 million doses, 15 to 150 gms) within one day while 20 L toxoid (about 10 million doses, 300 gms) requires two days for processing.
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8. The method as claimed in claim 1, wherein the monomeric purity of the tetanus toxoid improved by > 40% than conventional one.
9. A substantially purified preparation of tetanus toxoid made by the method as claimed in claim 1.
10. A substantially purified preparation of tetanus toxoid as claimed in claim 9, wherein it is highly purified monomeric tetanus toxoid being at least 80% monomeric.
11. A substantially purified preparation of tetanus toxoid as claimed in claim 9, wherein the monomeric tetanus toxoid preparation is free from higher order aggregates.
12. A substantially purified preparation of tetanus toxoid as claimed in claim 9, wherein the tetanus toxoid preparation is less than 6% dimeric.