WO2017009863A1 - A scalable, low variation and efficient method for purification of diphtheria toxoid and uses thereof - Google Patents
A scalable, low variation and efficient method for purification of diphtheria toxoid and uses thereof Download PDFInfo
- Publication number
- WO2017009863A1 WO2017009863A1 PCT/IN2016/050123 IN2016050123W WO2017009863A1 WO 2017009863 A1 WO2017009863 A1 WO 2017009863A1 IN 2016050123 W IN2016050123 W IN 2016050123W WO 2017009863 A1 WO2017009863 A1 WO 2017009863A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- diphtheria toxoid
- immunogenic
- purification
- scalable
- range
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
Definitions
- the present disclosure provides a method for purification of immunogenic diphtheria toxoid
- Diphtheria toxin is an exotoxin produced by the bacterium Corynebacterium diphtenae. It is produced as a single polypeptide that is readily nicked to form two subunits linked by a disulphide bond, fragment A ( -terminal, 21kDa), and fragment B (C-terminal, 37kDa), as a result of cleavage at residue 190, 192 or 193 (Moskaug et al, Biol. Chem., 1989, 264, 15709-15713; Collier et al, Biol. Chem., 1971, 246, 1496-1503).
- diphtheria was a leading cause of death among children until mass immunization with diphtheria vaccine, introduced in the late 1902s. Currently, about 4680 cases, and up to 2500 deaths per year are recorded world-wide. Present treatment for diphtheria includes therapeutic administration of antibodies as antitoxins that are specifically directed against the disease itself and prophylactic immunization with formaldehyde-inactivated diphtheria toxoid.
- diphtheria toxoid for vaccine preparation involves pilot studies for salting out experiments with ammonium sulphate and the purification process involves 4-5 days with variable yields and antigenic purity. Purification of diphtheria toxoid can also be achieved by chromatography to avoid contaminants; however it is limited by scalability and commercial viability issues.
- a scalable two-step method for purification of immunogenic diphtheria toxoid comprising the steps of: a) obtaining an immunogenic diphtheria toxoid containing material prepared from fermentation of Corynebacterium diphtheriae in deferrated papain digest medium; b) carrying out a first step of separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 100-500kDa to obtain a filtrate; c) carrying out a second step of separation of filtrate from step b) using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1800-2200Lf/mg p ' N, and wherein the percentage recovery of said purified
- a scalable one-step method for purification of immunogenic diphtheria toxoid comprising the steps of: a) obtaining an immunogenic diphtheria toxoid containing material prepared from semisynthetic medium; b) carrying out a separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1700-2600Lf/mg pN, and wherein the percentage recovery of said purified diphtheria toxoid is in the range of 85-90%.
- a scalable two-step method for purification of immunogenic diphtheria toxoid comprising the steps of: a) obtaining an immunogenic diphtheria toxoid containing material prepared from fermentation of Corynebacterium diphtheriae in deferrated papain digest medium; b) carrying out a first step of separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 100-500kDa to obtain a filtrate; c) carrying out a second step of separation of filtrate from step b) using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1800-2200Lf/mg p ' N, and wherein the percentage recovery of said purified
- a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein wherein membrane pore size in tangential flow filtration setup of first step of separation is 1 OOkDa.
- membrane pore size in tangential flow filtration setup of first step of separation is 300kDa.
- a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein said method further comprising of filter sterilizing said purified diphtheria toxoid with a 0.2 ⁇ filter.
- a scalable one-step method for purification of immunogenic diphtheria toxoid comprising the steps of: a) obtaining an immunogenic diphtheria toxoid containing material prepared from semi-synthetic medium; b) carrying out a separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1700-2600Lf/mg pN, and wherein the percentage recovery of said purified diphtheria toxoid is in the range of 85-90%.
- a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein said method further comprising of filter sterilizing said purified diphtheria toxoid with a 0.2 ⁇ filter.
- Diphtheria toxin was produced in a fermenter using deferrated papain digest medium by growing the organisms under controlled conditions with respect to temperature, pH, and aeration which can be automatically monitored and recorded. After determining the purity of the culture and quality of the toxin, the bacterial cells were separated using filters. The diphtheria toxoid is prepared by detoxification of toxin by the addition of formalin.
- the suitable membrane material for ultrafiltration cassettes can be of polyethersulphone or cellulose acetate or regenerated cellulose or cellulose triacetate or polyvinyl idene fluoride, preferably polythersulfone.
- the first step involves the separation of the diphtheria toxoid from higher molecular weight contaminants such as aggregates, with the diphtheria toxoid passing through the filtrate.
- the second step involves the concentration process to remove low molecular weight contaminants such as ammo acids and peptides from diphtheria toxoid. This two step process is extremely simple to operate and gives purified diphtheria toxoid with antigenic purity greater than 1500Lf/mg pN with a recovery of greater than 80%.
- the diphtheria toxoid for the first filtration step comes from fermentation of Coryne bacterium diphtheria in deferrated papain digest medium.
- the high molecular weight contaminants are retained by the first filter and the diphtheria toxoid passes through the filter and is obtained in permeate.
- the goal of the first filtration step is to retain more than 85% of high molecular weight contaminants, ideally >90% to >95% and a pore size can be selected accordingly.
- the first filtration step may be filtration through 500kDa, 300kDa or lOOkDa pore size membrane.
- the first filtration step is advantageously performed with a tangential flow (cross- flow) filtration (TFF) setup.
- TFF tangential flow filtration
- This arrangement helps to avoid clogging, which is typical for dead-ended filtration and minimizes the need for extensive pre-filtration steps.
- Continuous mode of diafiltration of the retained sample was done by maintaining the constant volume through the tangential flow filter. This permits efficient removal of filtrate components from the retentate and involves addition of fresh solvent (saline) during the first filtration step.
- the second ultrafiltration step uses a small molecular weight cut-off membrane filter than the first step.
- the permeate collected in the first ultrafiltration step is concentrated and retained in the second ultrafiltration step.
- the pore size for the second filtration is selected according to the size and characteristics of the diphtheria toxoid which are to be retained (the intact toxoid has a molecular weight of 58kDa, while the nicked toxoid contains two fragments, fragment A (21kDa) and fragment B (37kDa)).
- Low molecular weight contaminants may pass through the filter, but the goal of the second filtration step is to retain more than 85% of the diphtheria toxoid, ideally >90% to >95%, while removing low molecular weight proteins.
- a pore size can be selected accordingly, based on the diphtheria toxoid to be retained and the low molecular weight proteins, which are to be removed.
- the second ultrafiltration step may be filtration through ! OkDa or 30kDa pore size membrane.
- the second ultrafiltration step is advantageously performed with a TFF setup. As mentioned previously, it helps to avoid clogging and ideally performed with continuous diafiltration mode.
- the retentate obtained from the second filtration step contains the purified diphtheria toxoid.
- Table 1 Recovery and antigenic purity of three different lots of diphtheria toxoid produced from deferrated papain digest medium by two step ultrafiltration process.
- the retained diphtheria toxoid can be used as an immunogenic component in a vaccine preparation after sterile filtration with 0.2 ⁇ filter.
- Diphtheria toxin is produced in fermenter using semi-synthetic medium (medium prepared with enzymatic digest of casein) by growing the organisms under controlled conditions with respect to temperature, pH, aeration which can be automatically monitored and recorded. After determining the purity of the culture and quality of the toxin, the bacterial cells were separated using filters. The diphtheria toxoid is prepared by detoxification of toxin by the addition of formalin.
- the single ultrafiltration step uses a molecular weight cut-off membrane filter, which can retain diphtheria toxoid.
- the pore size for the filtration is selected according to the size and characteristics of the diphtheria toxoid which are to be retained (the intact toxoid molecular weight is 58kDa, while the nicked toxoid contains two fragments, fragment A (21kDa, and fragment B (37kDa)).
- Some low molecular weight contaminants may pass through the filter, but the goal of the filtration step is to retain more than 85% of the diphtheria toxoid, ideally >90% to >95%, while removing unwanted low molecular weight proteins.
- a pore size can be selected accordingly, based on the diphtheria toxoid to be retained and the low molecular weight proteins which are to be removed.
- the ultrafiltration step may be filtration through lOkDa or 30kDa pore size membrane.
- This ultrafiltration step is advantageously performed with a TFF (cross-flow) setup.
- TFF cross-flow
- this arrangement helps to avoid clogging and ideally performed with continuous diafiltration mode and buffer exchanged with saline.
- the retentate obtained contains diphtheria toxoid with antigenic purity and recoveries greater than 85%.
- the retained diphtheria toxoid can be used as an immunogenic component in a vaccine preparation after sterile filtration with 0.2 ⁇ filter.
- Table 2 Recovery and antigenic purity of three different lots of purified diphtheria toxoid from semi-synthetic media by ultrafiltration (one-step process).
Abstract
The present disclosure provides a scalable step-wise process for purification of diphtheria toxoid that is faster, and provides reduced process variability in toxoid retention.
Description
[001] The present disclosure provides a method for purification of immunogenic diphtheria toxoid,
BACKGROUND OF THE INVENTION
[002] Diphtheria toxin (DT) is an exotoxin produced by the bacterium Corynebacterium diphtenae. It is produced as a single polypeptide that is readily nicked to form two subunits linked by a disulphide bond, fragment A ( -terminal, 21kDa), and fragment B (C-terminal, 37kDa), as a result of cleavage at residue 190, 192 or 193 (Moskaug et al, Biol. Chem., 1989, 264, 15709-15713; Collier et al, Biol. Chem., 1971, 246, 1496-1503).
[003] Diphtheria was a leading cause of death among children until mass immunization with diphtheria vaccine, introduced in the late 1902s. Currently, about 4680 cases, and up to 2500 deaths per year are recorded world-wide. Present treatment for diphtheria includes therapeutic administration of antibodies as antitoxins that are specifically directed against the disease itself and prophylactic immunization with formaldehyde-inactivated diphtheria toxoid.
[004] Commercial purification of diphtheria toxoid for vaccine preparation involves pilot studies for salting out experiments with ammonium sulphate and the purification process involves 4-5 days with variable yields and antigenic purity. Purification of diphtheria toxoid can also be achieved by chromatography to avoid contaminants; however it is limited by scalability and commercial viability issues.
[0Θ5] There is a need in the art to develop economically feasible and industrially scalable process for purification of diphtheria toxoid for vaccine preparation that overcomes the limitations of the current practice in the art.
SUMMARY OF THE INVENTION
[006] In an aspect of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid, said method comprising the steps of: a) obtaining an immunogenic diphtheria toxoid containing material prepared from fermentation of Corynebacterium diphtheriae in deferrated papain digest medium; b) carrying out a first step of separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 100-500kDa to obtain a filtrate; c) carrying out a second step of separation of filtrate from step b) using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1800-2200Lf/mg p'N, and wherein the percentage recovery of said purified diphtheria toxoid is in the range of 85-95%.
[007] In an aspect of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid, said method comprising the steps of: a) obtaining an immunogenic diphtheria toxoid containing material prepared from semisynthetic medium; b) carrying out a separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1700-2600Lf/mg pN, and wherein the percentage recovery of said purified diphtheria toxoid is in the range of 85-90%.
[008] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
Δ
DETAILED DESCRIPTION OF THE INVENTION
[009] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0010] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
[0011 ] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
[0012] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".
[0013] Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
[0014] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.
[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0016] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid, said method comprising the steps of: a) obtaining an immunogenic diphtheria toxoid containing material prepared from fermentation of Corynebacterium diphtheriae in deferrated papain digest medium; b) carrying out a first step of separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 100-500kDa to obtain a filtrate; c) carrying out a second step of separation of filtrate from step b) using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1800-2200Lf/mg p'N, and wherein the percentage recovery of said purified diphtheria toxoid is in the range of 85-95%.
[0017] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein percentage recovery of diphtheria toxoid in said filtrate is in the range of 85-95%.
[0018] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein percentage recovery of diphtheria toxoid in said filtrate is at least 90%.
[0019] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein percentage recovery of diphtheria toxoid in said filtrate is at least 95%.
[0020] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein membrane pore size in tangential flow filtration setup of first step of separation is 1 OOkDa.
[0021] in an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein membrane pore size in tangential flow filtration setup of first step of separation is 300kDa.
[0022] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein membrane pore size in tangential flow filtration setup of first step of separation is lOOkDa.
[0023] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 2132 Lf/mg pN.
[0024] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 1841 Lf/mg pN.
[0025] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 1919 Lf/mg pN.
[0026] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein recovery of diphtheria toxoid is 90%.
[0027] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein recovery of diphtheria toxoid is 92%.
[0028] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein recovery of diphtheria toxoid is 88%.
[0029] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 2132 Lf/mg pN, and recovery is 90%.
[0030] in an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 1841 Lf/mg N, and recovery is 92%.
[0031] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 1919 Lf/mg pN, and recovery is 88%.
[0032] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein percentage recovery of diphtheria toxoid in said retentate is in the range of 85-95%.
[0033] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherem percentage recovery of diphtheria toxoid in said retentate is at least 90%.
[0034] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein percentage recovery of diphtheria toxoid in said retentate is at least 95%.
[0035] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein membrane pore size in tangential flow filtration setup of second step of separation is lOkDa.
[0036] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein membrane pore size in tangential flow filtration setup of second step of separation is 30kDa.
[0037] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, said method further comprising of filter sterilizing said purified diphtheria toxoid with a 0.2μηι filter.
[0038] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid, said method comprising the
steps of: a) obtaining an immunogenic diphtheria toxoid containing material prepared from semi-synthetic medium; b) carrying out a separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1700-2600Lf/mg pN, and wherein the percentage recovery of said purified diphtheria toxoid is in the range of 85-90%.
[0039] in an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein percentage recovery of diphtheria toxoid is at least 90%.
|Ό040] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein membrane pore size in tangential flow filtration setup is l OkDa,
[0041 ] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein membrane pore size in tangential flow filtration setup is 30kDa.
[0042] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 1785 Lf/mg pN.
[0043] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 2295 Lf/mg pN.
[0044] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 2571 Lf/mg pN.
[0045] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein recovery of diphtheria toxoid is 90%.
[0046] in an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein recovery of diphtheria toxoid is 87%,
[0047] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 1785 Lf/mg pN, and recovery is 90%,
[0048] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 2295 Lf/mg pN, and recovery is 87%.
[0049] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of diphtheria toxoid is 2571 Lf/mg pN, and recovery is 90%.
[0050] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, said method further comprising of filter sterilizing said purified diphtheria toxoid with a 0.2μηι filter.
[0051] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of purified diphtheria toxoid variation is in the range of 2%-8%.
[0052] In an embodiment of the present disclosure, there is provided a scalable two-step method for purification of immunogenic diphtheria toxoid as described herein, wherein recovery of purified diphtheria toxoid variation is less than 3%.
[0053] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein antigenic purity of purified diphtheria toxoid variation is in the range of 3%-19%.
[0054] In an embodiment of the present disclosure, there is provided a scalable one-step method for purification of immunogenic diphtheria toxoid as described herein, wherein recovery of purified diphtheria toxoid variation is less than 3%.
[0055] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible,
EXAMPLES
[0056] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this 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 to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary .
Example 1
Purification of diphtheria toxoid prepared from deferrated papain digest medium by ultrafiltration (two-step process)
[0057] Diphtheria toxin was produced in a fermenter using deferrated papain digest medium by growing the organisms under controlled conditions with respect to temperature, pH, and aeration which can be automatically monitored and recorded. After determining the purity of the culture and quality of the toxin, the bacterial cells were separated using filters. The diphtheria toxoid is prepared by detoxification of toxin by the addition of formalin.
[0058] The suitable membrane material for ultrafiltration cassettes can be of polyethersulphone or cellulose acetate or regenerated cellulose or cellulose triacetate or polyvinyl idene fluoride, preferably polythersulfone. The first step involves the separation of the diphtheria toxoid from higher molecular weight contaminants such as aggregates, with the diphtheria toxoid passing through the filtrate. The second step involves the concentration process to remove low molecular weight contaminants such as ammo acids
and peptides from diphtheria toxoid. This two step process is extremely simple to operate and gives purified diphtheria toxoid with antigenic purity greater than 1500Lf/mg pN with a recovery of greater than 80%.
[0059] The diphtheria toxoid for the first filtration step comes from fermentation of Coryne bacterium diphtheria in deferrated papain digest medium. The high molecular weight contaminants are retained by the first filter and the diphtheria toxoid passes through the filter and is obtained in permeate. The goal of the first filtration step is to retain more than 85% of high molecular weight contaminants, ideally >90% to >95% and a pore size can be selected accordingly. The first filtration step may be filtration through 500kDa, 300kDa or lOOkDa pore size membrane.
[0060] The first filtration step is advantageously performed with a tangential flow (cross- flow) filtration (TFF) setup. This arrangement helps to avoid clogging, which is typical for dead-ended filtration and minimizes the need for extensive pre-filtration steps. Continuous mode of diafiltration of the retained sample was done by maintaining the constant volume through the tangential flow filter. This permits efficient removal of filtrate components from the retentate and involves addition of fresh solvent (saline) during the first filtration step.
[0061] The second ultrafiltration step uses a small molecular weight cut-off membrane filter than the first step. The permeate collected in the first ultrafiltration step is concentrated and retained in the second ultrafiltration step. In general, the pore size for the second filtration is selected according to the size and characteristics of the diphtheria toxoid which are to be retained (the intact toxoid has a molecular weight of 58kDa, while the nicked toxoid contains two fragments, fragment A (21kDa) and fragment B (37kDa)). Low molecular weight contaminants may pass through the filter, but the goal of the second filtration step is to retain more than 85% of the diphtheria toxoid, ideally >90% to >95%, while removing low molecular weight proteins. A pore size can be selected accordingly, based on the diphtheria toxoid to be retained and the low molecular weight proteins, which
are to be removed. The second ultrafiltration step may be filtration through ! OkDa or 30kDa pore size membrane.
[0062] The second ultrafiltration step is advantageously performed with a TFF setup. As mentioned previously, it helps to avoid clogging and ideally performed with continuous diafiltration mode. The retentate obtained from the second filtration step contains the purified diphtheria toxoid.
[0063] Antigenic purity and recoveries are shown in Table 1 below.
[0064] Table 1 : Recovery and antigenic purity of three different lots of diphtheria toxoid produced from deferrated papain digest medium by two step ultrafiltration process.
[0065] The results in the above table revealed that two step ultrafiltration process for purifying diphtheria toxoid provides antigenic purities of >1500Lf/mg protein nitrogen and recoveries greater than 80%.
[0066] The retained diphtheria toxoid can be used as an immunogenic component in a vaccine preparation after sterile filtration with 0.2 ιη filter.
Example 2
Purification of diphtheria toxoid prepared from semi-synthetic medium by ultrafiltration (one-step process)
[0067] Diphtheria toxin is produced in fermenter using semi-synthetic medium (medium prepared with enzymatic digest of casein) by growing the organisms under controlled
conditions with respect to temperature, pH, aeration which can be automatically monitored and recorded. After determining the purity of the culture and quality of the toxin, the bacterial cells were separated using filters. The diphtheria toxoid is prepared by detoxification of toxin by the addition of formalin.
[0068] The single ultrafiltration step uses a molecular weight cut-off membrane filter, which can retain diphtheria toxoid. In general, the pore size for the filtration is selected according to the size and characteristics of the diphtheria toxoid which are to be retained (the intact toxoid molecular weight is 58kDa, while the nicked toxoid contains two fragments, fragment A (21kDa, and fragment B (37kDa)). Some low molecular weight contaminants may pass through the filter, but the goal of the filtration step is to retain more than 85% of the diphtheria toxoid, ideally >90% to >95%, while removing unwanted low molecular weight proteins. A pore size can be selected accordingly, based on the diphtheria toxoid to be retained and the low molecular weight proteins which are to be removed. The ultrafiltration step may be filtration through lOkDa or 30kDa pore size membrane.
[0069] This ultrafiltration step is advantageously performed with a TFF (cross-flow) setup. As mentioned above, this arrangement helps to avoid clogging and ideally performed with continuous diafiltration mode and buffer exchanged with saline. The retentate obtained contains diphtheria toxoid with antigenic purity and recoveries greater than 85%. The retained diphtheria toxoid can be used as an immunogenic component in a vaccine preparation after sterile filtration with 0.2μηι filter.
[0070] Antigenic purity and recoveries are shown in Table 2 below.
[0071] Table 2: Recovery and antigenic purity of three different lots of purified diphtheria toxoid from semi-synthetic media by ultrafiltration (one-step process).
3 2571 90
|Ό072] The results shown in Table 2 revealed that single ultrafiltration process for purifying diphtheria toxoid leads to antigenic purities >1 500Lf/mg pN and recoveries greater than 80%.
[0073] Overall, it can be appreciated from the present disclosure that the purification methodologies as described in detail herein are advantageous over currently practiced methods for a number of reasons, such as: (a) less time (1 day compared to 4-5 days required for conventional methods); (b) lower variability in toxoid yield; and (c) cost effective due to less time and use of fewer process steps.
Claims
1. A scalable two-step method for purification of immunogenic diphtheria toxoid, said method comprising the steps of:
a. obtaining an immunogenic diphtheria toxoid containing material prepared from fermentation of Corynebacterium diphtheriae in deferrated papain digest medium;
b. carrying out a first step of separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 100-500kDa to obtain a filtrate;
c. carrying out a second step of separation of filtrate from step b) using a tangential flow filtration setup having membrane pore size in the range of 10- 30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid, wherein the purity of diphtheria toxoid is in the range of 1800-2200Lf/mg pN, and wherein the percentage recovery of said purified diphtheria toxoid is in the range of 85-95%.
2. The method as claimed in claim 1 , wherein percentage recovery of diphtheria toxoid in said filtrate is in the range of 85-95%.
3. The method as claimed in claim 1 , wherein percentage recovery of diphtheria toxoid in said retentate is in the range of 85-95%.
4. A scalable one-step method for purification of immunogenic diphtheria toxoid, said method comprising the steps of:
a. obtaining an immunogenic diphtheria toxoid containing material prepared from semi-synthetic medium;
b. carrying out a separation of diphtheria toxoid using a tangential flow filtration setup having membrane pore size in the range of 10-30kDa to obtain a retentate comprising of purified immunogenic diphtheria toxoid,
wherein the purity of diphtheria toxoid is in the range of 1700-2600Lf/mg pN, and wherein the percentage recovery of said purified diphtheria toxoid is in the range of 85-90%.
5. The method as claimed in any of the claims 1. -4, wherein said purified immunogenic diphtheria toxoid is optionally further filter sterilized with a 0,2μηι filter.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN3601/CHE/2015 | 2015-07-14 | ||
| IN3601CH2015 | 2015-07-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2017009863A1 true WO2017009863A1 (en) | 2017-01-19 |
Family
ID=57757086
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IN2016/050123 WO2017009863A1 (en) | 2015-07-14 | 2016-05-03 | A scalable, low variation and efficient method for purification of diphtheria toxoid and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2017009863A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012136739A1 (en) * | 2011-04-07 | 2012-10-11 | Neovacs | Method for treating ifnalpha related conditions |
-
2016
- 2016-05-03 WO PCT/IN2016/050123 patent/WO2017009863A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012136739A1 (en) * | 2011-04-07 | 2012-10-11 | Neovacs | Method for treating ifnalpha related conditions |
Non-Patent Citations (2)
| Title |
|---|
| SUNDARAN B. ET AL.: "Tangential Flow Filtration Technology Applicable to Large Scale Recovery of Diphtheria Toxin", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 94, no. 2, 2002, pages 93 - 98 * |
| WORLD HEALTH ORGANIZATION: "Manual for the production and control of vaccines: diphtheria toxoid", 1977, pages 18, Retrieved from the Internet <URL:extranet.who.int/iris/restricted/ handle/10665/70058?locale=en&locale=es. extranet.who.int/iris/restricted/ bitstream/10665/70058/1/BLG_UNDP_77.1_Rev.1.pdf> * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8993294B2 (en) | Cross-flow membrane filtration-based process for protein recovery | |
| EP0157899B1 (en) | Multivalent pneumococcal vaccine and preparation thereof | |
| KR102539167B1 (en) | Alternating tangential flow rapid harvesting | |
| WO2005091801A3 (en) | Methods of protein fractionation using high performance tangential flow filtration | |
| US20160032268A1 (en) | Urease purification from jack beans or other organisms | |
| US10011662B2 (en) | Downstream process for purifying polysaccharides | |
| JPH03180182A (en) | Purification of phage dna | |
| EP0555135B1 (en) | Process for preparing highly purified fibrinogen | |
| WO2017009863A1 (en) | A scalable, low variation and efficient method for purification of diphtheria toxoid and uses thereof | |
| CA2924042C (en) | Method for the clarification of high density crude cell culture harvest | |
| US6103502A (en) | Ultrafiltration process for desalination and concentration of a peptide in a cell-free fermentation medium | |
| RU2762256C2 (en) | Method for purifying agent of interest | |
| SI9500265A1 (en) | Process for purification of the aqueous fermented broth filtrate of streptomyces sp. p 6621 ferm p 2804 by ultrafiltration | |
| JP5124475B2 (en) | Method for removing colored impurities from vancomycin in powder form | |
| CN106146608A (en) | A kind of extraction preparation method of apidaecin | |
| CA3100869A1 (en) | Method for preparing precursor of recombinant human insulin or analogue thereof | |
| EP1699811B1 (en) | A process for the preparation and purification of recombinant proteins | |
| CA2951735C (en) | Preparation methods for a novel generation of biological safe klh products used for cancer treatment, for the development of conjugated therapeutic vaccines and as challenging agents | |
| RU2283135C1 (en) | Method for releasing cleaned meningococcal polysaccharides of a and c serological group | |
| WO2017009864A1 (en) | A scalable, low variation and efficient method for purification of tetanus toxoid and uses thereof | |
| CN106432411A (en) | Method for extracting alanyl-glutamine from enzymatic conversion liquid | |
| IE45013B1 (en) | Improvements in or relating to the preparation of insulin | |
| CN104560893B (en) | It is a kind of to be used to cultivate culture medium of virus and preparation method thereof | |
| JPWO2020129986A1 (en) | How to make botulinum toxin | |
| JP6599870B2 (en) | Method for separating dicarboxylic acids from aqueous mixtures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16824004 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 16824004 Country of ref document: EP Kind code of ref document: A1 |