Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
  • B01D15/361—Ion-exchange
  • B01D15/362—Cation-exchange
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
  • B01D15/361—Ion-exchange
  • B01D15/363—Anion-exchange
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
  • B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/644—Coagulation factor IXa (3.4.21.22)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/10—Process efficiency

Definitions

• the invention relates to the methods to manufacture through fractionation and purification of human albumin, intravenous immunoglobulins, clotting factor VIII and clotting factor IX, from human plasma by sequential chromatography steps to produce highly pure, virus-free and therapeutically administrable proteins.
• the purification is carried out by an all-chromatography process that avoids the use of ethanol precipitation.
• Plasma protein fractionation is approximately an 1 1.8 billion dollar industry (Plasma Product Biotechnology meet 2011 , Dr. Jean-Francois Prost, "Will plasma products inevitably be replaced by a new generation of therapeutics?") supplying products to more than a million patients each year. It is estimated that more than 500 metric tons of human serum albumin and more than 40 tons of intravenous immunoglobulin are produced annually from more than 22 million liters of source and recovered plasma. Plasma contains about 60 g/L of protein, of which about 57 grams (not including processing losses) are used for many therapeutic products. Plasma- derived products, such as IVIG and albumin, are not expected to be manufactured by recombinant means. IVIG demand is now the primary driver of the plasma collection market, with demand growing 6-8% annually.
• the volume of IVIG was forecast to grow from about 82.3 metric tons in 2008 to about 107.9 tons by 2012, corresponding to an annual growth rate of 7% - the rate observed in the past ten years. According to this report, the demand of IVIG beyond 2012, is expected to grow depending upon the results of the Alzheimer's disease trials and the possible approval of IVIG for this new indication.
• the concentration of different proteins in the plasma varies over a very wide range, from less than 1 microgram per ml to 40 grams per litre. To develop separation and purification processes that can ensure production of several of these high and low level proteins from the same starting volume of plasma, is a challenge for the separation specialists.
• US 5,679,776 discloses a simple chromatographic process for purifying Factor VIII from total plasma.
• none of the above referred patents disclose a process that can simultaneously isolate and purify several proteins from the same starting volume from human plasma to therapeutic grade proteins by avoiding the use of ethanol precipitation step anywhere in the process.
• Patent US 4,639,513 discloses a method for producing intravenously injectable immunoglobulin G (IgG) comprising a particulate separation step, an ion exchange separation step and an affinity separation step, ethanol precipitation, Additionally useful high purity by-products such as prothrombin complex, transferrin and albumin recovery were published.
• these methods often employ a separation medium that can be designed to selectively adhere either the protein of interest or the impurities.
• US 5,061 ,789 discloses a method for isolating blood-clotting factor IX by first adsorbing it onto a matrix derivatised with alpha hydroxylamine groups, eluting the factor and adsorbing it onto a column matrix of sulphated carbohydrates to collect a pure form of Factor IX at high yields.
• US 5,138,034 discloses a process for isolation of the Factor IX by a sequence of steps involving ethanol precipitation, followed by treatment with an anion exchanger and affinity chromatography.
• US 5,286,849 discloses the process for purification of Factor IX from an impure protein fraction by the addition of a solvent-detergent solution to inactivate any viral contaminants and purify the Factor IX by chromatography on a sulphated polysaccharide resin.
• the purified factor IX by the above method is claimed to have a specific activity of atleast 85 units/mg.
• US 5,457,181 discloses a method for preparing a high purity Factor IX concentrate from the supernatant fraction of cryoprecipitated human plasma. A pre- purification is done with DEAE Sephadex chromatography. The resulting Factor IX has a specific activity of atleast 0.5IU/mg of protein.
• the invention describes the method to decrease the ratio of Factor IX to PHBP by contacting it with an ion exchange column and eluting the protein with 0.35M to 0.4M salt to separate protease from factor IX.
• EP617049 and US 5,378,365 discloses a process for the purification of factor IX, factor X and factor II from human plasma or fractions by chromatographic methods.
• US 5,245,014 discloses a method for isolating biological compounds such as proteins, especially coagulation factor VIII in high yield and almost free of other proteins, from body chromatographic and alcohol precipitation methods. However, purity of the obtained factor VIII seems to be a challenge.
• US 4,822,872 discloses a method of purifying an anti-hemophilic factor (AHF) comprising of a cryoprecipitation of F-VIII and adsorption of the F-VIII protein on a water insoluble carrier. But, this is a complex process involving the preparation of custom-made resins that is not easy for industrial scale manufacturing.
• AHF anti-hemophilic factor
• Other patents that discuss isolation and/or purification processes include WO2007136327; US20010051708.
• the present invention discloses an approach to overcome the challenge of utilization of a scarce and precious resource like human plasma, by using a sequential chromatography procedure that can help purify the four proteins Albumin, IgG, and Clotting factors (F-VIII and F-IX), with a potential to purify more proteins from the same starting volume of plasma. This will in turn reduce the cost and improve the affordability of these life-saving medicines for the patients.
• the present invention is an improved process for manufacturing plasma proteins using an all-chromatography process.
• the integrated process scheme simultaneously purifies plasma proteins like Albumin, IgG and clotting factors (F-VIII and F-IX), from the same starting volume, thereby reducing the cost, improving affordability and ensuring a more economical process.
• Figure 3A represents the High-performance liquid chromatography (HPLC) analysis of IVIG.
• Figure 3B represents the HPLC analysis of Albumin.
• Integration of the processes is important to maximally utilize the plasma and recover as many therapeutic products from it as possible with limited resources.
• Certain combinations of chromatography steps leading to the purification of the individual proteins, starting from the plasma raw material may be available in literature.
• the present invention discloses an integrated process that involves identification of different products that could be extracted at a particular stage of the process to derive the final product of therapeutic grade purity in minimal steps from the intermediate product. This process involves analysis of the identified intermediates and the suitability of a fraction to be used as starting material to obtain the desired final product.
• the present invention has the advantages of minimal duplication of equipment and maximal usage of the facility while purifying the desired products in parallel from a said starting volume of plasma and not sequentially, where purification of product 2 begins after the purification of product 1.
• the buffer salts are optimised for individual steps for a given product purification scheme.
• the suitability of the same buffers across the four product purification schemes laterally was also studied and an exercise at minimisation was attempted. This overlap of types of buffers also improves efficiency and economy of the process, thereby minimising the number of salts to be procured for the manufacture of the four products.
• the invention is schematically described in Figure 1.
• the purification scheme shows the preferred embodiment of the sequence of chromatography steps to be followed starting from blood plasma which can be fresh frozen, source plasma, recovered plasma or other variants of liquid or thawed plasma.
• the characteristic feature is the absence of ethanol precipitation and the simultaneous purification of several plasma proteins to the desired therapeutic grade quality, using an all- chromatography based process.
• Blood plasma can be fresh frozen, source plasma, recovered plasma or other variants of liquid or thawed plasma.
• the fresh frozen or source plasma is collected by a process called plasmapheresis.
• Plasmapheresis involves separation of blood into cellular and other components by any of the standard procedures or by the use of a specialized automated plasma collection system (like Haemonetics PCS-2 or other similar equipments).
• the automated collection system uses sterile disposable sets in a self-contained automated process that separates the plasma from the cellular blood components that are then returned to the donor.
• the plasma collected by plasmapheresis is stored frozen below -20°C, preferably below -40°C in sterile collection bottles.
• Production is initiated by thawing the frozen plasma in the bottles and pooling the liquid plasma in an appropriate collection vessel.
• the column is packed with any of the matrices like Cellufine, Sepharose, Sephacryl or any other commercial brands and packed to a height of in the range of 30 to 60cm.
• the column is run in a buffer composed of phosphate, citrate or other similar buffer salts that give a pH between 6.0 and 7.5.
• the buffer salt molarity does not exceed 0.2M, preferably less than 150mM.
• the column is loaded with around 50-150 liters of thawed plasma.
• the fraction I collected is further processed using a series of chromatography steps for purifying coagulation Factor VIII using an anion exchange resin packed in a column, the resin is chosen from one of the commercially available anion exchangers like DEAE, Q or related resins.
• - Salts like acetate, citrate or related salts are used for the equilibration buffer in the molarity range of 0.005M to 0.1 M containing salts like NaCI and other salts in the range of 0.01 M to 0.15M at pH in the range of 5.5 to 8.0.
• This column is washed with buffer and eluted using an elution buffer in the concentration range of 0.05M to 0.5M containing sodium chloride more specifically in the range of 0.05M to 0.15M.
• elution buffer in the concentration range of 0.05M to 0.5M containing sodium chloride more specifically in the range of 0.05M to 0.15M.
• sodium chloride is added to a final elution concentration in the range of 0.25M to 2.5M.
• the said cation exchange column comprising SP (sulpho propyl) or CM (caboxymethyl) or related resins is eluted with sodium acetate buffer containing 0.1 M to 1.5M sodium chloride.
• the eluate from this column can be optionally loaded onto a gel filtration column containing separation media in the range for high molecular weight separations
• the column can be run using the final formulation buffer. This helps in getting rid of any minor protein impurities in the FVIII sample and facilitates exchange of protein into the final formulation buffer.
• This is subjected to a second virus removal step by nanofiltration using commercially available molecular size cut-off filters followed by a third viral inactivation step after filling the liquid into the vials, freeze drying and then subjecting it to heat treatment at 80°C for 72 hrs.
• Fraction 3 One of the said 3 fractions (Fraction 3) collected from the initial gel filtration separation of plasma is further fractionated by loading onto an anion exchange column like DEAE or Q Sepharose, more preferably DEAE or any similar weak anion exchangers.
• the euglobulins are removed by precipitation and this sample is used as the starting material for the purification of IgG and Albumin.
• Desalting of the second lot of the sample is achieved by Sephadex-G25 chromatography using any anionic salt buffer like sodium acetate buffer in the required pH range of 6.5 to 8.0; or by other methods like diafiltration on a membrane filtration set-up can also be employed.
• the desalted and concentrated sample is subjected to low pH precipitation for the removal of euglobulins.
• the euglobulin precipitation step is carried out in the conductivity range of 0.5mS to 5mS, more preferably in the range of 0.6mS to 4mS and preferably in the pH range of 4 to 7.
• the plasma sample under these conditions is held at a temperature between 2 to 20 degrees centigrade, for a time of 2 hrs to 16hrs.
• the euglobulin pellet obtained after this step has a weight of about 20-45 gm/L of plasma.
• the column has a height between 5 and 25 cm and the column is equilibrated with a buffer made from salts like acetate, citrate or phosphate at concentration ranges from 5mM tolOOmM in the pH range of 4.0 to 7.0, more preferably in the range of 4.5 to 6.5.
• the elution is carried out with buffers preferably of the same salt in the concentration range of 50mM to 200mM, in the pH range of 4.0 to 7.0.
• Elution peak fraction obtained contains albumin as the major protein, while the flow through fraction contains IgG and thus these two different fractions are processed separately for further purification.
• the flow through fraction containing IgG, from anion exchange column is loaded onto another anion exchange column (Q, DEAE, TMAE or any other anionic resins from commercial vendors) to further purify the IgG from the other plasma proteins that are present in the sample.
• another anion exchange column Q, DEAE, TMAE or any other anionic resins from commercial vendors
• a column of height 5cm to 20cm is packed, more preferably between 6cm and 18cm, with the chosen anionic exchange resin.
• the column is equilibrated with a buffer of acetate, citrate, phosphate or any other suitable anionic salt with a molarity in the range of 5mM to 100mM, more preferably in the range of 10mM to 60mM, and of pH in the range of 5 to 8, more preferably between 5.5 and 7.5.
• the sample is loaded on a cation exchange column and the eluate containing IgG is collected and processed for virus removal.
• This sample is passed through a nanofilter to remove viruses and a series of ultrafiltration/ diafiltration steps are carried out to diafilter and concentrate the sample.
• This sample is formulated, sterile filtered and filled in vials.
• the solvent-detergent solution addition is done at concentrations required for viral inactivation.
• the final IgG sample obtained by this process has the required purity, efficacy and safety profile required for intravenous administration.
• the bulk solution is formulated as a 5% IgG solution containing 10% D-maltose in water in the pH range of 5.1 to 6.0.
• IgG The manufacturing process of IgG described above doesn't compromise on the biological activity of the IgG molecules. They are highly pure, functionally intact with normal IgG sub-class distribution and effector functions. The preparations are also safe with regard to pathogen safety and product and process related impurities.
• prekallikrein activator is 7.4IU/ml against a limit of ⁇ 35IU/ml activated coagulation factors has >400 sec clotting time against a limit of >150 sec
• IgA is 0.5mg against a limit of ⁇ 4mg/L
• IgM is 0.000009 mg/mL against a limit of ⁇ 0.1mg/mL.
• the eluate from the second anion exchange column (like DEAE) containing albumin, is loaded on an ion exchange column having groups like SP, CM or equivalent.
• - Column is equilibrated with a suitable buffer that is a salt of a weak acid- strong base like (Na or K salts of acetate, citrate or phosphate) at a concentration of 10mM to 100mM in the pH range of 4 to 6.
• a suitable buffer that is a salt of a weak acid- strong base like (Na or K salts of acetate, citrate or phosphate) at a concentration of 10mM to 100mM in the pH range of 4 to 6.
• the column is eluted with the same buffer containing NaCI in the range of 25mM to 0.2M, in the same pH range.
• Virus inactivation is carried out by caprylate addition and low pH treatment in a solution containing caprylate, at pH 4.5 for 10-12 hours.
• the albumin sample is concentrated and subjected to a heat treatment step.
• Heat treatment is carried out in the temperature range of 45°C and 65°C, more preferably 50°C - 60°C, for a period of 1 hr to 10hrs, more preferably 2 hrs to 6 hrs and then subjected to filtration.
• the filtrate obtained is diafiltered and concentrated before the addition of formulation excipients.
• the final formulated sample also contains 16mM each of N-acetyl tryptophanate and sodium caprylate. This is pasteurized at 60 degrees C for 10 - 12 hours.
• This formulated bulk solution is filled aseptically in vials, stoppered, sealed and then subjected to a second round of pasteurization at conditions similar to the initial pasteurization.
• the process for the isolation and purification albumin allows albumin to be purified to near homogeneity and predominantly in the monomeric form, with aggregate levels not exceeding 0.5%. This is ten-fold lower than that stated in the pharmacopoeia, where the specification limit for aggregates is expected to be not greater than 5%.
• the albumin preparation is purified to near homogeneity that is apparent from detection of only one principal band of 100% albumin in the electropherogram when investigated using Zone Electrophoresis method.
• the limit specified in the monograph allows not more than 5% of bands other than the principal band. According to this invention, there are no additional protein bands and the principal albumin band makes up 100% of the protein present in the sample.
• the pharmacopoeial limit is set to not exceed 200 ppb for Aluminium and ⁇ 0.05 mmol of Potassium per gm of protein.
• the column is eluted using buffers containing sodium chloride in the concentration range of 0.06M to 0.3M at the same pH.
• the eluate collected is concentrated on a tangential flow filtration set-up to a reduced volume so that viral inactivation can be accomplished.
• the anion exchange column like DEAE or equivalent is equilibrated with anionic buffers in the molarity range of 0.01 M to 0.15M in the pH range of 6.5 to 8.0 and eluted with sodium chloride in the range of 0.06M to 0.3M at the same pH.
• the affinity column is equilibrated with an anionic buffer in the molarity range of 0.01 M to 0.1 M and contained a divalent cation in the pH range of 5 to 9. This column is eluted with the same buffer containing sodium chloride from 0.05M to 1.5M.
• the eiuate from the affinity column is loaded onto a gel filtration chromatography column packed with resins like Sephadex, Superose, Superdex or Sephacryl in the separation range of 10 KD to 600KD, equilibrated with buffers containing sodium salts of citrate or acetate in the molarity range of 5mM to 100mM and a pH in the range of 6.0 to 8.0.
• the peak fraction containing Factor-IX is collected and subjected to concentration step.
• the factor IX protein sample is concentrated using ultra filtration and nanofiltered to remove viruses. Finally, this sample is sterile filtered and filled in vials for lyophilization.
• the contaminating levels of Heparin in the Factor IX preparation is around 0.0095 IU/ IU of Factor IX against the regulatory specified limit of not to exceed 0.5 IU of Heparin per IU of Factor IX. These results are formulated in Table 1 (Test for impurities in Factor IX preparation).
• the present invention is not limited to the products specified in the embodiments, but has the potential to purify several other plasma products by incorporating minor variations in the wash and elution conditions.
• the purification scheme of the present invention saves on the use of several thousands of liters of ethanol, a reagent that requires explosion-proof manufacturing facilities, affects protein quality (more aggregates, denaturants), activity (lower potency) and yield (lower recoveries).
• the quality control tests on the plasma products purified by this scheme show that the products fulfill the QC specifications exceedingly well.

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