By a News Reporter-Staff News Editor at Drug Week — According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Arunakumari, Alahari (Pennington, NJ); Ferreira, Gisela Maria Marques (Gaithersburg, MA), filed on February 28, 2014, was made available online on July 3, 2014 (see also Medarex, L.L.C.).
The assignee for this patent application is Medarex, L.L.C.
Reporters obtained the following quote from the background information supplied by the inventors: “The large-scale, economic purification of proteins is an increasingly important problem for the biopharmaceutical industry. Therapeutic proteins are typically produced using prokaryotic or eukaryotic cell lines that are engineered to express the protein of interest from a recombinant plasmid containing the gene encoding the protein. Separation of the desired protein from the mixture of components fed to the cells and cellular by-products to an adequate purity, e.g., sufficient for use as a human therapeutic, poses a formidable challenge to biologics manufacturers for several reasons.
“Manufacturers of protein-based pharmaceutical products must comply with strict regulatory standards, including extremely stringent purity requirements. To ensure safety, regulatory agencies, such as Food and Drug Administration (FDA), require that protein-based pharmaceutical products are substantially free from impurities, including both product related contaminants such as aggregates, fragments and variants of the recombinant protein and process related contaminants such as host cell proteins, media components, viruses, DNA and endotoxins. While various protein purification schemes are available and widely used in the biopharmaceutical industry, they typically include an affinity-purification step, such as Protein A purification in the case of antibodies, in order to reach a pharmaceutically acceptable degree of purity.
“Despite the advent of advanced chromatography and filtration methods, affinity chromatography is still often employed as a capture step to meet the purity, yield, and throughput requirements for biopharmaceutical antibody purification in order to achieve therapeutic grade purity. Despite a high binding affinity of Protein A chromatography for antibodies (about 10.sup.-8 M for human IgG), and ability to remove as much as 99.5% of impurities, affinity chromatography is an expensive purification step for use in purifying therapeutic proteins on a commercial scale. Not only is Protein A significantly more expensive than non-affinity media, it also has problems such as resin instability, difficulty with cleaning, ligand leakage, and potential immunogenicity of Protein A or Protein A related compounds contaminating the purified product. The high cost and instability of affinity media, however, increases the ultimate cost of protein-based therapeutics, particularly those requiring high doses and/or chronic administration. Chromatography alone can account for two thirds of downstream processing costs and, with respect to monoclonal antibodies, the resin cost for an affinity-capture column can overwhelm raw materials cost. (see Rathore et al, Costing Issues in the Production of Biopharmaceuticals, BioPharm International, Feb. 1, 2004).
“Even if Protein A affinity chromatography is used, adequate purity is often not achieved unless several purification steps are combined, thereby further increasing cost and reducing product yield. Since antibodies account for an increasingly large percentage of therapeutic biologics on the market and in development for the treatment of cancer, autoimmune disease, infectious disease, cardiovascular disease, and transplant rejection, there is a need for a process that can purify proteins using fewer steps and thus realizing lower cost.
“US Pat. Pub. No. 2003/0229212, which is hereby incorporated by reference in its entirety, describes a method for purifying antibodies from a mixture containing host cell proteins using non-affinity chromatography purification steps followed by a high-performance tangential-flow filtration (HPTFF) step. As determined by the reduction of CHOPs (Chinese Hamster Ovary cell Proteins), that purification process resulted in contaminant levels of about 144,780 ppm CHOPs after cation exchange purification, about 410 ppm CHOPs after anion exchange purification, and about 17-21 ppm CHOPs after HPTFF purification (final step), thereby providing a three-step non-affinity process. The purification step of HPTFF, which uses a charged membrane to separate impurities (without limit to relative size), such as proteins, DNA and endotoxins, and to eliminate protein oligomers and degradation products from the mixture containing the antibodies, was essential to achieve the final purity.
“Moreover, HPTFF has disadvantages, namely (1) it is an extra step in the development, optimization and scale-up of the protein therapeutic, which requires membrane cleaning, validation, commercial availability of large-scale GMP cassettes (which is not yet available for HPTFF), and additional buffers, equipment and greater process time, and (2) it increases costs while risking potential loss of product by compromising antibody integrity (by degradation or aggregation or other molecular change that affects molecular activity).
“Therefore, it would be desirable to obtain high purity of a protein therapeutic from a two-step, non-affinity process, which is not based on HPTFF, at a reduced cost in comparison with affinity-based purification and other multi-step purification processes. It would be advantageous if the non-affinity purification process could remove host cell proteins, nucleic acids, endotoxins, product-related contaminants, e.g., aggregated, oxidized, deamidated or degraded forms of the protein, and media additives, e.g., lipids, vitamins, insulin, methotrexate, amino acids, carbon sources such as glucose, etc.
“The development of a purification scheme applicable to various types of proteins, scaleable, controllable, and that employs cheaper, reusable resins will allow its integration into product development at a very early stage in overall drug development. This approach to the design of a purification scheme can minimize costly changes to manufacturing processes which may otherwise be necessary later in drug development or, worse, after approval. As the process is scaled-up and approaches GMP production conditions, additional inherent complexities arise, including those associated with resin packing and buffer preparation. The manufacturing process, and its capacity, can be improved by simplifying the purification scheme by eliminating process steps and maximizing throughput and productivity, while maintaining the integrity and purity of the molecule that is being purified. Therefore, it would be desirable and advantageous to start with a simple and efficient process that can produce a drug substance of high quality and safety.”
In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors’ summary information for this patent application: “The present invention is based on the unexpected finding that polypeptides and proteins, in particular recombinant proteins such as monoclonal antibodies, can be purified from a contaminated mixture comprising the molecule of interest and at least one contaminant, such as host cell proteins and other materials such as media components, by using a two-step purification process that does not include an affinity chromatography step or an in-process buffer exchange step (e.g., TFF or HPTFF). Rather, only pH manipulations are necessary from the first step to the second step. The two-step process of the present invention, therefore, substantially reduces the cost of purifying proteins that typically rely on affinity chromatography or multi-step methodologies to achieve similarly high levels of protein purity.
“In the present invention, proteins are highly purified to a therapeutic grade using the steps of cation exchange chromatography and hydrophobic charge induction chromatography, without regard to their order and without the use of affinity chromatography, in order to yield a high-purity protein composition that contains negligible amounts of impurities [e.g., host cell proteins, such as CHOPs in amounts less than 100 parts per million (ppm)]. In particular embodiments of the invention, described in the Example infra., HCP CHOP levels were reduced to less than 20 ppm. It is an advantage of a method of the present invention that the chromatography steps are interchangeable, thereby allowing practitioners to tailor the method to their needs. Furthermore, a purification process of the present invention can remove not only host cell protein of interest, but also nucleic acids, endotoxins, product-related contaminants, such as aggregated, oxidized, deamidated or degraded forms of the protein, and media additives, e.g., lipids, vitamins, insulin, methotrexate, amino acids, carbon sources such as glucose, etc.
“The present invention provides methods for the purification of recombinant proteins including, but not limited to antibodies, fusion proteins with Fc like regions, and antibody-like molecules, e.g. immunoadhesins, in order to achieve a highly pure protein composition suitable for use in preparing therapeutic grade compositions. As a result, it is an advantage of a method of the present invention that the highly pure protein composition is suitable for use in preparing therapeutic grade material and can be used directly in human therapy.
“Thus, in one embodiment, a method of the invention is directed to purifying a mixture that contains a target protein and one or more contaminants by (a) subjecting the mixture to an ion exchange purification step and a hydrophobic charge induction purification step, where there is no in-process tangential flow filtration step, and (b) isolating the target protein.
“In a particular embodiment, the method is directed to purifying a mixture that contains a target protein and one or more contaminants such as host cell proteins (e.g., CHOPs) and nucleic acids by subjecting the mixture to cation exchange chromatography and to hydrophobic charge induction chromatography, and isolating the target protein to a purity of 100 parts per million (ppm) or less of host cell protein and 10 pg/mg or less of nucleic acids.
BRIEF DESCRIPTION OF THE DRAWING
“FIG. 1 shows a flow diagram of a two-step purification process of the invention, where Panel A and Panel B refer to the same resins used in different sequential order. Panel A represents a first embodiment of the purification process of the invention, where cation exchange chromatography (CEXC) is the capture chromatography step followed by hydrophobic charge induction chromatography (HCIC) with only a pH manipulation in between the two chromatography steps. Panel B represents a second embodiment, where HCIC is the capture chromatography step followed by CEXC with only a pH manipulation in between the two chromatography steps. The approximate binding pH during capture was 6.2 for Panel A and 7.0 for Panel B.”
For more information, see this patent application: Arunakumari, Alahari; Ferreira, Gisela Maria Marques. Protein Purification Using Hcic and Ion Exchange Chromatography. Filed February 28, 2014 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2134&p=43&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626
Keywords for this news article include: Antibodies, Endotoxins, Immunology, Proinsulin, Amino Acids, Legal Issues, Blood Proteins, Medarex L.L.C., Immunoglobulins, Bacterial Toxins, Peptide Hormones.
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