US20230167153A1 - An improved process of purification of protein - Google Patents

An improved process of purification of protein Download PDF

Info

Publication number
US20230167153A1
US20230167153A1 US17/922,729 US202117922729A US2023167153A1 US 20230167153 A1 US20230167153 A1 US 20230167153A1 US 202117922729 A US202117922729 A US 202117922729A US 2023167153 A1 US2023167153 A1 US 2023167153A1
Authority
US
United States
Prior art keywords
less
protein
buffer
process according
interest
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US17/922,729
Other languages
English (en)
Inventor
Om NARAYAN
Tarun Kumar Gupta
Mayankkumar Thakkar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Acquiom Agency Services LLC
Original Assignee
Kashiv Biosciences LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kashiv Biosciences LLC filed Critical Kashiv Biosciences LLC
Assigned to KASHIV BIOSCIENCES, LLC reassignment KASHIV BIOSCIENCES, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GUPTA, TARUN KUMAR, NARAYAN, Om, THAKKAR, Mayankkumar
Publication of US20230167153A1 publication Critical patent/US20230167153A1/en
Assigned to ACQUIOM AGENCY SERVICES LLC, AS COLLATERAL AGENT reassignment ACQUIOM AGENCY SERVICES LLC, AS COLLATERAL AGENT SHORT FORM TERM LOAN INTELLECTUAL PROPERTY ASSIGNMENT Assignors: KASHIV BIOSCIENCES, LLC
Assigned to ACQUIOM AGENCY SERVICES, AS COLLATERAL AGENT reassignment ACQUIOM AGENCY SERVICES, AS COLLATERAL AGENT SHORT FORM TERM LOAN INTELLECTUAL PROPERTY SECURITY AGREEMENT Assignors: KASHIV BIOSCIENCES, LLC
Priority to US19/432,924 priority Critical patent/US20260116917A1/en
Pending legal-status Critical Current

Links

Images

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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE

Definitions

  • the present invention relates to hydroxyapatite chromatography to the purification of at least one antibody or fusion protein from a protein mixture containing low molecular weight impurities and basic variants.
  • the present invention provides a scalable purification process of antibody or fusion protein by using chromatography steps. More specifically the present invention provides a purified antibody composition which is pharmaceutically acceptable and substantially free of product and process related impurities.
  • Monoclonal antibodies as a class of therapeutic molecules are finding an increasing demand in the biotechnology industry for the treatment of diseases. Also, these antibodies are heterogeneous in their biochemical and biophysical properties due to multiple posttranslational modification and degradation events occurs during the production. With the advancements in upstream technologies, the capacity for monoclonal antibody (mAb) production has transformed from a few milligrams to grams per liter. These titers lead to enormous pressure on downstream processes (DSPs), which need to be reworked to achieve higher efficiency and better utilization of available resources. If any of these critical parameters are not defined during the facility design stage, collapse of the process can result, further resulting in commercial loss and delaying entry of the product into the market.
  • DSPs downstream processes
  • a key challenge associated to successful commercialization of antibodies and fusion proteins is to develop pure product with acceptable amount of removal of product and process related impurities to comply with regulatory requirement.
  • Product and process related impurities must be remained in the acceptable limit set by regulatory bodies for an approval.
  • the different kind of impurities often contain unwanted components, such as size variants or charge variants.
  • This formation of size variant, charge variant, and other undesired species for instance, Low molecular weight aggregates (LMWs) and Basic variants respectively, which can adversely affect product safety by causing complement activation or anaphylaxis upon administration.
  • LMWs Low molecular weight aggregates
  • Basic variants Basic variants respectively, which can adversely affect product safety by causing complement activation or anaphylaxis upon administration.
  • aggregate and charge variants formation may hinder manufacturing processes by causing decreased product yield, peak broadening, and loss of activity.
  • Hydroxyapatite chromatography is shown to be a method for chromatographically purifying monoclonal antibodies (Mab). Ceramic hydroxyapatite (CHT) chromatography is reported for the separation of HMW by using sodium chloride or calcium chloride.
  • LMWs low molecular weight aggregates
  • BVs Basic Variants
  • the present invention we also focus on other chromatographic techniques to separate other process and product related impurities, for instance, high molecular weight aggregate (HMWs) and acidic variants.
  • HMWs high molecular weight aggregate
  • ion exchange and hydrophobic interaction chromatography may induce the formation of aggregates due to an increased protein concentration or the required changes in buffer concentration and/or pH during elution.
  • antibodies show differences in isoelectric points that are too small to allow for their separation by ion-exchange chromatography. Tarditi, J. Immunol. Methods 599:13-20 (1992).
  • the present invention uses anion exchange which significantly reduce high molecular weight impurity and acidic variants.
  • Size exclusion chromatography is cumbersome and results in the significant dilution of the product, which is a hindrance in large-scale, efficiency-based manufacturing processes. Leakage of ligands from affinity chromatography columns can also occur, which results in undesirable contamination of the eluted product. Steindl, J. Immunol. Methods 235:61-69 (2000).
  • the present invention provides a scalable robust purification process which significantly reduces impurities associated with antibody or fusion proteins.
  • the present invention provides simple, scalable, CHT process to remove at least one impurity selected from LMW, and basic variant, by using phosphate elution gradient without using NaCl, CaCl 2 , or any other additives.
  • the purified protein mixture is free from or comprises minimal acceptable amount of process and product related impurities like host cell proteins, host cell DNA, leached proteins, half antibodies, clipped antibodies, dimers, tetramers, acidic or basic charge variants, aggregates, low molecular weight (LMW) species, and high molecular weights (HMW) species.
  • process and product related impurities like host cell proteins, host cell DNA, leached proteins, half antibodies, clipped antibodies, dimers, tetramers, acidic or basic charge variants, aggregates, low molecular weight (LMW) species, and high molecular weights (HMW) species.
  • LMW low molecular weight
  • HMW high molecular weights
  • the purification process provides the purity of monomer selected from more than 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99%.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and one or more low molecular weight (LMW) variants, the process comprising:
  • purified protein of interest is substantially pure and LMW's are below 0.4%, analysed by SE-HPLC Analysis.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and one or more basic variants (BV), the process comprising:
  • purified protein of interest is substantially pure and basic variants are below 15% analysed by CEX-HPLC Analysis.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and one or more low molecular weight (LMW) variants and one or more basic variants (BV), the process comprising:
  • purified protein of interest is substantially pure and LMW's are below 0.4%, analysed by SE-HPLC Analysis.
  • purified protein of interest is substantially pure and basic variants are below 15% analysed by CEX-HPLC Analysis.
  • the protein mixture eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis.
  • the protein mixture eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variant 2H1L, the process comprising:
  • the purified protein of interest is substantially pure and 2H1L is less than 3% measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises low 2H1L selected from about 3% or less, 2.6% or less, 2.5% or less, 2.3% or less, 2% or less, 1.7% or less, 1.5% or less, 1.4% or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variant HH, the process comprising:
  • the purified protein of interest is substantially pure and HH is less than 0.2% measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises low HH is selected from about 0.2% or less, 0.24% or less, 0.21% or less, 0.15% or less, 0.12% or less, 0.09% or less or 0.08% or less, 0.06% or less or 0.04% or less and 0.03% or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variant HC, the process comprising:
  • the purified protein of interest is substantially pure, and HC is less than 0.4 measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises low HC is selected from about 0.4% or less, 0.16% or less, 0.15% or less, 0.12% or less and 0.18% or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variant LC, the process comprising:
  • the purified protein of interest is substantially pure, and LC is reduced by 0.5% measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises low LC selected from about 0.5% or less, 0.4% or less, 0.3% or less and 0.2 or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variants are LC, HC, HH, and 2H1L, the process comprising:
  • the purified protein of interest is substantially pure and LC, HC, HH, 2H1L were reduced by 3% measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises LMWs selected from LC, HC, HH and 2H1L from 2.2% to 2.6% measured by CE-SDS.
  • the process provides linear elution gradient of CHT column wherein the concentration of phosphate is increased gradually from about 32 mM to 88 mM and thereby separate or reduce LMWs having molecular weight of about 23 kDa to about 125 kDa, and about 33% reduction in basic variant.
  • the process provides linear elution gradient of CHT column wherein the concentration of phosphate is increased gradually from about 40 mM to 96 mM and thereby separate or reduce LMWs having molecular weight of about 23 kDa to about 125 kDa, and about 33% reduction in basic variant.
  • the invention provides a scalable purification process at 50 L, 100 L, 200 L capable to provide substantial pure monomeric form of antibody or fusion protein and low acceptable amount of impurity selected from host cell proteins, host cell DNA, leached proteins, half antibodies, dimers, tetramers, acidic or basic charge variants, aggregates, low molecular weight (LMW) species, and high molecular weights (HMW) species.
  • impurity selected from host cell proteins, host cell DNA, leached proteins, half antibodies, dimers, tetramers, acidic or basic charge variants, aggregates, low molecular weight (LMW) species, and high molecular weights (HMW) species.
  • LMW low molecular weight
  • HMW high molecular weights
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and acidic species or variant thereof, the purification process comprising:
  • the eluted protein mixture obtained in step (d) comprises substantially purified protein of interest and less than 15% of acidic species or variant analysed by CEX-HPLC Analysis.
  • the process provides the protein mixture comprising the acidic variant is less than about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, 1% or less AV.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and high molecular weight (HMW) impurity, the purification process comprising:
  • step (d) wherein the eluted protein mixture obtained in step (d) comprises substantially purified protein of interest and less than 0.5% of HMW impurity analysed by SE-HPLC Analysis.
  • the process provides the protein mixture comprising HMW less than 0.5% or less, about 0.4% or less or 0.3% or less or 0.2% or less or 0.1% or less.
  • the invention provides a process of purifying the protein of interest from protein mixture comprising:
  • the eluted protein mixture is enriched with protein of interest, and substantially free of impurities HMW, LMW's, acidic & basic variants (BV).
  • the protein of interest is IgG1 antibody which binds to IgE.
  • the IgG1 antibody is biosimilar of Omalizumab.
  • FIG. 1 depicts the Process Chromatogram of AEX run.
  • FIG. 2 depicts CEX-HPLC Chromatogram of AEX run (Charge Variants).
  • FIG. 3 depicts SE-HPLC Chromatogram of AEX run (Size Variants).
  • FIG. 4 depicts zoomed view of SE-HPLC Chromatogram of AEX run (Size Variants).
  • FIG. 5 depicts process chromatogram of CHT run.
  • FIG. 6 depicts SE-HPLC Chromatogram of CHT run (Size Variants).
  • FIG. 7 CEX-HPLC Chromatogram of CHT run (Charge Variants).
  • the present invention provides a purification process which is capable to reduce various types of product and process related impurities.
  • the invention provides the reduction of few product related impurity by using CHT chromatography.
  • the invention provides the reduction of few product related impurity by using anion exchange chromatography.
  • the invention provides the reduction of product related impurity by using CHT and anion exchange chromatography.
  • the invention provides the reduction of product related impurity by using Affinity Chromatography, CHT and Anion Exchange Chromatography.
  • antibody includes an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • Omalizumab is a recombinant DNA-derived humanized IgGlK monoclonal antibody that selectively binds to human immunoglobulin (IgE).
  • the antibody has a molecular weight of approximately 149 kD.
  • Xolair® is produced by a Chinese hamster ovary cell suspension culture in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detectable in the final product.
  • Xolair® is a sterile, white, preservative-free, lyophilized powder contained in a single-use vial that is reconstituted with Sterile Water for Injection (SWFI), USP, and administered as a subcutaneous (SC) injection.
  • SWFI Sterile Water for Injection
  • SC subcutaneous
  • acidic variant or “acidic species” and “AV” used herein refer to the variants of a protein, e.g., an antibody or antigen-binding portion thereof, which are characterized by an overall acidic charge.
  • mAb monoclonal antibody
  • acidic species can be detected by various methods, such as ion exchange, for example, WCX-10 HPLC (a weak cation exchange chromatography), or IEF (isoelectric focusing).
  • Acidic variants of antibodies are formed through Chemical and enzymatic modifications such as deamidation and sialylation, respectively, result in an increase in the net negative charge on the antibodies and cause a decrease in pI values, thereby leading to formation of acidic variants.
  • C-terminal lysine cleavage results in the loss of net positive charge and leads to acidic variant formation.
  • Another mechanism for generating acidic variants is the formation of various types of covalent adducts, e.g., glycation, where glucose or lactose can react with the primary amine of a lysine residue during manufacturing in glucose-rich culture media or during storage if a reducing sugar is present in the formulation.
  • process-related impurity refers to impurities that are present in a composition comprising a protein but are not derived from the protein itself.
  • Process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatographic materials, and media components.
  • anion exchange chromatography or “anion exchange column” or “AEX” used herein is a form of ion exchange chromatography (IEX), which is used to separate molecules based on their net surface charge.
  • Anion exchange chromatography more specifically, uses a positively charged ion exchange resin with an affinity for molecules having net negative surface charges.
  • Anion exchange chromatography is used both for preparative and analytical purposes and can separate a large range of molecules, from amino acids and nucleotides to large proteins.
  • POROS 50 HQ used herein is a Thermo ScientificTM POROSTM Strong Anion Exchange Resins (POROS AEX resins) are designed for charge-based chromatographic separation of biomolecules including recombinant proteins, monoclonal antibodies.
  • Thermo ScientificTM POROSTM 50 HQ resin is functionalized with quaternized polyethyleneimine groups.
  • strong anion exchange When “strong anion exchange” is used in flow through process the equation changes, the impurities are differentiated from the protein of interest, i.e., strong anion exchange is generally known for removal of protein A contaminant, HCP, DNA or virus in antibody purification.
  • the sample and equilibration buffer are adjusted to conditions where contaminant molecules will still bind to the resin, but the protein of interest will not (because of the charge). This is achieved by increasing the salt concentration and/or increasing the pH of the buffers to a point below the pI of your molecule of interest.
  • flow-through mode or “flow-through” refers to purification process wherein antibody of interest does not bind to chromatography resin.
  • the at least 50% antibody of interest does not bind to chromatographic resin.
  • the at least 60% or 70% or 80% antibody of interest does not bind to chromatographic resin.
  • process and product related impurities bind the chromatographic resin.
  • the at least 50% process and product related impurities bind to chromatographic resin.
  • at least 60% or 70% or 80% process and product related impurities bind to chromatographic resin.
  • CHT or “ceramic hydroxyapatite chromatography” is a form of calcium phosphate used in the chromatographic separation of biomolecules.
  • Sets of five calcium doublets (C-sites) and pairs of —OH containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Repeating hexagonal structures can be seen in electron micrographs of the material. Space-filling models and repeat structure from Raman spectroscopy have also been constructed. Hydroxyapatite has unique separation properties and unparalleled selectivity and resolution. It often separates proteins shown to be homogeneous by electrophoretic and other chromatographic techniques.
  • CHT ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. It has been sintered at high temperatures to modify it from a crystalline to a ceramic form.
  • substantially pure antibody used herein includes an antibody that is substantially free of impurity selected from product or process related impurity.
  • antibody is free of acidic variant, basic variant, low molecular weight and high molecular weight
  • substantially pure antibody has purity less than about 99% or less than about 98% or less than about 97% or less than about 95% or less than about 92% or less than about 90% or less than about 88% or less than about 85% or less than about 82% less than about 80% or less than about 75% or less than about 70% or less than about 65% or less than about 60% or less than 50%.
  • Size variants refers to LMW, HMW, or aggregates.
  • LMW low molecular weight
  • LMW species which is a protein backbone-truncated fragments & considered as product-related impurities that contribute to the size heterogeneity of antibody.
  • LMW species often have low or substantially reduced activity relative to the monomeric form of the antibody and can lead to immunogenicity or potentially impact pharmacokinetic properties in vivo.
  • LMW species are considered critical quality attributes that are routinely monitored during drug development and as part of release testing of purified drug product during manufacturing.
  • the LMW has molecular weight selected from 23 kDa, 24 kDa, 25 kDa, 26 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 105 kDa, 110 kDa, 115 kDa, 120 kDa, 125 kDa, 126 kDa, and 127 kDa.
  • the LMW is selected from LC, HC, HH, and 2HL1.
  • Light Chain or “LC” of an antibody having a weight of 25 kDa is light or L chain.
  • a typical antibody contains two light chain, lambda ( ⁇ ) and kappa ( ⁇ ).
  • An antibody either has ⁇ chains or ⁇ chains, never one of each.
  • There is no functional difference has been found between antibodies having ⁇ or ⁇ light chains, and either type of light chain can be found in antibodies of any of the five major classes.
  • the ratio of the two types of light chain varies from species to species. However, sometime protein mixture comprises only one LC which is considered LMW impurity.
  • Heavy Chain or “HC” of an antibody having a weight of 50 kDa each is a heavy or H chain.
  • the heavy chains contain a series of immunoglobulin domains with one variable domain (VH) for binding an antigen and constant domains (CH1, CH2, etc.).
  • VH variable domain
  • CH1, CH2, etc. constant domains
  • the heavy chains with ⁇ and ⁇ have approximately 450 amino acids and the heavy chains with ⁇ and ⁇ have approximately 550 amino acids.
  • protein mixture comprises only one HC which is considered LMW impurity.
  • Two Heavy One Light Chain or “2H1L” or “H2L” in antibody species possess a single light chain.
  • the 2H1L species of antibody forms due to the ⁇ -elimination of a heavy chain cysteine residue, forming the inter-heavy and light chain disulfide bond, that leads to the formation of H2L species.
  • HMW high molecular weight
  • product-related impurities that contribute to the size heterogeneity of antibody products.
  • the formation of HMW species within a therapeutic antibody drug product as a result of protein aggregation can potentially compromise both drug efficacy and safety (e.g., eliciting unwanted immunogenic response).
  • HMW is considered critical quality attribute that are routinely monitored during drug development and as part of release testing of purified drug product during manufacturing.
  • the HMW relates to aggregates.
  • aggregates are classified based on types of interactions and solubility. Soluble aggregates are invisible particles and cannot be removed with a filter. Insoluble aggregates can be removed by filtration and are often visible to the human eye. Both types of aggregates cause problems in biopharma development. Covalent aggregates arise from the formation of a covalent bond between multiple monomers of a given peptide. Disulfide bond formation of free thiols is a common mechanism for covalent aggregation. Oxidation of tyrosine residues can lead to formation of bityrosine which often results in aggregation. Reversible protein aggregation typically results from weaker protein interactions they include dimers, trimers, multimers among others.
  • basic variant refers to variants can result from the presence of C-terminal lysine or glycine amidation, succinimide formation, amino acid oxidation or removal of sialic acid, which introduce additional positive charges or removal of negative charges; both types of modifications cause an increase in pI values.
  • high salt buffer refers to high strength or high molality buffer.
  • Buffer A and Buffer B is interchangeable with “first buffer” or “second buffer” respectively in CHT chromatography.
  • viral reduction/inactivation is intended to refer to a decrease in the number of viral particles in a particular sample (“reduction”), as well as a decrease in the activity, for example, but not limited to, the infectivity or ability to replicate, of viral particles in a particular sample (“inactivation”).
  • Such decrease in the number and/or activity of viral particles can be on the order of about 1% to about 99%, preferably of about 20% to about 99%, more preferably of about 30% to about 99%, more preferably of about 40% to about 99%, even more preferably of about 50% to about 99%, even more preferably of about 60% to about 99%, yet more preferably of about 70% to about 99%, yet more preferably of about 80% to 99%, and yet more preferably of about 90% to about 99%.
  • CE-SDS is used herein to analyze LMW is selected from LC, HC, HH, and 2HL1.
  • analytical HPLC comprises CEX HPLC used for the estimation of charge variants and SE-HPLC for the estimation of HMWs and LMWs.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and one or more low molecular weight (LMW) variants the process comprising:
  • purified protein of interest is substantially pure and LMW's are below 0.4%, analysed by SE-HPLC Analysis.
  • the protein mixture eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis.
  • the LMW has molecular weight selected from 23 kDa, 24 kDa, 25 kDa, 26 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 105 kDa, 110 kDa, 115 kDa, 120 kDa, 125 kDa, 126 kDa, and 127 kDa.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and one or more basic variants (BV), the process comprising:
  • purified protein of interest is substantially pure and basic variants are below 15% analysed by CEX-HPLC Analysis.
  • the protein mixture eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and one or more low molecular weight (LMW) variants and one or more basic variants (BV), the process comprising:
  • purified protein of interest is substantially pure and LMW's are below 0.4%, analysed by SE-HPLC Analysis;
  • purified protein of interest is substantially pure and basic variants are below 15% analysed by CEX-HPLC Analysis.
  • the protein mixture eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis.
  • the protein mixture eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variant 2H1L, the process comprising:
  • the purified protein of interest is substantially pure and 2H1L is less than 3% measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises low 2H1L selected from about 3% or less, 2.6% or less, 2.5% or less, 2.3% or less, 2% or less, 1.7% or less, 1.5% or less, 1.4% or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variant HH, the process comprising:
  • the purified protein of interest is substantially pure and HH is less than 0.2% measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises low HH is selected from about 0.2% or less, 0.24% or less, 0.21% or less, 0.15% or less, 0.12% or less, 0.09% or less or 0.08% or less, 0.06% or less or 0.04% or less and 0.03% or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variant HC, the process comprising:
  • the purified protein of interest is substantially pure, and HC is less than 0.4 measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises low HC is selected from about 0.4% or less, 0.16% or less, 0.15% or less, 0.12% or less and 0.18% or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variant LC, the process comprising:
  • the purified protein of interest is substantially pure, and LC is reduced by 0.5% measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises low LC selected from about 0.5% or less, 0.4% or less, 0.3% or less and 0.2 or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and low molecular weight (LMW) variants are LC, HC, HH, and 2H1L, the process comprising:
  • the purified protein of interest is substantially pure and LC, HC, HH, and 2H1L were reduced by 3% measured by CE-SDS.
  • the protein mixture eluted from CHT column comprises LMWs LC, HC, HH and 2H1L from 2.2% to 2.6% measured by CE-SDS.
  • the process provides linear elution gradient of CHT column wherein the concentration of phosphate is increased gradually from about 32 mM to 88 mM and thereby separate or reduce LMWs having molecular weight of about 23 kDa to about 125 kDa, and about 33% reduction in basic variant.
  • the process provides linear elution gradient of CHT column wherein the concentration of phosphate is increased gradually from about 40 mM to 96 mM and thereby separate or reduce LMWs having molecular weight of about 23 kDa to about 125 kDa, and about 33% reduction in basic variant.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and one or more low molecular weight (LMW) variants and one or more basic variants (BV), the process comprising:
  • purified protein of interest is substantially pure and LMW's are below 0.4%, analysed by SE-HPLC Analysis.
  • the purification process reduces LMW at least by about less than about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55% and about 50% or less.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and one or more basic variants (BV), the process comprising:
  • purified protein of interest protein is substantially pure, and basic variants are below 15% analysed by CEX-HPLC Analysis.
  • the purification process reduces basic variant at least by about 5% or about 10% or about 15% or about 20% or about 25% or about 30% or about 31% or about 32% or about 33% or about 34%, about, 35%, about 40% or about 45% and about 50%.
  • the purification process reduces basic variant at least by about 33%.
  • the purification process reduces basic variant at scale of 5 L, or 50 L or 100 L or 200 L at least by about 33%.
  • the invention provides a scalable purification process at 5 L, 50 L, 100 L, and 200 L capable to provide substantial pure monomeric form of antibody or fusion protein and low acceptable amount of impurity selected from host cell proteins, host cell DNA, leached proteins, half antibodies, dimers, tetramers, acidic or basic charge variants, aggregates, low molecular weight (LMW) species, and high molecular weights (HMW) species.
  • LMW low molecular weight
  • HMW high molecular weights
  • the invention provides a robust standardized purification process which is capable to utilize from small (5 L) to high scale 50 L-200 L or higher with same efficiency and provide uniform result, thereby also providing for reliable product quality.
  • a further advantage herein is the ability to develop effective and reliable regulatory compliant (such as in terms of US FDA and/or EMA standards) protein of interest such as antibody or fusion protein.
  • the loading, washing and elution steps of CHT column completes within about 30 min to about 105 min. In certain embodiment, the loading, washing and elution steps of CHT column completes within 104 min.
  • the mammalian cell is a CHO cell.
  • the loading, washing, and elution buffer does not have any additive selected from sodium chloride, calcium chloride, and glycine.
  • the suitable buffer is a Polar Protic Molecule, Sodium Phosphate, Tris, and HEPES etc.
  • the loading, washing and elution buffer have pH ranging from about 6.5 to about 7.2.
  • the loading, washing and elution buffer have pH 7.0 ⁇ 0.2.
  • the loading and washing buffer has conductivity range from about 3 to about 7 mS/cm.
  • the loading and washing buffer have conductivity about 6 mS/cm.
  • the elution phosphate gradient is a linear gradient wherein the concentration of phosphate is increased gradually from about 5% to 25% of buffer B.
  • the protein mixture eluted from CHT column comprises LMW below 0.4%, analysed by SE-HPLC Analysis.
  • the protein mixture eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis.
  • the protein mixture eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV.
  • the protein mixture eluted from CHT column comprises less than 10% BV.
  • the process provides the linear elution gradient wherein the concentration of phosphate is increased gradually from about 24 mM to 100 mM.
  • the present invention provides a process of purifying an antibody binds to IgE from protein mixture comprising said antibody and one or more low molecular weight (LMW) variants and one or more basic variants (BV), the process comprising:
  • the purified antibody is substantially pure and LMW's are below 0.4%, analysed by SE-HPLC Analysis;
  • purified antibody is substantially pure and basic variants are below 15% analysed by CEX-HPLC Analysis.
  • the antibody eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis.
  • the antibody eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV.
  • the protein of interest is IgG1 antibody or fusion proteins.
  • the protein of interest is IgG1 antibody which binds to IgE.
  • the IgG1 antibody is biosimilar of Omalizumab.
  • the purification process provides the purity of monomer selected from more than 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99%.
  • the invention provides a pharmaceutical purified composition of Omalizumab comprising substantially purified monomer of said Omalizumab and LMW less than about 0.2% measure by SE-HPLC.
  • the invention provides a pharmaceutical purified composition of Omalizumab purified by CHT chromatography comprising substantially purified monomer of said Omalizumab and LMW less than about 0.2% measure by SE-HPLC.
  • the invention provides a pharmaceutical purified composition of Omalizumab purified by CHT chromatography comprising substantially purified monomer of said Omalizumab and LMWs selected from LC, HC, HH and 2H1L from 2.2% to 2.6% measured by CE-SDS.
  • the invention provides a pharmaceutical purified composition of Omalizumab comprising substantially purified monomer of said Omalizumab and basic variant less than 5% measure by CEX-HPLC.
  • the invention provides a pharmaceutical purified composition of Omalizumab purified by CHT chromatography comprising substantially purified monomer of said Omalizumab and basic variant less than 5% measure by CEX-HPLC.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and acidic species or variant thereof, the purification process comprising:
  • the eluted protein mixture comprises substantially pure monomer of the protein of interest and less than 15% of acidic species or variant analysed by CEX-HPLC Analysis.
  • the process provides the protein mixture comprising the acidic variant is less than about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, 1% or less AV.
  • the invention provides a pharmaceutical purified composition of Omalizumab comprising substantially purified monomer of said Omalizumab and acidic variant less than 12% measure by CEX-HPLC.
  • the invention provides a pharmaceutical purified composition of Omalizumab purified by AEX chromatography comprising substantially purified monomer of said Omalizumab and acidic variant less than 12% measure by CEX-HPLC.
  • the invention provides a pharmaceutical purified composition of Omalizumab purified by AEX chromatography comprising substantially purified monomer of said Omalizumab and acidic variant less than 10% measure by CEX-HPLC.
  • the present invention provides a process of purifying an protein of interest from the protein mixture comprising protein of interest and high molecular weight (HMW) impurity, the purification process comprising:
  • the eluted protein mixture comprises substantially pure monomer of the protein of interest and less than 0.5% of HMW impurity analysed by SE-HPLC Analysis.
  • the process provides the protein mixture comprising HMW less than 0.5% or less, about 0.4% or less or 0.3% or less or 0.2% or less or 0.1% or less.
  • the invention provides a pharmaceutical purified composition of Omalizumab purified by AEX chromatography comprising substantially purified monomer of said Omalizumab and HMW less than 0.5% measure by SE-HPLC.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and acidic species or variant thereof, the purification process comprising:
  • step (d) wherein the eluted protein mixture obtained in step (d) comprises substantially pure monomer of the protein of interest and less than 15% of acidic species or variant analysed by CEX-HPLC Analysis.
  • the process provides the protein mixture comprising the acidic variant is less than about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, 1% or less AV.
  • the purification process reduces acidic variant at least by 40% preferably by 50% in protein mixture obtained in flow through mode of strong anion exchange.
  • the present invention provides a process of purifying a protein of interest from protein mixture comprising protein of interest and high molecular weight (HMW) impurity, the purification process comprising:
  • step (d) wherein the eluted protein mixture obtained in step (d) comprises substantially pure monomer of the protein of interest and less than 0.5% of HMW impurity analysed by SE-HPLC Analysis.
  • the process provides the protein mixture comprising HMW less than 0.5% or less, about 0.4% or less or 0.3% or less or 0.2% or less or 0.1% or less.
  • the purification process reduces HMW at least by 80% preferably by 90% in protein mixture obtained in flow through mode of strong anion exchange.
  • the present invention surprisingly found the removal of acidic variants and HMW through strong anion exchange column by performing the column in flow-through mode wherein the buffer solution pH is 7.0 to 7.3 marginally below than the pI of the omalizumab.
  • the optimization of the desired pH of buffer leads to the substantial binding at least more than 40% of acidic variant or HMW to strong anion exchange. In certain embodiment the more than 80% HMW binds to strong anion exchange.
  • the present invention provides the purified antibody composition obtained from strong anion exchange wherein the acidic variants are less than 15% preferably less than 12% and HMW less than 0.5% preferably 0.3% which is under the acceptable limitation of regulatory bodies.
  • the present invention is very useful in reducing the burden in downstream processing by avoiding the use of multiple columns.
  • the present invention avoids the use HIC, and multimodal chromatography.
  • the invention provides a process of purifying the protein of interest from protein mixture comprising:
  • the eluted protein mixture is enriched with protein of interest and substantially free of impurities HMW, LMW's & basic variants (BV).
  • the invention provides an anion exchange process to produce an antibody composition enriched antibody of interest and substantially reduced acidic variants and HMW or aggregates.
  • the invention provides an anion exchange process performed in negative mode to produce an antibody composition enriched antibody of interest and substantially reduced acidic variants comprises about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, 1% or less AV.
  • the invention provides an anion exchange process performed in negative mode to produce an antibody composition enriched antibody of interest and substantially reduced HMW comprises about 0.5% or less HMW.
  • the low HMW composition comprises about 0.4% or less HMW, 0.3% or less HMW, 0.2% or less HMW, 0.1% or less HMW.
  • the invention provides a CHT column chromatography process to produce an antibody composition enriched antibody of interest and reduced basic variants and LMW impurities.
  • the invention provides the protein mixture eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis.
  • the invention provides the protein mixture eluted from CHT column comprises LMW below 0.4%, analysed by SE-HPLC Analysis.
  • the invention provides the protein mixture eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV.
  • the invention provides the protein mixture eluted from CHT column comprises less than 10% BV.
  • the invention provides the protein mixture eluted from CHT column comprises less than 5% BV.
  • the invention provides a process of purifying the Omalizumab at 50 L scale or 200 L scale from protein mixture comprising:
  • the eluted protein mixture is enriched with the Omalizumab and substantially free of impurities LMW's & basic variants (BV).
  • the CHT washing removes loosely bound impurities.
  • the invention provides an anion exchange process to produce Omalizumab composition enriched antibody of interest and substantially reduced acidic variants and HMW or aggregates.
  • the invention provides an anion exchange process performed in negative mode to produce an antibody composition enriched the Omalizumab and substantially reduced acidic variants comprises about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, 1% or less AV.
  • the invention provides an anion exchange process performed in negative mode to produce an antibody composition enriched the Omalizumab and substantially reduced HMW comprises about 0.5% or less HMW.
  • the low HMW composition comprises about 0.4% or less HMW, 0.3% or less HMW, 0.2% or less HMW, 0.1% or less HMW analysed by SE-HPLC Analysis.
  • the invention provides a CHT column chromatography process to produce an antibody composition enriched the Omalizumab and reduced basic variants and LMW impurities.
  • the invention provides the protein mixture eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis.
  • the invention provides the protein mixture eluted from CHT column comprises LMW below 0.4%, analysed by SE-HPLC Analysis.
  • the invention provides the protein mixture eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV.
  • the invention provides the protein mixture eluted from CHT column comprises less than 10% BV.
  • the invention provides the protein mixture eluted from CHT column comprises less than 5% BV.
  • the antibody is capable to bind IgE and thereby capable to treat allergic diseases, asthma, nasal polyps and urticaria.
  • the antibody is omalizumab.
  • the purified pharmaceutical composition of biosimilar of Omalizumab comprising substantially purified monomer and impurity acidic variant less than 12%, basic variant less than 5%, LMW less than 0.4% and HMW less than 0.5%.
  • antibodies are selected from IgG1, IgG2, IgG3, IgG4 and fusion proteins.
  • the antibodies are selected from Etanercept, Rituximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Ibritumomab tiuxetan, Omalizumab, Cetuximab, Bevacizumab, Natalizumab, Eculizumab, Certolizumab pegol, Ustekinumab, Canakinumab, Golimumab, Ofatumumab, Tocilizumab, Denosumab, Belimumab, Ipilimumab, Brentuximab vedotin, Pertuzumab, Trastuzumab emtansine, Raxibacumab, Obinutuzumab, Siltuximab, Ramucirumab,
  • the protein of interest is IgG1 antibody which binds to IgE.
  • the IgG1 antibody is biosimilar of Omalizumab.
  • the chromatographic step performed in sequence after affinity chromatography are adjustable or replaceable with each other.
  • CHT chromatography can be performed after affinity chromatography.
  • CHT can be performed between affinity chromatography and AEX chromatography.
  • CHT can be performed after affinity chromatography.
  • CHT can be performed between affinity chromatography and AEX chromatography.
  • affinity chromatography is selected from Protein A or Protein G.
  • affinity chromatography rein is selected from Mabselect, Mabselect SuRe, Mabselect SuRe LX, Prosep Ultra Plus, Eshmuno A.
  • the Affinity chromatography resin is Mabselect Sure LX.
  • the equilibration buffer or loading buffer or wash buffer is selected from Sodium Phosphate, Tris-HCl, Tris-Acetate, HEPES, Glycine-NaOH.
  • the loading buffer is Tris Acetate or Tris-HCl.
  • the equilibration buffer or loading buffer or wash buffer used in combination with a salt used in combination with a salt.
  • the salt is selected from sodium Chloride, Potassium Chloride.
  • the salt is Sodium Chloride.
  • the equilibration buffer has concentration range from about 5 mM to about 40 mM. In certain embodiment, the equilibration buffer has concentration range from about 10 mM to about 25 mM. In preferred embodiment, the equilibration buffer concentration is about 20 mM.
  • the equilibration buffer or loading buffer or wash buffer optionally comprises a salt selected from about 50 mM to about 400 mM.
  • the equilibration buffer comprises a salt buffer concentration selected from about 100 mM to about 200 mM.
  • the equilibration buffer concentration is about 150 mM.
  • the equilibration buffer concentration is about 100 mM.
  • the equilibration buffer or loading buffer or wash buffer has conductivity range from about 10 mS/cm to about 20 mS/cm. In an embodiment, the equilibration buffer or loading buffer or wash buffer conductivity is about 15.0 mS/cm to 18.0 mS/cm. In another embodiment, the equilibration buffer or loading buffer or wash buffer conductivity is about 10.0 mS/cm to 13.0 mS/cm.
  • the pH of the equilibration buffer or loading buffer or wash buffer is selected from about 6.5 to about 7.5. In preferred embodiment, the equilibration buffer pH is about 7.0.
  • the loading buffer has concentration range from about 5 mM to about 40 mM. In an embodiment, the loading buffer has concentration range from about 10 mM to about 30 mM. In preferred embodiment, the loading buffer concentration is about 20 mM.
  • the affinity chromatography has at least one wash buffer. In another embodiment, the affinity chromatography has three wash buffers.
  • the first wash buffer has concentration range from about 5 mM to about 40 mM. In certain embodiment, the first wash buffer has concentration range from about 10 mM to about 25 mM. In preferred embodiment, the first wash buffer concentration is about 20 mM.
  • the second wash buffer is selected from sodium phosphate, Tris-HCl, Tris-Acetate, HEPES, and Glycine-NaOH.
  • second wash buffer used in combination with a salt.
  • the salt is selected from sodium chloride, potassium Chloride. In preferred embodiment, the salt is Sodium Chloride.
  • the second wash buffer has concentration range from about 5 mM to about 40 mM. In certain embodiment, the second wash buffer has concentration range from about 10 mM to about 25 mM. In preferred embodiment, the second wash buffer concentration is about 20 mM.
  • the second wash buffer has a salt buffer concentration range from about 0.5 M to about 1.5 M. In preferred embodiment, the second wash buffer concentration is about 1.0 M.
  • the second wash buffer has conductivity range from about 70 mS/cm to about 120 mS/cm. In an embodiment, the second wash buffer has conductivity range from about 80 mS/cm to about 100 mS/cm. In preferred embodiment, the second wash buffer conductivity is about 90 mS/cm.
  • the pH of the second wash buffer is selected from about 6.5 to about 7.5. In preferred embodiment, the second wash buffer pH is about 7.0.
  • the second wash buffer further comprises a surfactant which is selected from Polysorbate 20, Polysorbate 80, and Triton X-100, In an embodiment, the preferred surfactant is Polysorbate 20.
  • the percentage of the surfactant in the second wash buffer is from about 0.01% to about 1%. In preferred embodiment, the surfactant in the second wash buffer is 0.1% (w/v).
  • the third wash buffer has concentration range from about 5 mM to about 40 mM. In certain embodiment, the third wash buffer has concentration range from about 10 mM to about 30 mM. In preferred embodiment, the third wash buffer concentration is about 20 mM.
  • the third wash buffer has concentration range from about 5 mM to about 40 mM. In certain embodiment, the third wash buffer has concentration range from about 10 mM to about 40 mM. In preferred embodiment, the third wash buffer concentration is about 30 mM.
  • the third wash buffer has conductivity range from about 0.5 mS/cm to about 2.5 mS/cm. In preferred embodiment, the third wash buffer conductivity is less than 2.5 mS/cm.
  • the pH of the third wash buffer is selected from about 5 to about 6. In preferred embodiment, the third wash buffer pH is about 5.5.
  • the elution buffer is selected from Acetic acid, Phosphoric acid, Sodium acetate, and HCl. In preferred embodiment, the elution buffer is Acetic acid.
  • the elution buffer is selected from Acetic acid, Phosphoric acid, Sodium acetate, and HCl. In preferred embodiment, the elution buffer is Sodium Acetate.
  • the elution buffer has concentration range selected from about 25 mM to about 250 mM.
  • the elution buffer has concentration range selected from about 100 mM to about 250 mM. In an embodiment, the elution buffer has a concentration range about 125 mM to 200 mM.
  • the elution buffer has conductivity range from about 0.2 mS/cm to about 1.0 mS/cm. In an embodiment, the elution buffer has conductivity range from about 0.5 mS/cm to about 1.0 mS/cm. In an embodiment, the elution buffer has conductivity range from about 0.5 mS/cm to about 0.6 mS/cm. In an embodiment, the elution buffer has conductivity range from about 0.2 mS/cm to about 0.3 mS/cm.
  • the pH of the elution buffer is selected from 2.5 to about 3.5. In preferred embodiment, the elution buffer pH is about 3.5.
  • the elution buffer has conductivity range from about 0.2 mS/cm to about 0.3 mS/cm.
  • the pH of the elution buffer is selected from 2.5 to about 3.5.
  • the elution buffer pH is about 3.0.
  • elution is performed in linear gradient. In certain embodiment, the elution is performed in step gradient.
  • the elution peak Collection starts at an ascending value of about 2.5 AU/cm and ends at a descending value of about 2.5 AU/cm.
  • the elution peak Collection starts at an ascending value of about 0.25 AU/cm and ends at a descending value of about 0.25 AU/cm.
  • the invention provides the antibody composition having a turbidity selected from less than about 100 NTU, less than about 50 NTU, less than about 30 NTU, less than about 10 NTU obtained from Affinity Chromatography wherein the elution buffer has a concentration of about 200 mM.
  • the invention provides a purification process of antibodies or fragment thereof by using affinity chromatography wherein the elution is performed at low salt concentration.
  • the invention provides a purification process of antibodies or fragment thereof by using affinity chromatography wherein the elution is performed at low salt concentration, which does not reduce the turbidity compared to elution performed with high salt concentration of the of the eluted protein mixture during viral inactivation.
  • the equilibration is performed for about 3 CV's to about 10 CV's. In a preferred embodiment, the equilibration is performed for about 5 CV's. In an embodiment, the equilibration is performed until the equilibration buffer conductivity end point is achieved.
  • the amount of protein loaded onto the column during loading is at a range of about 10 g/L to about 45 g/L. In an embodiment, the amount of protein loaded onto the column during loading is at a range from about 10 g/L to about 50 g/L.
  • the first wash is performed for at least 1 to about 5 CV's. In preferred embodiment the first wash is performed for 3 CV's. In an embodiment, the first wash is performed until the buffer conductivity end point is achieved.
  • the second wash is performed for at least 1 CV to about 5 CV's. In preferred embodiment, the second wash is performed for 3 CV's. In an embodiment, the second wash is performed until the buffer conductivity end point is achieved.
  • the third wash is performed for at least 4 CV's to about 8 CV's. In preferred embodiment, the third wash is for 5 CV's. In an embodiment, the third wash is performed until the buffer conductivity end point is achieved.
  • the residence time of the protein in the column during protein A purification has a range from about 2 minutes to about 6 minutes. In preferred embodiment, the residence time of the protein in the column is about 4 minutes.
  • the anion exchange chromatography resin is selected from Capto Q, DEAE Sepharose fast flow, Fractogel EMD DEAE(M), Toyopearl DEAE-650, Q Sepharose Fast Flow, POROS XQ, POROS 50 HQ, POROS 50 PI, and POROS 50 D.
  • the anion exchange chromatography resin is strong anion exchange POROS 50 HQ.
  • the equilibration buffer or loading buffer is selected from Sodium Phosphate, Tris-HCl, HEPES, Glycine-NaOH, and Tris-Acetate. In certain embodiment, the equilibration buffer or loading buffer is Tris Acetate or Tris-HCl.
  • the equilibration buffer has a concentration range from about 40 mM to about 60 mM. In a certain embodiment, the loading buffer concentration is about 50 mM. In a certain embodiment, the loading buffer concentration is about 20 mM.
  • the equilibration buffer has conductivity range from about 1.5 mS/cm to about 3.5 mS/cm. In a certain embodiment, the equilibration buffer conductivity is less than 2.6 mS/cm.
  • the pH of the equilibration buffer is selected from about 6.5 to about 7.5. In a certain embodiment, the loading buffer pH is about 7.0 to about 7.5. In an embodiment, the pH of loading buffer is 7.2-7.4.
  • the equilibration buffer conductivity is ⁇ 2.0 mS/cm.
  • the pH of the equilibration buffer is selected from about 6.5 to about 7.5.
  • the loading buffer pH is about 7.0 to about 7.5. In an embodiment, the pH of loading buffer is 7.2-7.3.
  • the loading buffer has a concentration range from about 40 mM to about 60 mM. In a certain embodiment, the loading buffer concentration is about 50 mM.
  • the loading buffer has a concentration range from about 10 mM to about 30 mM. In a certain embodiment, the loading buffer concentration is about 20 mM.
  • the loading buffer has conductivity range from about 1.5 mS/cm to about 3.5 mS/cm. In a certain embodiment, the loading buffer conductivity is less than 2.6 mS/cm.
  • the pH of the loading buffer is selected from about 6.5 to about 7.5.
  • the loading buffer pH is about 7.0 to about 7.5. In an embodiment, the pH of loading buffer is 7.2-7.3.
  • the loading buffer conductivity is about 3.0 mS/cm.
  • the pH of the loading buffer is selected from about 6.5 to about 7.5.
  • the loading buffer pH is about 7.0 to about 7.5. In an embodiment, the pH of loading buffer is 7.2-7.3.
  • the invention provides protein peak collection criteria selected from the ascending value of about 2.5 AU/cm and ends at a descending value of about 1.5 AU/cm.
  • the invention provides protein peak collection criteria selected from the ascending value of about 1.5 AU/cm and ends at a descending value of about 1.5 AU/cm.
  • the invention provides the antibody composition comprising antibody of interest and about 10% to 12% acidic variant obtained from AEX chromatography wherein the peak collection criteria is selected from about 2.5 AU/cm to about 1.5 AU/cm.
  • the invention provides protein peak collection criteria selected from the ascending value of about 1.5 AU/cm and ends at a descending value of about 1.5 AU/cm.
  • the washing buffer is selected from sodium phosphate, Tris-HCl, HEPES, Glycine-NaOH, and Tris-Acetate.
  • the washing buffer has concentration range from about 40 mM to about 60 mM. In certain embodiment, the washing buffer concentration is about 50 mM.
  • the washing buffer has concentration range from about 10 to about 30 mM. In certain embodiment, the washing buffer concentration is about 20 mM.
  • the washing buffer has conductivity range from about 1.5 mS/cm to about 3.5 mS/cm. In preferred embodiment, the washing buffer conductivity is less than 2.6 mS/cm.
  • the washing buffer conductivity is ⁇ 2.0 mS/cm.
  • the pH of the washing buffer is selected from about 6.5 to about 7.5. In certain embodiment the washing buffer pH is about 7.2.
  • the regeneration buffer is selected from Sodium Phosphate, Tris-HCl, HEPES, Glycine-NaOH, Tris-Acetate.
  • the regeneration buffer has concentration range from about 5 mM to about 30 mM. In certain embodiment, the regeneration buffer concentration is about 20 mM.
  • the regeneration buffer also contains a salt selected from Sodium Chloride, Potassium Chloride, and Calcium Chloride.
  • the salt in the regeneration buffer is Sodium Chloride.
  • the salt in the regeneration buffer has concentration range from about 0.5M to about 1.5 M. In certain embodiment, the salt in the regeneration buffer has concentration of about 1.0 M.
  • the regeneration buffer has conductivity range from about 80 mS/cm to about 90 mS/cm. In certain embodiment, the regeneration buffer conductivity is about 85 mS/cm.
  • the regeneration buffer has conductivity range from about 90 mS/cm to about 110 mS/cm. In certain embodiment, the regeneration buffer conductivity is about 100 mS/cm.
  • the pH of the regeneration buffer is selected from about 6.5 to about 7.5. In preferred embodiment, the regeneration buffer pH is about 7.0.
  • the pH of the regeneration buffer is selected from about 6.5 to about 7.5. In preferred embodiment, the regeneration buffer pH is about 7.2.
  • the elution is performed in a flow through mode.
  • the sanitization buffer is selected from NaOH, Iso-propyl alcohol, benzyl alcohol. In certain embodiment, the sanitization buffer is NaOH.
  • the sanitization buffer has concentration range from about 300 mM to about 1500 mM. In certain embodiment, the regeneration buffer concentration is about 500 mM.
  • the loading is performed for at least about 5 CV's or more. In a certain embodiment the loading is performed for about 30 CV's.
  • the equilibration is performed for at least about 3 CV's to about 10 CV's. In a certain embodiment, the equilibration is performed for about 5 CV's.
  • the equilibration is performed until the equilibration buffer conductivity end point is achieved.
  • the amount of protein loaded onto the column during loading is selected from less than about 150 g/L, less than about 130 g/L, less than about 120 g/L, less than about 110 g/L, less than about 100 g/L.
  • the washing is performed for at least about 5 CV's.
  • the washing is performed for at least about 2 CV's.
  • the regeneration is performed for at least 2 CV's to about 5 CV's. In a certain embodiment, the regeneration is performed for about 3 CV's.
  • the regeneration removes most of the impurities. In preferred embodiment, the regeneration removes most of the HMWs, and acidic charged variant based impurities.
  • the sanitization is performed for at least 2 CV's to about 5 CV's. In a certain embodiment, the sanitization is performed for about 3 CV's.
  • the sanitization buffer is held in the column for about 15 minutes to about 60 minutes. In certain embodiment, the sanitization buffer is held in the column for about 20 minutes.
  • the residence time of the protein in the column during AEX purification has a range from about 2 to about 6 minutes. In a certain embodiment, the residence time of the protein in the column is about 4 minutes.
  • Ceramic Hydroxy apatite is CHT (Ceramic Hydroxy apatite)—Type 1 and CHT (Ceramic Hydroxy apatite) XT.
  • the loading buffer, washing buffer, equilibration buffer and elution buffer is prepared by combining Buffer A and Buffer B.
  • buffer A is polar protic molecule.
  • the polar protic molecule is selected from water (H—OH), acetic acid (CH3CO—OH) methanol (CH3-OH), ethanol (CH3CH2-OH), n-propanol (CH3CH2CH2-OH), n-butanol (CH3CH2CH2CH2-OH).
  • the polar protic molecule is water.
  • buffer B is selected from Sodium Phosphate, Tris, HEPES, Glycine-NaOH.
  • the buffer B is Sodium Phosphate.
  • the concentration range of buffer B is from about 20 mM to about 40 mM. In certain embodiment, the concentration of buffer B is about 32 mM.
  • the concentration range of buffer B is from about 20 mM to about 40 mM. In certain embodiment, the concentration of buffer B is about 40 mM.
  • the concentration range of equilibration buffer and loading buffer is from about 20 mM to about 40 mM. In certain embodiment, the concentration of equilibration buffer and loading buffer is about 32 mM.
  • the equilibration buffer, loading buffer and wash buffer are same in strength. In an embodiment, the strength of elution buffer is higher than loading buffer.
  • the equilibration buffer and loading buffer has concentration range from about 20 to about 50 mM. In certain embodiment, the equilibration buffer concentration is about 40 mM.
  • the equilibration buffer has a concentration from about 20 mM to 50 mM. In certain embodiment, the equilibration buffer has a concentration of about 32 mM. In an embodiment, the pH of the equilibration buffer or loading buffer or washing buffer or elution buffer is selected from about 6.5 to about 7.5.
  • the buffer pH is about 7.0 ⁇ 0.2.
  • the equilibration buffer has conductivity range from about 3.0 to about 7.0 mS/cm. In preferred embodiment, the equilibration buffer conductivity is about 6.0 mS/cm.
  • the pH of the equilibration buffer is selected from about 6.5 to about 7.5.
  • the equilibration buffer pH is about 7.0 ⁇ 0.2.
  • the equilibration is performed with isocratic gradient by using combination of at least two buffers where buffer A concentration is at least 10% of buffer B concentration. In preferred embodiment, buffer A concentration is at least 8% of buffer B concentration.
  • the loading buffer has concentration range from about 30 to about 60 mM. In preferred embodiment, the loading buffer concentration is about 40 mM.
  • the loading buffer has a concentration from about 10 mM to 40 mM. In certain embodiment, the loading buffer has a concentration of about 32 mM.
  • the loading buffer has conductivity range from about 3.0 to about 7.0 mS/cm. In preferred embodiment, the loading buffer conductivity is about 6.0 mS/cm.
  • the pH of loading buffer is selected from about 6.5 to about 7.5.
  • the loading buffer pH is about 7.0 ⁇ 0.2.
  • the loading is performed with isocratic gradient by using combination of at least two buffers where buffer A concentration is at least 10% of buffer B concentration. In preferred embodiment, buffer A concentration is at least 8% of buffer B.
  • the washing buffer has concentration range from about 30 mM to about 60 mM. In preferred embodiment, the washing buffer concentration is about 40 mM.
  • the washing buffer has a concentration from about 10 mM to 40 mM. In certain embodiment, the washing buffer has a concentration of about 32 mM.
  • the washing buffer has conductivity range from about 3.0 mS/cm to about 7.0 mS/cm. In preferred embodiment, the washing buffer conductivity is about 6.0 mS/cm.
  • the pH of the washing buffer is selected from about 6.5 to about 7.5.
  • washing buffer pH is about 7.0 ⁇ 0.2.
  • the washing is performed with isocratic gradient by using combination of at least two buffers where buffer A concentration is at least 10% of, buffer B or less. In preferred embodiment, buffer A concentration is at least 10% of, buffer B concentration.
  • the elution buffer has concentration range from about 10 mM to about 400 mM. In preferred embodiment, the elution buffer has a concentration range from about 32 mM to about 104 mM.
  • the elution buffer has concentration range from about 10 mM to about 400 mM. In preferred embodiment, the elution buffer has a concentration range from about 24 mM to about 100 mM.
  • the elution buffer has concentration range from about 10 mM to about 400 mM. In preferred embodiment, the elution buffer has a concentration range from about 32 mM to about 88 mM.
  • the elution buffer has concentration range from about 10 mM to about 400 mM. In preferred embodiment, the elution buffer has a concentration range from about 40 mM to about 96 mM.
  • the elution buffer has conductivity range from about 6 mS/cm to about 12 mS/cm.
  • the pH of the elution buffer is selected from about 6.5 to about 7.5. In an embodiment, the elution buffer pH is about 7.0 ⁇ 0.2.
  • the elution is performed in linear gradient. In certain embodiment, the elution is performed in step gradient.
  • the elution is performed with linear gradient by using combination of at least two buffers wherein buffer A concentration is at least 10% of buffer B concentration.
  • buffer A concentration is at least 10% of buffer B concentration.
  • buffer B is gradually increased from about 10% to about 24% and optimized concentration of buffer B accordingly.
  • elution is performed in isocratic gradient with 15% of buffer B. In such embodiment, elution is performed in isocratic gradient with 20% of buffer B. In such embodiment, elution is performed in isocratic gradient with 15%, 16%, 17%, 18%, 19%, 20% of buffer B.
  • the elution is performed with linear gradient by using combination of at least two buffers wherein buffer A concentration is at least 8% of buffer B concentration.
  • buffer A concentration is at least 8 of, buffer B % concentration.
  • buffer B is gradually increased from about 8% to about 22% and optimized concentration of buffer B accordingly.
  • the elution peak collection starts at an ascending value of about 1.5 AU/cm and ends at a descending value of about 2.0 AU/cm for minimising LMWs.
  • the elution peak collection starts at an ascending value of about 1.0 AU/cm and ends at a descending value of about 1.5 AU/cm for minimising LMWs.
  • the CHT is performed in bind and elute mode.
  • the CHT is performed with buffer or solvent free from calcium chloride.
  • the concentration of the elution buffer is higher than wash buffer.
  • the elution buffer comprises at least 8% the concentration of the 400 mM salt buffer. In an embodiment, the elution buffer comprises at least 10% the concentration of the 400 mM salt buffer. In an embodiment, the elution buffer comprises at least 15% the concentration of the 400 mM salt buffer. In an embodiment, the elution buffer comprises at least 20% the concentration of the 400 mM salt buffer. In an embodiment, the elution buffer comprises at least 26% the concentration of the 400 mM salt buffer.
  • the elution is performed for about 5 CV's to about 10 CV's.
  • the elution is performed for about 10 CV's to about 15 CV's.
  • the elution is performed for about 15 to about 20 CV's or more. In a preferred embodiment, the elution takes place for about 19 CV's.
  • the equilibration is performed for about 3 CV's to about 10 CV's. In a preferred embodiment, the equilibration takes place for about 5 CV's.
  • the equilibration is performed until the equilibration buffer conductivity end point is achieved.
  • the amount of protein loaded onto the column during loading is in the range from about 5 g/L to about 25 g/L. In an embodiment, amount of protein loaded onto the column during loading is 12.5 g/L.
  • the washing is performed for at least about 2 CV's to about 5 CV's. In preferred embodiment, the washing is performed for about 3 CV's. In an embodiment, the washing is performed until the buffer conductivity end point is achieved.
  • the residence time of the protein in the CHT column during equilibration washing and loading is in the range from about 2 minutes to about 6 minutes. In preferred embodiment, the residence time of the protein in the column during equilibration, washing and loading is about 4 minutes.
  • the residence time of the protein in the column during elution has a range from about 2 minutes to about 6 minutes. In preferred embodiment, the residence time of the protein in the column during elution is about 4 minutes.
  • a monoclonal antibody molecule capable to bind to IgE molecule expressed in Chinese Hamster Ovary (CHO) cell line is captured using Protein A (Mab Select Sure LX, GE Healthcare) packed in VL 11/250 or C10/20 column. The residence time is 4 min for all the phases. After equilibration with Tris Acetate+100 mM NaCl, pH 6.8-7.2 clarified harvest is loaded at ⁇ 45 mg/mL of the resin. After loading, the column is washed with equilibration buffer (wash 1) followed by wash 2 and wash 3 buffer as mentioned in Table 1.
  • Affinity chromatography step is operated in bind and eluate mode and collection is done from 50 mAU (0.25 AU/cm) ascending to 50 mAU (0.25 AU/cm) descending of the peak.
  • Protein A output is analysed with SE-HPLC, CEX-HPLC for size and charge variants.
  • the experimental design for Protein A step is summarized in Table 1.
  • Eluted protein is further subjected to viral inactivation and neutralization. After neutralization, protein has been filtered by 0.2 ⁇ m filter.
  • the eluted protein mixture is further purified using Anion Exchange Chromatography resin (Poros HQ, Thermofisher) packed in C10/20 column.
  • the residence time is 4 min for all the phases.
  • After equilibration with Tris Acetate, pH 7.0-7.3 Neutralized Protein A output is loaded at ⁇ 100 mg/mL of the resin.
  • the Load is diluted with water to meet the (mS/cm) conductivity specification i. e. ⁇ 3.0 mS/cm before introducing it into the AEX column.
  • AEX step is operated in Flow-through (negative) mode and collection is done from 500 mAU ascending to 300 mAU descending of the peak.
  • the column is washed using Tris Acetate, pH 7.0-7.3.
  • AEX output is analysed with SE-HPLC, CEX-HPLC for size and charge variants.
  • the experimental design for POROS 50 HQ step is summarized in Table 2.
  • Table 3 shows 99% purity of main peak and 98% reduction of HMW which is determined by SE-HPLC.
  • Table 4 shows the results of Cation Exchange-High performance Liquid Chromatography comparing the main peak with acidic variants.
  • Table 4 shows 74%% purity of main peak and approximately 55% reduction of acidic variants which is determined by CEX-HPLC.
  • a monoclonal antibody capable to bind to IgE expressed in Chinese Hamster Ovary (CHO) cell line is captured using Protein A (Mab Select Sure LX, GE Healthcare) packed in VL 11/250 column. Eluted protein is further subjected to viral inactivation and neutralization. After neutralization, protein has been filtered by 0.2 ⁇ m filter.
  • the protein mixture is further purified using Anion Exchange Chromatography resin (Poros HQ, Thermofisher) packed in C10/20 column.
  • a monoclonal antibody capable to bind to IgE expressed in Chinese Hamster Ovary (CHO) cell line is captured using Protein A (Mab Select Sure LX, GE Healthcare) packed in VL 11/250 column.
  • Eluted protein is further subjected to viral inactivation and neutralization. After neutralization, protein has been filtered by 0.2 ⁇ m filter.
  • the eluted protein mixture is further purified using Anion Exchange Chromatography resin (Poros HQ, Thermofisher) packed in C10/20 column.
  • composition obtained from CHT has substantially reduced approx. 100% LMW and purity of protein is 99.99% by SE-HPLC.
  • composition obtained from CHT has substantially reduced approx. 81% LMW.
  • Table 9 shows 84% purity of main peak and approximately 56% reduction of LMW which is determined by CEX-HPLC.
  • a monoclonal antibody molecule capable to bind to IgE molecule expressed in Chinese Hamster Ovary (CHO) cell line is captured using Protein A (Mab Select Sure LX, GE Healthcare) packed in Chromatographic 100/250 column. The residence time is 4 min for all the phases. After equilibration with Tris HCl+150 mM NaCl, pH 6.8-7.2 clarified harvest is loaded at ⁇ 45 mg/mL of the resin. After loading column is washes with equilibration buffer (wash 1) followed by wash 2 and wash 3 buffer as mentioned in Table 10. Affinity chromatography step is operated in bind and eluate mode and collection is done from 0.25 AU/cm ascending to 0.25 AU/cm descending of the peak. Protein A output is analysed with SE-HPLC, CEX-HPLC for size and charge variants. The experimental design for Protein A step is summarized in Table 10.
  • Eluted protein is further purified using Anion Exchange Chromatography resin (POROS 50 HQ, Thermofisher) packed in Chromatographic 100/250 column. The residence time is 4 min for all the phases. After equilibration with Tris HCl, pH 7.1-7.3 Neutralized Protein A output is loaded at 70 to 145 mg/mL of the resin. The Load is diluted with water to meet the (mS/cm) conductivity specification i. e. ⁇ 2.0 mS/cm before introducing it into the AEX column.
  • POROS 50 HQ Anion Exchange Chromatography resin
  • AEX step is operated in Flow-through (negative) mode and collection is done from 2.0 AU/cm ascending to 2.5 AU/cm descending of the peak.
  • the column is washed using Tris HCl, pH 7.1-7.3.
  • AEX output is analysed with SE-HPLC, CEX-HPLC for size and charge variants.
  • the experimental design for POROS 50 HQ step is summarized in Table 11.
  • Table 12 shows 99% purity of main peak and 96% reduction of HMW which is determined by SE-HPLC.
  • Table 13 shows 67% purity of main peak and approximately 25% reduction of acidic variants which is determined by CEX-HPLC.
  • a monoclonal antibody capable to bind to IgE expressed in Chinese Hamster Ovary (CHO) cell line is captured using Protein A (Mab Select Sure LX, GE Healthcare) packed in Chromatographic 100/250 column. Eluted protein is further subjected to viral inactivation and neutralization followed by depth filtration. After depth filtration, protein has been filtered by 0.2 ⁇ m filter.
  • the protein mixture is further purified using Anion Exchange Chromatography resin (Poros HQ, Thermofisher) packed in Chromatographic 100/250 column.
  • Anion Exchange Chromatography resin Pan HQ, Thermofisher
  • a monoclonal antibody molecule capable to bind to IgE molecule expressed in Chinese Hamster Ovary (CHO) cell line is captured using Protein A (Mab Select Sure LX, GE Healthcare) packed in Chromatographic 250/250 column. The residence time is 4 min for all the phases. After equilibration with Tris HCl+150 mM NaCl, pH 6.8-7.2 clarified harvest is loaded at ⁇ 45 mg/mL of the resin. After loading column is washes with equilibration buffer (wash 1) followed by wash 2 and wash 3 buffer as mentioned in Table 18. Affinity chromatography step is operated in bind and eluate mode and collection is done from 0.25 AU/cm ascending to 0.25 AU/cm descending of the peak. Protein A output is analysed with SE-HPLC, CEX-HPLC for size and charge variants. The experimental design for Protein A step is summarized in Table 18.
  • Eluted protein is further purified using Anion Exchange Chromatography resin (POROS 50 HQ, Thermofisher) packed in Chromatographic 350/250 or 200/350 column. The residence time is 4 min for all the phases. After equilibration with Tris HCl, pH 7.1-7.3 Neutralized Protein A output is loaded at 70 to 145 mg/L of the resin. The Load is diluted with water to meet the (mS/cm) conductivity specification i. e. ⁇ 2.0 mS/cm before introducing it into the AEX column.
  • POROS 50 HQ Anion Exchange Chromatography resin
  • AEX step is operated in Flow-through (negative) mode and collection is done from 2.0 AU/cm ascending to 2.5 AU/cm descending of the peak.
  • the column is washed using Tris HC, pH 7.1-7.3.
  • AEX output is analysed with SE-HPLC, CEX-HPLC for size and charge variants.
  • the experimental design for POROS 50 HQ step is summarized in Table 19.
  • Table 20 shows 99% purity of main peak and 91% reduction of HMW which is determined by SE-HPLC.
  • Table 21 Shows the Results of Cation Exchange-High Performance Liquid Chromatography Comparing the Main Peak with Acidic Variants.
  • Table 21 shows 74% purity of main peak and approximately 46% reduction of acidic variants which is determined by CEX-HPLC.
  • a monoclonal antibody capable to bind to IgE expressed in Chinese Hamster Ovary (CHO) cell line is captured using Protein A (Mab Select Sure LX, GE Healthcare) packed in Chromatographic 250/250 column. Eluted protein is further subjected to viral inactivation and neutralization followed by depth filtration. After depth filtration, protein has been filtered by 0.2 ⁇ m filter.
  • the protein mixture is further purified using Anion Exchange Chromatography resin (Poros HQ, Thermofisher) packed in Chromatographic 350/250 or 200/250 column.
  • Anion Exchange Chromatography resin Pan HQ, Thermofisher

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
US17/922,729 2020-05-01 2021-05-01 An improved process of purification of protein Pending US20230167153A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US19/432,924 US20260116917A1 (en) 2020-05-01 2025-12-24 Process of purification of protein

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
IN202021018731 2020-05-01
IN202021018735 2020-05-01
IN202021018714 2020-05-01
IN202021018731 2020-05-01
IN202021018735 2020-05-01
IN202021018714 2020-05-01
PCT/IB2021/053658 WO2021220251A1 (en) 2020-05-01 2021-05-01 An improved process of purification of protein

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2021/053658 A-371-Of-International WO2021220251A1 (en) 2020-05-01 2021-05-01 An improved process of purification of protein

Related Child Applications (5)

Application Number Title Priority Date Filing Date
US18/416,844 Continuation US12139510B2 (en) 2020-05-01 2024-01-18 Process of purification of protein
US18/416,846 Continuation US12435106B2 (en) 2020-05-01 2024-01-18 Process of purification of protein
US19/092,641 Continuation US12600747B2 (en) 2020-05-01 2025-03-27 Process of purification of protein
US19/282,184 Continuation US20250353877A1 (en) 2020-05-01 2025-07-28 Process of purification of protein
US19/432,924 Continuation-In-Part US20260116917A1 (en) 2020-05-01 2025-12-24 Process of purification of protein

Publications (1)

Publication Number Publication Date
US20230167153A1 true US20230167153A1 (en) 2023-06-01

Family

ID=78373378

Family Applications (6)

Application Number Title Priority Date Filing Date
US17/922,729 Pending US20230167153A1 (en) 2020-05-01 2021-05-01 An improved process of purification of protein
US18/416,846 Active US12435106B2 (en) 2020-05-01 2024-01-18 Process of purification of protein
US18/416,844 Active US12139510B2 (en) 2020-05-01 2024-01-18 Process of purification of protein
US18/893,058 Active US12378282B2 (en) 2020-05-01 2024-09-23 Process of purification of protein
US19/092,641 Active US12600747B2 (en) 2020-05-01 2025-03-27 Process of purification of protein
US19/282,184 Pending US20250353877A1 (en) 2020-05-01 2025-07-28 Process of purification of protein

Family Applications After (5)

Application Number Title Priority Date Filing Date
US18/416,846 Active US12435106B2 (en) 2020-05-01 2024-01-18 Process of purification of protein
US18/416,844 Active US12139510B2 (en) 2020-05-01 2024-01-18 Process of purification of protein
US18/893,058 Active US12378282B2 (en) 2020-05-01 2024-09-23 Process of purification of protein
US19/092,641 Active US12600747B2 (en) 2020-05-01 2025-03-27 Process of purification of protein
US19/282,184 Pending US20250353877A1 (en) 2020-05-01 2025-07-28 Process of purification of protein

Country Status (6)

Country Link
US (6) US20230167153A1 (https=)
EP (1) EP4142792A4 (https=)
JP (2) JP7817743B2 (https=)
AU (1) AU2021262609A1 (https=)
CA (1) CA3182368A1 (https=)
WO (1) WO2021220251A1 (https=)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12378282B2 (en) 2020-05-01 2025-08-05 Kashiv Biosciences, Llc Process of purification of protein

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023007516A1 (en) * 2021-07-29 2023-02-02 Dr. Reddy’S Laboratories Limited Method to control high molecular weight aggregates in an antibody composition
US20240400612A1 (en) * 2021-09-28 2024-12-05 Kashiv Biosciences, Llc An improved process for purification of fusion protein
WO2023112000A2 (en) * 2021-12-17 2023-06-22 Kashiv Biosciences, Llc An improved method for separation of low molecular weight proteins

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110129468A1 (en) * 2008-02-29 2011-06-02 Biogen Idec Ma Inc. Purified immunoglobulin fusion proteins and methods of their purification
WO2013096791A1 (en) * 2011-12-23 2013-06-27 Genentech, Inc. Process for making high concentration protein formulations
WO2013180647A1 (en) * 2012-05-31 2013-12-05 Agency For Science, Technology And Research Chromatographic purification of immunoglobulin g preparations with particles having multimodal functionalities
US20140288272A1 (en) * 2013-03-15 2014-09-25 Alderbio Holdings Llc Antibody purification and purity monitoring
US20190256556A1 (en) * 2016-06-17 2019-08-22 Genentech, Inc. Purification of multispecific antibodies

Family Cites Families (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5328834A (en) 1989-09-08 1994-07-12 Unisyn Technologies, Inc. Method for preparing immunoglobulin fragments
CA2259618A1 (en) 1996-07-05 1998-01-15 The Governing Council Of The University Of Toronto Genetic sequences and proteins related to alzheimer's disease, and uses therefor
US8703126B2 (en) 2000-10-12 2014-04-22 Genentech, Inc. Reduced-viscosity concentrated protein formulations
WO2002082066A1 (en) 2001-03-28 2002-10-17 Cetek Corporation Concentration and identification of moderate-to-strong hits in natural products by capillary electrophoresis
ES2322945T5 (es) * 2001-12-21 2012-10-17 Immunex Corporation Métodos para purficación de proteína
US9415102B2 (en) 2002-09-06 2016-08-16 Alexion Pharmaceuticals, Inc. High concentration formulations of anti-C5 antibodies
MXPA05010555A (es) 2003-04-04 2006-03-09 Genentech Inc Formulaciones de proteina y anticuerpo de alta concentracion.
PL1615945T3 (pl) 2003-04-09 2012-03-30 Ratiopharm Gmbh Sposoby glikopegylacji i białka/peptydy wytwarzane tymi sposobami
RU2359270C2 (ru) 2003-07-29 2009-06-20 Зольвай Фармасьютиклз Гмбх Способ анализа панкреатита и содержащих его композиций
US7452697B2 (en) * 2003-09-25 2008-11-18 Allergan, Inc. Chromatographic method and system for purifying a botulinum toxin
KR101221862B1 (ko) * 2003-10-27 2013-01-14 와이어쓰 엘엘씨 하이드록시아파타이트 크로마토그래피를 사용한 고분자량응집체의 제거
TW200621282A (en) 2004-08-13 2006-07-01 Wyeth Corp Stabilizing formulations
ZA200710330B (en) 2005-06-17 2009-12-30 Wyeth Corp Methods of purifying FC region containing proteins
KR20080025174A (ko) 2005-06-23 2008-03-19 메디뮨 인코포레이티드 응집 및 단편화 프로파일이 최적화된 항체 제제
AR058568A1 (es) 2005-12-20 2008-02-13 Bristol Myers Squibb Co Metodos para producir una composicion con moleculas ctla4-ig a partir de un medio de cultivo
US9309316B2 (en) 2005-12-20 2016-04-12 Bristol-Myers Squibb Company Stable subcutaneous protein formulations and uses thereof
DK1969007T3 (da) 2005-12-20 2013-11-25 Bristol Myers Squibb Co Sammensætninger og fremgangsmåder til fremstilling af en sammensætning
CN101511387A (zh) 2006-09-08 2009-08-19 惠氏公司 使用亲和色谱法纯化蛋白质的精氨酸洗涤液
US20100129379A1 (en) 2006-09-25 2010-05-27 John Carpenter Stabilized antibody formulations and uses thereof
WO2008085988A1 (en) * 2007-01-05 2008-07-17 Amgen Inc. Methods of purifying proteins
CN102164954B (zh) * 2008-09-23 2018-04-06 弗·哈夫曼-拉罗切有限公司 促红细胞生成素的纯化
US8058407B2 (en) 2008-10-31 2011-11-15 Wyeth Llc Purification of acidic proteins using ceramic hydroxyapatite chromatography
AU2010221156A1 (en) 2009-03-06 2011-09-22 Genentech, Inc. Antibody formulation
PT3345593T (pt) 2009-11-13 2023-11-27 Sanofi Aventis Deutschland Composição farmacêutica compreendendo despro36exendina- 4(1-39)-lys6-nh2 e metionina
WO2011067283A1 (en) 2009-12-01 2011-06-09 Novo Nordisk A/S Novel peptidyl alpha-hydroxyglycine alpha-amidating lyases
AR080428A1 (es) 2010-01-20 2012-04-11 Chugai Pharmaceutical Co Ltd Formulaciones liquidas estabilizadas contentivas de anticuerpos
HUE030100T2 (en) 2010-02-19 2017-04-28 Xencor Inc New CTLA4-IG immunoadhesins
WO2011121020A1 (en) 2010-03-30 2011-10-06 Octapharma Ag A process for purifying vitamin k dependent proteins such as coagulation factor ix
KR101976853B1 (ko) 2010-05-25 2019-05-09 제넨테크, 인크. 폴리펩티드의 정제 방법
WO2013071396A1 (en) 2011-11-18 2013-05-23 Université de Montréal Oral leptin formulations and uses thereof
GB2491168B (en) 2011-05-26 2014-09-10 Thermo Electron Mfg Ltd Method and apparatus for improved resolution chromatography
DK2729482T3 (en) 2011-07-08 2018-05-07 Merck Sharp & Dohme PROCEDURE FOR CLEANING FC-FUSION PROTEIN
SG10201606218WA (en) * 2011-07-29 2016-09-29 Eleven Biotherapeutics Inc Purified Proteins
US9291630B1 (en) 2011-08-29 2016-03-22 Scientific Protein Laboratories, Llc RP-HPLC method for the analysis and quantification of pancreatin active pharmaceutical agents
JP6092892B2 (ja) 2011-12-15 2017-03-08 プレステージ バイオファーマ プライベート リミテッド 抗体の精製方法
WO2013158279A1 (en) 2012-04-20 2013-10-24 Abbvie Inc. Protein purification methods to reduce acidic species
WO2013169338A1 (en) 2012-05-11 2013-11-14 Medimmune, Llc Ctla-4 variants
WO2013176754A1 (en) 2012-05-24 2013-11-28 Abbvie Inc. Novel purification of antibodies using hydrophobic interaction chromatography
PE20150964A1 (es) 2012-09-07 2015-07-25 Coherus Biosciences Inc Formulaciones acuosas estables de adalimumab
SI2895188T1 (en) 2012-09-11 2018-05-31 Coherus Biosciences, Inc. CORRECTED QUALITY ETHANERCEPT WITH HIGH QUALITY AND EXCELLENT EXPERIENCE
US20150284673A1 (en) 2012-12-13 2015-10-08 Sapphire Energy, Inc. Harvesting algae from water
KR101569783B1 (ko) 2013-06-05 2015-11-19 한화케미칼 주식회사 항체의 정제 방법
RS57864B1 (sr) 2013-06-17 2018-12-31 De Staat Der Nederlanden Vert Door De Minister Van Vws Ministerie Van Volksgezondheid Welzijn En Spo Postupci za sprečavanje agregacije virusnih komponenti
CN103405756B (zh) 2013-07-10 2014-12-24 浙江众益制药股份有限公司 药物组合物复方消化酶胶囊(ii)及其制备方法
US20160228371A1 (en) 2013-10-18 2016-08-11 Abbvie Inc. Stable solid units and methods of making the same
CN103667225B (zh) 2013-11-23 2016-08-17 青岛康原药业有限公司 一种制备弹性蛋白酶的方法
PL3116891T3 (pl) 2014-03-10 2020-07-27 Richter Gedeon Nyrt. Oczyszczanie immunoglobuliny z zastosowaniem etapów wstępnego oczyszczania
WO2016009049A1 (en) 2014-07-18 2016-01-21 Sandoz Ag Methods for purifying tnfr:fc
ES2733298T3 (es) 2014-07-18 2019-11-28 Sandoz Ag Cuantificación de TNFR2:Fc plegado erróneamente
CN107250798A (zh) 2014-12-05 2017-10-13 雀巢产品技术援助有限公司 用于检测患者样品中的生物制品的间接均相迁移率变动试验
KR20180048731A (ko) 2015-08-20 2018-05-10 제넨테크, 인크. 재조합 폴리펩티드를 제조하기 위한 fkpa의 정제 및 그의 용도
BR112018009881A2 (pt) 2015-11-18 2018-11-13 Merck Patent Gmbh separação de proteína aperfeiçoada em cromatografia de troca iônica
ES2979210T3 (es) 2015-12-15 2024-09-24 Oncoc4 Inc Anticuerpos monoclonales anti-CTLA4 humano quiméricos y humanizados y usos de los mismos
US11661438B2 (en) 2015-12-21 2023-05-30 Pfizer, Inc. Purification of antibody drug conjugates using a sodium phosphate gradient
EP3436473A4 (en) 2016-03-29 2019-10-23 Navya Biologicals Pvt. Ltd. METHOD FOR PURIFYING FC FUSION PROTEINS
EP3443346B1 (en) 2016-04-13 2023-08-30 Medimmune, LLC Use of amino acids as stabilizing compounds in pharmaceutical compositions containing high concentrations of protein-based therapeutic agents
TWI734775B (zh) 2016-04-26 2021-08-01 美商美國泰福生技股份有限公司 細胞培養基
CN106190935A (zh) 2016-07-13 2016-12-07 江南大学 一种产木聚糖酶的重组菌及其应用
JP7116059B2 (ja) 2016-11-21 2022-08-09 ジャスト－エヴォテック バイオロジックス、インコーポレイテッド アフリベルセプト製剤及びその使用
CA3051466A1 (en) 2017-01-31 2018-08-09 Vanderbilt University Generation of human allergen-and helminth-specific ige monoclonal antibodies for diagnostic and therapeutic use
US10646569B2 (en) 2017-05-16 2020-05-12 Bhami's Research Laboratory, Pvt. Ltd. High concentration protein formulations with reduced viscosity
PE20200737A1 (es) 2017-06-30 2020-07-23 Spark Therapeutics Inc Metodos de purificacion de columna de vector aav
JP2020531557A (ja) * 2017-08-30 2020-11-05 アレス トレーディング ソシエテ アノニム タンパク質の精製方法
DK3517543T3 (da) 2018-01-30 2020-12-07 Bachem Ag Fremstilling af glucagonpeptider
DK4364724T3 (da) 2018-05-10 2025-12-22 Regeneron Pharma Formuleringer med høj koncentration af VEGF-receptorfusionsprotein
JP7449243B2 (ja) 2018-05-25 2024-03-13 ドクター レディズ ラボラトリーズ リミテッド 安定な融合タンパク質製剤
JP2021525255A (ja) 2018-05-25 2021-09-24 ドクター レディズ ラボラトリーズ リミテッド ＣＴＬＡ４−Ｉｇ融合タンパク質製剤
CN109142728A (zh) 2018-09-04 2019-01-04 深圳市鸿美诊断技术有限公司 一种定量测定粪便中胰弹性蛋白酶1的试剂盒及其应用
TWI871300B (zh) * 2019-01-28 2025-02-01 美商安進公司 藉由將藥物物質和藥物產品過程整體化的生物製劑製造之連續製造過程
JP2022531317A (ja) 2019-05-03 2022-07-06 ジェネンテック， インコーポレイテッド 精製プラットフォームから得られた組成物中の酵素加水分解活性速度を低下させる方法
AR119264A1 (es) 2019-06-05 2021-12-09 Genentech Inc Método para reutilización de cromatografía
CA3147265A1 (en) 2019-07-14 2021-01-21 Kashiv Biosciences, Llc A process for separation and quantitation of proteins using capillary electrophoresis
EP4021497B1 (en) 2019-08-30 2026-02-25 Kashiv Biosciences, LLC Novel formulation of highly concentrated pharmacologically active antibody
KR102154952B1 (ko) 2020-02-21 2020-09-10 프레스티지바이오로직스 주식회사 아달리무맙의 Non-Protein A 정제방법
US20230166200A1 (en) 2020-05-01 2023-06-01 Kashiv Biosciences, Llc An improved process of purification of protein
CA3182315A1 (en) 2020-05-01 2021-11-04 Kashiv Biosciences, Llc An improved process of affinity chromatography
AU2021262609A1 (en) 2020-05-01 2022-12-22 Kashiv Biosciences, Llc An improved process of purification of protein
WO2021240458A2 (en) 2020-05-28 2021-12-02 Kashiv Biosciences, Llc An improved process of storing and preparing the protein
WO2022195505A1 (en) 2021-03-16 2022-09-22 Kashiv Biosciences, Llc Novel formulation of fusion protein
CA3188632A1 (en) 2021-03-23 2022-09-29 Kashiv Biosciences, Llc An extraction process of pancrelipase and evaluation threof
CA3176410A1 (en) 2021-09-24 2023-03-24 Kashiv Biosciences, Llc An improved process for preparation of stable protein solution
US20240400612A1 (en) 2021-09-28 2024-12-05 Kashiv Biosciences, Llc An improved process for purification of fusion protein
AU2022358612A1 (en) 2021-09-28 2024-04-11 Kashiv Biosciences, Llc An improved process of purification of fusion protein
EP4408856A4 (en) 2021-09-28 2025-11-12 Kashiv Biosciences Llc IMPROVED PROCESS FOR PROTEIN PURIFICATION
AU2022359021A1 (en) 2021-10-08 2024-05-02 Kashiv Biosciences, Llc Separation of pre-peak in fusion protein sample by using size exclusion high performance liquid chromatography
EP4392156A4 (en) 2021-10-08 2025-06-25 Kashiv Biosciences, LLC Separation of pre-peak and post-peak in fusion protein sample by using size exclusion high performance liquid chromatography
WO2023112000A2 (en) 2021-12-17 2023-06-22 Kashiv Biosciences, Llc An improved method for separation of low molecular weight proteins
CA3255003A1 (en) 2022-03-23 2023-09-28 Kashiv Biosciences, Llc IMPROVED METHOD FOR SEPARATION AND ANALYSIS OF PANCREATIC ENZYMES

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110129468A1 (en) * 2008-02-29 2011-06-02 Biogen Idec Ma Inc. Purified immunoglobulin fusion proteins and methods of their purification
WO2013096791A1 (en) * 2011-12-23 2013-06-27 Genentech, Inc. Process for making high concentration protein formulations
WO2013180647A1 (en) * 2012-05-31 2013-12-05 Agency For Science, Technology And Research Chromatographic purification of immunoglobulin g preparations with particles having multimodal functionalities
US20140288272A1 (en) * 2013-03-15 2014-09-25 Alderbio Holdings Llc Antibody purification and purity monitoring
US20190256556A1 (en) * 2016-06-17 2019-08-22 Genentech, Inc. Purification of multispecific antibodies

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12378282B2 (en) 2020-05-01 2025-08-05 Kashiv Biosciences, Llc Process of purification of protein
US12435106B2 (en) 2020-05-01 2025-10-07 Kashiv Biosciences, Llc Process of purification of protein
US12600747B2 (en) 2020-05-01 2026-04-14 Kashiv Biosciences, Llc Process of purification of protein

Also Published As

Publication number Publication date
WO2021220251A1 (en) 2021-11-04
US12378282B2 (en) 2025-08-05
US12600747B2 (en) 2026-04-14
US20250282814A1 (en) 2025-09-11
EP4142792A4 (en) 2024-11-27
JP2026032129A (ja) 2026-02-25
EP4142792A1 (en) 2023-03-08
US20240158437A1 (en) 2024-05-16
US20250011395A1 (en) 2025-01-09
AU2021262609A1 (en) 2022-12-22
US12435106B2 (en) 2025-10-07
CA3182368A1 (en) 2021-11-04
JP2023523823A (ja) 2023-06-07
US20250353877A1 (en) 2025-11-20
JP7817743B2 (ja) 2026-02-19
US12139510B2 (en) 2024-11-12
US20240239838A1 (en) 2024-07-18

Similar Documents

Publication Publication Date Title
US12378282B2 (en) Process of purification of protein
KR101683415B1 (ko) 단일클론항체에 대한 정제 방법
JP5687469B2 (ja) タンパク質精製方法
US9249182B2 (en) Purification of antibodies using hydrophobic interaction chromatography
CN114375307B (zh) 产生抗α4β7抗体的方法
WO2002072615A1 (en) Method of purifying protein
US20250296014A1 (en) An improved process of purification of protein
JP2025166120A (ja) 抗体精製方法における宿主細胞タンパク質含有量を減少させるための方法及び宿主細胞タンパク質含有量を減少させた抗体組成物
US20240343758A1 (en) Process for purification of protein
JP2017507132A (ja) 抗体プロセス
US20250099873A1 (en) Process of affinity chromatography
US20260116917A1 (en) Process of purification of protein
US20250296015A1 (en) An improved process of purification of protein
US20260103542A1 (en) Process of purification of protein
US20260117000A1 (en) Process of purification of protein

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

AS Assignment

Owner name: KASHIV BIOSCIENCES, LLC, NEW JERSEY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NARAYAN, OM;GUPTA, TARUN KUMAR;THAKKAR, MAYANKKUMAR;REEL/FRAME:063246/0097

Effective date: 20221128

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

AS Assignment

Owner name: ACQUIOM AGENCY SERVICES LLC, AS COLLATERAL AGENT, COLORADO

Free format text: SHORT FORM TERM LOAN INTELLECTUAL PROPERTY ASSIGNMENT;ASSIGNOR:KASHIV BIOSCIENCES, LLC;REEL/FRAME:071150/0372

Effective date: 20250402

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

AS Assignment

Owner name: ACQUIOM AGENCY SERVICES, AS COLLATERAL AGENT, COLORADO

Free format text: SHORT FORM TERM LOAN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNOR:KASHIV BIOSCIENCES, LLC;REEL/FRAME:071824/0222

Effective date: 20250402

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION COUNTED, NOT YET MAILED

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION