Description:
“METHOD FOR COST EFFECTIVE PURIFICATION COMMERCIAL PRODUCTION OF RECOMBINANT HUMAN ERYTHROPOIETIN”

FIELD OF INVENTION
The invention generally relates to the field of biochemistry/biopharmaceutical and in particular, relates to a method of purification of recombinant human erythropoietin (rHuEPO) from a cell culture harvest.
BACKGROUND
Erythropoietin (EPO) is a hormone that plays a crucial role in the production of red blood cells (erythropoiesis) by stimulating the bone marrow to produce more red blood cells. Synthetic version of the naturally occurring erythropoietin (EPO) protein, i.e., recombinant human erythropoietin (rHuEPO), of desired isoform in purified form, has wide usage in the medical field.
Achieving consistent purity levels of a desired isoform of rHuEPO is crucial in obtaining an effective drug. The purification of erythropoietin (EPO) on a commercial scale typically involves a series of chromatographic and filtration steps such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, and many more. However, as rHuEPO and its analogues contain sugar moieties in their glycan chains, there is generally heterogeneity in their isoforms which poses challenges for the purification process.
Various works in this field have contributed significantly to the advancement of the purification method of rHuEPO. For example, a method of rHuEPO purification disclosed in US-granted patent US7619073B2 published in 2009, for producing recombined erythropoietin having a purity (?98%), the method consisting of at least five chromatographic purification steps, i.e., at least two anion exchange chromatography, one hydrophobic interaction chromatography; one affinity chromatography; and one hydroxyapatite chromatography. Further, European granted patent EP1453857B1, published on August 13, 2014, discloses a method for recovering and purifying recombinant human erythropoietin (rHuEPO) from a cell culture medium that contains host cells. The method as disclosed, recites a combination of anion exchange chromatography, reverse phase chromatography, anion exchange chromatographic, and one or more size exclusion chromatographic. Further, WIPO published (June 5, 2003) patent application WO2003045996A1 discloses a method for recovering and purifying recombinant human erythropoietin (rHuEPO) from a cell culture medium containing host cells. The method involves several chromatographic steps; including anion exchange chromatography, reverse phase chromatography using a polystyrene resin, another round of anion exchange chromatography, and one or more size exclusion chromatographic steps.
As can be seen in all the aforementioned methods, the conventional purification processes involve at least five to eight chromatographic purification steps, which neither results in high product yield or any significantly high purity nor are efficient in terms of cost and resources. Consequently, the above-discussed methods are not convenient when it comes to scaling up the production as the resulting product is also less commercially viable. Further, in the purification process, it is essential to obtain a bioactive finished product that is commercially viable, which can be obtained by a streamlined and convenient purification method.
Thus, there is a pressing need for an efficient method of purification for commercially viable rHuEpo with fewer chromatography steps. There is also a need for a purification method for rHuEPO that can substantially increase the yield of the end product as compared to the conventional processes, takes less processing time, and is available at a significantly lower cost in an industrial setting.

OBJECT OF THE INVENTION
The object of the present invention, therefore, is to provide a unique and efficient purification method for purifying the rHuEpo using only two chromatographic steps.
Another object of the present invention is to increase reproducibility/productivity with advanced and increased process efficiency.
Still, another object of the present invention is to develop a method for purifying the rHuEpo which can be viably and economically scaled up to large-scale production and can be implemented for the industrial production of rHuEPO.
Additionally, the method aims to yield pure and commercially viable bioactive rHuEPO as compared to conventional processes with minimal cost and less processing time.

SUMMARY
Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of embodiments, along with the accompanying drawing figures in which like numerals represent like components.
Throughout this specification, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, or group of elements, but not the exclusion of any other element, or group of elements.
The present invention pertains to an efficient purification method for purifying recombinant proteins, particularly rHuEPO. In one aspect of the present invention, the method of purification of rHuEPO from cell culture is disclosed, the method comprises harvesting the cell culture to provide a clarified harvest (cell-free filtrate), subjecting the clarified harvest to an Anion Exchange (AEx) Negative mode chromatography Step-1 to obtain a flow-through (containing rHuEPO). The obtained flow-through is further subjected to an Anion Exchange (AEx) Positive mode Step-2 chromatography to obtain an elute containing purified human recombinant erythropoietin (rHuEPO).
In another embodiment, the method as disclosed comprises adjusting the pH of the clarified harvest in a range of 5.0-9.0 and conductivity in a range of 5-17mS/cm of clarified harvest before loading said clarified harvest onto an AEx chromatography column- Step-1.
The present invention in an embodiment discloses viral inactivation and neutralization of the flow-through obtained from AEx Negative mode chromatography Step-1. The viral inactivation and neutralization are achieved by adjusting the pH of the flow-through in a range of 3.2-4.5 and incubating the flow-through for 30-120 min at room temperature, which is followed by adjusting the pH of the flow-through in the range of 7.0 to 7.2. The viral inactivation and neutralization may be advantageously carried out before buffer exchange/diafiltration and Step-2 AEx positive mode chromatography.
Further, in another embodiment, the viral inactivated and neutralized flow-through is subjected to diafiltration via a polyethersulfone (PES) membrane having a cut-off range of 3-10 KDa, followed by the buffer exchange with 25mM Tris-HCl buffer, wherein the Tris-HCl buffer has a pH range of 7.0- 9.0 and a conductivity range of 2.2- 4mS/cm,. The diafiltration and buffer exchange may be advantageously carried out before Step-2 AEx positive mode chromatography.
In yet another embodiment the method comprises loading the diafiltered neutralized flow-through onto the Step-2 AEx chromatography column and eluting the rHuEPO by Tris NaCl elution buffer. The Tris NaCl elution buffer may have a pH range of 5.0-9.0 and conductivity in the range of 30-60mS/cm at 8-150C.
In one embodiment, the method of purification rHuEPO of the present invention may optionally comprise additional chromatographic steps, e.g. hydrophobic interaction Chromatography (HIC) Step-3 in a Negative mode.
In one embodiment, the method of purification rHuEPO of the present invention may comprise filtration steps such as diafiltration, ultrafiltration, or Nano-filtration, and at least one or more virus inactivation steps.
The efficient purification method of the present invention disclosed herein results in rHuEPO with purity 98.0 % to 99.8%, preferably 98.5%, more preferably 99, and most preferably 99.5%.

BRIEF DESCRIPTION OF DRAWINGS
The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure. The diagrams are for illustration only, which thus is not a limitation of the present disclosure.
For a fuller understanding of the disclosure, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:
Figure 1 depicts a flow diagram 100 depicting a purification method of rHuEPO, in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION
The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and the following description. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the present disclosure herein may be employed.
At the outset, for ease of reference, certain terms used in this application and their meanings as used in this context are set forth. To the extent a term used herein is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in at least one printed publication or issued patent. Further, the present techniques are not limited by the usage of the terms shown below, as all equivalents, synonyms, new developments, and terms or techniques that serve the same or a similar purpose are considered to be within the scope of the present claims.
The articles “a” and “an” as used herein mean one or more when applied to any feature in embodiments of the present invention described in the specification and claims. The use of “a” and “an” does not limit the meaning to a single feature unless such a limit is specifically stated. The article “the” preceding singular or plural nouns or noun phrases denotes a particular specified feature or particular specified features and may have a singular or plural connotation depending upon the context in which it is used. The adjective “any” means one, some, or all indiscriminately of whatever quantity.
The present invention provides an efficient purification of recombinant proteins. In one embodiment, the purification process is provided especially for rHuEPO and its desired isoforms. The method of purification consists of two chromatography steps interspersed with other steps (filtration, Buffer Exchange, viral inactivation) as required by the sample being processed.
The method is advantageously carried out by using fewer chromatographic steps preferentially a two-step chromatographic process. The preferred approach in this invention is to perform anion exchange chromatography (AEx) in negative mode as an initial step as opposed to capture chromatography which is used in the state of the art. This allows loading clarified harvest (cell-free filtrate) directly onto the column, even high amounts of the sample, without clogging or overloading the column. Additionally, the buffer composition of the chromatographic method does not cause excessive aggregation of components present in the clarified harvest. In addition, this step reduces further preparation steps and enables the use of large sample volumes with various components alongside the target rHuEPO.
In a preferred embodiment, rHuEPO may be produced in Chinese Hamster Ovary (CHO) cells using appropriate culture conditions, and process parameters such as temperature, conductivity, pH, etc.,.
Upstream processing to obtain rHuEPO from cell culture
In the method of the present invention, the host cells, capable of expressing rHuEPO and secreting the erythropoietin into the culture medium, are cultured in a protein-free and free-of-animal components culture medium.
In an embodiment, host cells for culturing are generally selected from a group comprising, mammalian cells or HEK293 cells for the production of a stable and high titer of recombinant proteins. In a preferred embodiment, host cells are Chinese Hamster Ovary (CHO) cells, in particular, CHO cells having EPO gene construct, producing rHuEPO may be cultured in CHO cells selected from the group comprising genetically modified CHO-DG44, CHO-S, CHO-K1, GS-CHO cells. Cultivation of the cells is performed in a culture medium, which is not limited to protein-free and free of animal components and may be selected from the group comprising CDM4Mab, Dynamis™, MAMPF77, and ActiPRO medium.
The cells are primarily grown in a flask and then scaled up to obtain a seed culture for inoculating the bioreactor. In another embodiment, a cell culture mode for the production of rHuEPO may be carried out as per the requirement of the invention, and the culture mode may be selected from and not limited to a group comprising serum-free culture mode, suspension culture mode, perfusion culture mode, and Fed-batch culture mode. In a preferred embodiment, the Fed-batch mode is selected for the production of rHuEPO, wherein cell culture is operated with two stages; a primary feed and a secondary feed stage. The primary feed is designed to support and achieve the desired cell growth and the secondary feed is designed to achieve the higher cell viability and desired glycosylation of the rHuEPO.
Primary & Secondary Feed
Cell cultures are fed with the primary feed on the third, fourth, and fifth days. The Primary feed comprises 3% Cell Boost 7a (Cytiva), and 0.3% Cell Boost 7b (Cytiva). Cell culture is fed with the secondary feed from the sixth day onwards till the cell viability reaches up to 70%. The secondary feed is 0.2% to 5% cell Boost 5 (Cytiva) plus a 20% mixture of Glucose, Galactose, and mannose (GGM). In one embodiment, the secondary feed comprises 0.3%, 0.4%, 0.5%, 0.7%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5% of the Cell Boost 5 plus 20%GGM. The secondary feed helps the cells to maintain cell viability and at the same time, support desired glycosylation. The GGM is designed in a manner to maintain the residual glucose at a level ranging from 1g/L to 5g/L on each culture day, preferably at 1g/L, 2g/L, 3g/L, 4g/L, 5g/L. Cell culturing is carried out for 16 days, preferably for 15 days, preferably for 14 days, preferably for 13, preferably for 12, preferably for 11, or preferably for 10 days. The most preferred is 12 days.
In an embodiment, cell culture harvesting, step 102, may be performed subsequently to a production phase lasting and limited to at most eight days. Herein, the cells are separated from the cell culture medium employing centrifugation followed by 0.2µm filtration but not limited to ultrafiltration, and diafiltration.
In yet another embodiment, alternatively, the cells may be removed from the cell culture medium via depth filtration. Depth filtration may be employed to remove insoluble particles present in the cell culture such as cells, debris, aggregates, and impurities.
In a preferred embodiment, rHuEPO titre obtained from harvesting the cell culture is in a range of 500-1200mg/L, preferably 600-1200mg/L, preferably 700-1200mg/L, preferably 800-1200mg/L, preferably 900-1200mg/L, preferably 1000-1200mg/L.
In another preferred embodiment, the clarified harvest/cell-free filtrate/protein solution/rHuEPO solution obtained from the upstream processing is further employed for preparing a load (protein solution) for Step-1 chromatography 104.
Downstream processing to obtain rHuEPO
In a preferred embodiment, the purification process in the present invention involves chromatographic steps as described below:
Anion Exchange (AEx) Negative mode chromatography Step 1 (104)
In a preferred embodiment, the process of the present invention in particular comprises an anion exchange (AEx) Negative mode Step-1 chromatography 104 as the first step. The anion exchange chromatography in negative mode Step-1 chromatography 104 is usually performed by equilibrating and loading an anion exchange chromatography column, followed by collecting the flow-through (solution containing rHuEPO) and subsequent separation of impurities from the desired rHuEPO.
Load preparation:
In another preferred embodiment, before loading, the pH of the clarified harvest is adjusted in a range of mildly acidic to alkaline pH, e.g. in a range of pH 5.0 to 9.0, preferably in a range of 6.0 to 7.0, more preferably in a range of 7.0 to 8.0, most preferably at or about 7.4 by using but not limited to 5N HCl (Hydrochloric Acid) and/or 5N NaOH (Sodium Hydroxide) solution. The conductivity of the clarified harvest is adjusted in the range of 5-17mS/cm, preferably in a range of 6-15mS/cm, 7-14mS/cm, 8-13mS/cm, 9-12 mS/cm, most preferably 10-11mS/cm by using but not limited to 4M NaCl (Sodium Chloride).
In an embodiment, the anion exchange chromatography may be carried out, with a quaternary polyethyleneimine or quaternary ammonium resin, Such as POROS™ 50 HQ Strong Anion Exchange Resin (Thermo Fisher SCIENTIFIC), or a resin having similar characteristics such as POROS™ XQ Strong Anion Exchange Resin (Thermo Fisher SCIENTIFIC), POROS™ 50 PI Strong Anion Exchange Resin (Thermo Fisher SCIENTIFIC), UNOsphere Q is a strong anion exchange resin (Bio-Rad) or other quaternary ammonium (Q) based resins including but not limited to Q Sepharose 6FF (Cytiva), Nuvia HP-Q Resin (Bio-Rad), and Macro-Prep High Q (Bio-Rad). The preferred resin for Step-1 chromatography 104 is POROS™ 50 HQ strong anion exchange resin.
In an embodiment, the anion exchange chromatography resin/column is equilibrated, loaded, and washed by using a buffer having a mildly acidic to alkaline pH, e.g. pH in the range of 5.0 to at or about 9.0, or 6.0 to 7.0, or 7.0 to 8.0, or 7.5 to 8.0, and most preferably at pH in a range of 7.2-7.5. Suitable equilibration buffers include but are not limited to, Tris (hydroxymethyl) aminomethane (THAM) hydrochloride, borate buffer, Tris (2-Amino-2-hy droxymethyl-propane-1,3-diol), Sodium phosphate, ammonium acetate, tricine (N-(Tri(hydroxymethyl)methyl) (glycine), bicine (2-(bis(2-hydroxyethyl)amino) ethanoic acid), TES (2-[Tris-(hydroxymethyl)methylamino]-1-ethane sulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS (N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid).
In another embodiment, the preferred buffer for equilibration and striping for Step 1 chromatography 104 may include 25mM-100mM Tris-Cl, preferably 50mM-75mM Tris-HCl, preferably 40-60mM Tris-HCl and most preferably 45-55mM Tris-HCl, at a pH in the range of 5.0-9.0, 6.0-7.0, 7.0-8.0, or 7.5-8.0, and most preferably at pH in a range of 7.2-7.5, and having the conductivity in a range of 5-17 mS/cm, 6 to 15 mS/cm, or 7 to 14 mS/cm, 8 to 13 mS/cm, 9 to 12 mS/cm, most preferably 10 to 11mS/cm.. The Striping buffer comprises 50mM Tris-HCl, at a pH of 7.4 + 1M NaCl having conductivity in a range of 90-100mS/cm.
In a preferred embodiment, Step-1 chromatography 104 of the purification process may be employed for the removal of product-related or culture process-related impurities from the cell culture harvest.
In an embodiment, step recovery of rHuEPO in Step-1 104 chromatography is in the range of 50-70%, preferably in the range of 60-70% and purity is in the range of 75-80%.
Virus Inactivation and Neutralization
In yet another embodiment, another additional step that may be performed as a key step prior to Step-2 106 chromatography may be virus inactivation and neutralization of the flow-through obtained from Step-1 chromatography 104 via incubation which is designed to be performed at a specific pH treatment. For example, the pH of the flow-through is adjusted in the range of 3.2 to 4.5, preferably 3.5-4.5, and most preferably 4.0-4.5 by using but not limited to glacial acetic acid.
The incubation time for the same may be preferably in the range of 30-120 min, or at least in the range 40-70 min, or at least in the range 50-100 min, or at least in the range of 60-90 min, most preferably the range of 45-60 min. Further, incubation is performed at low temperatures such as 25°C, 10°C or less, or 4°C or less. For example, the flow-through is incubated at a pH of about 4.0 for about 45-60 min at room temperature. The virus inactivation and neutralization step may be preferably carried out after Step-1 chromatography 104 and prior to Step-2 chromatography 106.
In an embodiment, the pH of virus inactivated and neutralized flow-through is adjusted in the range of 7.0-7.5, most preferably pH in a range of 7.2-7.5 using but not limited to 1M Tris solution prior to buffer exchange/ diafiltration.
Buffer Exchange/diafiltration
In a preferred embodiment, prior to the step-2 chromatography 106 (particularly prior to a step of anion exchange positive binding mode chromatography), it is desirable to carry out the filtration step which can be carried out by, not limited to, the ultrafiltration step and/or a diafiltration of the flow-through. The ultrafiltration and/or the diafiltration may be preferably carried out using a Polyethersulfone (PES) membrane having a cut-off of 3-15 KDa, most preferably a PES membrane having a cut-off of 10 kDa.
It is preferred to perform the buffer exchange during ultrafiltration and/or diafiltration of the flow-through, for example, the buffer may be selected from the group consisting of sodium phosphate, sodium citrate, MES (2-(N-morpholino) ethanesulfonic acid), Bis-Tris, TES, HEPES, preferably 10mM-25mM Tris-Cl. The pH of the buffer may be preferably in the range of 7.0 to 9.0, more preferably 7.5 to 8.5, and the conductivity is preferably in the range of 2.2-4mS/cm, more preferably at 3-3.5mS/cm, most preferably at 3mS/cm. The diafiltration step preferably, is performed prior to Step-2 chromatography 106.
However, the present invention also encompasses purification processes wherein no ultrafiltration steps and/or diafiltration steps are performed prior to Step-1 chromatography 104. The diafiltered virus inactivated neutralized flow-through obtained after the above step is the desirable load for Step-2 chromatography106.
Anion Exchange (AEx) Positive Binding mode Chromatography (Step-2 chromatography)
In a preferred embodiment, the process of the present invention in addition comprises an anion exchange positive binding mode chromatography Step-2 106. The anion exchange chromatography in positive binding mode is usually performed by process steps such as equilibrating and loading the column, followed by at least one wash of the column, and subsequently elution of rHuEPO. The Step-2 106 chromatography is carried out, preferably with the same resin as used in Step-1 chromatography 104.
In an embodiment, the anion exchange chromatography resin/column is preferably equilibrated, loaded, and washed by using/passing an equilibration buffer having a mildly alkaline pH, e.g. pH may be in a range of 6.5-9.0, preferably 7.0-8.0, most preferably pH at or about 8.0. Suitable buffers include but are not limited to borate buffer, Tris (2-Amino-2-hy droxymethyl-propane-1,3-diol), Sodium phosphate, ammonium acetate, tricine (N-(Tri(hydroxymethyl)methyl)glycine), bicine (2-(bis(2-hydroxyethyl)amino)ethanoic acid), TES, HEPES, TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid).
In an embodiment, an equilibration buffer for Step-2 chromatography 106 may be employed for equilibrating the column, said buffer comprising 10mM-50mM Tris-Cl, preferably 20-40mM Tris-Cl, most preferably 25-30mM Tris-Cl, having pH in the range of 7.0-9.0 more preferably in the range of 7.5-8.5, most preferably at pH 8.0 and having conductivity in the range of 2.2-4mS/cm, more preferably in the range of 3-3.5mS/cm, most preferably at 3mS/cm. Wash buffer II used in Step-2 chromatography 106 may comprise 20-50mM of sodium acetate (C2H3NaO2) having pH in a range of 3.0- 4.5, preferably 3.5-4.0, most preferably 3.8-4.0. The washing step using wash buffer II significantly removes the lower glycosylated form of rHuEPO in Step-2 chromatography 106 and thus aids in recovering the desired isoforms of rHuEPO
In a preferred embodiment, elution from the ion exchange column/resin may be obtained by increasing the conductivity of the mobile phase through the addition of salt, preferably but not limited to NaCl. Suitable buffers include but are not limited to, for example, borate buffer, triethanolamine/iminodiacetic acid Tris, ammonium acetate, tricine, bicine, TES, HEPES, TAPS, and the preferable buffer is Tris.
In an embodiment, the elution buffer comprises 25-50mM Tris, and 100-1000mM NaCl having pH in the range of 5.0-9.0, or 6.0-7.0, or 7.0-8.0, or preferably 7.5 to 8.0, and most preferably at pH 7.4 The conductivity of the elution buffer is in a range of 30 -60mS/cm, 40 -50 mS/cm, and most preferably at 45 mS/cm at an on-column temperature in a range of 8-150C, 8-120C and most preferably at 100C. The high salt concentration in the elution buffer enables the significant removal of aggregates and yields highly purified and bioactive rHuEPO.
In a preferred embodiment, Step-2 chromatography 106 is employed to selectively elute different charge isoforms mainly originating from different sialylation levels of the glycan moieties of the rHuEPO during the cell culture process.
The term "isoform," as used here, denotes a subset of the glycoproteins characterized by an identical amino acid sequence yet differing isoelectric points. These disparities reflect the variability in EPO glycosylation. Individual EPO molecules can exhibit variations in the extent, complexity, nature, antennarity, and order of attached -glycosyl, -sialyl, and -acetyl groups. Additionally, charged inorganic entities such as phosphate and sulphate may also influence the distinctive nature of a particular isoform. Consequently, the glycoprotein isoforms of the present invention are defined by their unique isoelectric points and identical amino acid sequences. Each isoform may indeed encompass multiple diverse EPO molecules in a stringent chemical context.
The isoform, as described in this invention, may comprise multiple glycoprotein forms with the same or very similar amino acid sequence and isoelectric point. These forms may also differ in other modifications carrying charges, such as acetylation and sulfation. The term "very similar amino acid sequence" implies that the protein's amino acid sequence includes those sequences functionally equivalent to the wild-type amino acid sequence, thereby exerting the same function. Specifically, a "very similar amino acid sequence" shares sequence homology, preferably sequence identity, with a reference amino acid sequence of at least 70%, preferably at least 80%, at least 90%, at least 95%, and most preferably at least 98% over a stretch of consecutive amino acids representing at least 50%, preferably at least 70%, at least 80%, at least 90%, at least 95%, and more preferably 100% of the entire reference amino acid sequence.
Isoforms
Both human erythropoietin and recombinant erythropoietin (expressed in mammalian cells) contain three N-linked and one O-linked oligosaccharide chains which together comprise about 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues located at positions 24, 38, and 83 while O-linked glycosylation occurs at a serine residue located at position 126. European Pharmacopoeia 5.2 has reported 1- 8 isoforms in the recombinant erythropoietin. The isoforms numbered 3 to 6 comprise up to 100% of the purified isoforms.
The EPO isoforms primarily result from variations in their glycosylation, featuring different numbers of negatively charged terminal sialic acid residues. EPO variants with an increased count of sialic acid residues, resulting in a more acidic isoelectric point (pI), exhibit enhanced biological activity and therapeutic efficacy. This is attributed to the ability of terminal sialic acid residues on glycostructures to impede the rapid clearance of rHuEPO in vivo through the asialoglycoprotein receptor route.
In the present invention, using this purification process, the inventors are able to purify rHuEPO containing these 4 major isoforms (isoforms numbered 3-6 as referred in the European Pharmacopeia). These 4 major isoforms comprise over 95% of the total isoforms in the purified rHuEPO of the present invention. The purified isoform distribution is similar and/or comparable to the isoform distribution pattern of the commercially available EPO.
Step-2 Chromatography 106 ensures high purity and specific bioactivity of rHuEPO. The step yield is in a range of 40-50% with selective isoforms of rHuEPO achieving desired protein purity of preferably over 98%, and more preferably over 99 %, aggregate less than 0.2%, and sialic acid content is in a range of 11-14 sialic acid per molecule of rHuEPO.
Hydrophobic Interaction (HIC) Negative mode Chromatography Step- 3 (Optional Step)
In another embodiment, the process of the present invention optionally/alternatively comprises hydrophobic interaction (HIC) Negative mode Step-3 chromatography after Step-2 chromatography 106. The hydrophobic interaction chromatography (HIC) in negative mode is usually performed by process steps such as equilibrating and loading the HIC column, followed by collecting flow-through (solution having rHuEPO), and subsequent elution of rHuEPO. This optional chromatography Step-3 is configured to help remove excess HCPs and HCDs when the culture process is run below 70% cell viability.
In an embodiment, the HIC is carried out, preferably with a cross-linked agarose resin, such as Phenyl Sepharose 6 Fast Flow Resin (Cytiva), or a resin having similar characteristics such as including but not limited to Butyl Sepharose 4 Fast Flow (Cytiva), OLIGO™ R3 (Thermo Fisher), Reverse Phase resins like SOURCE 15 and SOURCE 30 RPC and a likes.
In an embodiment, the HIC resin/column is preferably equilibrated, loaded, and washed using a buffer having a mildly acidic to alkaline pH, e.g. pH in a range of 5.0-9.0, or 6.0-7.0, or 7.0-8.0, or 7.5-8.0, and most preferably at pH 7.4. Suitable equilibration buffers include Tris (hydroxymethyl) amino methane (THAM) hydrochloride, borate buffer, Tris (2-Amino-2-hydroxymethyl-propane-1,3-diol), Sodium phosphate, ammonium acetate, tricine (N-(Tri(hydroxymethyl) methyl) (glycine), bicine (2-(bis(2-hydroxyethyl)amino) ethanoic acid), TES, HEPES, TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid). The most preferred buffer for equilibration comprises 25-75mM Tris-Cl, preferably 50-75mM Tris-Cl,, most preferably 50mM Tris-Cl,, having pH in a range of 5.0-9.0, or 6.0-7.0, or 7.0-8.0, or 7.5-8.0, and most preferably at pH 7.4. The conductivity of the equilibration buffer may be in the range of 30 -60 mS/cm, preferably in a range of 40-50mS/cm, or in a range of 50-60mS/cm, most preferably at 50 mS/cm. The Tris-HCL is combined with but not limited to 500-1000mM NaCl.
In an embodiment, the conductivity of the elute obtained from Step-2 chromatography 106 is adjusted in a range of 30-60 mS/cm, preferably in a range of 40-50mS/cm, or in a range of 50-60mS/cm, most preferably at 50mS/cm at pH in a range of 5.0-9.0, or 6.0-7.0, or 7.0-8.0, or 7.5-8.0, and preferably at pH 7.4.
In an embodiment, the elute obtained from Step-2 chromatography 106 is loaded to the column and a flow-through (containing rHuEPO) is collected. After loading, 2-10CV elution buffer (25-75mM Tris-Cl having pH in the range of 5.0-9.0 and conductivity in the range of 30-60mS/cm using 500-1000mM NaCl buffer) is passed through the column. The bounded impurities (if present) are eluted by passing 2-10CV of 30% isopropanol (IPA) solution through the column.
In another embodiment, the column is regenerated by passing 0.5N NaOH solution through the column further, passing WFI through the column till the conductivity equals that of WFI passed, and then the column is stored in 20% of ethanol (EtOH).
Advantages of the present invention
The present invention herein offers a highly efficient purification procedure, minimizing the number of chromatographic steps to a minimum of two. Notably, it eliminates the need for costly and resource/time-intensive other chromatographic purification steps such as affinity purification, particularly immunoaffinity purification, pseudo-affinity chromatography, reverse phase chromatography, and more.
In the present invention chromatography Step-I conducted in negative mode with specific pH and conductivity of buffer, efficiently eliminates the maximum amount of impurities present in the load. These impurities bind to the column in chromatography Step-I allowing the rHuEPO to flow through. This step ensures the removal of maximum impurities, enhancing the robustness of the protein. Step I also eliminates the need for an additional ultrafiltration and diafiltration step for purification, thereby minimising protein losses. Step I results in an overall increase in productivity, subsequently reducing both the time and cost of the entire procedure.
As a result, the first chromatography Step itself provides a significant recovery of rHuEPO in a range of 50%-70%, preferably in the range of 60-70%, and purity in a range of 75%-80%.
Chromatography Step-II
The purification process of the present invention, avoids potentially denaturing conditions, such as the use of urea-containing elution buffers. It also circumvents the high cost of equipment and consumables, as well as the frequent replacement of consumables, which would otherwise render the process expensive and result in significantly longer analysis times. Additionally, the process prevents issues such as poor resolution and increased losses, as observed in reverse-phase methods. Moreover, the utilization of pseudo-affinity resins, like blue Sepharose, is eschewed due to their inherent limitations. These resins lack high specificity, leading to potential non-specific binding. They also exhibit limited capacity and slow flow rates, thereby prolonging the separation process. Furthermore, similar resins may not be compatible with certain sample types or buffer systems, thus restricting their applicability.
The interplay of pH and conductivity of buffer used in Chromatography Step-I and Step-II ensures the isolation of desirable isoforms of rHuEPO with high bioactivity and purity. The process ensures high purity and specific bioactivity of EPO, with a desired protein purity of over 90%, preferably over 98%, and more preferably over 99%.
EXAMPLES
The following experiment illustrates the process of the present invention and in no way is intended to limit the disclosure.
EXAMPLE 1
UPSTREAM PROCESS
EPO Production – Cell culture
The recombinant human EPO is produced in the genetically modified dihydrofolate reductase deficient (DHFR-) Chinese Hamster Ovary (CHO-DG44 cells). The cultivation of the CHO-DG44 cells is carried out with a protein-free- and animal-derived component-free (ADCF) medium,. The cells are grown in the CDM4Mab or equivalent cell culture medium at 36°C in a humidified atmosphere of 5% CO2 saturation, on an orbital shaker platform rotating at 100-125 rpm and serially passage into fresh medium every 2-3 days to generate the seed culture for rHuEPO production in a bioreactor. The cells are assessed for rHuEPO secretion in the cell supernatant at regular intervals.
EPO Production in Fed-Batch
The production of rHuEPO is carried out in Fed-batch mode wherein culture, is operated with primary and secondary feeds. The primary feed in the fed-batch mode is carried out with combination of Cell Boost 7a and Cell Boost 7b from Cytiva. The culture is fed with primary feed on Days 3, 4, and 5 of the fermentation. The secondary feed containing 1% Cell Boost™ 5 [procured from Cytiva]) + 20% mixture of glucose, galactose, and mannose (GGM) is fed to the culture from day 6 onwards. The Cells are grown in a production bioreactor for 12+ 1 days with feeding till the cell viability reaches 70%. The GGM is used to maintain the residual glucose at a level of 4g/L on each culture day.
Harvesting the Cell Culture 102
Harvesting of the cell culture 102 is carried out after the production phase lasting at most on the eighth day. The cells/ cell debris is separated by centrifugation followed by filtration with 0.2 µm membrane or by depth filtration. rHuEPO titer in the harvest is in a range of 500-1200mg/L. The cell-free filtrate is then subjected to a purification procedure starting with the first chromatographic step.
DOWNSTREAM PROCESS
Load preparation- The pH of the clarified harvest is adjusted in a slightly alkaline range using 5N HCl/5N NaOH solution. The conductivity of the load sample is kept under 17mS/cm using 4M NaCl solution.
Anion Exchange (Negative mode) Step-1 chromatography
Buffer Concentration conductivity pH
Equilibration buffer 30-70mM Tris-HCl 8-12mS/cm 7.5.0-8.0
Stripping Buffer Equilibration buffer + 1M NaCl 90-100mS/cm 7.4

The column is equilibrated with 3 column volume (CV) of the equilibration buffer or till the UV baseline is zero and the pH & conductivity of the column are matched with that of the equilibration buffer. The clarified harvest is loaded onto the column, and the column is washed with not less than 2CV of equilibration buffer. The flow-through is collected in a separate container for further processing with Step 2 Anion Exchange (AEx) Positive mode chromatography 106. The preferred resin is quaternary polyethyleneimine based Strong Anion Exchange Resin having 50 µm particle size. The impurities in the column are eluted by washing with striping buffer wash with not less than 2CV. Afterward, the water for injection (WFI) is passed through the column till the conductivity of the column equals that of the input WFI and the column is stored after passing 20 % ethanol through the column with not less than 2CV. The obtained rHuEPO in the flow-through by Step-1 chromatography 104 is assessed for purity and yield by SDS-PAGE and RP-HPLC quantification respectively. The recovery of rHuEPO in flow-through for Step-1 chromatography 104 is found to be in a range of 60-70% and the purity is in a range of 75-80%.
Virus inactivation and Neutralization
The pH of the flow-through of Step-1 chromatography 104 is adjusted to slightly acidic with 17.4M glacial acetic acid in a range of 3.2 to 4.5. The viral inactivation in the flow-through is carried out by holding the flow-through at this pH 4.0 for 30–120 min at room temperature (25oC). The pH of the flow-through is then adjusted to a slightly neutral to alkaline range i.e., 7.0-7.2 with 1M Tris solution. The viral inactivated rHuEPO-containing flow-through is diafiltered on a PES membrane of cut-off 10kDa with 25mM Tris-Cl pH 8.0-8.2, conductivity 2.2-4mS/cm to prepare the solution for Step-2 - Anion Exchange (AEx) Positive mode chromatography, Step 106.
Anion Exchange (Positive binding mode) chromatography (Step-2 Chromatography)
Buffer Concentration conductivity pH
Equilibration buffer (wash buffer -I) 10-50mM Tris-HCl 3mS/cm 8.0
Sodium acetate buffer (wash buffer -II) 20-50mM under 5mS/cm 3.8-4.0
Elution buffer 20-50mM Tris and 100-1000mM NaCl 40-50mS/cm 7.4

The column is equilibrated by passing the column with not less than 2CV of Tris-HCl buffer (equilibration buffer) at a pH range of 8.0 (conductivity 3mS/cm). The post pH, conductivity, pressure, and volume at the end of the buffer pass are recorded. The diafiltered neutralized rHuEPO-containing flow-through is loaded onto the column. The flow-through is collected in a separate container with the UV absorbance peak ascending from 0.1 AU until it descends to 0.1 AU or the baseline.
This is followed by the first wash with equilibration buffer, the equilibration buffer is passed through the column until the absorbance on the system comes to baseline. The column is then washed with wash buffer II. The wash buffer-II (Sodium acetate buffer) is passed through the column till the absorbance on the system comes to baseline. The elution buffer (Tris NaCl buffer) is passed through the column and eluting fractions (containing rHuEPO) are collected until the peak of the fraction to be collected reaches the baseline.
EXAMPLE-2
Quality Assessment
The obtained rHuEPO as per example 1 is further characterized by SDS-PAGE, Western BLOT, IEF, RP-HPLC, SEC-HPLC, sialic acid content, Total protein by UV, and peptide mapping.
The purity of the rHuEPO achieved by the above method is >99% with less than 0.2% aggregate. The sialic acid content was in the range of 11-14 sialic acid per mole of EPO, The molecular weight of rHuEPO was approx. 38KDa as assessed by SDS-PAGE. The IEF revealed four major and one minor isoform isoforms. Selective isoforms comprise isoform 1 (0-5%), isoform 2 (5-20%), isoform 3 (10-35%), isoform 4 (15-40%) and isoform 5 (10-35%). In the final product host cell proteins were less than 100ng/mg protein (40-50ng/mg EPO) and host cell DNAs were less than 100pg/dose.
Hydrophobic interaction Chromatography (HIC) (Negative mode) Step -3 (optional)
Column Equilibration- the column is passed with not less than 2CV of Equilibration buffer (Tris-Cl with neutral to basic pH, with NaCl and conductivity under 70mS/cm). The resin used is Phenyl Sepharose 6FF
Process step: The conductivity, of rHuEPO-containing elute obtained from Step-2 Chromatography 106 is adjusted under 70mS/cm and pH is set from neutral to basic (pH 7.0-9.0). The Load rHuEPO-containing elute is loaded onto the column and the flow-through is collected in a separate container. The column is passed with less than 2CV of equilibration buffer through the column. The bounded impurities are eluted by passing with less than 2CV of 30% isopropanol through the column. The column is regenerated by passing 0.5N NaOH solution through the column. The water for injection (WFI) is passed through the column till the conductivity of the column equals that of the input WFI and the column is stored after passing 20 % ethanol through the column with not less than 2CV.
, Claims:We Claim:

1. A method of purification of human recombinant erythropoietin (rHuEPO) from a clarified cell culture harvest, comprising:
subjecting the clarified harvest to Step-1 Anion Exchange (AEx) Negative mode chromatography (104) to obtain a flow-through containing rHuEPO; and
subjecting the flow-through of step I chromatography to Step-2 Anion Exchange (AEx) Positive mode chromatography (106) to obtain an elute containing purified human recombinant erythropoietin (rHuEPO).
2. The method as claimed in claim 1, wherein the cell culture and harvesting (102) comprises:
preparing a culture of genetically modified CHO-DG44 (Chinese Hamster Ovary) cells in a culture medium, wherein the culture medium is devoid of protein and animal components;
feeding the culture medium with a primary and a secondary feed, wherein the primary feed comprises 3% Cell Boost 7a plus 0.3% Cell Boost 7b, and the secondary feed comprises 0.5% to 1% Cell Boost 5 plus 20% mixture of Glucose, Galactose, and mannose (GGM); and subjecting the culture medium to centrifugation followed by filtration to obtain the clarified harvest.
3. The method as claimed in claim 1, wherein the Step 1 chromatography (104) comprises adjusting a pH of the clarified harvest in a range of 7.0-8.0 and conductivity in a range of 10-11 mS/cm before loading said clarified harvest on to an AEx chromatography column.
4 The method as claimed in claim 1, wherein the flow-through is subjected to viral inactivation and neutralization by adjusting the pH of the flow-through in the range of 3.5-4.5 and incubating for 60-90 min at room temperature/250C followed by adjusting the pH of the flow-through in the range of 7.2 to 7.5 prior to Step-2 Chromatography (106).
5. The method as claimed in claim 4, wherein the viral inactivated and neutralized flow-through is further subjected to diafiltration via a 10 KDa polyethersulfone (PES) membrane and a buffer exchange with 25mM Tris-HCl at pH 8.0 and a conductivity range of 3.0mS/cm, prior to Step-2 chromatography (106).
6. The method as claimed in claim 1 and 5, wherein the Step-2 chromatography (106) comprises loading the diafiltered flow-through of step -1 chromatography onto the Anion Exchange chromatography column, removing the lower sialated rHuEPO by washing the column with Sodium Acetate buffer at pH 3.85 and eluting the desired isoforms of rHuEPO by Tris-HCl plus NaCl elution buffer at pH 7.4, conductivity 45mS/cm and an on-column temperature range of 8-12 degree C.
7. The method as claimed in claim 1, wherein the Step-1 chromatography (104) and Step-2 chromatography (106) are performed in the presence of a strong Anion Exchange resin having quaternized polyethyleneimine functional group and a pore size of 50µm.
8. The method as claimed in claim 1, wherein the Step-1 chromatography (104) yields 60%-70% rHuEPO with purity in a range of 75%-80%,
9. The method as claimed in claim 1, wherein step yield of the Step-2 chromatography (106) is in the range of 40-50% with desired isoforms of rHuEPO and purity in a range of 99-100 %, aggregates in a range of 0-0.2%, sialic acid content in a range of 12-14 sialic acid per mole of rHuEPO.
10. The method as claimed in claim 1, further comprises an optional hydrophobic interaction (HIC) negative mode chromatography Step - 3 chromatography using Phenyl Sepharose 6FF resin.
11. A purified human recombinant erythropoietin (rHuEPO) obtained by the method as claimed in any one of claims 1-10.