Patent Application: US-74678708-A

Abstract:
a method of purifying a recombinant interferon protein involves providing an aqueous mixture of the recombinant protein and contaminating proteins ; precipitating the contaminating proteins from the aqueous mixture at a ph in a range of from 0 . 5 to 6 ; separating the aqueous mixture from the precipitated contaminating proteins ; and , eluting the separated aqueous mixture through a cation exchange column using a mobile phase with a salt or ph gradient , the gradient being from lower salt concentration or ph to higher salt concentration or ph , to produce a recombinant interferon protein fraction separated from other components of the aqueous mixture . the method provides for the recovery of recombinant interferon proteins in better yield and purity .

Description:
expression plasmid was purified with a maxi - prep plasmid purification kit ( qiagen , mississauga , on , canada ). f17 serum - free culture media and blasticidin were obtained from invitrogen ( carlsbad , calif .). pluronic ™ f68 and glutamine were from sigma - aldrich ( st . louis , mo .) and tryptone n1 from organotechnie ( la courneuve , france ). reagents for ifn - α2b purification and electrophoresis include anhydrous citric acid and tri - na citrate ( emd chemicals inc , darmstadt , germany ) 0 . 45 mm filtering units ( millipore , bedford , mass . ), nacl ( sigma - aldrich , st . louis , mo . ), fractogel ™ so 3 − ( m ) ( merck kgaa , darmstadt , germany ), econo - pac ® 10 columns ( bio - rad laboratories ), bradford reagent ( biorad , hercules , calif .) 2 μm filters ( pall corp , ann arbor , mich . ), nupage bis tris 4 - 12 % gradient gels , mes 20 × buffer ( invitrogen , carlsbad , calif . ), and coomassie stain ( sigma - aldrich , st . louis , mo .). trypsin ( promega , madison wis . ), neuraminidase , dithiothreitol , iodoacetamide and guanidine hcl ( sigma - aldrich , st . louis , mo . ), o - glycosidase ( roche ), tris hcl , ( bio - rad , mississauga , on ), high purity acetonitrile , formic acid and ammonium bicarbonate ( vwr international , montreal , qc ) and centricon ™ 3 , 000 mwl centrifugal filters ( millipore , bedford , mass .) were used for glycosylation analysis . the ifn - αantibody and elisa kit are from pbl biomedical laboratories ( new brunswick , n . j ., usa ) and bacterially produced ifn - α2b from cell sciences inc ( norwood , mass ., usa ). pnifty2 - 56k - seap plasmid is from invivogen ( san diego , usa ). the ifn - α2b gene was synthesized with human - optimized codons ( geneart ag , regensburg , germany ) according to the genbank accession no . ay255838 . the synthetic cdna was inserted as a bamhi / ecori fragment downstream of the cytomegalovirus ( cmv ) promoter into the pyd7 expression plasmid . this plasmid is a derivative of the previously described ptt vector [ 35 ] encoding the original functional elements in addition to a blasticidin resistance cassette . engineering of an ifn - α2b expressing hek293 - 6e stable clone and fed - batch production a hek293 cell line constitutively expressing the ebna1 protein of ebv ( clone 6e ) was used to generate ifn - producing clones . hek293 - 6e and ifn - producing clone are grown in suspension in serum - free f17 culture media supplemented with 1 % pluronic ™ f68 . cultures are grown at 37 ° c . and 5 % co 2 under constant agitation ( 120 rpm ). hek293 - 6e were transfected as previously described [ 35 ] with pvul - linearized pyd7 / ifn - α2b and selected in the presence of 2 μg / ml of blasticidin . the blasticidin resistant cells were next seeded into 96 well plates at 1 cell / well without blasticidin . after 3 - 4 weeks , emerging clones were expanded ( in the absence of blasticidin ) and tested for ifn - α2b expression by dot blot . the selection of ifn - producing clones was based on the levels of ifn - α2b expression and growth properties of the clones . the highest producers were amplified as suspension cultures and tested for ifn - α2b accumulation over 4 days of culture . one clone , identified as d9 , was selected because it is stably producing high ifn - α2b levels while maintaining a high growth rate ( doubling time of 24 hours ). the d9 clone was deposited at the international depositary authority of canada , national microbiology laboratory , public health agency of canada , 1015 arlington street , winnipeg , manitoba , canada , r3e 3r2 , under accession no . 021208 / 03 on dec . 5 , 2008 , the contents of which are herein incorporated by reference . for ifn - α2b production , cells are seeded at a density of 0 . 25 × 106 cells / ml in f17 antibiotic - free media in shaker flasks . twenty - four hours post seeding , the cultures are fed with 0 . 5 % peptones [ 35 , 46 ] and the cells are grown for an additional 7 - 8 days . optional addition of glucose ( 5 - 25 mm ) and glutamine ( 1 - 5 mm ) is performed 4 days post seeding . the culture medium of a fed - batch culture is collected by centrifugation at 1000 g for 10 min . the supernatant is then acidified to ph 3 . 6 - 3 . 8 with 1 m citric acid . acidification causes the formation of a precipitate which is removed by centrifugation . the clarified supernatant is then filtered on 0 . 45 mm filtering unit . purification of ifn - α2b from the filtered supernatant is performed on an äkta explorer ™ system ( ge healthcare , baie d ′ urfé , qc , canada ). the supernatant is loaded at a flow rate of 10 ml / min on a fractogel ™ so 3 − cation exchange column , previously equilibrated with 0 . 1 m tri - na citrate buffer ph 3 . 5 containing 0 . 35 m nacl . following a wash with 2 column volumes of the equilibration buffer , the ifn - α2b is then eluted with a ph gradient . the ph of the mobile phase is increased from ph 3 . 5 to ph 6 . 0 with 0 . 1 m tri - na citrate buffer ph 6 . 0 , plus 0 . 35 m nacl . the fractions containing ifn - α2b are pooled . an additional desalting step is performed on econo - pac ® 10 columns according to the manufacturer &# 39 ; s specifications . for the determination of glycosylation by enzymatic digestion the purified ifn - α2b , is desalted in 0 . 1 m nh 4 hco 3 buffer ph 5 and lyophilized , whereas for bioassays the purified ifn - α2b is desalted in pbs and sterile filtered . ifn - α2b recovered from the so 3 − column was quantified by measuring absorption at 280 nm in a spectrophotometer , with a nanodrop ™ nd - 1000 ( fisher scientific , montreal , qc , canada ), with a bradford assay and by elisa according to the manufacturer &# 39 ; s protocol . the concentration in the harvest was measured with elisa and used to calculate the percent recovery . to assess the purity level of ifn - α2b , 3 mg were analyzed by sds - page followed by coomassie staining . as hek293 - produced ifn - α2b migrates as two bands on sds - page , n - terminal amino acid sequences from both bands were obtained by automated sequencing performed at our sequencing facility . enzymatic treatments with neuraminidase and o - glycosidase were performed to remove sialic acid and o - linked sugars respectively . sequential digestions were performed in 50 mm phosphate buffer ph 5 . 0 on 100 μg of purified / desalted ifn - α2b . removal of sialic acid was done with 0 . 5 iu of neuraminidase for 1 h at 37 ° c . followed by addition of 15 mu of o - glycosidase . non - glycosylated recombinant ifn - α2b from e . coli and , glycosylated and deglycosylated hek293 - produced ifn - α2b were resolved on sds - page in parallel to compare migration profiles . migration profiles of glycosylated and deglycosylated hek293 - produced ifn - α2b were compared to non - glycosylated ifn - α2b produced in e . coli . the protein solution ( about 1 μg / μl in pbs buffer ) was desalted by filtration on a 3 , 000 mwl centricon ™ filter and diluted to its original concentration with deionized water . the solution was adjusted to 20 % acetonitrile , 0 . 2 % formic acid just prior to infusion at 1 μl / min into the electrospray interface of a q - tof 2 hybrid quadrupole time - of - flight mass spectrometer ( waters , milford , mass .). the mass spectrometer was set to acquire one spectrum every 2 seconds over the mass range , m / z 800 - 2600 . the protein molecular weight profile was generated from the mass spectrum using maxent ™ ( waters ). purified ifn - α2b was reduced , alkylated and digested with trypsin according to standard protocols . in summary , approximately 100 μg of the protein was dissolved in 1m tris hcl , 6m guanidine hcl , ph 7 . 5 containing 2 mm dithiothreitol ( dtt ) and incubated at 500 ° c . for 1 hour . the reduced cysteines were converted to carboxyamidomethyl derivatives using 10 - fold excess of iodoacetamide over dtt . the protein solution was then concentrated on a 3 , 000 mwl centricon ™ and diluted to 100 μl using 50 mm ammonium bicarbonate . this process was repeated a second time . trypsin ( 5 μg ) was added to the sample , which was then incubated overnight at 370 ° c . the tryptic digest was analyzed and fractionated by lc - ms using an agilent ™ 1100 hplc system coupled with the q - tof2 mass spectrometer . approximately , 60 μg of the protein digest was injected onto a 4 . 6 mm × 250 cm jupiter , 5 μm , 300 å , c18 column ( phenomenex , torrance , calif .) and resolved using the following gradient conditions : 5 % to 60 % acetonitrile , 0 . 2 % formic acid in 45 minutes , increasing to 95 % after 50 minutes ( 1 ml / min flow rate ). approximately , 60 μl / min of the hplc eluate was directed to the mass spectrometer while the remainder was collected in 1 minute fractions . the q - tof2 mass spectrometer was set to acquire 1 spectrum per second ( m / z 150 - 2000 ) whilst cycling between a low and high offset voltage within the collision cell ( 10 v and 35v , respectively ). this enabled the simultaneous detection of intact peptide and glycopeptide ions in the higher m / z regions ( low offset mode ) as well as the unique glycan oxonium ions in the lower regions of the spectrum ( high offset mode ). by screening the fractions in this manner it was possible to determine that only two of them ( fractions 25 - 26 and 26 - 27 minutes , respectively ) contained glycopeptides . glycopeptides were interrogated by collision induced dissociation ( cid ) to determine their amino acid sequence and glycan composition and by electron transfer dissociation ( etd ) to identify the site of linkage . etd preserves delicate modifications intact during the fragmentation process and is ideal for identifying the linkage sites of o - glycans [ 47 - 49 ]. the glycopeptide - containing fractions were infused at 1 μl / min into the electrospray ionization source of a ltq xl linear ion trap ( thermo fisher scientific ) capable of performing etd . the cid collision voltage was adjusted for optimum production of peptide fragment ions from the multiply charge glycopeptide precursor ions ( typically 25 - 30 v ). etd was performed using fluoranthene as the anionic reagent and with supplementary activation enabled . the optimal etd reaction time for these glycopeptides was 350 msec . a seap reporter gene assay based on expression plasmid containing an ifn - inducible promoter ( pnifty2 ) was used to assess the biological activity of glycosylated hek293 - produced ifn - α2b in comparison to non - glycosylated ifn - α2b . this experiment was performed as previously reported [ 50 ]. briefly , hek293 cells were transfected with the pnifty2 reporter plasmid , which encodes the secreted embryonic alkaline phosphatase ( seap ) under the control of the human isg56 promoter . transfected cells were plated in 96 well plates at a cell density of 105 cells / ml and stimulated , 24 h post transfection , with ifn - α2b at the indicated concentrations . following an additional 48 h period of incubation , the supernatants were collected and assayed for seap activity . the specific hydrolysis of paranitrophenyl phosphate ( pnpp ) was measured as a function of time to determine seap activity induced with ifn treatments , according to our previously described procedure [ 50 ]. seap activity is expressed the increase in absorbance at 410 nm over time . generation of a stable ifn - expressing hek293 cell clone and production of ifn - α2b in fed - batch cultures the expression plasmid pyd7 encoding the human ifn - α2b gene codon - optimized for expression in human cells ( fig1 a ) is derived from the previously described ptt vector [ 35 ]. the signal peptide sequence , cysteine residues involved in intramolecular cystine formation , and the threonine of the consensus sequence for o - glycosylation of human ifn - α2b are highlighted ( fig1 b ). the calculated molecular weight of the mature core protein ( a . a . 24 - 188 ) of ifn - α2b is 19 , 269 da . in order to generate ifnα2b - producing cells , hek293 were transfected with linearized pyd7 - ifnα2b and selected in the presence of blasticidin . the d9 clone , which stably produces ifn - α2b was isolated as described in materials and methods . the production of ifn - α2b with the d9 clone was performed in fed - batch culture . a coomassie - stained gel of daily samples from the culture media sampled daily for a period of 9 days shows that the levels of ifn - α2b plateau at 8 days ( fig2 a ), time at which cell - derived contaminating proteins begin to accumulate significantly . this is also the period of culture corresponding to a decline in cell number and viability ( fig2 b ). therefore , fed - batch productions were harvested at this point . it is noteworthy that , early during production , hek293 - produced ifn - α2b migrates with an apparent molecular weight of 2 kda greater than its predicted mass calculated from the amino acid sequence ( 19 . 3 kda ), while at around day 4 , a less abundant band of about 19 . 5 kda appears . purification of recombinant ifn - α2b by cation exchange chromatography and analysis by gel filtration and sds - page at the end of the production phase , the ifn - α2b is purified as described in material and methods . the ifn - α2b elutes in a single peak at ph 4 . 5 - 4 . 6 from the cation exchange column ( fig3 a ). the electrophoretic profiles of proteins contained in the harvest , the acid precipitate , the clarified harvest and eluted fractions , are shown on a coomassie - stained gel ( fig3 b ). the low level of ifn - α2b in the acid precipitate highlights the efficacy of acidification step to selectively remove protein contaminants . the absence of ifn - α2b in the flow through and in the wash suggests that ifn - α2b strongly binds to the so 3 − column . according to a conservative estimate performed by densitometric analysis of the sds - page resolved purified material , the purity of ifn - α2b exceeds 98 % after the so 3 − column and the final desalting step . the ph - dependence of precipitation of ifn - α2b is shown in fig4 . the ph from a d9 cells harvest was lowered incrementally by 0 . 5 ph units using citric acid or hcl . the resulting precipitates were washed with pbs and resuspended in 100 μl of 1 × lds buffer per ml of harvest . two ml equivalents of each precipitate were ran in parallel with the harvest ( the ph of which was 7 . 3 ). no significant precipitate was detectable at ph about 6 . 0 . also , ifn - α2b does not significantly precipitate at ph above 0 . 5 . following desalting in pbs , purified ifn - α2b was loaded on a superdex ™ 75 gel filtration column . the peak elution volume is almost identical to that of ovalbumin , a 44 kda protein , indicating that hek293 - produced ifn - α2b forms dimers at neutral ph ( fig5 a ). a coomassie - stained sds - page gel of ifn - containing fractions shows species with different electrophoretic mobilities ( fig5 b ), reflecting some glycosylation heterogeneity in the purified material . under reducing conditions , purified ifn - α2b migrates as a major band of approximately 21 kda , whereas under non - reducing conditions , ifn - α2b migrates with an apparent molecular weight of about 17 kda , a greater electrophoretic mobility typical of the presence of intramolecular disulfide bridges ( fig5 c ). the absence of dimers ( i . e . about 42 kda band ) in non - reducing conditions , indicate that the formation of dimers is independent of intermolecular disulfide bridges . the d9 clone produces hundreds of milligrams of ifn - a2b per liter of culture that are efficiently recovered ifn - α2b in the crude harvests of fed - batch cultures was quantified by elisa . the average concentration from two independent productions is 237 ± 11 mg / l , with a maximum of 316 mg / l when extra glucose and glutamine are added during production ( table 1 ). ifn - α2b recovered from the so 3 − column measured by elisa correlated well with measures obtained with a bradford assay and by absorbance at 280 nm using ifn - α2b molar extinction coefficient . the concentrations of ifn - α2b measured by elisa in the harvest and in the recovered fraction from the so 3 − column were used to determine the recovery . the mean concentration of ifn - α2b shows that between 70 and 80 % of the ifn - α2b produced could be recovered , for two independent productions for each condition ( table 1 ). these results are comparable in terms of volumetric productivity and recovery to some productions of non - glycosylated ifn - α2b performed in e . coli and in the methyltrophic yeast pichia pastoris ( table 2 ). one of the major therapeutic interests for producing ifn - α2b in mammalian cells is to generate a fully active and glycosylated protein . the apparent molecular weight of the major 21 kda product observed on sds - page suggests either that ifn - α2b produced in hek293 undergoes post - translational modifications or less likely , that the signal peptide is incorrectly processed . there is also a less abundant product of around 19 . 5 kda on sds - page . in order to ascertain that the signal peptide is accurately processed , n - terminal sequencing was performed on both products . the sequences obtained were identical and read c - d - l - p - q - t , as expected for a correctly processed signal peptide . we next determined whether ifn produced in hek293 is , o - glycosylated [ 36 ] and sialylated as previously reported for ifn - α2b produced by human peripheral blood leucocytes [ 37 ]. we performed a sequential digestion of purified ifn - α2b with neuraminidase and o - glycosidase to respectively remove sialic acid residues and o - linked saccharides . each digestion successively increase the electrophoretic mobility of purified ifn - α2b to generate a deglycosylated product that migrates as fast as non - glycosylated recombinant ifn - α2b produced e . coli ( fig6 ), demonstrating that ifn - α2b produced in hek293 cells is o - glycosylated and sialylated . note here the quasi absence of the lower about 19 . 5 kda product in the lane containing the non - digested ifn . we found that the majority of this product is lost during the purification process , as most of it remains bound to the column ( data not shown ). a minor band with lower electrophoretic mobility was still visible after glycosidases treatment , suggesting that this species might be fucosylated . a detailed mass analysis and glycosylation pattern of the purified ifn - α2b was next performed by mass spectroscopy . an electrospray ionization ( esi ) mass spectrum exhibiting the glycoform profiles associated with each charge state of purified ifn - α2b is shown ( fig7 a ). the masses of the principal glycoform of this protein correspond to the mature ifn - α2b peptide chain plus the glycans indicated ( fig7 b ). the most intense peak at 20 , 213 da appears to be composed of the mature peptide chain plus a single core type - 1 disialylated glycan ( hex1hexnac1sa2 ). a ms / ms analysis of the tryptic glycopeptides confirms the composition of this glycan . the sialylated ( mono and disialylated ) glycoforms appear to constitute 75 % of the total species . this percentage is likely to be underestimated , as some of the other peaks that cannot be assigned easily may be sialylated as well . the disialylated type 1 glycoform represents 50 % of the total peak area while the monosialylated glycoform is 10 % of the total . using electron transfer dissociation , we also show that the glycan is linked to the expected threonine residue at position 106 ( fig8 ). we next tested the purified glycosylated ifn - α2b produced in hek293 for biological activity in comparison to non - glycosylated form produced in e . coli . using a reporter gene assay we show that hek - produced ifn - α2b is biologically active as it induces the production of a secreted alkaline phosphatase ( seap ) reporter enzyme under the control of the human isg56 promoter ( fig9 ). in this assay , his assay shows that hek - produced ifn - α2b is at least as active as bacterially produced ifn - α2b .) we describe here the generation of a hek293 cell clone ( d9 ) stably producing up to 316 mg of glycosylated human recombinant ifn - α2b per litre of serum - free culture in a simple fed batch culture maintained for only 7 - 8 days . this is the highest volumetric production of ifn - α2b reported for a mammalian system . in addition , ifn - α2b productivity of the d9 clone is stable for over 4 months without selection pressure . we have further developed a rapid and reliable method for the efficient recovery of biologically active ifn - α2b . we also provide an exhaustive analysis of its glycosylation , demonstrating by mass spectrometry that ifn - α2b produced in hek293 cells is o - glycosylated and extensively sialylated . we show that the o - glycosylation of ifn - α2b produced in hek293 cells is heterogeneous but similar to ifn - α2b produced by human peripheral blood leucocytes . to date , the production of recombinant ifn - α2b and other cytokines in mammalian systems , particularly the development of clones stably expressing a cytokine of interest , has not been well exploited due to limitations in the volumetric productivity . one of the possible causes maybe that many cytokines exhibit strong anti - proliferative and cytotoxic activities on diverse cell lines [ 38 , 39 ], therefore strongly selecting for clones that show little or no cytokine expression . the d9 clone nonetheless grow almost as well as parental cells indicating that hek293 cells can adapt to proliferate in the presence of high levels of ifn - α2b . this adaptability of hek293 cells to a growth inhibitory cytokine suggests that they may be suitable hosts for the large - scale production of other interferons and cytokines . clearly , a production capacity of & gt ; 300 mg / l of ifn - α2b with more than 70 % recovery and & gt ; 98 % purity is a strong argument in favour of using hek293 cells for the large - scale productions of human recombinant cytokines . these results can be advantageously compared in terms of purified ifn - α2b obtained in e . coli t ( 300 mg / l ) [ 17 ] and the methyltrophic yeast pichia pastoris [ 40 ], two hosts insensitive to the growth inhibitory activity of ifn - α2b . however the productivity reported by srivastava et al [ 19 ] is 20 - fold greater than that reported here for the d9 clone . although we believe that the production capacity of hek293 cells for ifn - α2b can be improved , we doubt that such productivity can ever be achieved in mammalian cells , at least for a cytokine . however , the reported ifn - α2b recovery from prokaryotic systems ranges from 7 . 5 - 58 % ( table 2 ), which is lower than what we were able to achieve (& gt ; 75 %). purifications of recombinant proteins from prokaryotes usually require extraction from inclusion bodies and complicated refolding procedures , which reduce recovery yields [ 41 ]. protein refolding is a critical step in the processing of biotherapeutics , as incompletely refolded species lower specific activity and may trigger an immune response . antibodies to recombinant prokaryotic ifn - α2b have been detected in hcv patients with acquired resistance to ifn - α2b treatment [ 24 , 25 ], although it is not clear whether denatured ifn - α2b played a role in this case . because the vast majority of biotherapeutics including growth factors , cytokines and antibodies are secreted proteins , mammalian systems , unlike prokaryotes , allow for productions in perfusion as well as the development of non - denaturing purification procedures . the first and foremost advantage for the production of human recombinant proteins in mammalian systems is to generate proteins with the necessary posttranslational modifications required for full biological activity . n - glycosylation in particular , is often required for proper protein folding [ 42 ], protein - protein interactions , stability and optimal pharmacokinetics [ 43 ]. although o - glycosylation is less critical for structure and function of proteins , it has been shown for example to increase the serum half - life of igfbp6 by 2 , 3 folds over the non - glycosylated protein [ 44 ] and protect against proteolysis [ 45 ]. in a recent randomized study , o - glycosylated ifn - α2b was show to have an increased serum half life in comparison to non - glycosylated ifn - α2b [ 30 ]. we show here that human recombinant ifn - α2b produced in hek293 cells is o - glycosylated and sialylated . despite heterogeneity in the glycan structures , the nature and distribution of glycan moieties are quite similar to ifn - α2b naturally produced by human leukocytes [ 37 ]. approximately 50 % of our purified protein is disialylated , while another 30 % is monosialylated in comparison to 50 % and 10 % respectively for leukocyte - produced ifn . finally , we show that hek - produced ifn - α2b is biologically active and is more potent than non - glycosylated e . coli - produced ifn . the present invention demonstrates that the hek293 cell line is a suitable host for the high volumetric production of glycosylated human recombinant ifn - α2b and potentially other cytokines . the contents of the entirety of each of which are incorporated by this reference 1 . kaluz s , kabat p , gibadulinova a , vojtassak j , fuchsberger n , kontsek p : interferon alpha2b is the predominant subvariant detected in human genomic dnas . acta virol 1994 , 38 : 101 - 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