( 12 ) Patent Application Publication ( 10 ) Pub . No . : US 2018 / 0230445

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US 20180230445A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2018 /0230445 A1 JALURIA et al. (43 ) Pub . Date : Aug . 16 , 2018 ( 54) MANUFACTURING OF ALKALINE Publication Classification PHOSPHATASES (51 ) Int. CI. C12N 9 / 16 (2006 . 01) (71 ) Applicant: Alexion Pharmaceuticals , Inc. , New A61P 19 / 08 (2006 . 01) Haven , CT (US ) (52 ) U . S . CI. CPC ...... C12N 9/ 16 ( 2013 . 01 ) ; C12Y 301 /03001 (72 ) Inventors : Pratik JALURIA , Madison , CT (US ) ; ( 2013 . 01 ) ; A61K 38 / 00 ( 2013 .01 ) ; C12N Siguang18 SUI, Cheshire , CT (US ) 2500 /22 (2013 .01 ) ; A61P 19/ 08 (2018 .01 ) (21 ) Appl. No. : 15 /751 , 498 (57 ) ABSTRACT A method for producing a recombinant polypeptide, com ( 22 ) PCT Filed : Aug . 16 , 2016 prising : (a ) providing a 100 L to 25 ,000 L fed -batch biore actor comprising ( i) cells capable of expressing the recom ( 86 ) PCT No . : PCT/ US2016 / 047166 binant polypeptide asfotase alfa (SEQ ID NO : 1) , and (ii ) a $ 371 ( C ) ( 1 ), culture medium suitable for conducting such expression the (2 ) Date : Feb . 9 , 2018 culture medium comprising about 25 uM to about 300 uM zinc; ( b ) culturing the cells under conditions suitable to express the recombinant asfotase alfa wherein the pH of the culture medium is about 6 . 7 to about 7 . 1 , and wherein zinc Related U .S . Application Data is added into said culture medium such that the zinc con ( 60 ) Provisional application No. 62 / 206 , 001 , filed on Aug . centration in the culture medium is maintained at a concen 17 , 2015 . tration of about 25 uM to about 300 uM of zinc . Patent Application Publication Aug. 16 , 2018 Sheet 1 of 54 US 2018 /0230445 A1

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FIG . 53 US 2018 /0230445 A1 Aug . 16 , 2018

MANUFACTURING OF ALKALINE BRIEF SUMMARY PHOSPHATASES [0005 ] Disclosed herein are compositions of alkaline phosphatases ( e . g ., asfotase alfa ) which have specific char SEQUENCE LISTING acteristics ( e . g . , particular glycan structures , particular total [0001 ] The amino acid sequences listed in the accompa sialic acid content ( TSAC ) values, etc . ) and manufacturing nying sequence listing are shown using standard three - letter processes utilized to produce the alkaline phosphatases ( e . g . , code for amino acids, as defined in 37 C . F . R . 1 .822 . The asfotase alfa ) having such specific characteristics. Such Sequence Listing is submitted as an ASCII text file , created alkaline phosphatases ( e . g . , asfotase alfa ) are suited for use on Aug . 16 , 2016 , named 0351 WO _ SL . txt and 6 ,604 bytes in therapy , for example, for treatment of conditions associ in size , which is incorporated by reference herein . ated with decreased alkaline phosphatase protein levels and /or functions (e . g ., insufficient cleavage of inorganic FIELD OF THE INVENTION pyrophosphate (PPi ) ) in a subject, for example , a human subject. [ 0002 ] The disclosure is directed to a method for produc [0006 ] In one aspect, the present disclosure provides a ing a recombinant polypeptide , comprising : ( a ) providing a method for producing a recombinant polypeptide having 100 L to 25 ,000 L fed -batch bioreactor comprising ( i ) cells alkaline phosphatase function . In various embodiments , the capable of expressing the recombinant polypeptide asfotase alkaline phosphatase function may include any functions of alfa (SEQ ID NO : 1 ) , and ( ii ) a culture medium suitable for alkaline phosphatase known in the art , such as enzymatic conducting such expression , the culture medium comprising activity toward natural substrates including phosphoetha about 25 uM to about 300 uM zinc ; ( b ) culturing the cells nolamine (PEA ), inorganic pyrophosphate (PPi ) and pyri under conditions suitable to express the recombinant asfo doxal 5 ' - phosphate (PLP ) . Such recombinant polypeptide tase alfa ; wherein the pH of the culture medium is about 6 . 7 can comprise asfotase alpha (SEQ ID NO : 1 ) . to about 7 . 1 , and wherein zinc is added into said culture [0007 ] In some embodiments , the method disclosed herein medium such that the zinc concentration in the culture further comprises adding zinc into said culture medium for medium is maintained at a concentration of about 25 uM to producing the recombinant polypeptide. Zinc may help about 300 uM of zinc . improving the activity and /or stability of the recombinant polypeptide. In some embodiments, zinc may be added to BACKGROUND provide a zinc concentration of from about 1 to about 300 [0003 ] Hypophosphatasia (HPP ) is a life - threatening , uM in said culture medium . In one embodiment, zinc may be genetic and ultra - rare metabolic disorder that results in a added to provide a zinc concentration of from about 10 to failure to produce functional tissue nonspecific alkaline about 150 UM ( e . g . , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , phosphatase ( TNSALP ). It leads to the accumulation of 110 , 120 , 130 , 140 , or 150 uM ) in the culture medium . In unmineralized bone matrix ( e . g . rickets , osteomalacia ), char some embodiments , zince is added to provide a zinc con acterized by hypo -mineralization of bones and teeth . When centration in the culture medium of from about 25 uM to growing bone does not mineralize properly , an impairment about 150 uM , or about 60 uM to about 150 uM . In one of growth is a result that disfigures joints and bones . This particular embodiment, zinc is added to provide a zinc result in turn impacts motor performance , respiratory func concentration in the culture medium of from about about 30 , tion , and may even lead to death . Different forms of HPP 60 , or 90 uM of zinc . In one particular embodiment, zinc is were discovered to include perinatal , infantile , juvenile , and added to provide a zinc concentration in the culture medium adult HPP . Recently , six clinical forms have been defined , of about 28 uM . In some embodiments , the zinc is added into most based upon age at symptom onset, to include perinatal , said culture medium in a bolus , continuously , or semi benign prenatal, infantile , juvenile , adult , and odonto - HPP. continuously . Asfotase alfa is an approved first - in - class targeted enzyme [0008 ] In some embodiments , the method disclosed herein replacement therapy designed to address defective endog further comprises controlling the pH of said culture medium enous TNSALP levels . For treating HPP with TNSALP, see for producing the recombinant polypeptide . For example, Whyte et al. , 2012 N Engl J Med . 366 : 904 - 13 . the pH may be set at about 6 .8 to about 7 . 0 . In one particular [ 0004 Asfotase alfa (STRENSIQR , Alexion Pharmaceu embodiment , the pH is set at about 6 . 9 . ticals , Inc . ) is a soluble fusion glycoprotein comprised of the [0009 ] In some embodiments , the method disclosed herein catalytic domain of human TNSALP , a human immuno further comprises adding at least one extra fresh culture globulin G1 Fc domain and a deca aspartate peptide ( i . e . , medium bolus feed to the original cell - containing culture D10 ) used as a bone -targeting domain . In vitro , asfotase alfa medium during culturing and /or polypeptide production . binds with a greater affinity to hydroxyapatite than does Such addition of fresh culture medium may improve activity soluble TNSALP lacking the deca - aspartate peptide , thus (e . g ., specific activity ) of the produced recombinant poly allowing the TNSALP moiety of asfotase alfa to efficiently peptide . In one embodiment, at least one, two , three, or four degrade excess local inorganic pyrophosphate (PPi ) and feed bolus ( es ) are added to the culture medium during restore normal mineralization . Pyrophosphate hydrolysis culturing . In one particular embodiment, at least four feed promotes bone mineralization , and its effects are similar boluses are added . In some embodiments , the feed bolus among the species evaluated in nonclinical studies . Initial addition ( s ) improves specific activity of the recombinant efficacy studies were conducted in a mouse model of HPP polypeptide . The cells disclosed herein for producing the ( Akp2 -/ - mice ). The Akp2- / - mouse model, created by recombinant polypeptide may be any cells ( e . g . , mammalian inactivating the TNSALP gene (Narisawa et al. 1997 Dev cells ) known in the art . In some embodiments , the cells are Dyn . 208 :432 - 46 ) , shares many common features of the selected from the group comprising CHO , NSO / 1 , PER . CO , human condition including accumulation of unmineralized COS - 7 , human embryonic kidney line (293 or 293 cells bone matrix . subcloned for growth in suspension culture ), BHK , TM4, US 2018 /0230445 A1 Aug . 16 , 2018

CVI, VERO - 76 , HeLa , MDCK , BRL 3A , W138 , Hep G2, from about 0 . 5 to about 1. 5 ; and (x ) molar ratio of zinc per MMT 060562 , TRI, MRC 5 , FS4 cells , and Hep G2 cells . In mole of recombinant polypeptide of from about 0 . 5 to about some embodiments , the cells are CHO cells. 3 . 0 . [0010 ] In some embodiments , the cells the cells are grown [ 0012 ] In one embodiment, the produced recombinant at a first temperature for certain of time for cell growth and protein has from about 0 . 7 to about 1 . 19 free cysteine per then shifted to a second temperature for polypeptide expres half molecule . sion . For example , in some embodiments , it is disclosed the [0013 ] In one embodiment, the produced recombinant method further comprises culturing the cells at a first tem protein has phosphorylation at Ser 93 at a percentage of from perature until reaching a cell density of at least about about 13 . 5 % to about 35 . 7 % . 2. 5x10º viable cells , and then shifting to a second tempera [ 0014 ] In one embodiment, the produced recombinant ture which is lower than the first temperature for recombi protein has no less than 90 .0 % main peak , no more than nant polypeptide expression . For example , in some embodi 6 . 0 % for acidic peaks , and no more than 4 . 0 % for basic ments , the first temperature is about 35° C . to about 37 .5° C . peaks on an AEX chromatogram . In one particular embodi In some embodiments , the second temperature is about 29° ment , the produced recombinant protein has no less than C . to about 35° C . In some embodiments , the first tempera 93 . 7 % main peak , no more than 4 . 9 % for acidic peaks , and ture is about 37° C . and the second temperature is about 30° no more than 3. 4 % for basic peaks on an AEX chromato C . In some embodiments , the first temperature is about 36 . 5° gram . C . and the second temperature is about 33° C . [0015 ] In some embodiments , the produced recombinant [0011 ] In another aspect , the present disclosure provides a protein has a total sialic acid content ( TSAC ) between about recombinant polypeptide produced by any one of methods 0 . 9 to about 3. 5 mol sialic acid /mol protein monomer. In one disclosed herein . Such produced recombinant polypeptide embodiment, the produced recombinant protein has a mean may have at least one of specific characteristics originated Total Sialic Acid Content ( TSAC ) value from about 1 . 2 to from the producing methods disclosed herein . Such charac about 3 . 0 mol sialic acid /mol per monomer. In one embodi teristics may include at least one selected from the group ment, the produced recombinant protein has a mean Total comprising: ( a ) a total sialic acid content ( TSAC ) between Sialic Acid Content value from about 1 . 9 to about 2 . 7 mol about 0 . 9 to about 3 . 5 mol sialic acid /mol protein monomer ; sialic acid /mol per monomer . In one particular embodiment, ( b ) an isoelectric focusing ( IEF ) between about 5 . 2 to about the produced recombinant protein has a mean Total Sialic 6 . 7 ; ( c ) a major glycan structure as shown in FIG . 41 or FIG . Acid Content value from about 1 .85 to about 2 . 28 mol sialic 42 ; ( d ) a 2 - AB labeled oligosaccharide chromatogram pro acid /mol per monomer. file as shown in FIG . 38 or 39 ; ( e ) a MALDI- TOF glyco [0016 ) In one embodiment, the produced recombinant peptide finger printing profile as shown in FIG . 40 , or 44 -49 ; protein has a binding molar ratio of magnesium ion of less ( f ) a major band on a reduced SDS - PAGE having a molecu than about 0 . 15 . In some embodiments, the produced recom lar weight of about 88 - 108 kDa and no less than about 85 % binant protein has a molar ratio ofmagnesium of about 0 . 05 of the total amount of the produced recombinant polypep to about 0 . 10 . In one particular embodiment, the produced tide; ( g ) a major band on a non - reduced SDS- PAGE having recombinant protein has a binding molar ratio of magnesium a molecular weight of about 194 to about 273 kDa and no ion of about 0 . 12 . less than about 85 % of the total amount produced recom [0017 ] In some embodiments , the produced recombinant binant polypeptide ; (h ) no less than about 95 . 0 % for dimers protein comprises at least about 95 . 0 % for dimers of the of the recombinant polypeptide and no more than about 5 . 0 % 0 for aggregates by size exclusion high pressure liquid recombinant polypeptide about 5 . 0 % or less polypeptide chromatography (HPLC ) ; ( i ) no less than about 95 . 0 % aggregates , i . e . , as measured by size exclusion HPLC . In one purity via reverse - phase high pressure liquid chromatogra embodiment, the produced recombinantprotein comprises at phy (RP -HPLC ) ; (i ) no less than about 90 . 0 % for main peak , least about 96 . 8 % dimers of the recombinant polypeptide no more than about 6 . 0 % for acidic peaks , and no more than and about 3. 2 % or less polypeptide aggregates, i. e ., as about 4 . 0 % for basic peaks via Anion Exchange Chroma measured by a size exclusion HPLC . In one particular tography ( AEX ) ; ( k ) a hydroxyapatite (HA ) binding per embodiment, the produced recombinant protein comprises at centage of about 75 to about 125 % ; ( 1 ) a product specific least about 97 .6 % dimers of the recombinant polypeptide activity (PNPP ) of about 620 to about 1250 units /mg ; ( m ) a and about 2 . 4 % or less aggregates , i . e . , as measured by size Km of about 13 to about 69 CPM in an inorganic pyrophos exclusion HPLC . phate (PPi ) hydrolysis assay ; (n ) a Kcat of about 65 to about [0018 ] In one embodiment, the produced recombinant 165 s - in an inorganic pyrophosphate (PPI ) hydrolysis protein has no less than 95 . 0 % purity as measured by assay ; ( o ) a pl range of about 6 . 45 to about 6 . 95 for all peaks RP - HPLC . In one particular embodiment, the produced on capillary electrophoresis ; ( p ) peaks on a MALDI- TOF recombinant protein has no less than 97 .6 % purity as mea mass spectrum as shown in FIG . 34A after deglycosylation ; sured by RP -HPLC . ( q ) peaks on a MALDI - ToF mass spectrum as shown in FIG . 100191 In some embodiments, the produced recombinant 34B after reduction and deglycosylation ; (r ) peaks on a protein has a Hydroxyapatite (HA ) binding percentage of MALDI- TOF mass spectrum as shown in FIG . 35 ; (s ) a about 75 to about 125 % . In one embodiment, the produced phosphorylation profile as shown in FIG . 36 ; ( t ) a si?lyated recombinant protein has a mean % hydroxyapatite binding glycans profile on a negative MALDI- ToF mass spectrum as of from about 85 % to about 97 % . In one particular embodi shown in FIG . 37A ; ( u ) a neutral glycans profile on a ment, the produced recombinant protein has a mean % positive MALDI- ToF mass spectrum as shown in FIG . 37B ; hydroxyapatite binding of from about 90 % to about 91 % . ( v ) a molar ratio of magnesium per mole of recombinant [0020 ] In one embodiment, the produced recombinant polypeptide of from about 0 .03 to about 0 . 15 ; ( w ) a molar protein has a specific activity (PNPP ) of about 620 to about ratio of calcium per mole of recombinant polypeptide of 1250 units /mg . In one particular embodiment, the produced US 2018 /0230445 A1 Aug . 16 , 2018 recombinant protein has a mean specific activity (PNPP ) of (0032 ] FIG . 5 is a graph showing the neural glycan profiles about 904 . 0 to about 907 . 7 U /mg . (by matrix assisted laser desorption /ionization -time of flight, [ 0021] In some embodiments , the recombinant polypep or MALDI- TOF ) of asfotase alfa produced by an exemplary tide is encoded by a polynucleotide encoding a polypeptide production process (# 1 ) with or without temperature shift comprising the sequence as set forth in SEQ ID NO : 1, or a ing . Reference represents a standard asfotase alfa produced sequence completely complementary to SEQ ID NO : 1 . by a previous 20K process. 10022 ] The recombinant polypeptide disclosed herein may [ 0033 ] FIG . 6 are graphs showing the impact of shifting be produced under an industrial or a commercial scale . For production temperature on the aggregate levels (panel A ) example, in some embodiments , the fed - batch reactor is 200 and TSAC (panel B ) of produced asfotase alfa . The error L to 20 ,000 L . In some embodiments , the fed -batch reactor bars represent standard deviation under each condition . is 2 , 000 L to 20 , 000 L . [0034 ] FIG . 7 is a graph showing the impact of culture pH [0023 ] In another aspect , the present disclosure provides a on the TSAC values of asfotase alfa produced by exemplary pharmaceutical formulation comprising a composition com Process # 2 after 10 or 12 days of culturing . prising the recombinant polypeptide disclosed herein , in [0035 ] FIG . 8 are graphs showing the impacts of medium combination with a pharmaceutically acceptable carrier, feed addition represented by different concentrations of diluent or excipient. glycose ( glc ) ; panel A ) and feed addition mode (panel B ) on [0024 ] In another aspect, the present disclosure provides a asfotase alfa TSAC in exemplary Process # 2 . method of using the recombinant polypeptide, or the phar [0036 ] FIG . 9 are graphs showing the impact of zinc maceutical formulation discussed herein to increase cleav supplementation on asfotase alfa activity (panel A ; zinc age of inorganic pyrophosphate (PPi ) in a subject. supplemented at Day 9 ) and the impact of zinc concentration [ 0025 ]. In another aspect, the present disclosure provides a on cell growth (panel B ) and viability (panel C ) in exem method of treating a subject, comprising administering to plary Process # 2 . the subject suffering from a condition associated with alka [0037 ] FIG . 10 are graphs showing the impact of pH and line phosphatase deficiency a therapeutically effective production temperature on asfotase alfa TSAC (panel A ) , amount of the recombinant polypeptide, or the pharmaceu protein A titer (panel B ), and volumetric activity (panel C ) tical formulation discussed herein . Such condition associ in exemplary Process # 3 . ated with alkaline phosphatase deficiency includes, for [0038 ] FIG . 11 are graphs showing the impact of growth example , hypophosphatasia (HPP ) and neurofibromatosis temperature on volumetric activity (panel A ) and asfotase type I (NF1 ) . Such hypophosphatasia (HPP ) may be any one alfa fragmentation (panel B , measured by SEC ) in exem of perinatal, infantile , juvenile , or adult HPP . Such condition plary Process # 3 . may be characterized with unmineralized bone matrix and / or [ 0039 ] FIG . 12 are graphs showing the impact of produc hypo -mineralization of bones and teeth . For example , such tion temperature and pH on asfotase alfa aggregation (panel unmineralized bone matrix may lead to rickets and /or A , measured by SEC ), % acidic peak ( panel B , measured by osteomalacia . AEX ) , asfotase alfa fragmentation (panel C , measured by [0026 ] In some embodiments , such subject is a mammal . SEC ), and % basic peak (panel D , measured by AEX ) in In some embodiment, such subject is a human . exemplary Process # 3 . [0040 ] FIG . 13 are graphs showing the impact of seeding BRIEF DESCRIPTION OF THE DRAWINGS density and temperature shift timing on volumetric activity [0027 ] In the appended drawings: (panel A ) and asfotase alfa fragmentation (panel B , mea [0028 ] FIG . 1 are graphs showing the comparisons of cell sured by SEC ) . Seeding stands for seeding density in exem growth (panel A , left ) and viability (panel A , right) and plary Process # 3 . overall glucose (panel B , left ) and lactate ( panel B , right) [0041 ] FIG . 14 are graphs showing the impact ofmedium concentrations between an exemplary production process feed ( amount and timing ) on asfotase alfa volumetric activ ( # 1 ) with or without temperature shifting for protein pro ity ( panel A ) , aggregation ( panel B , measured by SEC ) , % duction . Control represents the average result of two runs acidic peak ( panel C , measured by AEX ) , % basic peak with temperature shifting . (panel D , measured by AEX ) , and TSAC (panel E ) in 10029 ] FIG . 2 are graphs showing the comparisons of exemplary Process # 3 . Protein A bindable titer (panel A ), volumetric activity (panel [0042 ] FIG . 15 are graphs showing the impact of culture B ) and specific activity ( panel C ) of asfotase alfa produced medium pH on cell growth (panel A ) and viability (panel B ) using an exemplary production process ( # 1 ) with or without up to ten days in exemplary Process # 4 . temperature shifting . Control represents the average result of [0043 ] FIG . 16 are graphs showing the impact of produc two runs with temperature shifting . tion temperature on cell growth ( panel A ) and viability [0030 ] FIG . 3 are graphs showing the comparisons of ( panel B ) up to ten days in exemplary Process # 4 . AEX ( Anion Exchange Chromatography ) acidic peak ( % ) [0044 ] FIG . 17 are graphs showing the impact of culture ( panel A ) and SEC (Size Exclusion Chromatography) aggre medium pH on glucose (panel A ) and lactate ( panel B ) gate ( % ) ( panel B ) measurements of asfotase alfa produced concentrations in the culture medium up to ten days in using an exemplary production process (# 1 ) with or without exemplary Process # 4 . temperature shifting . [0045 ] FIG . 18 are graphs showing the impact of produc [ 0031] FIG . 4 are graphs showing the comparisons of tion temperature on glucose (panel A ) and lactate (panel B ) non -reduced LoC ( Lab -on - Chip ; panel A ), reduced LoC concentrations in culture medium up to ten days in exem ( panel B ) , and TSAC (total sialic acid content; panel C ) of plary Process # 4 . asfotase alfa produced using an exemplary production pro [0046 ] FIG . 19 are graphs showing the impact of culture cess ( # 1 ) with or without temperature shifting . medium pH on protein A titer (panel A ) , volumetric activity US 2018 /0230445 A1 Aug . 16 , 2018

(panel B ) , specific protein A productivity (panel C ) , and by different processes (blue line : a control process per specific activity (panel D ) up to ten days in exemplary formed for two bioreactor tanks with single bolus medium Process # 4 . feed ) ; red line : an improved process ( performed for six [ 0047 ] FIG . 20 are graphs showing the different impact of bioreactor tanks ) with four times ofmedium feeds; gray line : production temperature on protein A titer (panel A ) , volu previous 20K processes (performed for twenty bioreactor metric activity (panel B ), specific protein A productivity tanks ) as a standard ) . The solid gray line represents the (panel C ) , and specific activity (panel D ) in exemplary average of 20K processes, while the dotted lines represent Process # 4 . average + 1x standard deviation . [0048 ] FIG . 21 are graphs showing the impact of culture [0058 ] FIG . 31 are graphs comparing active titer (panel A ) medium pH (panel A ) and production temperature (panel B ) and total volumetric activity (panel B ) for different pro on asfotase alfa TSAC in exemplary Process # 4 . cesses (blue line : a control process (performed for two [0049 ] FIG . 22 are graphs showing the impact of culture bioreactor tanks ) with single bolus medium feed ) ; red line : medium pH (panel A ) and production temperature (panel B ) an improved process ( performed for six bioreactor tanks ) on AEX acidic peak of the produced asfotase alfa in exem with four times of medium feeds; gray line : previous 20K plary Process # 4 . processes (performed for twenty bioreactor tanks ) as a [0050 ] FIG . 23 are graphs showing the impact of culture standard ) . The solid gray line represents the average of 20K medium pH (panel A ) and production temperature (panel B ) processes, while the dotted lines represent average + 1x on asfotase alfa aggregation ( measured by SEC ) in exem standard deviation . plary Process # 4 . [ 0059 ] FIG . 32 is a graph comparing asfotase alfa specific [0051 ] FIG . 24 are graphs showing the different impact of activity from control processes ( gray ; single bolus medium culture medium pH (panel A ) and production temperature feed ) and improved processes ( dark gray ; four times of (panel B ) on LoC main Peak ( % , non - reduced condition ) medium feeds ). measurement of produced asfotase alfa in exemplary Pro [0060 ] FIG . 33 is a graph comparing asfotase alfa specific cess # 4 . activity from a previous Process Y (lower line ; without [0052 ] FIG . 25 are graphs showing the impact of culture further zinc supplement) and a further improved process medium pH on neutral glycan profiles of produced asfotase ( upper line ; showing the average of 30 - 90 uM zinc supple alfa (measured by MALDI- TOF ) in exemplary Process # 4 . ment conditions and the culture time extended to 14 days ) . The top panel shows all MALDI- ToF peaks for asfotase alfa [0061 ] FIG . 34 are graphs showing MALDI- ToF mass produced at 33° C . for 10 days but under different culture spectrum data for produced asfotase alfa after deglycosy medium pH . The value of each peak was calculated and lation (panel A ) and for produced asfotase alfa which was compared in the bottom panel. reduced and deglycosylated ( panel B ) . [0053 ] FIG . 26 are graphs showing the impact of produc [0062 ] FIG . 35 is a graph showing the MALDI- ToF mass tion temperature on neutral glycan profiles of produced spectrum of produced asfotase alfa . FIG . 35 discloses asfotase alfa (measured by MALDI- TOF ) in exemplary “ TYNTNAQVPDSAGTATAYLCGVK ” as residues 83 - 105 Process # 4 . The top panel shows all MALDI- ToF peaks for of SEQ ID NO : 1, “ SAGTA ” as residues 93 - 97 of SEQ ID asfotase alfa produced under different production tempera NO : 1 and “ AYLCGV ” as residues 99 - 104 of SEQ ID NO : ture at pH 6 . 9 for 10 days . The value of each peak was 1 , respectively , in order of appearance. calculated and compared in the bottom panel. [0063 ] FIG . 36 are graphs showing the MS /MS determi [0054 ] FIG . 27 are graphs showing the impact of culture nation of the phosphorylation site on asfotase alfa . medium pH (panel A ) and production temperature ( panel B ) [0064 ] FIG . 37 are graphs showing the negative MALDI on capillary isoelectric focusing peak percentages for ToF mass spectrum of si?lyated glycans (panel A ) and the iCE280 analysis of asfotase alfa quality in exemplary Pro positive MALDI- ToF mass spectrum of neutral glycans cess # 4 . ( panel B ) on produced asfotase alfa . [ 0055 ] FIG . 28 are graphs comparing cell growth (panel [0065 ] FIG . 38 is a graph showing the fluorescence chro A ) and viability (panel B ) for different processes (blue line : matogram of oligosaccharides of asfotase alfa . a control process (performed for two bioreactor tanks ) with single bolus medium feed ) ; red line : an improved process 10066 ] FIG . 39 is a graph showing the fluorescence chro (performed for six bioreactor tanks ) with four times of matogram of the asfotase alfa reference standard . medium feeds; gray line : previous 20K processes (per [0067 ] FIG . 40 are graphs showing mass spectrum of formed for twenty bioreactor tanks ) as a standard . The solid glycopeptides generated from asfotase alfa . gray line represents the average of 20K processes , while the [0068 ] FIG . 41 are representations of the proposed struc dotted lines represent average + 1x standard deviation . tures of major glycans of asfotase alfa [0056 ] FIG . 29 are graphs comparing glucose utilization (C7108H1100802206956 (Protein Part dimer ) ; or (panel A ) and lactate production (panel B ) for different C3554H550601103S28 (Monomer ) ) . The number of processes ( blue line : a control process (performed for two NeuAc per glycan (FA2G2 , FA2G1 and A2G2) is an esti bioreactor tanks ) with single bolus medium feed ) ; red line : mated number. an improved process ( performed for six bioreactor tanks ) [0069 ] FIG . 42 are representations of the estimated glycan with four times of medium feeds ; gray line : previous 20K structure on glycosylation sites of asfotase alfa . processes (performed for twenty bioreactor tanks ) as a [0070 ] FIG . 43 is a graph showing the representative standard . The solid gray line represents the average of 20K electropherogram of asfotase alfa . processes, while the dotted lines represent average + lx [ 0071] FIG . 44 are graphs showing glycopeptide mass standard deviation . fingerprints for asfotase alfa produced from 20K and 2K [0057 ] FIG . 30 are graphs comparing protein A titer (panel batches (N123 in T15 - 16 ) . 2K Batch Numbers: # 35 , # 36 and A ) and specific activity (panel B ) of asfotase alfa produced # 38 ; 20K Batch Numbers: # 40 , # 42 and # 34 . US 2018 /0230445 A1 Aug . 16 , 2018

[ 0072 ] FIG . 45 are graphs showing glycopeptide mass a mammalian cell culture ) . The bioreactor can be of any size fingerprints for asfotase alfa produced from 20K and 2K so long as it is useful for the culturing of cells. Typically, the batches (N213 in T26 - 27 ) . 2K Batch Numbers : # 35 , # 36 and bioreactor will be at least 1 liter and may be 10 , 100 , 250 , # 38 ; 20K Batch Numbers: # 40 , # 42 and # 34 . 500 , 1000 , 2500 , 5000 , 8000 , 10 , 000 , 12 , 0000 , 20 , 000 liters [0073 ] FIG . 46 are graphs showing glycopeptide mass or more, or any volume in between . The internal conditions fingerprints for asfotase alfa produced from 20K and 2K of the bioreactor , including , but not limited to pH and batches (N254 in T33 ) . 2K Batch Numbers : # 35 , # 36 and temperature , are typically controlled during the culturing # 38 ; 20K Batch Numbers : # 40 , # 42 and # 34 . period . The bioreactor can be composed of any material that [0074 ] FIG . 47 are graphs showing glycopeptide mass is suitable for holding mammalian or other cell cultures fingerprints for asfotase alfa produced from 20K and 2K suspended in media under the culture conditions of the batches (N286 in T35 ) . 2K Batch Numbers: # 35 , # 36 and present disclosure , including glass , plastic or metal. The # 38 ; 20K Batch Numbers: # 40 , # 42 and # 34 . term " production bioreactor ” as used herein refers to the [0075 ] FIG . 48 are graphs showing glycopeptide mass final bioreactor used in the production of the polypeptide or fingerprints for asfotase alfa produced from 20K and 2K protein of interest . The volume of the large - scale cell culture batches ( N413 in T45 - 46 ) . 2K Batch Numbers : # 35 , # 36 and production bioreactor is typically at least 500 liters and may # 38 ; 20K Batch Numbers : # 40 , # 42 and # 34 . be 1000 , 2500 , 5000 , 8000 , 10 , 000 , 12 ,0000 , 20 ,000 liters or [0076 ] FIG . 49 are graphs showing glycopeptide mass more , or any volume in between . One of ordinary skill in the fingerprints for asfotase alfa produced from 20K and 2K art will be aware of and will be able to choose suitable batches (N564 in T55 ) . 2K Batch Numbers : # 35 , # 36 and bioreactors for use in practicing the present disclosure . # 38; 20K Batch Numbers : # 40 , # 42 and # 34 . [0085 ] “ Cell density ” : The term “ cell density , " as used [0077 ] FIG . 50 are graphs comparing the ICP metal ion herein , refers to the number of cells present in a given molar ratio for zinc (panel A ) , magnesium ( panel B ) , and volume of medium . calcium (panel C ) of asfotase alfa produced from 2K and [0086 ] “ Cell viability " : The term “ cell viability ,” as used 20K batches. herein , refers to the ability of cells in culture to survive under [ 0078 ] FIG . 51A is graph comparing the viable cell den a given set of culture conditions or experimental variations. sity ( VCD ) through culture time under different pH condi The term as used herein also refers to that portion of cells tions . FIG . 51B is graph comparing the cell viability through which are alive at a particular time in relation to the total culture time under different pH conditions . number of cells , living and dead , in the culture at that time. [0079 ] FIG . 52A is a graph showing the glucose concen [00871 “ Culture " and " cell culture ” : These terms, as used tration in the bioreactors through the elapsed culture time. herein , refer to a cell population that is suspended in a FIG . 52B is a graph showing the lactate concentration in the medium ( see definition of " medium " below ) under condi bioreactors through the elapsed culture time. tions suitable for survival and/ or growth of the cell popu [ 0080 ] FIG . 53 is a graph comparing specific activity lation . As will be clear to those of ordinary skill in the art, profiles under different pH conditions. these terms as used herein may refer to the combination comprising the cell population and the medium in which the DETAILED DESCRIPTION population is suspended . [0088 ] “ Fed -batch culture” : The term “ fed -batch culture ,” Definitions as used herein , refers to a method of culturing cells in which [ 0081] “ About” , “ Approximately” : As used herein , the additional components are provided to the culture at some terms “ about” and “ approximately ” , as applied to one or time subsequent to the beginning of the culture process. The more particular cell culture conditions , refer to a range of provided components typically comprise nutritional supple values that are similar to the stated reference value for that ments for the cells , which have been depleted during the culture condition or conditions. In certain embodiments , the culturing process . A fed - batch culture is typically stopped at term “ about” refers to a range of values that fall within 25 , some point and the cells and / or components in the medium 20 , 19 , 18 , 17 , 16 , 15 , 14 , 13 , 12 , 11 , 10 , 9 , 8 , 7 , 6 , 5 , 4 , 3 , are harvested and optionally purified . Fed -batch culture may 2 , 1 percent or less of the stated reference value for that be performed in the corresponding fed -batch bioreactor. culture condition or conditions . [0089 ] “ Fragment ” : The term “ fragment, ” as used herein , 10082 ] “ Amino acid ” : The term “ amino acid , " as used refers to a polypeptide and is defined as any discrete portion herein , refers to any of the twenty naturally occurring amino of a given polypeptide that is unique to or characteristic of acids that are normally used in the formation of polypep that polypeptide . The term as used herein also refers to any tides , or analogs or derivatives of those amino acids . Amino discrete portion of a given polypeptide that retains at least a acids of the present disclosure can be provided in medium to fraction of the activity of the full - length polypeptide . In cell cultures . The amino acids provided in the medium may some embodiments the fraction of activity retained is at least be provided as salts or in hydrate form . 10 % of the activity of the full - length polypeptide . In various [0083 ] “ Batch culture” : The term “ batch culture, " as used embodiments the fraction of activity retained is at least 20 % , herein , refers to a method of culturing cells in which all of 30 % , 40 % , 50 % , 60 % , 700 / % , 80 % 0 or 90 % of the activity the components that will ultimately be used in culturing the of the full - length polypeptide . In other embodiments the cells , including the medium (see definition of 'medium ” fraction of activity retained is at least 95 % , 96 % , 97 % , 98 % below ) as well as the cells themselves, are provided at the or 99 % of the activity of the full - length polypeptide . In one beginning of the culturing process. A batch culture is typi embodiment, the fraction of activity retained is 100 % of the cally stopped at some point and the cells and / or components activity of the full - length polypeptide . The term as used in the medium are harvested and optionally purified . herein also refers to any portion of a given polypeptide that 10084 ] “ Bioreactor” : The term " bioreactor ” as used herein includes at least an established sequence element found in refers to any vessel used for the growth of a cell culture ( e. g ., the full - length polypeptide. In some embodiments , the US 2018 /0230445 A1 Aug . 16 , 2018 sequence element spans at least 4 - 5 amino acids of the cells and / or components in the medium are typically har full - length polypeptide . In some embodiments , the sequence vested on a continuous or semi- continuous basis and are element spans at least about 10 , 15, 20 , 25 , 30 , 35 , 40 , 45 , optionally purified . 50 or more amino acids of the full -length polypeptide. [0095 ] “ Polypeptide” : The term “ polypeptide, " as used [0090 ] “ Integrated Viable Cell Density ” : The term “ inte herein , refers a sequential chain of amino acids linked grated viable cell density , " as used herein , refers to the together via peptide bonds . The term is used to refer to an average density of viable cells over the course of the culture amino acid chain of any length , but one of ordinary skill in multiplied by the amount of time the culture has run . the art will understand that the term is not limited to lengthy Assuming the amount of polypeptide and / or protein pro chains and can refer to a minimal chain comprising two duced is proportional to the number of viable cells present amino acids linked together via a peptide bond . over the course of the culture , integrated viable cell density 0096 ] “ Protein ” : The term “ protein ," as used herein , is a useful tool for estimating the amount of polypeptide refers to one or more polypeptides that function as a discrete and / or protein produced over the course of the culture . unit . If a single polypeptide is the discrete functioning unit [0091 ] “ Medium ” , “ cell culture medium ” , and “ culture and does not require permanent physical association with medium ” : These terms, as used herein , refer to a solution other polypeptides in order to form the discrete functioning containing nutrients which nourish growing mammalian unit, the terms “ polypeptide” and “ protein ” as used herein cells . Typically , these solutions provide essential and non are used interchangeably . essential amino acids , vitamins , energy sources , lipids, and [0097 ] “ Recombinantly -expressed polypeptide ” and trace elements required by the cell for minimal growth “ recombinant polypeptide” : These terms, as used herein , and / or survival. The solution may also contain components refer to a polypeptide expressed from a host cell that has that enhance growth and / or survival above the minimal rate , been genetically engineered to express that polypeptide . The including hormones and growth factors . The solution is , e . g . , recombinantly - expressed polypeptide can be identical or formulated to a pH and salt concentration optimal for cell similar to a polypeptide that is normally expressed in the survival and proliferation . The medium may also be a mammalian host cell. The recombinantly - expressed poly " defined media ” — a serum - free media that contains no pro peptide can also be foreign to the host cell , i. e. , heterologous teins , hydrolysates or components of unknown composition . to peptides normally expressed in the host cell. Alterna Defined media are free of animal - derived components and tively, the recombinantly -expressed polypeptide can be chi all components have a known chemical structure. meric in that portions of the polypeptide contain amino acid 10092 ] “ Metabolic waste product” : The term “ metabolic sequences that are identical or similar to polypeptides nor waste product, ” as used herein , refers to compound pro mally expressed in the mammalian host cell , while other duced by the cell culture as a result of normal or non -normal portions are foreign to the host cell . metabolic processes that are in some way detrimental to the [ 0098 ] “ Seeding” : The term " seeding ,” as used herein , cell culture , particularly in relation to the expression or refers to the process of providing a cell culture to a biore activity of a desired recombinant polypeptide or protein . For actor or another vessel . The cells may have been propagated example , the metabolic waste products may be detrimental previously in another bioreactor or vessel . Alternatively , the to the growth or viability of the cell culture , may decrease cells may have been frozen and thawed immediately prior to the amount of recombinant polypeptide or protein produced , providing them to the bioreactor or vessel. The term refers may alter the folding, stability , glycosylation or other post to any number of cells , including a single cell. translational modification of the expressed polypeptide or [ 0099 ] “ Titer” : The term “ titer ," as used herein , refers to protein , or may be detrimental to the cells and / or expression the total amount of recombinantly - expressed polypeptide or or activity of the recombinant polypeptide or protein in any protein produced by a cell culture divided by a given amount number of other ways . Exemplary metabolic waste products of medium volume. Titer is typically expressed in units of include lactate , which is produced as a result of glucose milligrams of polypeptide or protein per milliliter of metabolism , and ammonium , which is produced as a result medium . of glutamine metabolism . In one embodiment, methods are [0100 ] Acronyms used herein include, e. g ., VCD : Viable taken to slow production of, reduce or even eliminate Cell Density ; IVCC : Integral of Viable Cell Concentration ; metabolic waste products in cell cultures . TSAC : Total Sialic Acid Content; HPAE - PAD : High - Per [ 0093] “ Osmolality ” and “ osmolarity ” : Osmolality is a formance Anion Exchange Chromatography with Pulsed measure of the osmotic pressure of dissolved solute particles Amperometric Detection ; SEC : Size Exclusion Chromatog in an aqueous solution . The solute particles include both ions raphy ; AEX : Anion Exchange Chromatography ; LOC : Lab and non - ionized molecules . Osmolality is expressed as the on - Chip ; and MALDI- TOF : Matrix Assisted Laser Desorp concentration of osmotically active particles (i . e . , osmoles ) tion / Ionization - Time of Flight. dissolved in 1 kg of solution ( 1 mOsm / kg H , O at 38° C . is [0101 ] The present disclosure provides a method of cul equivalent to an osmotic pressure of 19 mm Hg) . “ Osmo turing cells ( e . g . , mammalian cells including but not limited larity , ” by contrast , refers to the number of solute particles to Chinese Hamster Ovary (CHO ) cells ) expressing a recom dissolved in 1 liter of solution . When used herein the binant protein . The present disclosure provides manufactur abbreviation “ mOsm ” means “ milliosmoles/ kg solution ” . ing systems for the production of an alkaline phosphatase [0094 ] “ Perfusion culture” : The term “ perfusion culture , ” ( e . g ., asfotase alfa ) by cell culture. In certain embodiments , as used herein , refers to a method of culturing cells in which systems are provided that minimize production of one or additional components are provided continuously or semi more metabolic products that are detrimental to cell growth , continuously to the culture subsequent to the beginning of viability , and / or protein production or quality . In particular the culture process. The provided components typically embodiments , the cell culture is a batch culture , a fed - batch comprise nutritional supplements for the cells , which have culture , a culture or a continuous culture . Other embodi been depleted during the culturing process . A portion of the ments of the disclosure are discussed in detail below . Those US 2018 /0230445 A1 Aug . 16 , 2018 of ordinary skill in the art will understand , however, that L1 -S485 is the soluble part of the human tissue non -specific various modifications to these embodiments are encom alkaline phosphatase enzyme, which contains the catalytic passed within the scope of the disclosure . function . The second portion contains amino acids L486 K487 as a linker . The third portion (Fc ) containing amino Proteins acids D488 -K714 is the Fc part of the human Immunoglobu [0102 ] The present disclosure relates to expression of an lin gamma 1 ( IgG1) containing hinge , CH , and CH? alkaline phosphatase , asfotase alpha , protein in cell culture. domains . The fourth portion contains D715 - 1716 as a linker . In certain embodiments , such asfotase alpha , after being The fifth portion contains amino acids D717 -0726 (D10 ) , manufactured by the methods disclosed herein , can be used which is a bone targeting moiety that allows asfotase alfa to to treat or prevent alkaline phosphatase -related diseases or bind to the mineral phase of bone. In addition , each poly disorders . For example , such asfotase alpha may be admin peptide chain contains six potential glycosylation sites and istered to a subject having decreased and /or malfunctioned eleven cysteine (Cys ) residues . Cys102 exists as free cys endogenous alkaline phosphatase , or having overexpressed teine. Each polypeptide chain contains four intra -chain dis ( e . g . , above normal level) alkaline phosphatase substrates. In ulfide bonds between Cys122 and Cys184 , Cys472 and some embodiments , the asfotase alpha in this disclosure is a Cys480 , Cys528 and Cys588 , and Cys34 and Cys692. The recombinant protein . In some embodiments , the asfotase two polypeptide chains are connected by two inter - chain alpha is a fusion protein . In some embodiments , the asfotase disulfide bonds between Cys493 on both chains and between alpha in this disclosure specifically targets a cell type , tissue Cys496 on both chains. In addition to these covalent struc ( e . g . , connective , muscle , nervous , or epithelial tissues ) , or tural features, mammalian alkaline phosphatases are thought organ ( e . g . , liver, heart, kidney , muscles , bones , cartilage , to have four metal- binding sites on each polypeptide chain , ligaments , tendons , etc . ). Asfotase alfa is a soluble Fc fusion including two sites for zinc , one site for magnesium and one protein consisting of two sTNALP - Fc -D10 polypeptides site for calcium . each with 726 amino acids as shown in SEQ ID NO : 1 . Underlined asparagine ( N ) residues correspond to potential Asfotase Alfa glycosylation sites ( i. e . , N 123 , 213 , 254 , 286 , 413 & 564 ) . [0104 ] In one embodiment , the alkaline phosphatase pro Bold underlined amino acid residues (L496 -K497 & D715 tein ( e. g ., the bone -targeted SALP fusion protein ) is asfotase 1716 ) correspond to linkers between SALP and Fc, and Fc and alfa ( i. e . , sTNALP -Fc -D10 ; SEQ ID NO : 1 ) . Specifically, D10 domains, respectively . asfotase alfa is a complex soluble glycoprotein with a

( SEQ ID NO : 1 ) 10 20 30 40 50 60 LVPEKEKDPK YWRDQAQETL KYALELQKLN TNVAKNVIMF LGDGMGVSTV TAARILKGQL 70 80 90 100 110 120 HHNPGEETRL EMDKEPFVAL SKTYNTNAQV PDSAGTATAY LCGVKANEGT VGVSAATERS 130 140 150 160 170 180180 RCNTTQGNEV TSILRWAKDA GKSVGIVTTT RVNHATPSAA YAHSADRDWY SDNEMPPEAL 190 200 210 220 230 240 SOGCKDIAYO LMHNIRDIDV IMGGGRKYMY PKNKTDVEYE SDEKARGTRL DGLDLVDTWK 250 260 270 280 290 300 SFKPRYKHSH FIWNRTELLT LDPHNVDYLL GLFEPGDMQY ELNRNNVTDP SLSEMVVVAI 310 320 330 340 350 360 OILRKNPKGF FLLVEGGRID HGHHEGKAKO ALHEAVEMDR AIGOAGSLTSALCORCO ! ! SEDTLTVVTASEPTLYVEI 370 380 390 400 410 420 DHSHVFTFGG YTPRGNSIFG LAPMLSDTDK KPFTAILYGN GPGYKWVGGE RENVSMVDYA 430 440 450 460 470 480 HNNYQAQSAV PLRHETHGGE DVAVFSKGPM AHLLHGVHEQ NYVPHVMAYA ACIGANLGHC 490 500 510 520 530 540 APASSLKDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPTV 550 560 570 580 590 600 KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE 610 620 630 640 650 660 KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 670 680 690 700700 710 720 TPPVLDSDGS FFLYSKLTVD KSRWOOGNVF SCSVMHEALH NHYTOKSLSL SPGKDIDDDD

DDDDDD

[ 0103 ] Each polypeptide or monomer is composed of five polypeptide length of 726 amino acids. Asfotase alfa is an portions . The first portion ( SALP ) containing amino acids Fc - fusion protein composed of 3 domains . From the N - ter US 2018 /0230445 A1 Aug . 16 , 2018 minus to the C terminus, asfotase alfa comprises : ( 1 ) the regulatory elements is first transferred to a host cell . Usually , soluble catalytic domain of human tissue non - specific alka a second gene is transferred that confers to recipient cells a line phosphatase ( TNSALP ) (UniProtKB / Swiss - Prot Acces selective advantage . In the presence of the selection agent, sion No. P05186 ) , ( 2 ) the human immunoglobulin G1 FC which is typically applied a few days after gene transfer , domain (UniProtKB /Swiss - Prot Accession No. P01857 ) and only those cells that express the selector gene survive . Two ( 3 ) a deca aspartate peptide ( D ) used as a bone- targeting popular genes for selection are dihydrofolate reductase domain (Nishioka et al. 2006 Mol GenetMetab 88 :244 - 255 ) . (DHFR ) , an enzyme involved in nucleotide metabolism , and The protein associates into a homodimer from two primary glutamine synthetase (GS ) . In both cases , selection occurs in protein sequences . This fusion protein contains 6 confirmed the absence of the appropriate metabolite (hypoxanthine and complex N - glycosylation sites . Five of these N - glycosy thymidine, in the case of DHFR , glutamine in the case of lation sites are located on the SALP domain and one on the GS ) , preventing growth of nontransformed cells . In general, Fc domain . Another important posttranslational modifica for efficient expression of the recombinant protein , it is not tion present on asfotase alfa is the presence of disulfide important whether the biopharmaceutical- encoding gene bridges stabilizing the enzyme and the Fc -domain structure . and selector genes are on the same plasmid or not. A total of 4 intra -molecular disulfide bridges are present per 0109 Following selection , surviving cells may be trans monomer and 2 inter -molecular disulfide bridges are present ferred as single cells to a second cultivation vessel , and the in the dimer . One cysteine of the alkaline phosphatase cultures are expanded to produce clonal populations . Even domain is free . tually , individual clones are evaluated for recombinant pro [0105 ] Asfotase alfa may be used as an enzyme- replace tein expression , with the highest producers being retained ment therapy for the treatment of hypophosphatasia (HPP ) . for further cultivation and analysis . From these candidates , In patients with HPP , loss -of - function mutation ( s ) in the one cell line with the appropriate growth and productivity gene encoding TNSALP causes a deficiency in TNSALP characteristics is chosen for production of the recombinant enzymatic activity , which leads to elevated circulating levels protein . A cultivation process is then established that is of substrates , such as inorganic pyrophosphate ( PPi) and determined by the production needs. pyridoxal- 5 '- phosphate (PLP ) . Administration of asfotase alfa to patients with HPP cleaves PPi, releasing inorganic Cells phosphate for combination with calcium , thereby promoting [0110 ] Any mammalian cell or non -mammalian cell type , hydroxyapatite crystal formation and bone mineralization , which can be cultured to produce a polypeptide , may be and restoring a normal skeletal phenotype. For more details utilized in accordance with the present disclosure . Non on asfotase alfa and its uses in treatment, see PCT Publica limiting examples of mammalian cells that may be used tion Nos. WO2005103263 and WO2008138131, the teach include , e. g . , Chinese hamster ovary cells + / - DHFR (CHO , ings of which are incorporated herein by reference in their Urlaub and Chasin , 1980 Proc. Natl. Acad . Sci . USA , entirety . In another embodiment , asfotase alfa may be used 77 : 4216 ) ; BALB / c mouse myeloma line (NSO / 1 , ECACC as an enzyme- replacement therapy for the treatment of Accession No: 85110503 ) ; human retinoblasts (PER . C6 Neurofibromatosis type I (NF1 ) . For more details on asfo (CruCell , Leiden , The Netherlands) ); monkey kidney CVI tase alfa and its uses ( together with the uses of other alkaline line transformed by SV40 (COS - 7 , ATCC CRL 1651 ) ; phosphatases ) in treatment of NF1, see PCT Publication No. human embryonic kidney line (293 or 293 cells subcloned WO 2013 /058833 , which is incorporated herein by reference for growth in suspension culture , Graham et al. , 1977 J . Gen in its entirety . Virol. , 36 : 59 ) ; baby hamster kidney cells ( BHK , ATCC CCL 10 ) ; mouse Sertoli cells ( TM4, Mather, Biol. Reprod. , Manufacturing Process 23 : 243 - 251 ( 1980 ) ) ; monkey kidney cells ( CVI ATCC CCL [0106 ] The alkaline phosphatase protein (e .g ., asfotase 70 ) ; African green monkey kidney cells (VERO -76 , ATCC alfa ) may be produced by mammalian or other cells using CRL - I 587 ) ; human cervical carcinoma cells (HeLa , ATCC standard methods known in the art. Such cells may be grown CCL 2 ) ; canine kidney cells (MDCK , ATCC CCL 34 ) ; in culture dishes, flask glasses , or bioreactors . Specific buffalo rat liver cells (BRL 3A , ATCC CRL 1442 ) ; human processes for cell culture and producing recombinant pro lung cells (W138 , ATCC CCL 75 ) ; human liver cells (Hep teins are known in the art , such as described in Nelson and G2, HB 8065) ; mouse mammary tumor (MMT 060562, Geyer, 1991 Bioprocess Technol. 13 : 112 - 143 and Rea et al. , ATCC CCL51 ) ; TRI cells (Mather et al ., 1982 , Annals N . Y . Supplement to BioPharm International March 2008 , 20 - 25 . Acad . Sci. 383 : 44 -68 ) ; MRC 5 cells ; FS4 cells ; and a human Exemplary bioreactors include batch , fed -batch , and con hepatoma line (Hep G2 ) . In a particular embodiment, cul tinuous reactors . In some embodiments , the alkaline phos turing and expression of polypeptides and proteins occurs phatase protein is produced in a fed -batch bioreactor . from a Chinese Hamster Ovary (CHO ) cell line. [0107 ] Potential variability in the cell culture process [0111 ] Additionally , any number of commercially and physicochemical environment includes, for example , non - commercially available recombinant cell lines that changes in pH , temperature, cell culture media composition , express polypeptides or proteins may be utilized in accor raw material lot - to - lot variation , medium filtration material, dance with the present disclosure. One skilled in the art will bioreactor scale difference, gassing strategy (air , oxygen , appreciate that recombinant cell lines might have different and carbon dioxide) , etc . As disclosed herein , the glycosy nutrition requirements and /or might require different culture lation profiles of manufactured alkaline phosphatase protein conditions for optimal growth and polypeptide or protein may be affected by alterations in one or more parameters . expression , and will be able to modify conditions as needed . 10112 As noted above , in many instances the cells will be Development for Cell Culture Processes selected or engineered to produce high levels of protein or 0108 ] For recombinant protein production in cell culture , polypeptide . Often , cells are genetically engineered to pro the recombinant gene with the necessary transcriptional duce high levels of protein , for example by introduction of US 2018 /0230445 A1 Aug . 16 , 2018

a gene encoding the protein or polypeptide of interest and / or asfotase alfa is first set at about 36 . 5° C . and then shifted to by introduction of control elements that regulate expression about 33° C . In yet another embodiment, the temperature for of the gene (whether endogenous or introduced ) encoding producing asfotase alfa is first set at about 37 .0° C . and then the polypeptide of interest. shifted to about 33° C . In yet a further embodiment, the temperature for producing asfotase alfa is first set at about Temperature Shifting 36 . 5° C . and then shifted to about 30° C . In other embodi [0113 ] Run times of cell culture processes, especially ments, multiple ( e . g . , more than one ) steps of temperature non - continuous processes ( e . g . , fed -batch processes in bio shifting may be applied . For example , the temperature may reactors ) , are usually limited by the remaining viability of be lowered from 37° C . first to 33° C . and then further to 30° the cells , which typically declines over the course of the run . Therefore , extending the length of time for cell viability is [0115 ] The time for maintaining the culture at a particular desired for improving recombination protein production . temperature prior to shifting to a different temperature may Product quality concerns also offer a motivation for mini be determined to achieve a sufficient (or desired ) cell density mizing decreases in viable cell density and maintaining high while maintaining cell viability and an ability to produce the cell viability , as cell death can release sialidases to the protein of interest . In some embodiments , the cell culture is culture supernatant, which may reduce the sialic acid content grown under the first temperature until the viable cell of the protein expressed . Protein purification concerns offer density reaches about 10 % cells/ mL to about 107 cells/ mL yet another motivation for minimizing decreases in viable ( e .g . , 1x10° , 1 .5x10° , 2 .0x10° , 2 .5x10° , 3 . 0x10° , 3. 5x10°, cell density and maintaining high cell viability. Cell debris 4 .0x10 % , 4 . 5x105, 5 . 0x105, 5 .5x10 % , 6 .0x10 % , 6 .5x10 % , 7 . 0x and the contents of dead cells in the culture can negatively 105, 7 .5x105 , 8 .0x105 , 8 .5x105 , 9 .0x105 , 9 .5x10 “ , 1 .0x10 " , impact one ' s ability to isolate and /or purify the protein 1 .5x106 , 2 .0x106 , 2 .5x106 , 3. 0x106 , 3. 5x106 , 4 .0x106 , 4 .5x product at the end of the culturing run . Thus, by keeping 106, 5 . 0x106, 5 . 5x106, 6 .0x10 " , 6 . 5x106, 7 .0x106 , 7 . 5x106 , cells viable for a longer period of time in culture , there is a 8 .0x10 ", 8 .5x10 ", 9 .0x10® , 9. 5x10 ", 1x107 cell/ mL , or reduction in the contamination of the culture medium by more ) before shifting to a different temperature. In one cellular proteins and enzymes ( e . g . , cellular proteases and embodiment, the cell culture is grown under the first tem sialidases ) that may cause degradation and ultimate reduc perature until the viable cell density reaches about 2 . 5 to tion in the quality of the desired glycoprotein produced by about 3. 4x10 cells /mL before shifting to a different tem the cells . perature . In another embodiment, the cell culture is grown [ 0114 ] Many methodsmay be applied to achieve high cell under the first temperature until the viable cell density viability in cell cultures. One involves lowering culture reaches about 2 . 5 to about 3 . 2x10 cells /mL before shifting temperature following initial culturing at a normal tempera to a different temperature . In yet another embodiment, the ture . For example , see Ressler et al . , 1996 , Enzyme and cell culture is grown under the first temperature until the Microbial Technology 18 : 423 -427 ) . Generally , the mamma viable cell density reaches about 2 . 5 to about 2 . 8x10 % lian or other types of cells capable of expressing a protein of cells /mL before shifting to a different temperature . interest are first grown under a normal temperature to [ 0116 ] In some embodiments , the cell culture is grown increase cell numbers. Such “ normal” temperatures for each under 37° C . until the viable cell density reaches about cell type are generally around 37° C . ( e . g . , from about 35° 2 . 5 - 2 . 8x100 cells /mL before shifting to 30° C . for protein C . to about 39° C ., including, for example , 35 .0° C ., 35 .5° production . In other embodiments , the cell culture is grown C . , 36 .0° C . , 36 .5° C . , 37 . 0° C . , 37 . 5° C . , 38 . 0° C . , 38 . 5° C . , under 37° C . until the viable cell density reaches about and /or 39 .0° C .) . In one particular embodiment, the tem 2 .5 - 3. 4x10 cells /mL before shifting to 30° C . for protein perature for producing asfotase alfa is first set at about 37° production . C . When a reasonably high cell density is reached , the PH culturing temperature for the whole cell culture is then [0117 ] Alteration of the pH of the growth medium in cell shifted ( e . g . , decreased ) to promote protein production . In culture may affect cellular proteolytic activity , secretion , and most cases lowering temperature shifts the cells towards the protein production levels .Most of the cell lines grow well at non - growth G1 portion of the cell cycle , which may increase about pH 7 - 8 . Although optimum pH for cell growth varies cell density and viability , as compared to the previous relatively little among different cell strains , some normal higher- temperature environment. In addition , a lower tem fibroblast cell lines perform best at a pH 7 . 0 - 7 . 7 and trans perature may also promote recombinant protein production formed cells typically perform best at a pH of 7 . 0 - 7 . 4 ( Eagle , by increasing the cellular protein production rate , facilitating 1973 The effect of environmental pH on the growth of protein post - translational modification ( e . g . , glycosylation ) , normal and malignant cells . J Cell Physiol 82 : 1 - 8 ) . In some decreasing fragmentation or aggregation of newly -produced embodiments , the pH of the culture medium for producing proteins , facilitating protein folding and formation of 3D asfotase alfa is about pH 6 . 5 - 7 . 7 ( e . g . , 6 .50 , 6 .55 , 6 .60 , 6 .65 , structure ( thus maintaining activity ), and / or decreasing deg 6 .70 , 6 .75 , 6 . 80 , 6 . 85 , 6 . 90 , 6 . 95 , 7 . 00 , 7 .05 , 7 . 10 , 7 . 15 , 7 . 20 , radation of newly produced proteins . In some embodiments , 7 . 25, 7 . 30 , 7 . 35 , 7 . 39, 7 . 40 , 7 . 45, 7 .50 , 7 .55 , 7 .60 , 7 . 65 , and the lower temperature is from about 30° C . to about 35° C . 7 .70 ) . In some embodiments , the pH of the culture medium ( e . g . , 30 .0° C ., 30 .5° C ., 31 . 0° C . , 31 .5° C ., 32 . 0° C . , 32 .50 for producing asfotase alfa is about pH 7 . 20 - 7 .60 . In other C ., 33 .0° C ., 33. 5° C ., 34 .0° C ., 34 .5° C . , and /or 35 .0° C . ). embodiments , the pH of the culture medium for producing In other embodiments , the temperature for producing asfo asfotase alfa is about pH 6 . 9 - 7 . 1 . In one particular embodi tase alfa is first set to from about 35 . 0° C . to about 39 .0° C . ment, the pH of the culture medium for producing asfotase and then shifted to from about 30 .0° C . to about 35 .0° C . In alfa is about pH 6 . 9 . In another embodiment, the pH of the one embodiment, the temperature for producing asfotase culture medium for producing asfotase alfa is about pH 7 . 30 . alfa is first set at about 37 . 0° C . and then shifted to about 30° In yet another embodiment, the pH of the culture medium C . In another embodiment, the temperature for producing for producing asfotase alfa is about pH 7 . 39 . US 2018 /0230445 A1 Aug . 16 , 2018

[0118 ] All references cited herein are incorporated by for growth . Samples were taken daily for counts and viabil reference in their entirety . ity tests ( also for all following expansion steps ) . Cells were [0119 ] Although this disclosure has been described in some detail by way of illustration and example for purposes expanded through several steps and inoculated into a 1 , 000 of clarity of understanding, it is apparent to those skilled in L seed bioreactor (N - 3 low level ) , a 1, 000 L seed bioreactor the art that certain minor changes and modifications will be ( N - 2 high level ), and a 4 ,000 L seed bioreactor (N - 1 ) and practiced . Therefore, the description and examples should then a 20 , 000 L production bioreactor . After production of not be construed as limiting the scope of the disclosure . asfotase alfa , the harvest clarification process was employed to remove intact cells and cell debris by sterile filtration . The EXAMPLES harvest was then ultrafiltered (Post Harvest UF ) for concen Example 1 . General Manufacturing Process for tration and buffer dilution . Further processes included , for Asfotase Alfa example , viral inactivation (to chemically inactivate viral [ 0120 ] As described herein , a manufacturing process to particles ), MabSelect Sure chromatography, hydrophobic produce alkaline phosphatases (e . g. , asfotase alfa (sTNALP interaction chromatography (HIC ), post HIC UF/ DF (UF / Fc -D10 ) ) has been developed . DF2 ) , capto adhere mixed -mode chromatography, virus fil TABLE 1 Differences between exemplary manufacturing processes (upstream )

Process X Process Y Process Z Parental Cell CHO ??? line Production HyClone SFM4CHO HyClone HyClone SFM4CHO + medium SFM4CHO BD select Feed CHO Feed ( 0 . 5 % ) CHO Feed ( 2 % , ice. , CHO Feed ( 2 % ) + 4 x medium feed ) Cell Boost 2 + 5 ( 9 % ) pH set point 6 .90 6 . 90 6 . 90 Temperature 37 .0° C . then shifted to 37 .0° C . then shifted 37 .0° C . then shifted to 30° C . to 30° C . 30° C . DO 40 % 40 % 40 % Supplement N / A N / A ZnSO4 Harvest time 240 + 12 hr 240 + 12 hr 240 12 hr

[ 0121 ] Stable CHO cell lines expressing asfotase alfa were tration (by size exclusion ), formulation (UF /DF3 ) and bulk developed using the GS gene expression system . Secondary fill . Multiple manufacturing processes were performed clones were derived from high producing primary clones in a single round of limited dilution cloning and a final cell line including, for example , 2 ,000 L - scale processes and the was selected . following scale - up to the 20 , 000 L production scale . Exem [ 0122 ] An exemplary manufacturing process, Process X , plary differences between the 2 ,000 L (2K ) and the 20 ,000 is described herein . A vial of the Master Cell Bank was L ( 20K ) processes are summarized in Tables 2 and 3 . The thawed and the entire volume of the vial was re - suspended . 2 ,000 L (2K ) scale process had a more pronounced lag -phase The entire volume was transferred to a 250 mL shake flask and more varied late stage viability (data not shown ). TABLE 2 Inoculum Expansion Parameter Comparison between Exemplary 2 . 000 L Process and 20 . 000 L Process Parameter 2 ,000 L Process 20 ,000 L Process Culturing Vessels Shake flasks, cell bags Shake flasks , roller bottles, cell bags , expansion bioreactors Temperature 37° C . 36 . 5 - 37 .5° C . Medium HyQSFM4CHO with 4 mM HyQSFM4CHO with 4 mM Glutamine and 100 nM Glutamine and 100 nM Methotrexate Methotrexate Seed density target Thaw : 0 .50 x 106 viable cells /mL Thaw : 0 .50 x 106 viable Passaging : 0. 40 x 10 viable cells /ML cells /mL Passaging : 0 . 35 x 10 viable cells/ mL Passage length 3 - 4 days 3 - 4 days Time to production Fixed ( 7 passages , 21- 28 days ) Varies ( 11 Passages , with bioreactor ability to maintain at 100 L high volume stage ) Cell density at time of 21. 00 x 106 viable cells /mL 21 .40 x 106 viable cells/ mL passage US 2018 /0230445 A1 Aug . 16 , 2018

TABLE 3 Production Bioreactor Parameter Comparison between Exemplary 2 , 000 L Process and 20 , 000 L Process Parameter 2 ,000 L Process 20 ,000 L Process Temperature 37° C . at inoculation ; 37° C . at inoculation 30° C . once cell density is 2 . 5 - 2 . 8 x 106 30° C . Once cell density is 2 . 5 - 3 . 4 x viable cells/ mL 100 viable cells /mL PH pH allowed to drift to 7 .10 on day 1 , to 6 .90 + 0 . 10 7 . 00 on day 2 and then controlled at 6 . 90 from day 3 - harvest Dissolved oxygen 40 % 40 % Seed density 0 .50 x 106 viable cells /mL 0 .55 x 106 viable cells /mL Feed timing Single bolus feed prior to temperature Single bolus feed prior to shift temperature shift Foam control Foam - away foam Antifoam - C as required Culture duration Day 10 or when viability < 50 % Day 10 or when viability < 60 %

[0123 ] The overall yields of 2K processes and 20K pro cesses as well as the qualities of the corresponding produced TABLE 4 -continued asfotase alfa were compared . Analytical methods used to Process Parameter in An Exemplary Process compare the product characterization include , e. g . , SEC HPLC , RP -HPLC , and other methods to measure specific Process activity , protein concentration , pH , and total sialic acid Parameter Process # 1 content ( TSAC ) of produced asfotase alfa . In addition , medium Glutamine impurities and safety tests were also performed to measure , Seeding 5 .5e " cells /mL e . g ., residual DNA , residual Protein A , host cell proteins, density bioburden and endotoxin in asfotase alfa produced from PH 6 .90 w /o dead band different processes . Three additional tests , i . e ., isoelectric DO 40 % ( Air 0 . 0016 VVM + O2 as needed ) Temperature 37 .0° C . - > 30° C . (when VCD is 25 - 32e cells /mL ) focusing ( IEF ) , sodium dodecyl sulfate polyacrylamide gel Feed 5 mL/ L CHO feed when VCD is 25 - 32es cells /mL electrophoresis (SDS -PAGE ) , and oligosaccharide mapping , Other If [ gln ] < 0 . 18 g / L ( 1 . 2 mm ) , increase [ gln ] to 0 . 29 g / L were also performed to compare drug substances from 2K supplements ( 2 . 0 mM ) prior to temperature shift. and 20K scale batches. Results of these three tests showed Harvest 240 = 12 h (within 30 hrs of viability < 60 % ) comparable profiles for these batches . In conclusion , the criteria asfotase alfa quality between the 2 , 000 L and 20 ,000 L scales was comparable across all batches . Example 2 . Impact of Temperature Shifting on Methods Asfotase Alfa Productivity and Quality [0124 ] Among different manufacturing processes adopted Cell Culture Experiment Design and practiced for producing sTNALP -FC - D10 , temperature shifting is generally found to affect the productivity and final [0126 ] Two blocks of experiments were implemented to asfotase alfa quality . Temperature shift from growth tem evaluate the impact of temperature shift on asfotase alfa perature (comparatively high temperature ) to production productivity and quality attributes for an exemplary process . temperature (comparatively low temperature ) was imple As shown in Table 5 , two bioreactor runs ( i. e ., # 1 and # 2 ) mented in all processes . were conducted with temperature shifting (37° C . to 30° C .) [0125 ] To compare the effects of temperature shifting and two other runs (i . e ., # 3 and # 4 ) were conducted without versus no shifting , duplicate sTNALP - FC - D , production temperature shifting from 37° C . bioreactor runs were conducted , wherein there was or was not a temperature shift. Sartorius 2 L and 10 L bioreactors TABLE 5 were used for different production processes. In this exem plary process , raw materials used in the production biore Bioreactor conditions for Process # 1 actors included , e . g ., production medium , 10 % Sodium carbonate , CHO feed , and glutamine stock solution , as Bioreactor ID Condition summarized in Table 4 . SFM4CHO refers to serum free # 1 Temp Shift 1 # 2 Temp Shift 2 medium for CHO . # 3 No Temp Shift 1 # 4 No Temp Shift 2 TABLE 4 Process Parameter in An Exemplary Process [0127 ] Cell density and viability were counted using a cell Process counter (i .e . , ViCell VR , Beckman Coulter ). pH and off- line Parameter Process # 1 gas was measured using PHOx , and major metabolites Cell line Cell line X ( derived from CHO ) including glucose and lactate were measured using a sensor Production CM70 ( SFM4CHO + Pluronic + Sodium bicarbonate ) + (Nova Profile 100 , Nova Biomedical, Waltham , Mass. ) . Enzymatic activity was measured using a standard method US 2018 /0230445 A1 Aug . 16 , 2018 with the modification that each sample was diluted only ( FIG . 2A ) . With the more rapid viability decline under the once , instead of three times , prior to enzymatic activity no - temperature -shift condition , similar protein A bindable measurement . titers were achieved on day 10 under both conditions . The overall specific protein A bindable productivities were 6 . 4 Harvest and Purification Methods pg /cell /day for the temperature - shift condition , and 6 .7 [0128 ] Fifty microliters of day 10 ( 240 + 4 hours ) samples were harvested from all bioreactors using syringes. Post cell pg/ cell/ day for the no -temperature -shift condition . Interest removal by centrifugation ( 3000xg , 5 min ), supernatants ingly , though similar protein A bindable titer was observed were clarified using 0 . 22 um bottle - top filters, and stored at under both conditions , the volumetric activity under the - 80° C . prior to purification . A single step of high through no - temperature -shift condition was considerably lower than put protein A purification was applied in this study . Samples the temperature - shift condition ( FIG . 2B ). In addition , the were buffer exchanged to low salt buffer ( 5 mM NazP04, pH specific activity under the no - temperature - shift condition 7 .4 ) prior to analytical and protein characterization analysis . was significantly lower than the temperature -shift condition throughout the culture duration ( FIG . 2C ). This data indi Analytical and Protein Characterization Methods cates that a temperature shift from 37° C . to 33° C . is optimal [0129 ] Quality attributes analyzed in this study included for maintaining the specific activity for Process # 1. All asfotase alfa aggregation , fragmentation , charge distribu quality attributes quantified in Process # 1 are summarized in tion , total sialic acid content (TSAC ) , and neutral glycan Table 6 . TABLE 6 Quantified Quality Attributes for Process # 1 LOC LOC AEX AEX AEX ( % ( % SEC SEC SEC Sample ( % ( % ( % Main , Main , ( % ( % ( % ID Condition Basic ) Main ) Acidic ) NR ) R ) Agg ) Dimer ) Fragment) TSAC # 1 Temp shift 0 .6 98 . 5 90 . 9 5. 9 100. 0 10 .3 89. 6 0. 0 1. 9 # 2 Temp shift 0 . 2 98 . 8 2 97 . 1 100 . 0 10 . 9 89 . 1 0 . 0 2 . 0 # 3 No Temp 0 .6 99 .1 0 . 2 100 . 0 96 . 8 24 .0 76 .0 0 . 0 3. 9 Shift # 4 No Temp 0 . 6 99 . 2 0 . 2 100 .0 98 . 3 25. 6 74. 4 0 . 0 3 . 9 Shift species levels . The aggregate level was estimated by the [0132 ] AEX results indicated that fewer acidic species percentage of aggregate peaks to the total protein quantified were generated from the no - temperature - shift condition in in SEC . The fragment level was estimated by the percentage comparison to the temperature- shift condition (FIG . 3A ) . of fragment to the total protein quantified in LoC . The However, significantly higher aggregates were quantified by charge distribution was estimated by the percentage of the SEC results under the no -temperature - shift condition ( FIG . basic peaks, main peak and acid peak to the total protein , 3B ) . Under the no - temperature - shift condition , the non respectively , quantified in AEX . The total sialic acid content reduced LoC mean peak percentage was higher ( FIG . 4A , ( TSAC ) was quantified by HPAE - PAD . Detection ofneutral also in the " LoC ( % Main , NR ) " column in Table 6 ) , while glycan species was performed by MALDI- TOF mass spec the reduced LoC mean peak percentage was lower (FIG . 4B , trometry . also in the “ LoC ( % Main , R ) ” column in Table 6 ) . [0133 ] Sialylation was significantly increased by approxi Results mately one fold under the no - temperature - shift condition ( FIG . 4C ) . Cell Culture Performance [0134 ] Analysis of neutral glycans by MALDI- TOF [0130 ] In Process # 1 , the temperature set point was shifted exhibited no higher -order mannose species or atypical gly from 37° C . to 30° C . within 5 hours after the viable cell can species detected under the no - temperature - shift condi density (VCD ) reached 25 - 32x10 $ cells /mL . Without the tion ( FIG . 5 ). However , the no -temperature - shift condition temperature shift , the cell culture reached a higher peak resulted in less amount of A2 ( the predominant afucosylated VCD but experienced a more rapid viability decline ( FIG . glycan species under the control condition with temperature 1A ). Higher overall glucose consumption was observed shifting ), higher fucosylation (higher ratio of FA2 to A2 ), under the no - temperature -shift condition as well (FIG . 1B ) . and an increase in higher -order glycans including FA3G3 , A glucose bolus addition was applied on day 5 and day 8 FA4G3, FA4G1L1, and FA4G4L1 ( FIG . 5 ). under the no - temperature -shift condition according to the 10135 ] The the impact of not shifting temperature on process description . Interestingly, lactate was still con asfotase alfa productivity and quality in Process # 1 is sumed , but at a considerably lower rate under the no summarized in Table 7 . Without the temperature shift, the temperature- shift condition than the temperature -shift con produced asfotase alfa had a much lower specific activity . dition (FIG . 1B ) . Meanwhile , the higher production temperature under the [ 0131] A higher peak VCD under the no - temperature - shift no - temperature - shift condition led to higher sialylation and condition led to early production of protein A bindable titer higher fucosylation . US 2018 /0230445 A1 Aug . 16 , 2018

TABLE 7 tion , total sialic acid content (TSAC ) , neutral glycan profile , and specific activity . Both the aggregate and fragment levels Impact of Not Shifting Temperature on were determined by relative peak areas quantified using gel Asfotase Alfa Productivity and Quality permeation HPLC (GP - HPLC ) . In addition , the fragment Process # 1 - No Temp Shift level was determined using either SDS -PAGE or Lab - on Parameters Chip (LoC ) capillary electrophoresis . The charge distribu Protein A titer No impact Volumetric activity Significantly lower volumetric activity tion was estimated using anion - exchange ( AEX ) chroma Specific activity Significantly lower specific activity tography . TSAC was quantified by high -performance anion AEX acidic peak Less acidic peak exchange chromatography with pulsed amperometric detec SEC aggregate Further evaluation needed tion (HPAE - PAD ). Detection of neutral glycan species was LOC Fragment Further evaluation needed TSAC Significantly higher TSAC performed by matrix -assisted laser desorption / ionization Neutral glycan Higher fucosylation and more higher order (MALDI ) mass spectrometry . Specific activity was mea glycans sured following the paranitrophenylphosphate (PNPP ) based alkaline phosphatase enzymatic assay . Example 3 . Initial Upstream Process Parameter Results Assessment for Exemplary Process # 2 1 . Growth and Production Temperature [0136 ] This example summarizes the initial assessment of upstream manufacturing process parameters for an exem [ 0140 ] The impact of temperature on product fragmenta plary asfotase alfa manufacturing process # 2 and their tion has been discussed . For asfotase alfa produced by the potential to impact critical quality attributes of produced chosen cell clone grown at 36 . 5° C ., the main asfotase alfa asfotase alfa . Some upstream process parameters include % band was calculated to be 95 . 9 % , with approximately 4 % as aggregation , % fragmentation , sialylation , glycosylation , fragments , measured by SDS -PAGE . When temperature was charge distribution , and specific activity . shifted from 36 .5° C . to 33 .0° C . on day 5 , the same clone produced nearly no asfotase alfa fragments (main band Cell Culture calculated to be > 990 % ) . Other secondary clones showed [ 0137 ] All production processes referenced in this study very similar trends in the reduction of fragments via tem were conducted either in shake flasks or bioreactors . Post perature shifting from 36 . 5° C . to 33 . 0° C ., though not all thaw , cells were expanded through a series of shake flasks secondary clones were able to reduce the fragment level to and spinner flasks prior to the inoculation of the production be below 1 % . Therefore , the growth temperature and pro bioreactor . The production bioreactor ( 2 L , 5 L , 10 L , or 200 duction temperature can impact fragmentation . In addition , L ) was scaled using consistent power per volume and sparge the timing associated with conducting the temperature shift gas volume per liquid volume per minute (VVM ), unless could also impact asfotase alfa quality . otherwise specified . The temperature of the production bio [0141 ] Higher aggregate levels detected by GP -HPLC reactor was controlled at 36 . 5° C . from 0 to approximately were observed under lower production temperatures during 120 hours , and shifted to 33 .0° C . at approximately 120 secondary clone screening in 2 L bioreactors. FIG . 6A hours , unless otherwise specified . The dissolved oxygen set showed the aggregate level of asfotase alfa produced by 6 point was maintained at 30 % . Approximately 2 . 7 % ( v / v ) of secondary clones when the temperature was shifted to 33° C . CPN feed per bolus was added at the 96 hour ( hr ) , the 144 compared to material produced by 10 secondary clones hour, and the 192 hour when the culture was harvested on when the temperature was not shifted ( i . e . , maintained at day 10 (240 hr + 6 hr ), while an additional bolus feed was 36 . 5° C . ) . These data suggest temperature shifting from given at 240 hr when the culture was harvested later than day 36 . 5° C . to 33 .0° C . can cause an increase in aggregate level. 10 . [0142 ] In addition , the impact of temperature shifting on TSAC was investigated during primary clone selection and Harvest and Purification initial process development (FIG . 6B ) . A primary clone was [0138 ] Samples harvested from production bioreactors used to investigate the impact of production temperature were clarified by 0 . 22 um filtration post centrifugation at ( day 5 onwards ) on sialylation , or TSAC in 2 L bioreactors . 3000xg for 5 minutes . Purification of the clarified filtered In this study , the production temperature was shifted from harvest was performed and the extent of the purification was 36 . 5° C . to 30 . 0° C . on day 5 under condition 1 , from 36 .5° dictated by the study and sample test requirements . Either C . to 33 .0° C . on day 5 under condition 2 , and the control one chromatography step (protein A ) or two sequential was run without a temperature shift . Samples were harvested when viability was between 60 % and 80 % , and those chromatography steps (protein A followed by hydrophobic cultures were run for 11 to 18 days prior to harvest depend interaction chromatography (HIC ) purification were imple ing on viability . As shown in FIG . 6B , implementing a mented for most samples tested . Additionally , samples were temperature shift resulted in lower TSAC as compared to the buffer exchanged into a low salt buffer ( 5 mM Na PO 4, , pH7 . 4 ) prior to analytical testing . control under constant temperature under similar viability , and shifting to a lower temperature corresponded to a lower TSAC . The desired minimum TSAC value herein is 1 . 8 . As Analytical Characterization sialylation ( or TSAC ) will change the charge profile of the (0139 ] Quality attributes referenced in this study were product , and convert neutral glycans to negatively charged identified as subsets of broader quality metrics such as glycans , these three quality attributes ( production tempera purity, potency , and structure . The quality subsets described ture , aggregation , and TSAC ) are considered to be closely include % aggregation , % fragmentation , charge distribu related . US 2018 /0230445 A1 Aug . 16 , 2018 14

2 . pH 500 uM , both the growth and the viability were dramatically [ 0143 ] Preliminary results indicated that changing the pH affected . Therefore , the optimal concentration of total zinc set point from 6 . 90 to 7 . 00 led to increased TSAC ( FIG . 7 ) . supplementation was under 500 uM . Thus , it was estimated Using the chosen secondary clone , two 2 L bioreactors with that the optimal zinc concentration to be supplemented at pH set point 6 .90 and 7 .00 , respectively, were used to day 0 may be between about 25 uM and about 300 uM investigate the impact of pH on asfotase alfa sialylation . ( considering both cell growth / viability and protein function Samples were harvested from bioreactors on day 10 and day issues ) . In fact, zinc concentration at 150 uM may be even 12 , respectively . It was observed that the TSAC dropped better than 300 uM since the former resulted in less cell from 1 . 9 on day 10 to 1 . 4 on day 12 under pH 6 . 90 condition . growth inhibition after day 5 (FIG . 9A ) . It is also surprising Though the same trend in declining TSAC values was that although 20 uM zinc is theoretically sufficient for observed under pH 7 . 00 conditions , the TSAC values under producing functional asfotase alfa ( i. e ., 2 zinc ions per active pH 7 .00 on both days ( 2 .6 on day 10 , and 2 . 1 on day 12 ) enzyme) , the actual zinc supplementation may require sig were higher than those under the lower pH condition . These nificantly higher zinc concentrations ( e . g . , 150 uM ) in data suggested that TSAC declines with culture duration and alkaline phosphates ( e. g ., asfotase alfa , TNALP , PALP , a lower pH set point results in a lower TSAC value . GCALP, IAP, or fusion / variant proteins thereof) manufac turing processes. 3 . Medium Feed Addition 5 . Seeding Density [0144 ] Preliminary data indicated that delaying the first bolus addition or increasing the glucose concentration in the [0149 ] Generally , higher seeding density leads to a higher medium may lead to significantly higher TSAC in compari peak viable cell density , which is primarily due to certain son to the control condition (FIG . 8A ). nutrient component constraints in the production medium . 0145 ] In addition , how the medium feed is delivered As feeding and temperature shift is based on culture dura ( either continuously or by bolus addition ) also had an impact tion , seeding density in conjunction with other upstream on TSAC . Data from both 2 L and 10 L scales indicated process parameters , especially temperature shift timing , significantly higher TSAC was observed when the feed was could potentially impact asfotase alfa quality. added in bolus mode, in comparison to the continuous addition mode ( FIG . 8B ) . Therefore , the CPN feed is deter 6 . Harvest Timing mined to be supplemented as bolus . In addition , no impact [0150 ] The harvest timing in this exemplary process was on TSAC was observed when the feed was delivered through 240 6 hr. The impact of harvest timing on TSAC has been top port drop addition or by subsurface ( dip ) addition . demonstrated ( see , e . g . , FIG . 7 ) . In addition , harvest timing is also considered to be associated with viability ( see , e. g ., 4 . Zinc Sulfate Addition FIG . 14 ), and the viability decline toward the end of the [ 0146 ] Zinc ions are known to be essential for alkaline culture indicates that harvest timing could potentially impact phosphatase ( e . g ., asfotase alfa ) stability as it helps to asfotase alfa quality . maintain their structure and activity . For example , two zinc [0151 ] Various upstream process parameters were atoms associate with one placental alkaline phosphatase assessed , including control of seeding density , temperature, molecule (Helene Le Du et al. 2001 J. Biol. Chem . 276 : pH , DO , gassing strategy , agitation , CPN feed , glucose 9158 - 9165 ) . Based on this ratio , for the titer of 1 g / L asfotase addition , galactose addition , zinc addition , and harvest tim alfa produced by the exemplary manufacturing process ing . More upstream process parameters , including % aggre developed in small -scale models , approximately 20 uM zinc gation , % fragmentation , sialylation , glycosylation , charge is needed for asfotase alfa activity . distribution , and specific activity , were evaluated , e . g . , as [0147 ] It was observed that as cell culture progressed , discussed below . protein A - bindable asfotase alfa was being produced con [0152 ] Temperature had a strong impact on aggregation , tinuously, but the corresponding volumetric activity did not fragmentation , and sialylation . pH also impacted asfotase synchronize with the protein A titer, which indicated that the alfa sialylation . Gassing strategy or / and agitation seemed to protein being produced was inactive (FIG . 9A ). Further impact viability . CPN feed addition seemed to have an more , while the volumetric activity decreased from day 5 to impact on sialylation . Zinc addition was shown to be essen day 9 , the protein activity was immediately restored upon tial for maintaining enzymatic activity , and the amount of zinc sulfate supplementation on day 9 . This data suggested zinc addition ( 150 uM ) was determined based on the studies that zinc needs to be supplemented into the production presented . Harvest timing was shown to be associated with medium . In this study, the zinc sulfate concentration was viability and TSAC decline towards the end of the culture. increased by 25 uM through bolus addition on day 9 . Given that protein A titer was approximately 1000 mg/ L on day 9 , Example 4 . Further Upstream Process Parameter this zinc bolus addition increased the ratio of zinc ion to asfotase alfa by at least 2 : 1 . Assessment for Exemplary Process # 3 [0148 ] In the following study, the cytotoxicity level of zinc [0153 ] This example summarizes further characterization sulfate on cell growth and viability was investigated using a of upstream processes that were performed for the purpose mini- bioreactor system in batch mode, in which zinc sulfate of screening production bioreactor process parameters . For in different concentrations was added into the culture initial assessments , a list of upstream process parameters medium at day 0 . When zinc sulfate concentration was were tested in this exemplary process , include production below 300 uM , little impact on growth or viability was culture pH , temperature , seeding density , gassing strategy , observed ( FIGS . 9B and 17C ) . However, in conditions agitation , CPN feed , glucose addition , galactose addition , where the zinc concentration was greater than or equal to and harvest timing . US 2018 /0230445 A1 Aug . 16 , 2018 15

Acronyms on day O an day 4 , and 1. 0 g / L daily from day 5 to day 9 . A [01541 CPP : critical process parameter , a process param portion of the culture was harvested on day 10 (240 + 4 hr ) eter (process input) that has an impact on a critical quality and day 11 ( 264 + 4 hr ). attribute and therefore will be monitored and controlled to ensure the process produces the desired quality . Harvest and Purification [0155 ] CQA : critical quality attribute [0161 ] Samples harvested from production bioreactors were clarified by 0 .22 um filter post centrifugation at 3000xg [0156 ] HPLC: High Performance Liquid Chromatography for 5 minutes . Purification of the clarified filtered harvest [0157 ] LOQ : Limit of Quantification was performed . The extent of the purification was dictated [0158 ] P /V : Power per Volume by the study and sample test requirements . Either one step (protein A ) or two sequential steps ( protein A and HIC ) Materials and Methods purification were implemented . Additionally , samples were buffer exchanged into a low - salt containing buffer ( 5 mM 0159 ] Process parameters that were shown in initial Na3PO4, pH7. 4 ) prior to analytical testing . experiments to have a potential impact were further inves tigated . Six blocks of bioreactors were conducted in the following process characterization study ( Table 8 ) . Three Analytical Characterization levels for each process parameter were tested with the [0162 ] Quality attributes referenced in this study include median level as the current set point ( the control) in con % aggregation , % fragmentation , charge distribution , total sideration of the controlling capability at manufacturing and sialic acid content ( TSAC ) , and neutral glycan profile . Both process parameter range testing . In block 1 to 5 , three - level the aggregate and fragment levels were determined by full factorial design with two replicate controls was imple relative peak areas quantified using size exclusion chroma mented . In block 6 , four combinations of extreme P / V and tography (SEC ). In addition , the asfotase alfa purity (main sparge VVM plus two controls were conducted . peak ) was also determined using Lab -on - Chip (LoC ) capil lary electrophoresis . The charge distribution was estimated TABLE 8 using anion - exchange chromatography ( AEX ) . TSAC was quantified by High -Performance Anion Exchange Chroma Investigated Process Parameters and Levels tography with Pulsed Amperometric Detection (HPAE Investgated Levels PAD ) . Detection of neutral glycan species was performed by Matrix Assisted Laser Desorption / Ionization - Time of Flight Block Process Parameter Low Median High (MALDI - TOF) mass spectrometry . Volumetric activity was Gluscose addition threshold ( g / L ) 1. 5 2. 0 3 . 0 measured following the paranitrophenylphosphate (PNPP ) Galactose addition ( g / L ) 10 15 based alkaline phosphatase enzymatic assay . 2 pH set point 6 .75 6 . 90 7 . 10 Production temperature ( ° C .) 30 . 0 33. 0 35 . 0 w Growth temperature 35 . 0 36 . 5 37 . 5 Statistical Analysis and Data Visualization DO set point ( % ) 1515 30 60 4 Feed amount ( % ) 67 100100 133 Feed initiation (day ) 3 4 5 [0163 ] The potential of tested process parameters to 5 Seeding density (105 cells /mL ) 4 . 0 5 . 5 8 . 0 impact productivity and each quality attribute were evalu Temp shift timing (hr ) 108 120 132 ated using the statistical software package JMP ( Cary , N . C . ) . 6 P / V ( W /m3 ) 9 - > 41 17 - > 81 26 - > 121 Three continuous parameters ( three levels for two tested Maximum sparge 0 .015 0 .020 0 .025 process parameter , respectively , and two levels for harvest ) were analyzed . Individual p - values calculated by two sided t -test were reported , and 0 .05 was chosen as the significance threshold . Contour plots generated by JMP were used to Cell Culture visualize the potential impact of tested parameters on each [0160 ] All production processes referenced in this study quality attribute . were conducted either in 2 L or 10 L bioreactors . Post thaw cells were expanded through a series of shake flasks and Results spinner flasks prior to the inoculation of the production bioreactor. The production bioreactor ( 2 L or 10 L ) was [0164 ] As the acceptable range for each CQA in this scaled using PNV and sparge VVM , unless otherwise speci exemplary process was still under investigation , an exem fied . Under the control condition , the cell culture process plary experimental range was generated and used for com parameters were as follows. Culture pH set point was 6 . 90 parison purposes ( Table 9 ) . The data source stemmed from with 0 .05 dead band . The temperature was controlled at all of the day 10 results obtained in this study. For the 36 .5° C . from 0 to 120 hours , and shifted to 33° C . at 120 impurity measurement including fragment and aggregate by hours . The dissolved oxygen set point was maintained at SEC and basic and acidic peak by AEX , the average plus two 30 % . The initial PN was 17 W / m (0 - 96 hr) and shifted to fold of standard deviation was used as the upper threshold . 81 W /m3 prior to the first bolus feed addition at 96 hr. 2 . 67 % As the calculated upper threshold for fragment measured by ( v / v ) of CPN feed per bolus was added at 96 hr, 168 hr, and SEC and basic peak measured by AEX is lower than the LOQ 192 hr. Glucose was maintained at s2 g / L . If glucose was ( 1 % ) of SEC and AEX assay, the upper threshold was set to below 2 g / L , daily bolus addition was applied to increase be 1 % for both attributes. As for TSAC , the average , minus glucose concentration to be in the range of 1 . 8 to 2 . 0 g / L . and plus two fold of standard deviation , was used as the Galactose was supplemented throughout the culture: 2 . 0 g / L experimental range. US 2018 /0230445 A1 Aug . 16 , 2018 16

TABLE 9 TABLE 10 Significance of Direct Impact of Each Process Small Scale Experimental Ranges for CQA Parameter on COA Analyzed by JMP Critical Quality Attributes SEC AEX Basic Acidic p - value TSAC Aggregate Fragment Basic Acidic peak peak Glucose 0 . 41 0 .63 0 . 98 0 .67 Aggregate Fragment AEX AEX Galactose 0 . 04 0 .37 - 0 .. 14 0 . 44 pH 0 .00 0 .63 0 .00 0 .05 0 .29 TSAC SEC ( % ) SEC ( % ) ( % ) ( % ) Production temp 0 .00 0 . 00 0 . 00 0 . 64 0 . 00 DO 0 . 72 0 .73 0 . 72 0 . 64 0 .79 Average ( Avg ) 22. . 2 8 . 0 0 .. 5 0 . 4 8 . 6 Growth temp 0 . 16 0 . 47 0 . 01 0 . 26 0 .04 Standard deviation 0 . 4 0 . 8 80 . 2 0 . 1 0 . 8 Feed amount 0 . 03 0 .00 0 . 14 0 . 70 0 . 00 ( stdev ) Feed timing 0 .37 0 .00 0 .71 0 .03 0 . 00 Avg - 1 x stdev 1. 8 7 . 3 0 . 3 0 .3 7 . 8 Seeding density 0 . 81 0 . 34 0 . 00 0 . 31 0 . 49 Temp shift timing 0 .06 0 . 64 0 . 00 0 . 70 0 . 26 Avg - 2 x stdev 1 .. 55 6 . 5 0 . 1 0 .2 2 7 .0 Avg + 1 x stdev 2 . 5 8 . 8 00 . 7 00 .. 5 9 . 44 Avg + 1 x stdev 2 . 9 9 .6 0 . 8 0 .6 10 .2 (0166 ) Table 11 summarizes the average quality attribute Experimental range value obtained at the minimum and maximum tested process 1. 5 to 92 . 5 9 .6 51. 0 51. 0 s10 .2 parameter level. For instance , in the glucose and galactose addition study , three bioreactors used 1 . 5 g / L as the glucose addition threshold , and each of them had a different galac [ 0165 ] The significance of the direct impact of each pro tose addition level . The CQA value at 1 .5 g / L of glucose cess parameter on each CQA is summarized in Table 10 . The threshold condition was then calculated using the average p - value of PNV and sparge VVM impact was not calculated from those three bioreactor. In the following sections, day 10 due to lack of statistical power . Thep -values less than or data is used for quality discussion unless otherwise men equal to 0 .05 are considered as statistically significant. tioned . TABLE 11 Averaged Ouality Attribute Values under Tested Conditions Critical Quality Attributes Aggregate , Fragment, Basic peak , Acidic peak , Process Parameters and Tested Ranges TSAC SEC ( % ) SEC ( % ) AEX ( % ) AEX ( % ) Glucose threshold ( g / L ) Min 1 . 5 2 . 5 0 . 0 0 . 3 5 . 9 Max 3 . 0 0 . 0 0 . 4 5 . 5 Total galactose addition Min 0 . 0 0 . 3 5 . 6 ( g / L ) Max 15 tmto 0 . 0 0 . 5 * 5 . 7 * PH Min 6 .75 0 . 0 0 . 5 5 . 7 Max 7 . 10 NNWuviounVi 0 . 1 0. 3 6 . 1 Production Temp ( ° C .) Min 30 . 0 0 . 0 0. 3 8 . 5 Max 35 . 0 Nuwun 0 . 1 Dissolved Oxygen (% ) Min 15 0 . 5 0. 4 Max 60 7 . 6 0 .6 0 . 4 Dow:Ninovv Growth Temp (°C . ) Min 35 . 0 7 . 7 0 . 4 0 . 3 Max 37 . 5 AwwwN 7 . 6 0 . 8 0 .4 Feed amount (% ) Min 67 8 . 5 0 . 4 0 . 3 8 . 9 Max 133 co 0 . 4 0. 3 9 .4 Feed initiation ( day ) Min 3 o 0 . 4 0 .4 3 Max 5 o 8 . 2 0 . 4 Seeding ( 103 cells/ mL ) Min 4 . 0 a 0 . 6 0. 5 Max 8 . 0 ??? 0 . 8 0 .6 Ainbowaw Temp Shift Timing ( h ) Min 108 0 .6 Max 132 win 0 . 8 0 .5 OOO Harvest ( day ) Min 0 . 4 Max 19 8 .Na 1 0 . 5 0 . 4 8 . 9 Generation No. Min 38 8 . 8 0 . 4 0 . 4 8 .6 Max 48 No. 7 . 7 0 . 5 0 .4 8 . 7 ANNNNNNNNNNNNNHann 0 . 3 P /V (W /m2 ) Min 9 ( 0 - 96 hr ); 5 6 . 8 41 ( 96 - 240 hr) Max 26 ( 0 - 96 hr ); 5 0 . 3 7 . 3 121 ( 96 - 240 hr ) Maximum Sparge VVM Min 0 . 015 2 . 0 6 .. 7 00 . 33 0 . 4 7 . 1 Max 0 . 025 2 . 1 6 . 8 0 . 33 0 . 33 7 . 0 Note : When the average percentage of basic peak and acidic peak measured by AEX was calculated for the condition 15 g / L of total galactose addition , an outlier sample was excluded due to its low peak response in the assay . Its biological replicate sample from another bioreactor remained for the calculation purpose . US 2018 /0230445 A1 Aug . 16 , 2018 17

1 . pH the volumetric activity achieved a peak value at a growth [ 0167 ] Prior results indicated pH impacted TSAC of pro temperature of 36 . 5° C . ( FIG . 11A ) . duced asfotase alfa . The potential impact of culture pH and [0172 ] As for the impact on asfotase alfa quality , the production temperature on asfotase alfa productivity and growth temperature was shown to have a significant impact quality was further investigated in 2 L bioreactors . A full only on fragmentation as measured by SEC (FIG . 11B ). factorial design comprising three culture pH levels (6 .75 , Increasing the growth temperature from 35 .0° C . to 37 .5° C . 6 . 90 , and 7 . 10 ) and three production temperature levels resulted in a fragment level that was elevated from 0 . 3 % to ( 30 .0° C . , 33 . 0° C . , and 35 . 0° C .) was implemented , and 0 . 8 % . It is noted that those measured values were all below samples from all conditions were harvested at 240 + 4 hour , the LoQ ( 1 % ) of SEC assay. and 264 + 4 hours , respectively . Two -step purification ( ProA + [0173 ] As the impact on CQAs by growth temperature HIC ) was applied prior to analytical analysis . were all within the experimental range ( Table 9 and Table [0168 ] Culture pH set point in the tested range 6 . 75 to 7 . 10 11 ) , a growth temperature ranging from 35 . 0° C . to 37 . 5° C . was not considered to be a critical process parameter (CPP ) is unlikely to be a CPP . In various embodiments , alkaline in that all CQAs tested were within the small scale experi phosphatase ( e . g . , asfotase alfa ) is produced at a growth mental range specified in Table 9 . However, pH was shown temperature of 33 . 0° C . , 33 . 5° C . , 34 . 0° C . , 34 .5° C . , 35 . 0° to significantly affect several tested quality attributes . C ., 35 . 5° C . , 36 . 0° C . , 36 . 5° C . , 37 . 0° C . , 37 . 5° C ., 38 . 0° C ., Elevating the pH set point from 6 .75 to 7 . 10 led to TSAC or 38 . 5° C . In one particular embodiment, alkaline phos increasing from 1 . 9 to 2 . 7 at the post -shift control produc phatase ( e . g . , asfotase alfa ) is produced at a growth tem tion temperature of 33° C . (FIG . 10A ) . PH also had signifi perature of 35 .0° C . , 36 . 5° C ., 37. 0° C . or 37. 5º C . cant impact on fragmentation measured by SEC and basic peak measured by AEX ( Table 10 ) , but the impact was less 2 . 2 Production Temperature than or equal to 0 . 2 % ( Table 11) which was below the [0174 ] The impact of production temperature ( 30 .0° C ., HPLC -based assay variation of 0 . 5 % . Furthermore , under 33. 0° C . , and 35. 0° C . ) on CQA was investigated together the control condition ( pH 6 . 90 and production temperature with a culture pH set point in 2 L bioreactors . One -step 33 .0° C .) , the highest productivity in terms of both protein purification (ProA ) was applied prior to analytical analysis . A titer and volumetric activity was achieved ( FIGS . 10B and 10175 ] Production temperature significantly impacts 10C ) . CQAs especially TSAC , aggregation and fragmentation , as [0169 ] The data indicated that the impact of a pH set point measured by SEC , and acidic peak measured by AEX ( Table ranging from 6 .75 to 7 . 10 on CQA was all within the 10 and Table 11 ) . Elevating the production temperature from experimental range , suggesting production culture pH was 30 . 0° C . to 35 . 0° C . led to an average TSAC increase from unlikely to be a CPP . In various embodiments, alkaline 1 . 6 to 2 . 7 ( FIG . 10A ) , while lowering production tempera phosphatase ( e . g . , asfotase alfa ) is produced at a pH set point ture from 35 . 0° C . to 30 . 0° C . resulted in a large increase in of 6 .65 , 6 . 70 , 6 .75 , 6 .80 , 6 .85 , 6 . 90 , 6 . 95 , 7 .00 , 7 . 05 , 7 . 10 , aggregates as measured by SEC , from 3 . 8 % to 8 . 2 % ( FIG . 7 . 15 , 7. 2 , or higher. 12A ). In addition , the acidic peak measured by AEX increased from 3 . 7 % to 8 . 5 % with lowering the production temperature (FIG . 12B ) . This data suggested that a produc 2 . Temperature tion temperature higher than 30 .0° C . is probably beneficial [0170 ] Prior results indicated that temperature had a strong to reduce the risk of low TSAC and prevent increased impact on several parameters including aggregation , frag aggregation / acidic peak . Meanwhile , there seems to be a mentation , and TSAC . As previous processes employ a very subtle positive correlation between production tem temperature shift from 36 .5° C . to 33 . 0° C . at 120 hours , the perature and fragmentation , as measured by SEC ( FIG . investigation of the temperature impact was dissected into 12C ), and the basic peak , measured by AEX ( FIG . 12D ) . three parameters: growth temperature ( 0 to 120 hours ) , This data suggested a production temperature lower than 35° production temperature (120 hours to harvest ) , and tempera C . might be preferred to reduce the risk of asfotase alfa ture shift timing . These three temperature -related parameters fragmentation . were further investigated separately and summarized below . [0176 ] In terms of the impact on productivity , production temperature appears to have the largest impact on protein A 2 . 1 Growth Temperature titer and volumetric activity ( FIGS . 10B and 10C ) . In the range of 30 .0° C . to 35 .0° C . , increasing production tem [0171 ] The impact of growth temperature was investigated perature correlates with increasing productivity . However, together with the DO set point in 2 L bioreactors . Three under pH 7 . 10 , production temperature interacts with pH to levels of growth temperature ( 35. 0° C ., 36 .5° C . and 37 . 5° impact productivity, with the highest volumetric productiv C . ) were maintained from 0 to 120 hour under different test ity occurring at a production temperature of 33 . 0° C . Peak conditions , followed by a temperature shift to 33 .0° C . at productivity in regards to both volumetric activity and 120 hours . Samples from all conditions were harvested at protein A titer was observed at pH 6 . 90 under production 240 + 4 hours , and 264 + 4 hours . One -step purification (ProA ) temperature that is greater than or equal to 33 .0° C . Though was applied prior to analytical analysis . Growth temperature the production temperature was shown to have a significant impacts growth rate and viability . A higher growth tempera - impact on several CQAs ( Table 10 ) , the impact was still ture correlated to a faster growth rate in the early stages, within the experimental range ( Table 9 and Table 11 ) . followed by a rapid growth - rate decline post temperature Therefore , production temperature in the range of 30 . 0° C . shift , which resulted in lower peak VCD . In addition , to 35 .0° C . is unlikely to be a CPP . cultures with a higher growth temperature also had 2 - 4 % [0177 ] In various embodiments , alkaline phosphatase lower final viability on the day of harvest . Interestingly, ( e . g . , asfotase alfa ) is produced at a production temperature growth temperature impacts productivity significantly , and of 29 .0° C ., 29 . 5° C ., 30 .0° C ., 30 . 5° C ., 31. 0° C ., 31. 5° C . , US 2018 /0230445 A1 Aug . 16 , 2018

32 .0° C . , 32 . 5° C . , 33 . 0° C . , 33 . 5° C . , 34 . 0° C . , 34 . 5° C . , [0184 ] Though medium feed amount and initiation timing 35 .0° C ., 35 .5° C ., 36° C ., or a higher temperature . In one did not directly impact productivity , their interaction seemed particular embodiment, alkaline phosphatase ( e . g . , asfotase to have a subtle impact on volumetric activity ( FIG . 14A ) . alfa ) is produced at a production temperature of 30 .0° C . , Under the control condition , maximum volumetric activity 30 .5° C . , or 35 .0° C . was achieved . When 33 % less feed was provided , delaying feed initiation resulted in lower volumetric activity . 3 . Temperature Shift Timing [0185 ] The elevated acidic peak ( 10 . 1 % ) and aggregation ( 9 . 5 % ) observed under more ( 133 % ) and earlier ( first bolus [0178 ] The impact of temperature shift timing on CQA on day 3 ) feed condition is close to the small scale experi was investigated together with seeding density in 2 L mental range ( 10 . 2 % for acidic peak , and 9 . 6 % for aggre bioreactors. Three temperature shift times ( 108 hr, 120 hr gation , Table 9 ) , indicating that at least one parameter needs and 132 hr ) were tested , and samples from all conditions to be controlled tighter at scales, e . g . , 15 % variation of the were harvested at 240 + 4 hour and 264 + 4 hour. One- step medium feed amount per bolus, and / or relatively short purification (Protein A ) was applied prior to analytical addition time window ( 96 + 3 hr, 144 + 3 hr, and 192 + 3 hr ) . As analysis . the impact on CQAs by medium feed addition was all within [0179 ] Temperature shift timing has a subtle impact on the experimental range ( Table 9 and Table 11) ,medium feed volumetric activity ( FIG . 13A ) . At high seeding density , amount ranging from 67 % to 133 % and feed timing ( 96 + 24 delaying temperature shift timing from 108 hr to 132 hr hr, 144 + 24 hr, and 192 + 24 hr ) is unlikely to be a CPP . seems to decrease the volumetric activity . However , at lower 10186 ]. In various embodiments, alkaline phosphatase seeding density , maximum volumetric activity is observed ( e . g . , asfotase alfa ) is produced by a process in which extra under the later temperature shift condition . boluses of culture medium are added to the production [ 0180 ] In terms of asfotase alfa quality , temperature shift bioreactor. For example , one , two , three , four , five , six , or timing was shown to only significantly impact fragmenta more boluses of culture medium may be added . In one tion , as measured by SEC ( FIG . 13B ) . Under the latest particular embodiment, three boluses of culture medium are temperature shift condition (132 hr) , the average fragment added . In various embodiments , such extra boluses of cul level was 0 . 8 % , close to the small scale experimental range ture medium may be added in various amounts . For for fragmentation ( < 1 . 0 % ) . Therefore , a narrower tempera example , such boluses of culture medium may be added in ture shift time window is probably beneficial to reduce the an amount of about 20 % , 25 % , 30 % , 33 % , 40 % , 45 % , 50 % , risk of fragmentation . 60 % , 67 % , 70 % , 75 % , 80 % , 90 % , 100 % , 110 % , 120 % , [0181 ] As the impact on CQAs by temperature shift timing 125 % , 130 % , 133 % , 140 % , 150 % , 160 % , 167 % , 170 % , was all within the experimental range ( Table 9 and Table 11) , 175 % , 180 % , 190 % , 200 % , ormore , of the original volume temperature shift timing ranging from 108 hr to 132 hr is of culture medium in the production bioreactor. In one unlikely to be a CPP . particular embodiment, such boluses of culture medium may 10182 ] In various embodiments , alkaline phosphatase be added in an amount of about 33 % , 67 % , 100 % , or 133 % ( e . g ., asfotase alfa ) is produced by a process in which a of the original volume. In various embodiments , such addi temperature shifting occurs at about 100 hr, 105 hr, 108 hr, tion of extra boluses may occur at various times during the 110 hr, 115 hr, 120 hr, 125 hr, 130 hr , 132 hr, 135 hr, 140 hr, cell growth or protein production period . For example, 145 hr , or 150 hr post the starting point of growth ( e . g . , boluses may be added at day 1 , day 2 , day 3 , day 4 , day 5 , inoculation ) . In one particular embodiment, alkaline phos day 6 , day 7 , day 8 , day 9 , day 10 , day 11 , day 12 , or later phatase ( e . g . , asfotase alfa ) is produced by a process in in the process . In one particular embodiment, such boluses which a temperature shifting occurs at about 108 hr, 120 hr of culture medium may be added in every other day ( e . g ., at or 132 hr. In various embodiments , alkaline phosphatase ( 1 ) day 3 , day 5 , and day 7 ; ( 2 ) day 4 , day 6 , and day 8 ; or ( e . g . , asfotase alfa ) is harvested at a time point of about 200 ( 3 ) day 5 , day 7 , and day 9 . In practice , the frequency , hr, 210 hr, 220 hr, 230 hr, 240 hr, 250 hr, 260 hr, 264 hr, 270 amount, time point , and other parameters of bolus supple hr, 280 hr, 288 hr ( i . e . , 12 days ), or more than 12 days. In one ments of culture medium may be combined freely according particular embodiment , alkaline phosphatase ( e . g . , asfotase to the above limitation and determined by experimental alfa ) is harvested at a time point of about 240 , 264 , or 288 practice. hr. 5 . Seeding Density 4 . Medium Feed [0187 ] In this study, the impact of seeding density on CQA [0183 ] Previous results indicated that medium feed addi was investigated together with temperature shift timing in 2 tion impacts sialylation . As a control for this exemplary L bioreactors . Three seeding densities ( 4 .0x10 cells /mL , process , three boluses of medium were added to the pro 5 .5x10 cells/ mL and 8 .0x10 cells /mL ) were tested , and duction bioreactor on day 4 , day 6 , and day 8 , and the total samples from all conditions were harvested at 240 + 4 hour, amount was equivalent to 8 % ( v / v ) of the initial culture and 264 + 4 hour . One -step purification (Protein A ) was volume. With the number of boluses unchanged , three feed applied prior to analytical analysis . amounts were tested : 67 % , 100 % ( the control) , and 133 % . 10188 ] Temperature shift timing has a very strong impact In terms of bolus addition timing , three strategies were on productivity . As shown in FIG . 13A , increasing the investigated : ( 1) day 3 , day 5 , and day 7 ; ( 2 ) day 4 , day 6 , seeding density from 4 .0x10 cells/ mL to 8 . 0x10° cells /mL and day 8 ( the control) ; (3 ) day 5 , day 7, and day 9 . All led to a volumetric activity increase from 806 U /mL to 1012 bioreactors were conducted in 2 L bioreactors , and samples U /mL on average . from all conditions were harvested at 240 + 4 hours and [0189 ] In terms of asfotase alfa quality , seeding density 264 + 4 hours . One - step purification ( Protein A ) was applied was only shown to significantly impact fragmentation , as prior to analytical analysis . measured by SEC (FIG . 13B ) . Under the highest seeding US 2018 /0230445 A1 Aug . 16 , 2018

density condition ( 8 .0x10 cells/ mL ) , the average fragment be noted that HIC is able to increase sample purity without level was 0 .8 % , close to the small -scale experimental range impacting TSAC . TABLE 12 Analytical Result from Exemplary 2 L Bioreactor Batches Harvest AEX ( % ) SEC ( % ) # (Day ) Basic Main Acidic Aggregate Main Fragment TSAC Purification 10 10 0 . 2 90 . 8 9 . 1 8 . 0 91 . 6 0 . 4 2 . 1 ProA O 10 0 . 4 90 . 1 9 . 5 ? 91. 2 0 . 5 1 . 7 10 O0 . 3 90 . 7 9 . 0 92 . 0 0 . 6 2. 3 11 O 90 . 4 0 . 3 9 . 3 ? 91. 5 0 . 8 1. 8 0 .2 90 . 9 8 . 9 91. 2 0 . 3 1 . 7 o Ovv 9. 3 90 . 8 11 0 . 4 90 . 4 ? 0 . 4 10 0. 3 90. 9 8 . 8 91. 2 0. 5 11 0 . 4 90 . 3 9 . 3 8 .6 90 . 8 0 .6 10 0 . 5 91. 0 8 .6 8 .0 91 . 5 0 . 5 11 0 . 4 91. 6 8 . 0 7 . 2 92. 3 0 .5 10 0 . 5 91. 0 8 . 5 7 . 9 91 . 6 0 . 5 2 .5 11 0 . 5 92. 1 7 . 4 6 . 6 92 . 9 0 . 5 2 . 4 10 0 . 3 94 . 3 5. 4 90 . 7 0 . 2 2 . 1 11 0 .4 90 . 3 9 . 3 90 . 6 0 . 3 ? 10 0 . 4 91 . 0 8 . 7 90 . 8 0 . 3 ? 11 0 . 4 90 . 3 9 . 4 90 . 8 0 . 4 18 10 0 .5 93 . 4 66 . 1 94 . 1 0 . 0 Pro A + 18 11 0 . 5 92 . 8 6 . 6 93 . 6 0 . 0 1 . 5 HIC 19 1 100 0 .4 93. 7 5 . 9 cowooow 94 . 2 0 . 0 1 . 8 19 11 0 .5 92. 5 7 . 1 6 . 7 93 . 3 0 . 0 1. 6

for fragmentation ( < 1 . 0 % ) . Therefore , a tighter seeding [0192 ] In each individual study, harvest timing was treated density range may be beneficial in order to reduce the risk as the third parameter for statistical analysis . As shown in of fragment generation at scales . each individual study, delaying harvest from day 10 to day [0190 ] As the impact on CQAs by seeding density was all 11 led to approximately 10 - 15 % increased protein A titer and within the experimental range ( Table 9 and Table 11) , a volumetric activity . seeding density ranging from 4 .0x109 cells/ mL to 8 .0x10 % cells /mL is unlikely to be a CPP . In various embodiments , [0193 ] A significant impact ofharvesting timing on TSAC alkaline phosphatase ( e . g ., asfotase alfa ) is produced by a was observed in all five individual studies . Delaying harvest process in which cells are seeded in a density of about by one day resulted in a TSAC drop of approximately 1 .0x10 cells /mL , 1 .5x10 cells /mL , 2 .0x10 cells/ mL , 2 .5x 0 . 3 -0 . 4 moles of sialic acid per mole of protein on average , 10 cells /mL , 3 .0x10 cells /mL , 3 .5x10 cells /mL , 4 .0x10 although TSAC on both conditions were still within the cells /mL , 4 . 5x105 cells /mL , 5 . 0x105 cells /mL , 5 .5x105 cells/ small - scale experimental range ( 1 . 5 - 2 . 9 ) as specified in mL , 6 . 0x10 cells /mL , 6 .5x10 cells/ mL , 7 .0x10 cells /mL , Table 9 . Due to the significant impact of harvest timing on 7 .5x10 cells /mL , 8 .0x10 cells /mL , 8 .5x10 cells/ mL , 9 .0x TSAC , early harvest could be performed to increase TSAC . 10 % cells /mL , 9. 5x10 cells /mL , 1. 0x10 cells /mL , 1 .5x10 cells /mL , 2 .0x106 cells /mL , or a higher density . In one [0194 ] As for aggregation , as measured by SEC , and particular embodiment, in such process cells are seeded in a acidic peak , as measured by AEX , harvest timing was shown density of about 4 .0x105 cells/ mL , 5 . 5x105 cells /mL or to have statistically significant impact in three out of five 8 . 0x10 cells/ mL . studies ( Table 13 ) . [01951 . However, the magnitude of the impact on either 6 . Harvest Timing CRA was marginal, less than 1 % as compared to the control [0191 ] Prior data indicated that delaying harvest timing conditions in each individual study . In addition , all values was associated with a viability and TSAC decline , so harvest fell in the small - scale experimental ranges that are specified timing can have a potential impact on other CQAs. In all in Table 9 (59 . 6 % for aggregates and s10 . 2 % for acidic process characterization studies , samples were harvested peak ) . from day 10 ( 240 + 4 hr ) and day 11 (264 + 4 hr ) from all [0196 ] The fragment level was impacted by harvest timing bioreactors . Ten 2 L bioreactors served as the control con in one out of five studies ( shown in Table 13 ) . However, dition in multiple blocks of the process characterization delaying harvest by one day resulted in elevated fragment by study . As all were performed under the same conditions with only 0 . 1 - 0 . 2 % under two control conditions, and the frag current process parameter settings, they allowed for the ment level on day 11 was still below the LoQ ( 1 % ) of SEC investigation of harvest timing on asfotase alfa productivity assay . Similarly , the basic peak level was elevated from and quality . The analytical result from those ten 2 L biore 0 . 2 - 0 . 3 % on day 10 to 0 . 4 % on day 11 with a p -value of 0 . 03 actors is listed in Table 12 . It is noted that samples from two ( Table 13 ) . However , both values were still far below the bioreactors ( # 18 and # 19 ) were purified by a two - step LOQ ( 1 % ) of the AEX assay . Therefore , harvest timing , purification ( ProA + HIC ) , while those from the other eight ranging from day 10 ( 240 + 4 hr ) to day 11 ( 264 + 4 hr) , is bioreactors (# 10 - # 17 ) were purified only by ProA . It should unlikely to be a CPP . US 2018 /0230445 A1 Aug . 16 , 2018

TABLE 13 conditions were examined with the control production tem bioreactors ( # 10 - # 17 ) were purified only by ProA . It should Impact of Harvest Timing on COA from Individual Studies bioreactors ( # 10 -# 17 ) were purified only by ProA . It should bioreactors ( # 10 - # 17 ) were purified only by ProA . It should p - value for perature ( 33 . 0° C .) and four additional temperatures were harvest SEC AEX examined with the control culture pH (6 . 90 ) ( Table 16 ). timing TSAC Aggregate Fragment Basic Acidic TABLE 15 Reference 1 0 .00 0 . 84 — 0 . 55 0 . 61 Reference 2 0 .02 0 .00 0 . 37 0 . 13 0 . 01 Cell Culture Process Parameters for the Exemplary Process # 4 Reference 3 0 .02 0 . 21 0 . 34 0 .64 0 . 00 Reference 4 0 .00 0 . 00 0 . 21 0 . 03 0 . 00 Reference 5 0 .01 0 .01 0 . 00 0 . 33 0 . 43 Process Condition S et point Initial Working Volume 1 . 6 L Temperature 36 .5° C . ( 0 -120 hr ); 33 .0° C . (120 -240 hr ) PH 6 . 90 + 0 .05 Conclusion DO 30 % Glucose Addition If [glc ] 1 . 8 g / L , bolus addition daily to [0197 ] Tested process parameters in this study include the increase glucose concentration to 1 . 8 - 2 . 0 g / L culture pH set point, temperature , seeding density , medium Medium with galactose 2 . 7 % ( v / v ) bolus addition at 96 , 144 , 192 hr feed amount and timing, glucose addition , galactose addi CuSO4 addition 20 uM at 24 hr tion , and harvest timing . Table 14 summarizes each used process parameters . TABLE 16 TABLE 14 Tested conditions Summary of Process Parameters No . Culture pH Production Temperature Process parameter Tested ranges 6 .70 33 . 0° C . pH set point 6 .75 - 7 . 10 6 . 80 33 .0° C . Growth temperature ( ° C . ) 35 . 0 - 37 . 5 6 .90 33 . 0° C . Production temperature 30 . 0 - 35 . 0 7 .00 33. 0° C . (° C .) 7 . 10 33. 0° C . Temperature shift timing 108 - 132 6 . 90 31. 0° C . (hr ) 6 . 90 32. 0° C . Medium feed amount ( % ) 67- 133 6 . 90 35. 0° C . Medium feed initiation day 3- day 5 6 . 90 34 . 0° C . Seeding density (cells /mL ) 4 . 0 - 8 . 0 x 105 6 . 90 33. 0° C . Harvest timing ( day ) day 10 -day 11 [ 0200 ] Cell density and viability were counted using a ViCell VR . PH and off - line gases were measured using a Example 5 . Higher Resolution Investigation of the PHOx , and major metabolites including glucose and lactate Impact of pH and Production Temperature on were measured using a Nova Profile 100 (Nova Biomedical, Asfotase Alfa Quality Attributes for Exemplary Waltham , Mass . ) . Enzymatic activity was measured using Process # 4 standard methods with the modification that each sample [ 0198 ] Further to previous assessments , this study pro was diluted only once , instead of three times , prior to vides a higher - resolution examination of the impact of enzymatic activity measurement. culture pH and production temperature ( 2120 hr ) on asfotase Harvest and Purification alfa productivity and quality attributes in an exemplary [0201 ] Fifty microliters of day 10 ( 240 + 4 hours ) samples manufacturing process . Tested scenarios include: 1 ) differ were harvested from all bioreactors using syringes . Post cell ent pH levels ( i . e . , 6 .70 , 6 .80 , 6 . 90 , 7 .00 , and 7 . 10 ) and a removal by centrifugation ( 3000xg , 5 min ), supernatants same production temperature of 33 .0° C .; and 2 ) different were clarified using 0 . 22 um bottle - top filters , and stored at production temperature levels ( i. e ., 31 .0° C . , 32 . 0° C ., 33 .0° - 80° C . prior to purification . A single step of high - through C . , 34 . 0° C . , and 35 . 0° C . ) and a same pH set point of 6 . 90 . put plate - based purification (protein A ) was applied in this Samples from all conditions were harvested at 240 + 4 hours, study . Samples were buffer exchanged to a low salt buffer (5 and protein A -purified materials were tested for several mM Na3PO4, pH 7 . 4 ) prior to analytical and protein char quality attributes ( e . g . , percent fragmentation , percent acterization analysis . aggregation , charge distribution , total sialic acid content Analytical and Protein Characterization ( TSAC ), and neutral glycan coverage. This study confirmed [0202 ] Critical quality attributes analyzed in this study that the process run at a pH set point ranging from 6 . 70 to included percent aggregate , percent fragment, charge distri 7 . 10 and a production temperature ranging from 31. 0° C . to bution , TSAC , and neutral glycan distribution . The aggre 35 .0° C . maintained all CQAs . gate level was estimated by the percentage of aggregate peaks to the total protein quantified in SEC . The fragment Cell Culture level was estimated by the percentage of fragment to the [0199 ] All experiments conducted in this study used the total protein quantified in SEC , as well as in LoC . The Mobius 3 L disposable bioreactors (CR0003L200 ; EMD charge distribution was estimated by the percentage of the Millipore ) . A list of process parameters used in this study in basic peaks , main peak and acid peak to the total protein , presented in Table 15 . Further, four additional culture pH respectively , quantified by AEX . Sialylation level (or TSAC ) US 2018 /0230445 A1 Aug . 16 , 2018 21 was quantified by HPAE - PAD . Detection of neutral glycan tion temperatures between 30. 0° C . and 35 .0° C . and a species was performed by MALDI- TOF mass spectrometry . culture pH between 6 . 75 and 7 . 10 . However, the low pH Isoelectric focusing was performed using an iCE280 system . condition in this study (6 .70 ) appeared to have had a dramatic impact on the protein A titer producing a 35 % Results decline as compared to the control condition (pH = 6 .90 ) Cell Culture Performance (FIG . 19A ) and a much lower volumetric activity as com [ 0203] As noted , both culture pH and production tempera pared to all other examined pH conditions ( FIG . 19B ) . ture have an impact on cell culture performance . The low pH Culture pH did not appear to have an impact on specific condition (pH = 6 . 70 ) resulted in the lowest growth rate and protein A productivity (FIG . 19C ) . However , the low pH lowest maximum VCD , reaching a maximum density of condition generated the highest specific activity (FIG . 19D ) , 11 . 1x100 cells /mL (FIG . 15A ) on the day of harvest (day and a general trend between decreasing culture pH and 10 ) . Higher pH conditions (pH = 7 .00 and 7 .10 ) resulted in increasing specific activity was observed . 1 - 2 % lower viability than the other conditions on day 10 ( FIG . 15B ). [ 0208 ] The low production temperature conditions (31 . 0° [0204 ] Higher production temperatures were also associ C . and 32. 0° C . ) caused a large decline ( 26 % and 21 % ated with increased growth ( FIG . 16A ) and more rapid respectively ) in protein A titer , as compared to the control declines in viability towards the end of the culture ( FIG . 16B ) . Lower production temperature produced a lower peak conditions (FIG . 20A ). Additionally , increased production VCD but produced comparable viability to the control set temperature was associated with higher volumetric activity point of 33 .0° C . (FIG . 20B ) and slightly increased specific protein A produc [0205 ] Increasing the culture pH also caused much more tivity (FIG . 20C ) . No impact was observed on specific rapid consumption of glucose (FIG . 17A ) and caused both a activity from changing production temperatures (FIG . 20D ). greater production of lactate during the early stages of the culture (prior to the temperature shift ) and a greater overall [0209 ] All quality attributes from this study and the pre accumulation of lactate throughout the culture duration vious work examining the impact of culture pH and pro (FIG . 17B ) . duction temperature are summarized in Table 17 . TABLE 17 Quantified Quality Attributes for Varying pH and Production Temperature LOC LOC Production ( % ( % Temp - | AEX ( % ) _ Main , Main , SEC ( % ) # pH (°C . ) Basic Main Acidic NR ) R ) Aggregate Main Frag TSAC 20 6 . 70 33 . 0 0 . 2 93 . 2 6 . 5 98 .6 100 . 0 6 . 2 93. . 8 1 . 5 21 6 . 80 33 . 0 0 . 1 93 . 4 6 . 4 98 . 7 100 . 0 6 . 1 93 . 9 N 6 . 90 33 .0 0 . 1 93 . 1 97. 2 100 . 0 6 . 2 93 . 8 23 7 .00 33 .0 0 . 1 94 . 4 5 . 4 96 . 8 100 . 0 5 . 6 94 . 4 24 7 . 10 33 . 0 94 . 4 5. 5 97. 1 100 . 0 5 . 8 94 . 2 OOOOO0 2 .6 25 6 . 90 31. 0 0 . 1 92 . 3 7 . 6 97 . 0 100 . 0 7 . 0 93 . 0 0 26 6 .90 32 . 0 0 . 1 92 . 4 1. 5 97 . 2 100 . 0 6 . 7 93. 3 0 27 6 . 90 35 . 0 0 . 1 95 . 2 4 . 6 98 . 5 100 . 0 5 . 0 94 . 8 0 . 2 28 6 . 90 34 . 0 0 . 1 93 . 2 6 . 6 98 . 2 100 . 0 6 .6 93 . 2 0 . 2 29 6 .75 33 .0 0293 . 8 5 . 8 97 . 0 100 . 0 5 . 6 94. 4 0 30 6 .90 30 . 0 90 . 8 8 . 8 97. 1 100 . 0 8 . 5 91. 4 0 6 . 90 33 . 0 93 .4 100 . 0 100 . 0 5 . 9 94 . 1 0 in 32 6 .90 35 . 0 in 95 . 4 4 . 0 100 . 0 100 . 0 95 . 7 0 . 1 abáo 33 7 . 10 33 . 0 OOOOOO 94 . 0 5 . 8 100 . 0 100 . 0 5 . 3 94 . 7 0 NNAN [ 0206 ] Culture cells were grown for five days ( 120 hr ) [ 0210 ] As in previous studies , TSAC was affected by both prior to shifting grown temperature to production tempera culture pH and production temperature ( FIG . 21 ) . The ture . Both the glucose and lactate profiles were very con small - scale experimental range for TSAC was 1. 5 - 2 . 9, and sistent between day 0 and day 5 ( FIG . 18A and FIG . 18B ) . higher TSAC was more desirable within this range . Increas After the temperature shift , higher production temperatures ing either culture pH or production temperature was shown were associated with higher consumption of glucose . A to increase TSAC of the product. On one hand , increasing slight decrease in the maximum concentration of lactate was the culture pH set point from 6 .70 to 7 . 10 led to a TSAC observed for the 33 .0° C . production temperature condition , increase approximately from 1 . 5 to 2 .6 - 2. 8 , which was in the as compared to the other conditions. Additionally , a 35 . 0° C . small -scale experimental range . On the other hand , increas production temperature showed significantly decreased lac ing production temperature from 30 .0° C . to 35 . 0° C . led to tate consumption during days 9 and 10 of the culture, as a TSAC increase approximately from 1 . 4 to 2 .6 - 2 . 8 . As the compared to all other conditions. TSAC value ( 1 . 4 ) under a production temperature of 30 .0° [0207 ] It was previously observed that production tem C . was lower than the lower limit ( 1 . 5 ) of the small- scale perature had a more dramatic effect than culture pH on both experimental range , a production temperature of 30 .0° C . protein A titer and specific activity for the range of produc should probably be avoided in the production bioreactor. US 2018 /0230445 A1 Aug . 16 , 2018

This data indicates that TSAC may be increased by increas TABLE 18 ing culture pH or production temperature , or both . pI ranges for identified iCE280 peaks [ 0211 ] The impurity level, as presently measured by the acidic peak percentage using AEX , is presented in FIG . 22 . Peak ID pl range Increasing production temperature decreased the acidic peak Peak 1 56 .63 percentage in the product. Culture pH did not appear to have Peak 2 6 . 62 - 6 .67 Peak 3 6 .67 - 6 .72 any correlation with the impurity measured by AEX acidic Peak 4 6 . 71 - 6 . 77 peak . Although production temperature demonstrated an Peak 5 6 .77 - 6 . 82 impact on AEX acidic peak percentage , all values were Peak 6 26 . 83 within acceptable limits for purification (s10 .2 % ). The AEX basic peak was consistently below 1 % , the LOQ for the assay , for all conditions examined . Conclusions [ 0212 ] A previous study showed a strong correlation [0216 ] This study confirms previous results indicating that between AEX acidic peak and aggregate percentage , as culture pH (6 . 70 - 7 . 10 ) and production temperature ( 31. 0 measured by SEC . This trend was maintained for this study, 35 . 0° C . ) are unlikely to be CPPs in that the measured CQAS as shown in FIG . 23. Higher production temperature led to were all within the small scale range as previously specified . a lower aggregate percentage . All aggregate percentages Both culture pH and production temperature impact many were within the small -scale experimental range ( 59. 6 % ). cell culture performance parameters and quality attributes . Culture pH does not have a strong effect on the aggregate Notably, increasing either the culture pH or production percentage . Asfotase alfa fragments as measured by SEC temperature leads to an increased maximum VCD but also were below 1 % , the LoQ for the assay , for all conditions . causes a rapid decline in viability in the late stages of the culture . [ 0213] The main peak percentage measured by LoC was [0217 ] Both culture pH and production temperature can previously identified to be significantly impacted by pro impact productivity in terms of protein A titer and volumet duction temperature . A positive correlation between increas ric activity . A culture pH of 6 . 70 resulted in lower protein A ing production temperature and non -reduced main peak titer and volumetric activity. Increasing the culture pH set percentage was discovered (FIG . 24B ) . No clear impact of point resulted in decreased specific activity . Therefore , culture pH on LoC main peak percentage (non - reduced ) was though culture pH has a positive impact on protein A titer, found ( FIG . 24A ) . The reduced LoC analysis identified there was almost no difference in terms of volumetric 100 % main peak for all conditions. activity under a pH ranging from 6 .80 to 7 . 10 . In addition , increasing the production temperature led to an increased [0214 ] No new neutral species were observed for any of protein A titer and volumetric activity , but did not impact the conditions , as compared to the BDS materials described specific activity . in the exemplary process in Example 2 . However, a profile [0218 ] Increasing the culture pH or production tempera shift was seen between the low pH condition and higher pH , ture also led to higher TSAC values. Increasing the latter in which more higher -order glycoforms were observed from parameter also decreased aggregate and acidic peak forma the material produced at a higher culture pH ( FIG . 25 ) . A tion . Finally, culture pH and production temperature both similar trend was observed for production temperature with appear to shift the isoelectric focusing profile of the product. more higher order glycoforms present in the material pro These results indicate that a pH ranging from 6 .70 to 7 . 10 duced at the higher production temperature ( 34 . 0° C . and and a production temperature ranging from 31 .0° C . to 35 . 0° 35 .0° C .) (FIG . 26 ) . C . are acceptable , and tight controls of both process param eters are recommended to maintain process robustness and [ 0215 ) Peak determination from iCE280 chromatograms consistency in scaling up ( e. g . , to a 10 , 000 L scale - up was performed by identifying the most basic peak as Peak 6 process ) . and identifying the next four peaks as Peak 5 through 2 in reverse numerical order. Peak 1 then consisted of all peak Example 6 . Improving Active Asfotase Alfa Titer percentages identified below the pl of the next peak after by Extra Feed Supplementation Peak 2 . The resulting pl values for the identified peaks are [ 0219 ] The previous exemplary processes for producing listed in Table 18. Both culture medium pH and production asfotase alfa (sTNALP - Fc -D10 ) , as described in previous temperature appear to have an impact upon capillary iso Examples , were low - producing processes with an active electric focusing results ( FIG . 27 ) . Increasing either culture asfotase alfa titer of approximately 0 . 1 g / L at harvest . An pH or production temperature produced an increase in the alternative manufacturing process is exemplified herein for percentage of the low pl peaks , identified as Peak 1. Addi process improvement. Both upstream and downstream pro tionally , the related percentage of the high pl peaks , Peak 5 cesses were evaluated . [0220 ] The potential impact of upstream cell culture pro and 6 , was decreased with increasing culture pH or produc cess parameters on asfotase alfa quality and productivity tion temperature . These factors indicated a general shift were evaluated in a previous study . Cell culture parameters towards more acidic species associated with increasing pH including metal supplementation ( Zn2+ /Mg2 + /Ca2 + ) , tem or production temperature. This shift may be related to perature shift timing , CHO feed amount and harvest timing increased sialylation associated with both increased pH and were evaluated , in order to improve titer and / or volumetric production temperature , which would produce a greater activity without impacting any other critical quality attri percentage of acidic species. butes (CQAs ) . The data from the previous study indicated US 2018 /0230445 A1 Aug . 16 , 2018 that metal supplementation resulted in significantly lower tiple feedings at day 2 , 4 , 6 , and 8 ) . Table 19 summarizes the TSAC than the control condition , and therefore was cell culture process parameters used for these bioreactors . excluded in further evaluation . Delaying the temperature shift timing from 54 hours to 78 hours resulted in lower TABLE 19 specific activity and higher protein A titer. As a result, the overall volumetric activity under the later temperature shift Cell Culture Process Parameters conditions remained the same. Increasing the CHO feed Process Parameters Control Improved Process bolus amount from 1x ( 5 ml/ L on day 2 ) to 4x (5 ml/ L on day 2 , 4 , 6 , and 8 ) led to a significant increase in volumetric Initial Working Volume 10 L (Sartorius 10 L bioreactor) (media and inoculum ) activity without adverse impact on other tested quality Target Seeding Density 5 . 5 x 105 cells/mL attributes ( e . g ., TSAC , charge distribution , and neutral gly PH 6 .90 ( without dead band ) can distribution ) . Delaying the harvest day from day 10 to Temperature 37 .0° C ., then shift to 30. 0° C . when viable day 12 was associated with higher productivities ( e . g . , cell density is 25 - 32 x 103 cells/ mL (58 hr post inoculation ) volumetric activity , protein A titer, and specific activity ) and DO 40 % marginally lower TSAC . Based on this data , an increase in Agitation 309 rpm ( P / V = 81 W /m ? ) CHO feed amount was chosen in this study for further Gassing strategy Air flow 20 mL /min + Oxygen as needed evaluation at the 10 L scale . Overlay 450 mL/ min air CHO feed 5 mL / L bolus (50 mL ) 50 mL bolus * ( 50 mL ) on day 2 based on on day 2 , 4 , 6 , and 8 Materials and Methods initial working volume Glucose addition If [ glucose ) is below 2 g/ L , increase to 3 g/ L [ 0221 ] An upstream process for asfotase alfa ( sTNALP via bolus addition FC - D ) production was run with the following seed train Glutamine addition If [glutamine ) is below 0 .18 g/ L (1 .2 mm ) , and cell culture process : inoculum thaw of cells with target increase to 0 . 29 g / L ( 2 . 0 mM ) via bolus addition prior to temperature shift seeding density at 5x10 cells /mL ; inoculum expansion Base ( 10 % w / w Na2CO3) Add if needed ( flask / roller stage ) with target seeding density at 3 .5x100 Antifoam 5 mL on day 0 , then 1 mL as needed cells /mL ; inoculum expansion ( cell bag stage ) with target throughout culture seeding density at 3 .5x10 cells /mL ; inoculum expansion * each bolus feed contains 1 . 2 mM zinc chloride , comparing to the basal medium ( 100 L - low level) with target seeding density at 3 .5x10 containing only about 9 uM zinc chloride. Thus, each bolus feed increases about 6 UM of cells /mL ; inoculum expansion ( 100 L - high level ) with zinc concentration (0 . 5 v / v ) . target seeding density at 3 . 5x10 cells/ mL , 1000 L seed bioreactor ( N3 — low level) with target seeding density at [0223 ] The manufacturing process at 20 ,000 L scale 3 .5x10 cells /mL ; 1000 L seed bioreactor (N2 — high level ) employs a VCD based feeding strategy (single bolus at VCD with target seeding density at 3 .5x10 cells/ mL ; 4000 L seed about 25 . 0 -32 . 0x109 cells /mL , usually around day 2 of bioreactor (N1 ) with target seeding density at 3 .5x105 culture ). In this study, temperature shift timing was pro cells /mL ; 20000 L production bioreactor with target seeding grammed to occur at 58 hours post inoculation to meet the density at 5 .5x10 cells /mL . For this study, the small - scale VCD criteria (25 .0 -32 . 0x10 % cells/ mL ) based on averaged seed train process used as inoculum for the bioreactors for doubling time 24 . 5 hours calculated from manufacturing both control and improved bioreactor processes included : data at 20K bioreactor. This change was implemented to inoculum thaw ; Passage 1 ( 1x100 mL shake flask , target simplify the process and to allow tighter control of the seed at 5x10 cells /mL ) ; Passage 2 (2x100 mL shake flask , timing of feeding . target seed at 3 .5x10 cells /mL ); Passage 3 (2x1 L spinner, target seed at 3 . 5x10 cells/ mL ) ; Passage 4 ( 2x15 L spinner, target seed at 3 .5x10 cells /mL ) ; 8x10 L bioreactor , target Results seed at 5 .5x10 cells /mL . The 10 L bioreactors used in this study was the Sartorius BIOSTAT® B - DCU II bioreactor [0224 ] Cell culture performance of all bioreactors is sum system ( Sartorius Stedim Biotech ) and MFCS/ win 3 . 0 Mod marized in FIG . 28 through FIG . 31 . ule Shell 3 . 0 ( level 32 ) data acquisition software (Sartorius [0225 ] Growth dynamics and viability of the currently Stedim Biotech ). A list of raw materials used in the produc improved process were compared to the control and previ tion bioreactors includes : CM69 growth medium , 100 uM ous 20 ,000 L processes . The VCD over the course of the MTX (methotrexate ) , 200 mM L -Glutamine , CM70 growth culture were similar to the control and 20 , 000 L processes medium , Sigma CHO Feed , 10 % Sodium carbonate, and (FIG . 28A , error bars represent 1x standard deviation ). Cell FoamAway AOF (Gibco ). viability under the control processes and the improved 10222] A cell vial from a development working cell bank Process # 4 declined in similar rates, which is faster than the of asfotase alfa ( sTNALP - FC - D10) clone ( cell line X ) was rate under the previous 20 ,000 L - scale processes (FIG . thawed and passaged through the seed train through the N - 1 28B ) . Final viability was 73 % and 70 % , respectively, for the stage ( equivalent to the 4000 L seed bioreactor in the 20 ,000 control and the improved processes, while the 20 ,000 L L process previously used ). The N - 1 culture was conducted process typically remains about 80 % . This appears to be a in two 15 L spinners with a working volume of 15 L . Both scaling issue with the 10 L system , possibly due to agitation N - 1 cultures were used for inoculation . Agitation was set at rate . This was tested in a second material generation block 200 rpm with a total gas flow of 20 L /hr . Oxygen and carbon and data from that run indicated that the viability drop was dioxide gassing were adjusted according to measured dis likely due to a combination of the agitation rate and the solved oxygen and pH . Eight 10 L bioreactors were inocu ViCell cell counter settings being set to a generic CHO cell lated and harvested on day 10 . Two bioreactors were fed type , which had a broader acceptable cell diameter range using the control feeding strategy (one feeding ) , while six ( 5 - 50 uM ) than that for the cell type ( 11 - 30 uM ) in previous others were fed using the improved feeding strategy (mul processes . US 2018 /0230445 A1 Aug . 16 , 2018 24

[0226 ] Glucose utilization and lactate consumption pro Example 7 . Further Improving Asfotase Alfa files are shown in FIG . 29 . The improved process showed Activity by Zinc Supplementation similar glucose utilization and lactate profiles to that of the control. [0231 ] Based on manufacturing processes disclosed in [0227 ] Protein A titer of asfotase alfa and specific activity previous Examples , new studies were carried out to further throughout the culture are shown in FIG . 30 . Protein A titer optimize upstream process parameters . Exemplary Pro for both the control and improved process were comparable . cesses Z and Z ' are summarized and compared in Table 20 . TABLE 20 Differences between Exemplary Manufacturing Processes ( upstream ) Process X Process Y Process Z Process Z ' Parental Cell CHO CHO CHO ??? line Production HyClone HyClone HyClone HyClone medium SFM4CHO SFM4CHO SFM4CHO + BD SFM4CHO + BD select select Feed CHO Feed CHO Feed ( 2 %, ime. , CHO Feed ( 2 %) + CHO Feed ( 2 % ) + ( 0 . 5 % ) 4 x medium feed ) Cell Boost 2 + 5 Cell Boost 2 + 5 ( 9 % ) ( 9 % ) pH set point 6 . 90 6 . 90 6 . 90 6 . 90 Temperature 37 .0° C . then 37. 0° C . then shifted 37 .0° C . then shifted 37 .0° C . then shifted shifted to 30° C . to 30° C . to 30° C . to 30° C . DO 40 % 40 % 40 % 40 % Supplement N / A N / A ZnSO4 ZnSO4 ( 30 - 90 UM ) Harvest time 240 + 12 hr 240 + 12 hr 240 + 12 hr 288 + 48 hr

However , they were both lower than the titers typically seen [0232 ] Further optimizations have been carried out based at the 20 ,000 L process. This may be partially attributable to on previous Process Y . For example , new Process Z ' the scale differences seen with the viability , since the power included supplementing about 30 - 90 uM zinc sulfate into the to volume ratio used in this study was found to be 30 % culture medium and increasing total culture time from about higher than that used at manufacturing (confirmed by sub 240 hours ( i . e . , 10 days ) to about 288 hours ( i . e . , 12 days ) . sequence receipt of the PN ratio used at manufacturing A small scale shake flask study was carried out to test three scale ) . Specific activity (units /mg protein ) for the improved variants of Process Z ' ( supplementing 30 , 60 , or 90 uM zinc , process was on average 17 % higher (532 U /mg vs. 456 respectively ) . The 30 M supplementation was done by U /mg ) than control. These data , taken with the protein A titer front - loading into the culture medium . The 60 and 90 UM data , indicate that while the same amount of protein is supplementations involved supplementing zinc in two equal produced with each process , the protein produced was more boluses on day 2 and 6 ( for 60 UM condition ) , or three equal active when the culture is fed more . boluses on day 2 , 6 , and 10 ( for 90 UM condition ) . As shown [0228 ] Active titer and total volumetric activity ( shown in in FIG . 33 , zinc supplementation (red line ; showing the FIG . 31) indicate that the improved process generates average of three experiments with different zinc concentra approximately 22 -24 % more activity than the control pro tions ) effectively improved asfotase alfa specific activity , as cess at the 10 L scale . The improved process generated compared to the control Process Y (blue line ). This increase approximately the same active titer as seen at the 20 ,000 L reached its peak around the 288 -hour ( i. e . , 12 days ) time slot scale , while the control process was about 19 % lower . and decreased afterwards. Conclusion Example 8 . Characterization of Manufactured Asfotase Alfa [0229 ] This study documents the cell culture performance of an improved manufacturing process ( 10 L scale ) , which [ 0233 ] An exemplary characterization of manufactured utilizes an increased feed amount that led to an approxi asfotase alfa with multiple orthogonal analytical methods is mately 22 -24 % increase in active titer , while maintaining the illustrated herein . These methods were employed to evaluate protein A titer of the product . The comparison of asfotase identity , purity , size , structure, glycosylation , and charge alfa specific activity between control and improved pro profiles of produced asfotase alfa. Some methods were also cesses is shown in FIG . 32 . Clearly , all six replicate used to ensure lot- to - lot consistency . Additionally, product improved process conditions ( each with three extra feed related substances and product -related impurities were also additions) showed higher specific activity than the controls . characterized . [0230 ] Each bolus feed contained 1 . 2 mM zinc chloride, as compared to the basalmedium that contains only about 9 uM Structural Elucidation of Asfotase Alfa zinc chloride . Thus, each bolus feed resulted in an increase 102341 Matrix - Assisted - Laser -Desorption - Ionization of about 6 uM of zinc concentration ( 0 . 5 v / v ) in the whole Time- of- Flight (MALDI - TOF) Analysis of Molecular culture . Without being limited to any particular theory , it is Weights after Removal of Oligosaccharides postulated that the increase in specific activity by extra feed [0235 ] MALDI- TOF mass spectrometry analysis of asfo supplementation is at least partly due to the zinc supple tase alfa after removal of oligosaccharides was used to mentation . establish the identity of asfotase alfa . In addition , the pres US 2018 /0230445 A1 Aug . 16 , 2018 25 ence of any significant levels of posttranslational modifica - asfotase alfa is shown in FIG . 35 . Peaks with m /z of tions with molecular weights substantially different from the 180 , 157 , 90 , 223 and 60 ,540 correspond to the molecule that theoretical molecular weight of asfotase alfa would also be is singly , doubly or triply charged , respectively . Other low detected by this method . Removal of oligosaccharides was abundant peaks were formed due to fragmentation caused by achieved by digestion of asfotase alfa using PNGaseF with the harsh instrument settings. The measured molecular or without reduction of disulfide bonds . The samples were weight of 180 , 157 Da is significantly higher than the cal desalted and mixed with a 90 : 10 mixture of 2 , 5 -dihydroxy culated molecular weight of 161, 135 . 2 Da based on the benzoic acid and 2 -hydroxy - 5 -methoxybenzoic acid in known amino acid sequence . This result indicates extensive acetonitrile and trifluoreacetic acid . The samples were air posttranslational modifications of asfotase alfa , mainly gly dried and then analyzed using a MALDI- TOF mass spec cosylation , since MALDI- TOF and ESI - TOF analyses of trometer. Mass spectra were acquired in positive mode . The deglycosylated asfotase alfa did not detect significant levels instrument was externally calibrated using bovine serum of modifications. albumin (BSA ) . Mass spectrum of asfotase alfa after degly cosylation is shown in FIG . 34A . Peaks with m / z of 161, 140 , Size Exclusion Chromatography Multiple - Angle Light 80 ,571 and 53 , 748 correspond to the molecule that is singly, Scattering (SEC -MALS ) doubly or triply charged , respectively . The measured molecular weight of 161, 140 Da was in agreement with the [0238 ] SEC -MALS is used to separate proteins based on calculated molecular weight ( 161, 135 . 2 Da ) within the 1 % sizes and then determine the molecular weight of each peak . accuracy limit of the instrument. A mass spectrum of asfo Asfotase alfa was analyzed by SEC -MALS ( data not tase alfa after reduction and removal of oligosaccharide is shown ). The calculated molecular weight of the peak in the shown in FIG . 34B . Peaks with m / z of 80 ,545 , 40 , 267 , and retention timewindow of 7 . 0 - 8 . 2 minutes is 194 . 1 Da, which 26 ,808 correspond to the molecule that is singly, doubly or is in good agreement with the calculated molecular weight triply charged , respectively . The measured molecular weight of asfotase alfa based on the known amino acid sequence of 80 , 545 Da is in agreement with the calculated molecular and extensive glycosylation and also in agreement with the weight of 80 , 577 . 7 Da within the 1 % accuracy limit of the molecular weight observed by MALDI- TOF . instrument. The results confirmed the identity of asfotase alfa and demonstrated the lack of significant levels of Metal Analysis posttranslational modifications other than glycosylation . [0239 ] Inductively coupled plasma- mass spectrometry (ICP -MS ) was used to determine the content of zinc and Analytical Ultracentrifugation (AUC ) magnesium and inductively coupled plasma- atomic emis sion spectrometry ( ICP - AES ) was used to determine the [0236 ] AUC was used to determine the percentage of level of calcium . Asfotase alfa samples were digested using aggregates , dimer and fragments if present and thus the microwave digestion with heat with the addition of concen purity of asfotase alfa . In addition , it determines themolecu trated nitric acid first and then with the addition of hydrogen lar weight of asfotase alfa dimer , which is a characteristic of peroxide. The samples were then analyzed using an ICP- MS the molecule because of the unique amino acid sequence and instrument for zinc and magnesium and an ICP - AES instru posttranslational modifications . It is considered to be an ment for calcium . The determined molar ratios (mole of orthogonal method to size exclusion chromatography (SEC ) . ions/mole of asfotase alfa monomer ) were 2 .27 for zinc , 0 . 97 Asfotase alfa samples were diluted to approximately 1 for calcium and 0 . 12 for magnesium . The ratios of zinc and mg/ mL using 25 mM phosphate , 150 mM NaCl, pH 7 .4 . An calcium were in good agreement with the reported ratios in analytical ultracentrifuge was used to carry out sedimenta literature ( Stec et al. 2000 J Mol Biol 299 : 1303 - 1311 and tion velocity analysis . Double sector cells equipped with Mornet et al. , 2001 J . Biol Chem 276 :31171 - 31178 ) . Mornet quartz windows were used . The rotor was equilibrated under et al. ( 2001 ) teaches that calcium may occupy the magne vacuum at 20° C . and after approximately 1 hour the rotor sium binding site on TNALP and thus suggests that the ratio was accelerated to 36 , 000 RPM . Absorption scans at 280 nm of magnesium in the asfotase alfa molecule may be less than were acquired at 4 . 5 minute intervals for approximately 6 one ( 1 ) . However, the tested ratio of magnesium as 0 . 12 is hours. The data was first analyzed using DCDT to determine still surprisingly low . the apparent molecular weight and then using the c ( s ) method (SEDFIT software ) to determine the percentage of Determination of the Phosphorylation Site dimer, high molecular weight and low molecular weight species. The molecular weight of asfotase alfa was 211 kDa [0240 ] UPLC -MSC was used to determine the site and the and the purity was 96 . 8 % for the representative batch . percentage of phosphorylation . Asfotase alfa at a final con centration of 0 .5 mg/ mL was denatured and reduced in the MALDI- TOF of Intact Molecular Weight presence of 6M guanidine hydrochloride in 0 . 2 M Tris , pH , 8 . 6 , using 40 mM DTT at 37° C . for 1 hour. The reduced [ 0237] MALDI- TOF measurement of the molecular sample was alkylated by the addition of iodoacetic acid to a weight of asfotase alfa determines the size of the molecule final concentration of 50 mM and incubation at room including all posttranslational modifications, mainly , glyco temperature for 30 minutes . The sample was then buffer sylation . Asfotase alfa samples were desalted and mixed exchanged into 50 mM ammonium bicarbonate using dialy with matrix made from a 90 : 10 mixture of 2 , 5 -dihydroxy sis tubing with molecular weight cut -off of 10 kDa. The benzoic acid and 2 - hydroxy - 5 -methoxybenzoic acid . The sample was digested using trypsin at a final ratio ( w : w ) of samples were air -dried and then analyzed using an MALDI 1 : 50 trypsin ; protein at 37° C . for 24 hours. A UPLC system TOF mass spectrometer. Mass spectra were acquired in equipped with a reversed -phase C18 column and a O - TOF positive mode . The instrument was externally calibrated mass spectrometer were used to analyze the digested using bovine serum albumin (BSA ). A mass spectrum of samples . Tryptic peptide containing Ser93 was identified as US 2018 /0230445 A1 Aug . 16 , 2018

both phosphorylated and non -phosphorylated . As shown in TABLE 22 FIG . 36 , the MS/MS spectrum demonstrated that S93 is shifted by + 80 Da, the mass of a phosphate added by Theoretical and Observed Molecular Weights phosphorylation , as observed by the y12 and y13 ions. The of Neutral Glycans ( M + Na ) ? relative percentage of phosphorylation was determined Theoretical Error Relative using extracted ion chromatograms (EIC ' s ) peak areas as (m /z ) Glycoforms m /z (ppm ) Intensity Intensity 33 . 9 % . 933 .317 M3 933 . 301 16 383 .81 1136 . 401 A1 1136 . 4 0 . 6 2765 . 27 MALDI Analysis of Free Glycans 1257. 427 M5 1257 .435 - 8 . 2 792 .57 1282 .454 FA1 1282 . 465 - 10 . 7 1580 . 16 [0241 ] MALDI- TOF analysis of free glycans was used to 1298 . 453 A1G1 1298 . 454 - 0 . 6 840 . 8 9 determine oligosaccharide species and their relative percent 1339 .48 A2 1339 . 484 - 4 6704 .09 age . Oligosaccharides were released from asfotase alfa ref 1444 .507 FA1G1 1444 .522 - 14 .9 386 . 39 A 1485 .538 FA2 1485 . 545 - 7 . 1 8939 . 36 100 erence standard by PNGaseF digestion . The released gly 1501. 533 A2G1 1501 . 53 2 . 8 4852 .75 54 cans were purified using Top tip carbon reversed phase 1542 . 559 A3 1542. 556 3 . 4 1570 . 78 columns . The released glycans were mixed 1 : 1 with SDHB 1647 . 591 FA2G1 1647 . 599 - 8 . 3 3217 .43 in2mm matrix and then analyzed by MALDI- TOF. Analyses were 1663. 586 A2G2 1663 .582 3 . 6 2328 1688 .617 FA3 1688 .624 - 6 . 7 2752 . 37 performed in negative mode for glycans with sialic acid and 1704 .612 A3G1 1704 . 609 3 . 2 1998 . 15 then in positive mode for neutral glycans . A mass spectrum 1745 .639 A4 1745 . 624 14 . 8 803 acquired using the negative mode is shown in FIG . 37A . A 1809 .644 FA2G2 1809 .647 - 3 . 5 1704 .05 mass spectrum acquired using the positive mode is shown in 1850 .67 FA3G1 1850 .675 - 4 . 9 2024 . 14 1866 . 665 A3G2 1866 .656 1263 . 32 FIG . 37B . Oligosaccharide species are determined by 1891. 697 FA4 1891 . 7 - 3 . 3 1384 .76 matching the observed molecular weights with the theoreti 1907 .692 A4G1 1907 . 692 - 0 . 4 937 . 19 cal molecular weights of commonly -known oligosaccha 2012 .723 FA3G2 2012 . 728 - 5 . 1 1352 . 46 rides . The relative percentage was determined by dividing 2028 .718 A3G3 2028 . 719 - 1 . 2 992. 03 oAWOONao 2053. 75 FA4G1 2053 . 761 - 11 . 5 1404 . 04 the individual peak intensity by the total peak intensities of 2069 . 744 4G2 2069 .742 2 . 4 771 .77 all glycans . The major oligosaccharides are summarized in 2174 .776 FA3G3 2174 . 779 - 3 . 3 1463 . 26 Table 21 and Table 22 . Based on the molecular weights, the 2215 .793 FA4G2 2215 .814 - 20 . 7 1180 . 8 structure of the sialic acid was determined to be N -acetyl 2231 .797 A4G3 2231. 791 6 . 2 572 . 1 waoa neuraminic acid (Neu5Ac or NANA ) . TABLE 21 Theoretical and Observed Molecular Weights of Sialyated Glycans ( M - H ) - Theoretical Observed Error Relative m / z Glycoforms m / z (ppm ) Intensity Intensity 1768 .628 A2G1 + NANA 1768. 581 46 . 923 619 . 000 44 1914 .685 FA2G1 + NANA 1914 . 682 3 .816 646 . 122 46 1930 .680 A2G2 + NANA 1930 .677 3 . 596 774 .051 55 1971 .707 A3G1 + NANA 1971 .649 57 .426 195 .000 14 2076 .738 FA2G2 + NANA 2076 . 733 5 . 538 1402 . 453 100 2117 .765 FA3G1 + NANA 2117 .755 9 .504 359. 057 26 2133 .760 A3G2 + NANA 2133. 729 31 . 127 245 . 000 17 2279 .818 FA3G2 + NANA 2279 . 823 - 5 .668 421. 152 30 2295. 813 A3G3 + NANA 2295 . 825 - 12 .668 333. 253 2320 .844 FA4G1 + NANA 2320 . 75 94 .536 155 .000 2336 .839 A4G2 + NANA 2336 . 793 46 . 579 103 . 000 N 2441 . 870 FA3G3 + NANA 2441. 863 7 .559995 . 507 2482 .897 FA4G2 + NANA 2482 . 904 - 7 . 393 296 . 679 2498 .899 A4G3 + NANA 2498 . 827 71 . 854 158 . 291 2644 . 950 FA4G3 + NANA 2644 . 952 - 2 .229 289 .645 = 2660 . 945 A4G4 + NANA 2660 . 97 - 25 .771 165 . 363 0 2685 . 976 FA4G2 + GN + NANA 2685 .915 61. 386 52 . 974 2807 .003 FA4G4 + NANA 2807. 003 - 0 . 322 849 . 240 a 2848 .029 FA4G3 + GN + NANA 2848 .034 - 4 . 806 67 .026 3010 . 082 FA4G4 + GN + NANA 3010 .022 59 . 538 85 . 000 3026 .077 A4G4L1 + NANA 3025 . 985 91. 732 80 . 000 ANuoaau 3172 . 137 FA4G4L1 + NANA 3172. 11 26 . 443 277 . 854 3213 . 161 FA4G3L1 + GN + NANA 3213. 175 - 13 .611 15 .000 3537 . 267 FA4G4L2 + NANA 3537 . 181 86 . 085 72 . 238 3902 .399 FA4G4L3 + NANA 3902. 451 - 51. 951 21 . 992 US 2018 /0230445 A1 Aug . 16 , 2018 27

TABLE 22 - continued TABLE 23 -continued Theoretical and Observed Molecular Glycoforms and Their Relative Percentage by 2 - AB Labeling of Neutral Glycans ( M + Na ) Glycoforms Relative % Theoretical Error Relative ( m /z ) Glycoforms m /z (ppm ) Intensity Intensity FA4G1 3 . 42 FA3G3 1 . 71 2377 . 855 FA4G3 2377 . 864 - 8 . 9 853 . 1 10 A3G3/ A462 3 . 00 2393. 85 A4G4 2393 . 849 1 529 . 86 FA3G3 2 . 49 2418 . 877 FA4G1L1 2418 .954 - 76 . 6 202 FA3G3 3 . 01 2539 .904 FA4G4 2539 .918 - 14 . 3 1009 . 29 FA4G3 2. 13 2580 . 93 FA4G2L1 2580 . 949 - 18 . 8 188 . 9 FA4G3 0 . 89 2742 . 983 FA4G3L1 2742 . 985 - 1 . 9 255 FA4G4 5 . 35 2905 .036 FA4G4L1 2905 .048 - 12 . 1 301. 29 FA4G4L1 3 . 54 3270 . 168 FA4G4L2 3270 . 10266 . 1 204. 96 FA4G4L2 1 . 87 NwwNNA UNKNOWN 0 .61 2 - Aminobenzamide (2 - AB ) Labeled Oligosaccharides Profiling Glycopeptide Profiling [ 0242 ] Released oligosaccharides from asfotase alfa were [0243 ] A glycopeptide profile was used to determine the labeled by 2 - aminobenzamide ( 2 - AB ) and then analyzed by site -specific oligosaccharide distribution . Asfotase alfa normal phase HPLC with fluorescence detection to deter samples at a final concentration of 0 . 5 mg/mL were dena mine the types and the relative percentage of various oli tured and reduced in the presence of 6M guanidine hydro gosaccharides . Asfotase alfa samples were reduced using chloride in 0 . 2 M Tris , pH , 8 . 6 , using 40 mM DTT at 37° C . dithiothreitol (DTT ) at 37° C . for 1 hour and then digested for 1 hour . The reduced samples were alkylated by the using PNGaseF at 37° C . overnight to release N - linked addition of iodoacetic acid to a final concentration of 50 mM oligosaccharides . Protein from the digested sample was and incubation at room temperature for 30 minutes . The precipitated and separated from the released glycans by cold samples were then buffer - exchanged into 50 mM ammo ethanol and centrifugation . Supernatants containing the nium bicarbonate using dialysis tubing with a molecular released glycans were dried using a vacuum centrifuge , weight cut- off of 10 kDa . The samples were digested using reconstituted using 1 % formic acid and incubated at room trypsin at a final ratio (w :w ) of 1 :50 trypsin ; protein at 37° temperature for 40 minutes and then dried again . The C . for 24 hours . An UPLC system equipped with a reversed samples were then labeled using 2 -AB labeling reagents by phase C18 column and a Q - TOF mass spectrometer were following the manufacturer' s instruction (Ludger , Oxford used to analyze the digested samples. Data were acquired in shire , UK ) . The labeled glycans were cleaned up using the positive mode. Mass spectra across the elution time glycan S cartridges ( Prozyme, Hayward , Calif .) and then widows were averaged for each glycopeptide . Six glyco analyzed using HPLC with fluorescence detection with an peptides were generated from trypsin digestion of asfotase excitation wavelength of 330 nm and emission wavelength alfa , corresponding to six glycosylation sites per monomer of 420 nm . A fluorescence chromatogram from the analysis of asfotase alfa . Because the peptide moiety is the primary of asfotase alfa is shown in FIG . 38 . Identities of peaks were factor determining the retention times, glycopeptides are established by fraction collection ,mass spectrometry analy separated based on their unique amino acid sequence; there sis of molecular weights and matching the molecular fore , site - specific oligosaccharide distribution can be weights to the molecular weights of commonly -known oli obtained . Mass spectra of glycopeptides are shown in FIG . gosaccharide structures . Oligosaccharides identified by 40 . Oligosaccharide species associated with each glycosy 2 - AB labeling are summarized in Table 23 . A chromatogram lation site are summarized in Table 24 . obtained from analysis of an asfotase alfa reference standard is shown in FIG . 39 . TABLE 24 Oligosaccharide Species Associated with Each Glycopeptide TABLE 23 Theoretical Observed Glycoforms and Their Relative Percentage by 2 - AB Labeling Tryptic Molecular Molecular Peptides Retention Times Glycoforms Weights Weights Glycoforms Relative % T15 - 16 39 . 7 - 40 . 7 FA1 3078 . 34 3078 . 32 M3 0 .02 FA2 3281. 42 3281 . 40 A1 1 .90 FA2G1 3443 . 47 3443 .45 FA1 11 . 58 FA3 3484. 50 3484 . 48 A2 0 .26 FA2G2 3605 .53 3605 .51 FA2 25 .55 FA3G1 3646 . 55 3646 . 54 M5/ A3 5 . 18 FA4 3687 .58 3687 .57 A2G1 1 . 25 FA3G2 3808. 61 3808 .60 FA3 2. 67 FA4G1 3849 .63 3849 .62 FA2G1 4 . 58 FA3G3 3970 .66 3970 . 65 FA2G1 4. 59 176 - 27 17 . 4 - 17 . 9 A2 2755 . 12 2755 . 11 FA4 2 . 68 FA2 2901. 18 2901. 16 A2G2 2 . 46 FA2G1 3063. 23 3063 . 21 FAGG1 2 . 25 FA3 3104 . 76 3104 . 25 FA2G2 7 .03 FA2G2 3225 .28 3225 . 27 US 2018 /0230445 A1 Aug . 16 , 2018 28

TABLE 24 - continued TABLE 25 | Oligosaccharide Species Associated with Each Glycopeptide pl and Relative Percentage of Peaks Observed by cIEF Theoretical Observed Peaks p / Tryptic Molecular Molecular 6 .60 36 .64 Peptides Retention Times Glycoforms Weights Weights 6 .65 8 . 66 6 .68 11. 78 FA3G1 3266 . 31 3266 .29 6 . 73 16 .71 FA4 3307 . 34 3307 . 32 auAWNA 6 .80 11 .24 FA4G1 3469. 39 3469 . 38 6 . 85 15 . 27 FA4G2 3631. 43 3631 . 43 FA4G3 3793. 49 3793 . 47 T33 28. 6 - 29 . 9 A1 2191. 92 2191. 93 A2 2394 . 99 2395 .01 Product - Related Substance A2G1 2557. 05 2557 . 06 A3 2598. 07 2598 . 07 [0246 ] Structural heterogeneity of asfotase alfa due to A2G2 2719 . 10 2719 . 11 post - translational modification is expected as it is expressed A3G1 2760 . 13 2760 . 14 A4 2801. 15 2801 . 16 in CHO cell line. As described previously , heterogeneity of T35 99 . 5 - 102 . 8 A2 3496 . 66 3496 . 65 asfotase alfa is reflected as charge variants , where six peaks A3 3699. 74 3699 . 73 are observed when analyzed by cIEF . The consistent pattern FA3 3845 . 80 3845 .79 of charge profiles of asfotase alfa has been demonstrated in A4 3902 . 82 3902. 80 clinical and PV batches . As determined by AUC, SDS FA3G1 4007. 85 4007. 85 PAGE, SEC and AEX , using representative material, asfo FA4 4048 . 88 4048 . 87 tase alfa is at a minimum of 96 % purity , suggesting that FA4G1 4210 . 93 4210 .93 multiple peaks observed by cIEF are fully representative of T45 - 46 46 . 3 - 47 . 0 FA2 4548 . 04 4547 .99 the produced asfotase alfa and that all peaks are product FA2G1 4710 . 09 4710 .05 FA3 4751. 11 4751 . 08 related substance . FA2G2 4872 . 14 4872 . 10 FA3G1 4913 . 17 4913 . 13 Product- Related Impurities FA4 4954. 19 4954 . 21 FA3G2 5075 . 22 5075 . 18 [0247 ] Product- related impurities are molecular variants FA4G1 5116 . 25 5116 .21 that do not have the properties comparable to the desired FA4G2 5278 . 29 5278 . 26 asfotase alfa with respect to activity , efficacy , and safety T55 19 .6 - 20 .0 A1 2284 . 91 2284 . 92 (Guidance for Industry , Q6B specifications : test procedures A2 2487 . 99 2488 .00 and acceptance criteria for biotechnological / biological prod FA2 2634 .05 2634 . 06 ucts , August 1999 , ICH ) . Product - related impurities have FA2G1 2796 . 10 2796 . 12 been observed by three assays and the characteristics of each impurity are summarized in Table 26 . [ 0244 ] Based on observed molecular weights and other analysis results ( e . g ., mass spectrum data and 2 - aminoben TABLE 26 zamide (2 - AB ) labeled oligosaccharides profiling data ), Summary of Asfotase Alfa Product -Related Impurities structures of major glycans (24 % relative abundance ) of asfotase alfa were determined in FIG . 41 and FIG . 42 . Methods Impurities SDS - PAGE Bands with molecular weight other than the main band Capillary Isoelectric Focusing ( cIEF ) SEC Aggregates [0245 ] cIEF separates proteins based on their isoelectric AEX Truncated molecule points and was used to monitor asfotase alfa charge variants . Asfotase alfa samples at a final concentration of 0 . 5 mg/mL in a buffer containing urea ,methyl cellulose , sucrose , phar Characterization of SDS -PAGE Bands malytes, and p? markers were analyzed using a capillary electrophoresis system . The samples were focused for 0 . 1 [0248 ] Asfotase alfa was analyzed by both non - reduced minute at 1500 V and then for 14 minutes at 3000 V . The and reduced SDS -PAGE . The results of SDS -PAGE analysis separated protein variants were detected using UV absorp suggests that band 1 corresponds to intact asfotase alfa , band tion at 280 nm . An electropherogram from the representative 2 corresponds to intact asfotase alfa with only one enzyme batch of asfotase alfa is shown in FIG . 43 . Six peaks were arm , and band 3 corresponds to the reduced form (monomer ) observed by cIEF . Typically , Peak 1 has a broad pl range . of asfotase alfa ( data not shown ) . Additional shoulders were observed for peaks 4 and 5 . This [0249 ] As expected , peptides from both the enzyme por result demonstrated that asfotase alfa is highly heteroge tion and the IgG1- Fc portion were observed from the band neous with regard to charge , which was expected , because of on reduced SDS- PAGE . This result , in addition to the the presence of multiple glycosylation sites with various apparent molecular weight of the band on reduced SDS numbers and levels of sialic acids at each site . The pl of each PAGE, indicates that the main band corresponds to asfotase peak and their relative percentage of asfotase alfa of the alfa in its reduced form . Band characterization is summa representative batch are summarized in Table 25 . rized in Table 27 . US 2018 /0230445 A1 Aug . 16 , 2018

TABLE 27 Summary of SDS -PAGE Band Characterization Apparent MAIDI- TOF and LC SDS Band Molecular MS analysis of bands PAGE Description Weights after in -gel digestion Species Non Band 1 Approximately Confirmed band 1 as Asfotase alfa , based on the Reduced (Main band ) 200 - 220 kDa asfotase alfa (61 . 9 % apparent molecular weight sequence coverage ) and sequence coverage . Band 2 Approximately Confirmed band 2 is Presumably truncated 125 kDa asfotase alfa - related asfotase alfa based on the ( 13 . 8 % sequence apparent molecular weight coverage ) that is between asfotase alfa homodimer and its single polypeptide chain . Band 3 Approximately Confirmed band 3 is Probably a single 95 - 105 kDa asfotase alfa - related polypeptide chain of ( 7 . 4 % sequence asfotase alfa. coverage ) Reduced Main Band Approximately Confirmed the main Reduced asfotase alfa 95 - 105 kDa band as asfotase alfa ( single polypeptide chain ) (65 . 4 % sequence The hypothesized truncated coverage ) form detected by non reduced SDS- PAGE was not detected by reduced SDS - PAGE because of its low abundance .

Characterization of SEC Peaks molecule with the oligosaccharides . In addition , molecular weight analyses of asfotase alfa were also performed by [0250 ) SEC -HPLC was used as a purity assay by separat AUC and SDS -PAGE . Consistent higher molecular weights ing high molecular weight species ( aggregates ), asfotase alfa of asfotase alfa were observed by these orthogonalmethods , dimer , and low molecular weight species. On a typical SEC suggesting extensive glycosylation of the molecule at mul chromatogram , the most abundant peak corresponds to tiple sites , which was confirmed by analysis of glycosy asfotase alfa homo - dimer . The peak that eluted before the lation . Higher order structures of asfotase alfa were deter main peak corresponds to a high molecular weight species . mined by both far -UV and near -UV CD . The hydrodynamic The peak that eluted after the main peak corresponds to a size of the molecule can also be determined from the low molecular weight species . Fractions corresponding to analyses of both AUC and SEC -MALS , which further both high molecular weight species and the main peaks were confirmed that asfotase alfa exists as a covalent dimer. Low collected and analyzed by SDS- PAGE followed by in - gel amount of aggregates were detected by SEC , AUC and digestion and mass spectrometry analysis . Matching the SEC -MALS . The native disulfide bond structure and the observed peptide molecular weights to the theoretical location of free cysteine at 102 was confirmed by LC -MS molecular weights derived from the known amino acid analysis of the non - reducing peptide maps . The presence of sequence revealed sequence coverage of 86 . 6 % for the main only one major free cysteine was further confirmed by the peak and 70 . 7 % for the high molecular weight species. assessment of the total amount of free sulfhydryl. LC -MS Therefore , it is concluded that the high molecular weight analysis demonstrated that the active site serine residue was species represents product- related impurities . partially phosphorylated . Three metals including zinc , cal cium and magnesium , as reported in the literature , were Characterization AEX Peaks detected in asfotase alfa drug substance. Glycosylation of asfotase alfa was extensively analyzed by free glycan analy 0251] AEX was used to monitor the charge variants of sis using MALDI- TOF , normal -phase HPLC analysis of asfotase alfa . The five AEX fractions along with the starting 2 -AB labeled oligosaccharides, LC -MS analysis of glyco material were analyzed by GP -HPLC and by non - reduced peptides , and total sialic acid measurement . All six glyco and reduced SDS -PAGE (data not shown ). The data suggests sylation sites per monomer of asfotase alfa were found to AEX basic peaks contain mainly asfotase alfa and possibly associate with a heterogeneous population of oligosaccha a small percentage of high molecular weight species. AEX rides . Charge profile , heterogeneity caused mainly by the acidic peak contains asfotase alfa high molecular weight presence of sialic acid , was analyzed by cIEF , where six species . Basic and acidic species are product- related as peaks were observed . Product -related substance is reflected confirmed by peptide finger printing by MALDI- TOF analy by the observation of six peaks by cIEF . Low levels of sis. product - related impurities were detected by SEC , AEX and [ 0252] Asfotase alfa has been well characterized using SDS - PAGE . multiple analytical techniques . Its identity was confirmed by N - terminal sequencing, amino acid analysis, and molecular Example 9 . Comparability of Asfotase Alfa weight analysis by MALDI- TOF and ESI- MS . The purity of Manufactured at 2 ,000 L ( 2K ) and 20 ,000 L (20K ) the produced asfotase alfa was monitored by AUC, SDS Scales PAGE, GP - HPLC and AEX . The size of asfotase alfa was [0253 ] In this Example , asfotase alfa drug substance was evaluated by MALDI- TOF and SEC -MALS analysis of the manufactured using 2 , 000 L (2K ) and 20 ,000 L (20K ) US 2018 /0230445 A1 Aug . 16 , 2018 30 processes. To demonstrate comparability among asfotase glycosylation and activity . The test results are summarized alfa produced at different scales , three batches of asfotase in Table 29 . Manufacturing comparability was further evalu alfa produced using a 2K ( # 35 , # 36 and # 38 ) process and ated using a side - by -side forced degradation study, where three batches of asfotase alfa produced using a 20K process ( # 40 , # 42 , and # 34 ) were analyzed for their physicochemical the degradation pathways and kinetics of the above -men properties side -by -side , where possible , using the methods tioned 2K and 20K batches were compared . The samples described in Table 28 . The specific batches of 2K and 20K were incubated at 40° C . for 0 , 14 , 24 , and 48 hours and were were chosen based on clinical use , and previously - tested analyzed by the methods listed in Table 30 . The test results batches have proved batch - to - batch consistency for all 2K for the temperature forced degradation study are summa and 20K batches . rized in Table 31 . [0254 ] The tested physicochemical properties established [0255 ] The overall results demonstrate that asfotase alfa asfotase alfa purity , charge variants , size , identity , structure, manufactured using 2K and 20K processes are comparable . TABLE 28 Asfotase Alfa Physicochemical Tests Test Category Test Purity Analytical Ultracentrifugation Purity SDS -PAGE /LOC (Lab on Chip ) Purity SDS - PAGE Purity GP - HPLC Purity Anion Exchange Chromatography Charge Profile Capillary Isoelectric Focusing ( CIEF ) Size Size - Exclusion Chromatography -Multi - Angle Light Scattering ( SEC -MALS ) Size Intact Molecular Weight Analysis (MALDI - TOF -MS ) Identity Deglycosylated / Reduced & Deglycosylated Molecular Weight Analysis (MALDI - TOF -MS ) Identity Deglycosylated / Reduced & Deglycosylated Molecular Weight Analysis (ESI - ToF -MS ) Structure Circular Dichroism Spectrometry Structure Disulfide bonding and Free thiol by LC /MS Structure Metal Analysis (ICP -MS / ICP - AES ) Structure Phosphorylation Site Identification and Quantitation via UPLC -MSe Glycosylation MALDI- Free Glycan Glycosylation 2 -AB labeled N - linked Oligosaccharides profiling by HPLC Glycosylation Glycopeptide Profile and Site Occupancy (UPLC -QToF -MS ) Glycosylation Total Sialic Acid Content Activity pNPP Activity Activity HA Binding Activity PPi Enzyme Kinetic assay US 2018/ 0230445 A1 Aug . 16, 2018

20KSD NA 0.8 0.6 0.3 0. 0.1 0. 0. 0.4 0.4 0. 0.62 0.01 7.05 0.01 0.22 0.01 1.19 0.01 1.78 0.01 1.70 0.01 3.76

96.8 2.9 0.3 98.6 1.4 96.38 6.59 28.50 6.64 8.72 6.67 11.78 6.72 18.58 6.79 13.04 6.84 19.47 20KMean NA 968.? ? 100.0 _99.8? 100.0 100.0? ? 00 ? ?

2.9 0.3 4.1 0. 6.60 6.65 8.66 6.68 6.73 6.85 20K #34 101.6 96.96.8 100.0 99.9 100.0 100.0 98.6 96.63 36.64 11.78 16.71 6.80 11.24 15.27 8

2.3 0.1 1. 0. 6.59 6.63 6.67 6.72 6.79 6.84 #42 94.5 97.6 100.0 99.8 100.0 100.0 98.9 96.84 24.40 8.96 10.60 20.25 13.27 22.53

96.0 43. 0.6 98.2 1.8 0. 95.68 6.59 6.63 8.53 6.67 6.72 6.79 14.62 6.84 20.62 #40 107.6 96.0 100.0 _99.8 100.0 100.0 24.46 12.97 18.79

1.0 0.5 0. 0. 0. 0. 6.0 7.0 0.9 0.50 0.02 2.50 0.01 0.63 0.01 0.69 0.02 0.90 0.00 0.75 0.01 1.38 2KSD NA 1. 0. 0 TABLE29 AsfotaseAlfaPhysicochemicalSummary 2KMean NA 95.6? 3.37 0.9? 100.0 99.8 100.0 100.0 96.5 3.0 0.5 94.17 6.61 31.88 656. 10.19 6.69 13.16 6.74 17.67 6.80 11.09 6.85 16.01

2K #38 99.8 94.5 3.9 1.6 100.0 99.8 100.0 100.0 96.2 3.8 0. 93.62 6.62 33.09 6.66 10.24 6,70 13.62 6.76 17.09 6.80 11.21 6.85 14.74

3. 0.8 2.9 0. 6.59 6.64 9.54 6.68 6.73 6.80 6.85 #36 99.9 95.9 100.0 _99.8 100.0 100.0 97.1 94.60 29.01 13.49 18.71 11.78 17.47

2.9 0.3 4.2 1.5 6.61 6.65 6.69 73.6 6.80 846. 35# 93.5 96.5 100.0 99.8 100.0 100.0 96.1 94.29 33.55 10.79 12.36 17.21 10.29 15.81

Concentration(mg/mL) Aggregate(%)Fragment(%) Non-Reduced(%) Reduced(%) Non-Reduced(%) Reduced(%) Dimer(%)* %MainPeakArea. Test Analytical Ultracentrifugation %Monomerofasfotase alfa* %Dimerofasfotasealfa %Trimerofasfotasealfa SDS-PAGE/LOC SDS-PAGE GP-HPLC AEX CIEF Peak1 Peak2 Peak3 Peak4 Peak5 Peak6 US 2018 /0230445 A1 Aug . 16 , 2018 32

20KSD20KSD 3.6 72.7 1.23 0.11 (nm) 0. 50. 0.3 0.5 0. 0.3 0. 0.5 FarUV (nm) 0.01 0.004 0.001 0.006 0.006 0.004 0.005 0.001 324 226 27 NearUV 274 263 Decon

190.2 658.2 80574.76 290.5 285.5 268.3 261.0 257.0 292.7 286.5 280.5 0.88 0.202 0.046 0.188 0.148 0.062 0.353 0.998 20KMean 161137.28 NearUV (nm) FarUV (nm) 20KMean 180019 160881 80539 -7288 -8250 Decon

194.1 722.1 80574.77 290.5 285.0 268.5 261.0 257.0 293.0 286.5 280.0 0.87 0.201 0.046 0.193 0.141 0.059 0.358 0.998 20K20K #34#34 161138.69 NearUV (nm) FarUV (nm) 180157 161140 80545 -7320 -8400 Decon

189.4 579.1 80574.86 290.5 286.0 268.5 260.5 257.0 292.5 286.5 280.5 0.89 0.198 0.045 0.188 0.150 0.066 0.350 0.997 #42#42 161136.46 NearUV (nm) FarUV (nm) 80509 -7340 -8270 Decon ? 180252180252 160725160725 187.0 673.4 290.5 285.5 268.0 261.5 257.0 292.5 286.5 281.0 0.89 0.206 0.047 0.182 0.153 0.060 0.350 0.998 80574.65 (nm) (nm) #4040# 161136.69 NearUV FarUV 179649179649 160778160778 8056380563 -7520 -8450 Decon

2KSD 4.1 150.7 1.19 0.57 (nm)3 .0 0.3 0.3 0.3 0.3 0. 0.3 0.6 (nm) 0.01 0.008 0.002 0.004 0.004 0.002 0.004 0.003 2KSD 71 71 35 UVNear FarUV 365 355 Decon

0.196 189.2 963.5 290.7 285.3 268.2 261.2 256.7 293.0 286.8 280.7 0.88 0.190 047.0 0.147 0.062 0.355 0.997 80575.52 (nm) 161137.39 NearUV FarUV (nm) TABLE29-continued AsfotaseAlfaPhysicochemicalSummaryAsfotaseAlfaPhysicochemicalSummary 2KMean2KMean 180902 161050 80530 -7012 -7934 Decon

0.89 181.0 0.046 0.199 0.152 0.358 2K 193.6 1047.0 80576.18 290.5 285.0 268.0 261.0 257.0 293.0 286.5 280.0 0.061 0.997 #3838# 161138.33 NearUV (nm) FarUV (nm) 180869 161048 80562 -6830 -7680 Decon

188.5 1054.0 80575.16 290.5 285.5 268.5 261.5 256.5 293.0 287.0 281.0 0.88 0.193 0.046 0.196 0.144 0.064 0.357 1.000 #36#36 161136.05 NearUV (nm) FarUV (nm) 180853 160980 80493 -7210 -8180 Decon

185.5 789.6 291.0 285.5 268.0 261.0 256.5 293.0 287.0 281.0 0.9 0.197 0.049 0.192 0.146 0.060 0.351 0.995 80575.23 (nm) #3535# 161137.80 NearUV (nm) FarUV 180984 161122 80534 7550- -8410 Decon

MALDI-TOFMW. ESI-TOFMW(Da) (nm): (nm): Test Mw(kDa)High (Da) Amax1Amax2 Amax3 amax4 Amax5 Amin1 amin2 Amin3 SEC-MALS Mw(kDa)Dimer MolecularWeight Species ReducedandDeglyco Deglyco DeglycoandReduced CDFar-UVFeature signalintensity2210signalintensity1220 1210/1220ratio a-helix 3/10helix B-sheet Turns Poly(Pro)IIStructure Unorderedprotein Total Glycosylated Deglycosylated CircularDichroism Near-UVFeatures Deconvolution US 2018/ 0230445 A1 Aug . 16, 2018 33

0.01 0.05 0.05 0.05 0.08 0.06 0.07 0.06 0.06 0.06 0.07 0.07 0.06 0.06 0.06 0.07 0.06 0.06 0.07 0.07 0.07 0.07 0.07 20KSD 2114 314 1651 984 618 1352 350 1131 198 193 27 91) 1095

0.7 933.39 20KMean 13540 1071 15804 16462 3513 12112 4089 4626 1047 1011 40 4420 2138 zm/zm/z 1136.47 1257.48 1282.53 1298.55 1339.56 1444.58 1485.62 1501.61 1542.63 1647.67 1663.66 1688.70 1704.69 1745,70 1809.71 1850.74 1866.72 1891.77 1907.75 2012.78 2028.77 2053.81

0.7 933.38 1136.42 1257.43 1282.48 1298.48 1339.50 1444.52 1485.56 1501.56 1542.58 1647.61 1663.60 1688.64 1704.63 1745.64 1809.65 1850.69 1866.67 1891.71 1907.69 2012.72 2028.72 2053.75 20K #34 15068 1345 17669 15593 4077 12453 3729 5423 1140 1167 70 4365 3321 Im/z zm/ (M+Na) 70. m/z 933.38 1136.52 1257.53 1282.58 1298.63 1339.61 1444.65 1485.68 1501.67 1542.70 1647.74 1663.74 1688.76 1704.75 1745.75 1809.79 1850.81 1866.79 1891.84 1907.83 2012.86 2028.84 2053.88 #42 14424 1141 15217 16264 3611 13261 4428 5123 1181 1070 33 5356 1931 zm/ 0.70.7 933.4 m/z 1136.48 1257.49 1282.54 1298.51 1339.57 1444.58 1485.64 1501.61 1542.60 1647.66 1663.62 1688.69 1704.67 1745.70 1809.69 1850.72 1866.70 1891.74 1907.71 2012.73 2028.71 2053.77 #40 11128 728 14527 17530 2852 10623 4110 3332 820 795 17 3538 1161 m/

0.06 2KSD 490 462 1330 393 393 1357 354 679 144 272 f 515 200 0.05 0.01 0.05 0.05 0.03 0.01 0.03 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.02 0.01 0.02 0.01

0.6 m/z 933.40 1136.51 1257.55 1282.60 1298.57 1339.60 1444.68 1485.66 1501.66 1542.69 1647,72 1663.71 1688.76 1704.75 1745.77 1809.79 1850.82 1866.80 1891.84 1907.85 2012.88 2028.87 2053.90 TABLE29-continued AstotaseAlfaPhysicochemicalSummaryAsfotaseAlfaPhysicochemicalSummary 2KMean 14985 975 16224 17771 3754 13009 4500 6928 1260 1279 94 5463 3871 (M+Na)

0.6 933.3 2K m/z (M+Na) 1136.52 1257.54 1282.58 1298.56 1339.60 1444.64 1485.66 1501.67 1542,69 1647,73 1663.73 1688.76 1704.77 1745.79 1809.82 1850.83 1866.83 1891.86 1907.87 2012.89 2028.89 2053.91 38# 14976 442 15268 18066 3482 13817 4602 7307 1366 1463 92 5636 3661

0.6 m/z(M+Na) 933.36 1136.50 1257.51 1282.56 1298.54 1339.59 1444.69 1485.65 1501.65 1542.68 1647.71 1663.69 1688.75 1704.74 1745.76 1809.77 1850.80 1866.79 1891.83 1907.82 2012.86 2028.85 2053.89 #36 14499 1261 15660 17324 3576 11442 4107 6144 1096 966 100 4884 3893 Imz

.70.7 933.45 m/z 1136.52 1257.61 1282.66 1298.61 1339.60 1444.71 1485.66 1501.65 1542.70 1647.73 1663.72 1688.76 1704.75 1745.77 1809.79 1850.82 1866.79 1891.84 1907.85 2012.88 2028.86 2053.89 35# 15479 1222 17743 17922 ???4205 ??? 13768 4792 7332 1317 1408 90 5870 4060 (M+Na)

Analysis(LC/MS) TestDisulfideBonding andFreeThiol T16ssT21 T48ss T50ssT50 T52ssT58 T66ssT71 T13* T16* T21* T52* T71* T13ssT13 T13ssT16 T13ssT21 TotalFreeCysteine #FreeCysperhalf molecule MALDIFreeGlycan Species M3 A1 M5 FA1 A1G1 A2 FA1G1 FA2 A2G1 A3 FA2G1 A2G2 FA3 A3G1 A4 FA2G2 FA3G1 A3G2 FA4 A4G1 FA3G2 A3G3 FA4G1 US 2018 / 0230445 A1 Aug. 16 , 2018 34

|20KSD 0.08 0.08 0.07 0.05 0.09 0.10 0.05 0.06 08.0 0.13 0.06 0.32 0.08 037 0.11 0.14 0.40 054 0.10 0.67 0.51 0.30 0.60 0.10 0.40 0.64 0.83 0.28 0.17 0.12 0.05 0.26 0.39 0.31 0.13

20KMean 2069.81 2174.83 2215.84 2231.84 2377.87 2393.87 2418.98 2539.91 2580.92 2742.95 2904.89 3270.08 0.10?? 2.43 11.57 0.50 24.99 6.13 1.42 13.82 3.68 4.11 3.652.28 2.95 6.42 3.03 2.20 3.24 2.33 0.89 4.89 2.86 1.31 0.38

2069.74 2174.77 2215.80 2231.80 2377.84 2393.81 2418.93 2539.89 2580.94 2742.90 2904.89 3269.87 0.02 1.90 11.58 0.26 25.55 5.18 1.25 2.67 4.58 4.59 2.68 2.46 2.25 7.03 3.42 1.71 3.01 2.13 0.89 5.35 3.54 1.87 0.61 20K #34

2419.02 0.11 2.34 11.46 0.53 6.30 1.43 4.31 3.54 3.86 154. 2.24 19.3 6.80 2.13 2.36 3.41 2.36 0.93 4.78 2.64 1.13 0.29 #42. 2069.90 2174.92 2215.92 2231.90 2377.96 2393.98 2539.97 2580.97 2743.08 2904.94 3270.01 25.11

0.17 2.79 11.67 0.60 24.60 6.44 1.49 3.90 3.37 4.11 3.63 2.24 3.05 5.73 3.732.29 3.19 2.41 0.84 4.76 2.73 1.22 0.36 #40 2069.77 2174.78 2215.77 2231.80 2377.76 2393.78 2418.98 2539.82 2580.78 2742.77 2904.79 3270.63

0.02 0.02 2KSD 0.03 0.01 0.04 0.03 0.06 0.03 0.04 0.03 0.04 10.06 0.08 0.45 2.94 0.14 2.50 0.66 0.28 1.35 0.56 0.68 1.14 0.22 1.06 1.19 1.22S 0.46 0.17 0.27ITIES 0.27 0.21 0.32 0.17 0.05 TABLE29-continued AsfotaseAlfaPhysicochemicalSummary 2KMean 2069.93 2174.92 2215.93 2231.92 2377.98 2393.95 2418.97 2539.97 2581.00 2742.92 2904.83 3270.47 0.09 1.80 10.23 0.44 23.02 6.00 1.35 4.93 3.23 3.11 4.99 2.37 2.76 6.39 3.10 2.48 3.64 2.67 11.22 5.63 3.39 1.50 0.42

0.05 1.55 7.98 0.49 20.98 6.11 1.14 6.49 2.68 2.32 6.31 2.21 3.69 5.08 4.51 3.01 3.78 2.94 1.51 5.75 3.69 1.68 0.48 2K #38 2069.94 2174.94 2215.95 2231.96 2377.98 2393.99 2419.04 2539.98 2580.97 2742.94 2904.87 3270.54

2069.90 2215.92 2231.87 2378.01 2393.92 2418.91 2540.00 2581.05 2742.88 2904.79 3270.40 10.18 2.32 13.55 0.55 22.26 6.61 1.67 .24 3.2 3.43 4.31 2.62 1.61 6.71 2.46 2.15 3.45 2.41 1.15 5.39 3.05 1.34 0.41 #36 2174.90 -

0.05 1.52 9.16 0.29 25.81 5.3 1.23 4.1 3.8 3.57 4.35 2.28 2.99 7.39 2.33 2.28 3.68 2.65 0.99 5.74 43.3 1.49 0.38 #35 2069.96 2174.91 2215.93 2231.93 2377.96 2393.94 2418.96 2539.94 2580.97 2742.95 2904.83 3270.47 -

FA4G2. A2G2. Test A4G2 FA3G3 A4G3 FA4G3 A4G4 FA4G1L1 FA4G4 FA4G2L1 FA4G3L1 FA4G4L1 FA4G4L2 2ABlabeledGlycans PeakArea% M3 A1 FA1 A2 FA2 M5/A3 A2G1 FA3 FA2G1 FA2G1 FA4 FA3G1 FA2G2 FA4G1 FA3G3 FA3G3 FA4G3 FA4G3 FA4G4 FA4G4L1 FA4G4L2 UNKNOWN US 2018 /0230445 A1 Aug . 16 , 2018 35

20KSD20KSD mz (M+H) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.02 0.02 0.02

+H) 20KMean20KMean m/z 3078.32 3281.40 3443.45 3484.48 3605.51 3646.53 3687.56 3808.59 3849.62 3970.64 2755.11 2901.16 3063.22 3104.24 3225.28 3266.29 3307.32 3469.37 3631.43 3793.48 2191.92 2395.00 2557.06 2598.07 2719.11 2760.14 2801.16 3496.64 3699.73 3845.79 3902.80 4007.84 4048.87 4210.93 4547.99 4710.05 4751.07 (M

+H) m/z 3078.32 3281.40 3443.45 3484.48 3605.51 3646.54 3687.57 3808.60 3849.62 2755.11 2901.16 3063.21 3104.25 3225.27 3266.29 3307.32 3469.38 3631.43 3793.47 2191.93 2557.06 2598.07 2719.11 2760.14 2801.16 3496.65 3699.73 3845.79 3902.80 4007.85 4048.87 4210.93 4547.99 4710.05 4751.08 20K #34#34 3970.65 2395.01 (M

+H) #42#42 m/z 3078.33 3281.41 3443.47 3484.49 3605.52 3646.54 3687.57 3808.60 3849.63 3970.65 2755.12 2901.17 3063.22 3104.26 3225.28 3266.30 3307.33 3469.39 3631.44 3793.50 2191.93 2395.01 2557.07 2598.08 2719.12 2760.15 2801.17 3496.65 3699.73 3845.79 3902.81 4007.85 4048.88 4210.93 4548.01 4710.06 4751.09

m/z (M+H) 3281.38 3484.46 3605.49 3646.52 3808.57 2755.10 2901.15 3063.21 3307.30 2719.10 2760.12 2801.15 4007.83 4048.86 4210.92 #4040# 3078.31 3443.44 3687.55 3849.60 3970.62 3104.23 ND 3266.28 3469.36 3631.41 3793.46 2191.91 2394.99 2557.05 2598.06 3496.63 3699.71 3845.77 3902.78 4547.97 4710.03 4751.05

+H) 2KSD2KSD m/z 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 00.0 0.00 0.00 0.01 0.01 0.00 0.03 0.04 0.04 0.03 0.04 0.04 0.04 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

+H) 2KMean2KMean m/z 3078.32 3281.40 3443.45 3484.48 3605.51 3646.54 3687.56 3808.59 3849.62 3970.65 2755.11 2901.16 3063.21 3104.24 3225.28 3266.29 3307.32 3469.37 3631.43 3793.49 2191.94 2395.02 2557.08 2598.10 2719.13 2760.16 2801.18 3496.64 3699.73 3845.78 3902.80 4007.84 4048.87 4210.92 4547.99 4710.04 4751.06 TABLE29-continued AsfotaseAlfaPhysicochemicalSummaryAsfotaseAlfaPhysicochemicalSummary (M

2K #3838# m/z (M+H) 3078.31 3281.39 3443.45 3484.47 3605.51 3646.53 3687.55 3808.59 3849.61 3970.64 2755.10 2901.16 3063.21 3104.24 3225.27 3266.30 3307.32 3469.37 3631.42 3793.49 2191.92 2395.00 2557.05 2598.09 2719.10 2760.13 2801.15 3496.63 3699.72 3845.77 3902.80 4007.83 4048.86 4210.91 4547.97 4710.03 4751.05

#36#36 m/z (M+H) 3078.33 3281.40 3443.46 3484.49 3605.51 3646.54 3687.56 3808.60 3849.62 3970.65 2755.11 2901.16 3063.21 3104.25 3225.28 3266.30 3307.33 3469.38 3631.44 3793.49 2191.98 2395.06 2557.12 2598.14 2719.18 2760.20 2801.23 3496.65 3699.73 3845.78 3902.80 4007.84 4048.87 4210.93 4548.00 4710.05 4751.08

#3535# m/z (M+H) 3078.31 3281.40 3443.46 3484.48 3605.51 3646.54 3687.57 3808.59 3849.62 3970.65 2755.11 2901.16 3063.21 3104.25 3225.28 3266.29 3307.32 3469.38 3631.43 3793.48 2191.93 2395.00 2557.06 2598.07 2719.11 2760.14 2801.16 3496.64 3699.73 3845.79 3902.81 4007.85 4048.88 4210.93 4547.99 4710.04 4751.07

glycopeptidem/z(M+H) 3078.34 3281.42 3443.47 3484.50 3605.53 3646.55 3687.58 3808.61 3849.63 3970.66 2755.12 2901.18 3063.23 3104.26 3225.28 3266.31 3307.34 3469.39 3631.43 3793.49 2191.92 2394.99 2557.05 2598.07 2719.10 2760.13 2801.15 3496.66 3699.74 3845.80 3902.82 4007.85 4048.88 4210.93 4548.04 4710.09 4751.11

MS)UPLC-QToFOccupancy( Test SiteGlycopeptideProfileand ObservedinMassesGlycopeptide FA2 FA2G1 FA3 FA2G2 FA3G1 FA4 FA3G2 FA4G1 FA3G3 FA2 FA2G1 FA3 FA2G2 FA3G1 FA4 FA461 FA4G2 FA4G3 A2 A2G1 A3A2G2 A3G1 A4 A2 A3 FA3 A4 FA3G1 FA4 FA4G1 FA2 FA2G1 FA3 Reduced/AlkylatedTrypsinDigested PeptideGlycan T15-16FA1 T26-27A2 T33A1 Tryptic T35 T45-46

9 Time(min) 99.5-1028 Approximate Retention 39.7-40 17.4- 28.6-299 46.3-470 US 2018 /0230445 A1 Aug . 16 , 2018 9€

0.01 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 1.6 0.1 0.7 0.8 1.3 1.0 0.9 0.8 0.4 2.6 0.7 ObaNN 1.3 0.4 7.0 1. 0.9 0.8 0.5 1.3 0.4 0.5 20KSD 0.01 Relative%11 Observed - 353 -2834999228 2. = 339233 20KMean 4872.10 4913.13 4954.18 5075.18 5116.21 5278.26 2284.91 2487.99 2634.05 2796.11 Relative% Observed19993039729 10.0 17.6 11.5 9.3 10.7 7. 4. 4.54.2 3.7 10.7 7.5 5.0 12.1 4.7 7.88299393723 8.6 7.0 6.4 4.6 17.1 21.3 13.7 5.3 11.5 4.8 2.

#34 4872.10 4913.13 4954.21 5075.18 5116.21 5278.26 2284.92 2488.00 2634.06 2796.12 Relative% ve Auwovaa guvu nuwu 20K Observed 10- 18- 11 - - -

#42 4872.11 4913.14 4954.17 5075.20 5116.22 5278.27 2284.92 2488.00 2634.06 2796.12 Relative% 11 10 22 WA Observed -e18 - coroneogeo- 194513 10

40# 4872.08 4913.11 4954.18 5075.16 5116.18 5278.23 2284.90 2487.98 2634.05 2796.10 Relative% Observedfaseanna 12 18

0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 Relative% 1.7 1.3 1.6 8.2 1. 0.2 1.8 0.9 0.4 0.9 1.7 1.0 8.0 3.1 1. 0.7 2.1 1.0 5.0 8.0 1. 0.5 6.1 4.0 0.9 0.2 1.0 2KSD 1Observed - 2947823389992322332999999 TABLE29-continued SummaryPhysicochemicalAsfotaseAlfaAsfotaseAlfaPhysicochemicalSummary 2KMean 4872.09 4913.12 4954.15 5075.17 5116.20 5278.25 2284.92 2488.00 2634.06 2796.12 Relative% Observed1 6.4 17.732 9. 13.4 10.1 7.1 7.5 4.2ganzesgargs 5.3 3.4 10.4 6.3 4.0 15.3 6.0 7.0 10.9 8.4 5.8 42 3. 10.7 23.5 12.5nesa 100. 11.3 5.4 3.9

2K 2796.12 4954.13 5075.16 2487.99 2634.05 1 ?? ?? ?? ? ?? ? 4872.08 4913.11 5116.19 2284.91 1 #38 5278.25 Relative% 1 Observed --19 ------= 10= 24 = 11 15 ??

4872.10 4913.13 4954.16 5075.18 5116.21 5278.26 2284.92 2488.00 2634.06 2796.12 wa ??? ? ? ? ?? #36 Relative% 18 11 1 1 Observed - ?-11 12?? ? ?? ? ? ? ?? ?? 12 24

#35 4872.09 4913.12 4954.15 5075.17 5116.20 5278.25 2284.92 2488.00 2634.06 2796.12 Relative% 16 11 NO OA AAU 8 10 23 Observed 12 N? 14 12

4954.19 2284.91 2487.99 2634.05 4872.14FA2G2 FA3G14913.17 5075.22FA3G2 5116.25FA4G1 5278.29FA462 2796.10FA2G1 Glycan FA1 FA2 FA2G1 FA3 FA2G2 FA3G1 FA4 FA3G2 FA4G1 FA3G3 A2 FA2 FA2G1 FA3 FA2G2 FA3G1 FA4 FA4G1 FA4G2 FA4G3 Al A2 A2G1 A3 A2G2 A3G1 A4 Test FA4 A2 FA2 GlycopeptideProfileandSiteOccupancy(UPLC QTOF-MS) Site RelativeGlycoformContributions ateachN-LinkedOligosaccharide T26-27 T33

39.7-40T1516 19.6-200T55A1 RetentionTimeTryptic (min)Peptide Approximate 17.4-9 28.6-299 US 2018 /0230445 A1 Aug . 16 , 2018 37

20KSD 0.6 7.0 0.7 0.3 0.1 0.5 0.5 2.6 1.4 2.6 1. 0.8 0.3 0.9 8.0 5. 41. 0.50

20KMean 9.0 9.8 7.9 1.3 5.4 6.3 5.4 18.7 11.9 17.8 6.811.5 4.8 5.7 7.8 4.5 3.7 38.4 33.7 6.2 1.57

2.1 20K20K VOO? aaauwwavaroo ON

ooitavuw a coutea fuer a 1.5

WOOD Owvua ? ? ? ? ? ?? 1.1. #40 29 40 280026722579

2KSD 1. 1.4 1.3 0.4 3.0 1.7 0.6 0. 1.8 2. 1.0 0.1 2. 0.8 0.5 0.4 0.1 2.6 3.7 9.0 0.530.53

6.2 TABLE29-continued AsfotaseAlfaPhysicochemicalSummary 2KMean 7.6 12.7 8. 4.1 5.0 8.2 5.6 16.5 8.6 18.5 9. 14.8 4.8 8.6 4.2 4.2 36.0 40.1 5.6 2.20

1.88.1 2K ou #38 14 10 16 44 21372137

Wola o vooguvua uw EGAA 2.0 21902190

2.8 26112611

A2 A3 FA3 A4 FA3G1 FA4 FA4G1 FA2 FA2G1 FA3 FA2G2 FA3G1 FA4 FA3G2 FA461 FA4G2 Al A2 FA2 FA2G1 Test TotalSialic Acidper (mol/) LC-MSe PeakAreaT13 non-Phost T35 T45-46 T55 Monomer Phosphorylation

99.5-1028 46.3-470 19.6-200 US 2018 /0230445 A1 Aug . 16 , 2018 38

1.0 0.23 0.04 0.04 26.1 21.5 9.5 102.3 19.0 9% 20KSD20KSD ** * 5%* * *

20KMean20KMean 32.932.9 2.01 0.08 1.02 981.7 91 70.8 62.4 113% 131.4 115.5 114%

33.33.9 2.27 0.12 0.97 90.3 79.6 137.0 114.9 #34 1079.0 85 113% 119% 20K 14341324 9

32.0 1.88 0.08 1.04 41.1 38.3 100.0 99.0 142#42 1260 32.0 991.0 92 107% 101%

32.32.9 1.88 0.04 1.04 875.0875.0 81.0 69.3 125.7 116.0 #40 97 117% 108% 1401375 9

9% 2.32.3 0.20 0.02 0.05 8.1 5.9 2KSD2KSD 44,044.0 2 11.2 33% 19.0

TABLE29-continued AsfotaseAlfaPhysicochemicalSummaryAsfotaseAlfaPhysicochemicalSummary 2KMean2KMean 33.833.8 1.62 0.05 0.83 861.7861.7 91 giai94.5 75.2 129% 123.6 118.5 104%

2K 31.231.2 1.411.41 0.03 0.85 73.5 62.3 96% 43838# 967 812.0 89 s 8850€118% 103.7 107.6 I

35.735.7 1.81 0.05 0.87 896.0 88.9 81.7 109% 141.5 124.0 114% 136#36 1215 : SILICE *Monomerofasfotasealfaasreportedbyanalyticalultracentrifugationisequivalent/synonymoustodimer'GP-HPLCandSECMALS. 34.434.4 1.63 0.05 0.77 90.8 81.7 125.7 124.0 35# 1370 877.0 92 111% 101% PeptideMods:Cys102COMeS93P04;MolecularformulaC102H160N27041S1P1MonoisotopicMass2482.0723 *PeptideMods:Cys102COMe;MolecularformulaC102H159N2703891MonoisotopicMass2402.106

% Mg2+ Test PeakAreaT13 ICP-MS Zn2+ Ca2+ SpecificActivity (PNPP) (U/mg) %Binding PPI SampleKm SampleKcat S93Phost Phosphorylated IonMolarRatio HABinding Head-toheadReferenceKm Km%of:Reference Head-toheadReferenceKcat Kcat%ofReference US 2018 /0230445 A1 Aug . 16 , 2018 39

TABLE 30 body ( see Pekar and Sukumar 2007 Quantitation of aggre gates in therapeutic proteins using sedimentation velocity Asfotase Alfa Temperature Forced Degradation Tests analytical ultracentrifugation : Practical considerations that affect precision and accuracy. Analytical Biochemistry, 367 : Assay Time Points for each assay 225 - 237 ) , which is also a glycoprotein with a similar Category Assay TO T12 + 3 T24 + 3 T48 + 3 molecular weight. GP -HPLC data ( shown below ) indicates | comparable % of monomer , which further indicates that the Purity SDS -PAGE Reduced / X Non - Reduced slight difference among batches detected by AUC was due to Purity and HPLC Gel Permeation D X X X intra -assay variation ( such as difference between instrument Impurities cells ) . A small additional peak at 2 . 5 ( s ) was detected for Impurities AEX X X X asfotase alfa produced from exemplary Process # 35 , which Impurities RP - HPLC D X X X D was an artifact of the c ( s ) analysis . The shift of the # 35 peak , Impurities Peptide Mapping X X Potency Specific Activity (PNPP ) X X X corresponding to the dimer of asfotase alfa ( approximately 11 ( s ) ) , is considered within the error limits of the method . The asfotase alfa AUC data were acquired by the University TABLE 31 Summary of Asfotase Alfa Temperature Forced Degradation Results 2K 20K Test # 35 # 36 # 38 2K Mean 2K SD # 40 # 42 # 34 20K Mean 20K SD RP - HPLC % Main Peak Area ( RT - 21 . 2 minutes )

TO 99 .07 99 .61 99 . 45 99 . 38 0 . 28 99 .61 99 . 64 99 . 65 99 .63 0 . 02 T14 89 .21 88 . 98 80 . 86 86 . 35 4 . 76 90 . 88 91 .65 91. 35 91. 29 0 .39 T24 82. 96 83 .01 75 . 33 80 .43 4 . 42 87. 09 86 .68 86 . 32 86 . 70 0 . 39 T48 73. 28 72 . 24 67 .48 71 .00 3 .09 76 . 25 78 . 70 76 . 87 77 . 27 1 . 27 AEX % Main Peak Area

?? 93 . 90 94 . 60 94 . 20 94 . 23 0 . 35 96 . 00 96 . 80 96 . 50 96 . 43 0 . 40 T14 87 .00 86 .40 77 .70 83 .70 5 . 20 96 . 10 94 . 10 92 . 60 94 . 27 1 . 76 T24 76 . 40 77 . 20 67 . 30 73 .63 5 . 50 82. 40 83 .00 81. 70 82 .37 0 .65 T48 61. 47 63 . 89 56 . 87 60 . 74 3 . 57 68 . 80 72 . 10 69 .00 69 . 97 1 . 85 GP - HPLC % Main Peak Area ?? 96 . 0 96 . 2 95 . 9 96 . 0 0 . 2 97 . 5 98 . 2 97 . 8 97. 8 0 . 4 T14 84 . 7 85 . 3 74 . 3 81 .4 6 . 2 87 . 2 88 . 7 88 . 1 88 .0 0 . 8 T24 77 . 5 77 .6 67. 4 74 . 2 5 . 9 80 . 7 82 . 2 81 . 2 81. 4 0. 8 T48 63 . 8 62 .9 57 . 4 . 61 . 4 3. 5 68. 00 71 . 99 69 . 5 69 . 8 2 . 0 Specific Activity (PNPP ) 33333 ( U /mg ) TO 901 900 781 861 69 938 949 1084 990 T14 793 795 684 757 64 679 801 698 T24 747 793 600600 713 101 577 585 732 631 T48 524 543 458 508 45 386 505 369 420 SDS -PAGE Non - Reduced % Main Band ?? 100 . 0 100 . 0 100 . 0 100 . 0 0 . 0 100 . 0 100 . 0 100 . 0 100 . 0 0 . 0 T48 96 . 0 97 . 5 97 . 0 96 . 8 0 . 8 97 . 1 97 . 1 97 . 8 97 . 3 0 . 4 Reduced % Main Band TO 100 . 0 100 . 0 100 . 0 100 . 0 0 . 0 100 . 0 100 . 0 100 . 0 100 . 0 0 . 0 T48 100 . 0 100 . 0 100 . 0 100 . 0 0 . 0 100 . 0 100 . 0 100 . 0 100 . 0 0 . 0

Purity of Connecticut, Analytical Ultracentrifugation Facility , Bio technology Bioservices Center. This method distinguishes Analytical Ultracentrifugation (AUC ) asfotase alfa monomer from aggregates consisting of dimeric or larger species and their relative % based on 0256 ). The % monomer levels determined by analytical ultracentrifugation were comparable between the 2K and the continuous sedimentation coefficient distribution . 20K batches. All the values were in the range of 94 . 5 % to SDS- PAGE /LOC ( Lab on Chip ) 97 .6 % with a 20K mean of 96 . 8 % and a 2K mean of 95 .6 % . The difference is within the assay variability of AUC as [0257 ] The main band % determined by SDS -PAGE : previously demonstrated for the analysis of an IgG1 anti - LABCHIP (PerkinElmer ) GXII Protein Assay for both US 2018 /0230445 A1 Aug . 16 , 2018 40 non - reduced and reduced was comparable between the 2K retention times indicate the same species were observed . batches and the 20K batches. As summarized in Table 29 , This method distinguishes asfotase alfa from larger species the mean main band % was 100. 0 % ( non -reduced ) and and smaller fragments . 99 . 8 % ( reduced ) for the three 2K batches and 100 . 0 % ( non -reduced ) and 99. 8 % ( reduced ) for 20K batches . In Anion Exchange Chromatography (AEX ) addition , comparable band patterns were observed for the [0260 ] The % of the main peak by AEX for all 2K batches 2K and 20K batches as demonstrated by electropherograms and 20K batches were within a narrow range of 93 .62 % to of both the non - reduced and reduced analyses . This test 96 . 84 % as shown in Table 29 . The 2K mean was 94 . 17 % and separates the protein based on its molecular mass and the 20K mean was 96 . 38 % of main peak area . Slightly provides analysis of the purity of the intact protein expressed higher % of the main peak was observed for the 20K batches as a percentmain band . The assay was performed following due to a decrease in the late eluting peak . The slightly higher LabChip GXII Protein Assay Quick Guide , HT Protein level of the main peak in the 20K batches demonstrates Express LabChip Kit , Version 2 ( revised March 2010 ) . higher purity of the product . The AEX chromatogram showed overlaid peaks for 2K and 20K batches at the same SDS- PAGE retention times , suggesting that products from all batches [0258 ] The three 2K batches and three 20K batches were had the same species . This method separates protein in order analyzed side - by - side using SDS - PAGE. Analysis by den of increasing net surface anionic charge . Proteins separated sitometry resulted in the main band accounting for 100 % for are detected by ultraviolet absorbance at 280 nm and then all the batches for both the non -reduced and reduced SDS quantified as peak area percent. PAGE . In addition to the main band , a faint band below the quantitation limit with apparent molecular weight higher Charge Profile than that of the asfotase alfa dimer was observed for all [0261 ] Capillary Isoelectric Focusing ( cIEF ) batches . Only one band was observed for all batches by [0262 ] The peak profiles and the relative % of each peak reduced SDS -PAGE . This test separates the protein based on of the 2K and 20K batches were comparable when asfotase its molecular mass and provides analysis of the purity of the alfa was analyzed by cIEF . All samples exhibit six promi intact protein expressed as a percentmain band . The protein nent peaks ranging from isoelectric point (pl ) of ~ 6 . 57 to sample was separated on a gel, stained with Coomassie Blue ~ 6 . 85 and these peaks are referred to as peaks 1 , 2 , 3 , 4 , 5 Stain for visualization , destained and analyzed by densitom and 6 from left to right. Peak areas detected in 2K and 20K etry . Molecular weight of asfotase alfa , degradation prod batches for each charged variant species are comparable as ucts , and process related impurities were estimated by shown in Table 29 . Comparability between the cIEF peak % comparison with known molecular weight standards . Quan of the 2K and 20K batches was confirmed using a T - Test titative measurements of separated proteins are performed analysis of all batches as shown in Table 32 ( all p - values by scanning and analyzing with densitometric analysis . were > 0 . 05 indicating that any observed differences between lots is not significant) . The cIEF electropherograms of 2K GP -HPLC and 20K batches showed similar peak profiles in the samepi [ 0259 ] The purity of asfotase alfa was analyzed side - by ranges . cIEF provides semi- quantitative separation of side by GP - HPLC . The 2K mean ( 96 .5 % ) and the 20K mean charged -variant species based on protein isoform pi' s . The ( 98 . 6 % ) are shown in Table 29 . The % asfotase alfa of the separated proteins were detected at 280 nm absorbance . TABLE 32 T - Test of the cIEF Peak % of 2K and 20K Batches CIEF Peak Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 T - Test of Peak 2K # 35 33 . 55 10 .79 12 . 36 17 . 21 10 . 29 15 .81 % of 2K and BauBatches # 36 29 .01 9 . 54 13 .49 18. 71 11. 78 17 .47 20K batches # 37 27 . 97 11 . 38 12 . 25 18 . 96 11 .97 17 .47 ## 3838 33. 09 10 .. 2424 13 .62 17 . 09 11 .21 14 .74 # 39 27. 87 6 . 02 11 .96 12 . 93 15 .33 25 . 90 20K # 40 24 .46 8 .53 12 . 97 18. 79 14 .62 20 . 62 Batches # 41 35 . 52 6 . 87 15 . 94 11 . 42 12 .69 17 .56 # 42 24 . 40 8 . 96 10 .60 20 . 25 22 .53 # 43 32. 50 9 . 65 11 . 60 18. 00 11 .64 1660 # 34 36 .64 8 . 66 11 . 78 16 . 71 11 . 24 15 . 27 # 44 24 . 90 10 . 48 15 . 39 18 . 20 9 . 74 21 . 30 # 45 27. 40 10 . 10 11 .65 20 . 94 9 .50 20 . 40 T - Test P - Value 0 . 74 0 .57 0 .91 0 .65 0 .79 0 .67

20K batches is slightly higher than that of the 2K batches . Molecular Size However , batch data showed similar % dimer, suggesting that the slightly lower % dimer of 2K batches mainly due to Size Exclusion Chromatography - Multi- Angle Light differences in sample history such as material age . The Scattering ( SEC -MALS ) GP - HPLC chromatogram also showed comparable products [0263 ] The molecular weights of asfotase alfa determined from the 2K and 20K batches. Same peaks at the same by SEC -MALS of the 2K and 20K batches were compa US 2018 /0230445 A1 Aug . 16 , 2018 rable . The mean molecular weight of the 2K batches is 189 . 2 shown in Table 29 . The MALDI- ToF spectra for the 2K and kDa and the mean molecular weight of the 20K batches is 20 K batches detected similar peak profiles . Additional 189 . 2 kDa as shown In Table 29 . Similar peak profiles were masses present in the spectra were due to assay induced observed for all the batches on UV chromatograms ( the fragmentation , incomplete deglycosylation , and /or multiply sample from batch # 36 was run at a later date , due to sample charged ions. Intensity differences observed for identified availability, resulting in a non - perfect match of its peak species are a result of matrix crystallization and laser effects . profiles with the other samples ) . The SEC -MALS method [0267 ] The molecular weight for reduced and deglycosy combines an SEC column to separate protein species by size lated asfotase alfa was calculated from the primary amino with a MALS instrument to determine molecular weight of acid sequence of the polypeptide amino acid sequence of peaks . High variation in the molecular weights of the high 726 amino acids using NIST molecular weights and isotope molecular weight species ( listed in Table 29 ) , as determined percentages . Reduced and deglycosylated asfotase alfa by the light scattering signal and refractive index (RI ) , is due molecular weight was calculated to be 80 ,577 .68 Da. to higher method variation for determination of low abun [0268 ] The molecular weight values for reduced and dant species . Based on UV chromatograms, the relative deglycosylated asfotase alfa 20K batches compares with 2K retention times of the high molecular weight species to batches and all values were within 1 % of the calculated asfotase alfa are similar for all six batches , which indicate reduced and deglycosylated molecular weight (the mass that the molecular weights of the high molecular weight accuracy of the externally calibrated MALDI- TOF ) as shown species should also be similar. in Table 29 . The mean of 2K batches was 80 ,530 Da and the mean of the 20K batches was 80 ,539 Da . The MALDI- TOF Intact Molecular Weight Analysis (MALDI - TOF -MS ) spectra for the 2K and 20K batches detected comparable [0264 ] Themean molecular weight of the 2K batches was peak profiles. Additional masses present in the spectra are 180 , 902 Da and the mean molecular weight of the 20K due to assay induced fragmentation and or multiple charged batches was 180 , 019 Da as shown in Table 29 . The molecu ions. Intensity differences observed for identified species are lar weights of 20K batches and 2K batches are within the 1 % a result of matrix crystallization and laser effects . This mass accuracy of the externally calibrated MALDI- TOF and method identifies the molecule on the basis of molecular thus are comparable . The slightly higher trending of the 2K weight . Test samples were solid phase -extracted and mixed molecular weight is likely due to the presence of slightly with SDHB matrix solution and co - precipitated on the higher amount of larger oligosaccharides such as oligosac MALDI target . This dried sample was ionized with a laser charides with sialic acid . The MALDI- TOF Intact Molecular into a ToF mass spectrometer. An m / z spectrum was col weight ( IMW ) spectra for the asfotase alfa 2K and 20K lected from each sample and the intact m / z was converted to batches also detected the same peak profiles. Batch # 36 was molecular weight. analyzed on a separate occasion using a different instrument [0269 ] The molecular weights of deglycosylated asfotase calibration but was included for completeness of compara alfa of the 2K and 20K batches were comparable and all bility . Additional masses present in the spectra are due to values are within the 100 ppm of the calculated deglycosy assay induced fragmentation and or multiple charged ions . lated molecular weight (which is the mass accuracy of the Intensity differences observed for identified species are a externally calibrated ESI - TOF -MS ) as shown in Table 29 . result ofmatrix crystallization and laser effects . This method The mean molecular weight of the 2K batches was 161 , 137 . identifies the molecule on the basis of molecular weight. 39 Da and the mean molecular weight of the 20K batches Test samples were solid phase - extracted and mixed with was 161, 137 .28 Da and the values were consistent with the SDHB matrix solution and co - precipitated on the MALDI calculated deglycosylated molecular weight of 161 , 135 . 20 target . This dried sample was ionized with a laser into a ToF Da. The reduced and deglycosylated molecular weight val mass spectrometer. An m / z spectrum was collected from ues for the main peak of the 2K batches were comparable to each sample and the intact m / z was converted to molecular that of the 20K batches and all values are well within the 100 weight. ppm of the calculated reduced and deglycosylated molecular weight (which is the mass accuracy of the externally cali Identity brated ESI- TOF -MS ) as shown in Table 29 . The mean molecular weight of the 2K batches was 80 , 575 . 52 Da and Deglycosylated /Reduced & Deglycosylated Molecular the mean molecular weight of the 20K batches was 80 ,574 . 76 Da . The values are consistent with the calculated reduced Weight Analysis (MALDI - TOF -MS ) and deglycosylated molecular weight value for asfotase alfa [0265 ] The molecular weight for deglycosylated asfotase of 80 ,577 . 68 Da . This method identifies the molecule on the alfa was calculated from the primary amino acid sequence of basis of intactmolecular weight . Test samples were injected the two identical polypeptide amino acid sequences of 726 onto a C4 RP -HPLC column and eluted with an aqueous : amino acids using NIST molecular weights and isotope organic solvent gradient . The eluate was then electrosprayed percentages with 20 cysteines forming 10 disulfide bonds. into a ToF mass spectrometer and a spectrum from the upper Deglycosylated asfotase alfa molecular weight was calcu half of the chromatographic peak was deconvoluted to lated to be 161, 135 . 20 Da . provide the intact molecular weight. [0266 ] The molecular weights of deglycosylated asfotase [0270 ] The zinc and magnesium ion molar ratios deter alfa from the 20K batches and from the 2K batches were all mined by Inductively Coupled Plasma- Mass Spectrometry within 1 % of the calculated deglycosylated molecular ( ICP -MS ) for the three 20K and three 2K batches were weight ( the mass accuracy of the externally calibrated comparable as shown in Table 29 . The calcium ion molar MALDI- TOF ) as shown in Table 29 . The mean molecular ratios determined by Inductively Coupled Plasma- Atomic weight of 20K batches was 160 ,881 Da and the mean Emission Spectroscopy ( ICP - AES ) for the 20K and 2K molecular weight of the 2K batches was 161 , 050 Da as batches were comparable as shown in Table 29 . The ion US 2018 /0230445 A1 Aug . 16 , 2018 molar ratios for zinc and calcium were also in agreement MALDI - TOF and the results are shown in Table 29 . The with literature , where two zinc and one calcium were same glycan species at similar relative percentage was associated with each alkaline phosphatase enzyme monomer observed in the 20K batches and 2K batches . This method ( see E . Mornet et al. 2001 “ Structural evidence for a identifies the glycans associated with the drug substance by functional role of human tissue non -specific alkaline phos molecular weights . The glycans were enzymatically cleaved phatase in bone mineralization . ” JBC , 276 : 31171 - 31178 ; from the drug substance using PNGase - F digestion and and B . Stec , K M . Holtz and ER . Kantrowitz . 2000 “ A subsequent acidification . The glycans were then solid pase revised mechanism for the alkaline phosphatase reaction involving three metal ions ." JMB, 299 : 1303 - 1311 ) . The extracted and mixed with the super 3 , 4 - dihydroxybenzoic magnesium ion molar ratio for all batches was surprisingly acid matrix solution and co - precipitated on the MALDI lower than the expected value reported in literature . ICP target . This dried sample was ionized with a laser into a ToF AES was chosen for calcium analysis as ICP - AES does not mass spectrometer . An m / z (M + Na ) + spectrum was col suffer from the argon poly atomic interferences observed for lected . calcium isotopes in ICP -MS . The asfotase alfa ICP data were acquired commercially . These methods determined the 2 -AB Labeled N -Linked Oligosaccharides Profiling (HPLC ) amount of magnesium , zinc and calcium (ug /mL ) from [0273 ] The types of glycoforms and the relative percent which the molar ion ratios were calculated for as fotase alfa . age of each glycoform were analyzed by 2 - AB labeling and Phosphorylation Site Identification and Quantitation Via HPLC analysis . The results are shown in Table 29 . Peak at UPLC -MSe the same retention times and with similar relative percentage [0271 ] The site and the relative percentage of phosphory was observed in all batches (data not shown ) suggesting the lation were determined by UPLC -MS tryptic peptide map same types of oligosaccharides and similar levels . Batch # 36 ping . Alkaline phosphatase is known to contain a serine sample was run at a later date . As an example , the percentage residue in the active site that can form a serine - phosphate of the major glycoform FA2 was plotted against enzymatic intermediate (see pp 28 - 29 of Millán , José Luis . 2006 activities . No correlation was observed between percent FA2 “ Mammalian Alkaline Phosphatases. ” Wiley - VCH ) . The and Specific Activity ( PNPP ), HA Binding, Km , and Kcat same serine residue 93 was found to be phosphorylated in all ( data not shown ) . batches at a percentage in the range of 35 . 7 % to 31. 2 % as [ 0274 ] This assay characterizes the glycosylation pattern shown in Table 29 . The percentage of phosphorylation was by determining the oligosaccharides associated with the found not to correlate with asfotase alfa specific activity drug molecule on the basis of the retention time and peak (PNPP ) , HA binding , Km , and Kcat (data not shown ). Based area of the fluorescently tagged free - oligosaccharides . The on the lack of correlation , the phosphorylated intermediate free oligosaccharides from drug substance were released is not a rate limiting step for the activity of asfotase alfa . The enzymatically and tagged with 2 - aminobenzamide ( 2 - AB ) confirmation and identification of a phosphorylation site at by reductive amination . Samples were then injected on to an S93 in asfotase alfa was confirmed via UPLC -MSe peptide HPLC system with a Ludger column and the 2AB tagged mapping . The theoretical sequence of asfotase alfa and the oligosaccharides were separated and detected with a fluo predicted tryptic cleavages were used for the analysis . The rescence detector , 330 nm excitation 420 nm emission . The resultant LC -MSe data was collected and processed using chromatograms of each batch were compared and peak area Waters Biopharmalynx v . 1 . 2 . Biopharmalynx software (Wa of each resolved peak was used to determine relative amount ters) identified the 13th tryptic peptide from the primary of oligosaccharide from each batch . sequence ( T13 ), which contains S93 , the active site serine from the enzymatic portion of the molecule as having Cys102 capped with ac , and S93 existing as both phospho Glycopeptide Profile by UPLC -OTOF -AMS rylated and non -phosphorylated . Once identified as part of [0275 ] The oligosaccharide profile at each glycosylation the peptide map , extracted ion chromatograms (EIC ' s ) were site was evaluated by analysis of glycopeptides by UPLC pulled for each of these peptides and smoothed and inte MS . Tissue non - specific alkaline phosphatase ( TNAP ) has grated . From the resulting EIC peak areas , the ratio of five N - glycosylation sites ( see pp 54 -55 of Millan , 2006 phosphorylated peptide peak area to total peak area gives a Wiley - VCH ) . Asfotase alfa contains an additional N - glyco relative percentage of the phosphorylation at S93 . Confir sylation site in the Fc region providing a total of six mation of the + 80 Da associated with phosphorylated serine N - glycosylation sites per monomer . FIG . 44 through FIG . 49 side chain was made by examining the MSC fragmentation show the glycopeptide fingerprint profiles for N123 in pattern generated for the proposed phosphorylated T13 T15 - 16 , N213 in T26 -27 , N254 in T33 , N286 in T35 , N413 peptide as shown in FIG . 36 . This fragmentation pattern in T45 - 46 and N564 in T55 . UPLC - Q - ToF -MS glycopeptide confirmed that S93 was shifted by + 80 Da ( the mass of a fingerprints of the summed N - linked oligosaccharides pro phosphorylation ) observed by the y12 and y13 ions . Each vided detailed characterization of the N - glycosylation . Iden batch of asfotase alfa was reduced and alkylated then tification of glycopeptide species by mass analysis and digested with trypsin . Detection was performed by ESI- O relative quantitation of each species was performed by ToF -MS using a Waters Acquity UPLC and Synapt Q - ToF transforming the multiple charge states observed in the raw mass spectrometer , operating in positive ion electrospray glycopeptide mass fingerprint spectra into singly charged mode . and deisotoped spectra for each site . The results of these Glycosylation transformations are shown in Table 29 . It lists all masses observed for the glycoforms and their relative % for glyco N -Linked Oligosaccharides Mass Profiling forms contributing 25 % to the total glycoform content at (MALDI - TOF -MS ) each observed N - linked oligosaccharide site . In this assay , [ 0272 ] Released oligosaccharides from the three 2K asfotase alfa was reduced and alkylated then digested with batches and the three 20K batches were analyzed by trypsin . Detection was performed by ESI - Q - ToF -MS using US 2018 /0230445 A1 Aug . 16 , 2018 43 a Waters Acquity UPLC and Synapt Q - ToF mass spectrom PPi Enzyme Kinetic Assay Using Physiological Relevant eter, operating in positive ion electrospray mode . The theo Substance retical sequence of asfotase alfa and the predicted tryptic [0280 ] The kinetic parameters Km and Kcat were deter cleavages were used for the analysis . Molecular weights for mined by PPi Kinetic Assay . Samples were run on a number the theoretical glycopeptides were calculated using NIST of occasions head - to - head with the reference standard . For molecular weights and isotope percentages . Glycopeptides comparison , Km and Kcat are graphed as a percent of the are observed as collections of related glycoforms with reference standard value run at the time of sample testing closely related elution times and thus requires summation of ( Table 29 ) . All K , values were within 30 % of the reference spectra to ensure the complete glycopeptide complement for standard value run side - by -side and are considered compa a particular site is observed . Site - specific summed spectra rable . All Kcot values were within 20 % of the reference ( glycopeptide mass fingerprints ) were obtained for each of standard run side -by - side and are considered comparable . the most abundant peptide species containing each of the The Kn % of reference and Kcat % of reference for the 2K N - linked oligosaccharide sites in asfotase alfa for each batch batches were comparable to the 20K batches . PPi Kinetic tested . Assay measures the purified asfotase alfa potency ( enzy matic activity ) using PPi, a natural alkaline phosphatase Total Sialic Acid Content (7SAC ) substrate and determines kinetic constants Km and K . cat of [0276 ] Total sialic acid content was determined by HPAE PPi hydrolysis under physiological conditions (37° C . , PH PAD . As shown in Table 29 , sialic acid content for each 7 .4 ). batch is within the specification of 0 .9 - 3. 5 mole sialic acid per mole of monomer. Considering the variation of the sialic Temperature Forced Degradation acid content due to its relatively lower amount, the 2K [0281 ] To further ensure comparability of the 2K and the batches and 20K batches were comparable. No correlation 20K batches, side -by - side forced degradation study was was observed between TSAC value and Specific Activity performed to compare the degradation pathways and kinet ( PNPP ) , HA Binding , Km , and Kcat ics. Three 2K batches ( # 35 , # 36 , and # 38 ) and three 20K batches ( # 40 , # 42 , and # 34 ) , detailed in Table 33 , were used Activity for the study . Some batches required the use of drug product (DP ) material due to limited BDS sample availability . [ 0277] Specific Activity ( PNPP ) [0282 ] Batches # 35 , # 36 , # 40 , # 42 , and # 34 were diluted [0278 ] Specific activity of asfotase alfa was determined using asfotase alfa formulation buffer ( 25 mM sodium for the three 20K and three 2K batches . The mean specific phosphate and 150 mM sodium chloride , pH 7 . 4 ) to 40 activity of the 20K batches is 981. 7 U /mg and 861. 7 U /mg mg/ mL to match the protein concentration of batch # 38 . To for the three 2 K batches . The specific activity of all batches achieve substantial levels of degradations to establish deg was within the specification of 620 - 1250 U /mg and was radation kinetics, forced degradation conditions were chosen comparable for the 20K and 2K batches. This method is used based on preliminary forced degradation condition screen for the determination of asfotase alfa enzymatic activity ing . Aliquots ( 200 uL ) were created for each test assay at using PNPP as a substrate . Asfotase alfa is a recombinant each time point ( 0 , 14 , 24 and 48 hours ). The TO aliquots protein that has one domain from the human tissue non were held at 5° C . until testing . The rest of the sample specific alkaline phosphatase enzyme. This domain is cata aliquots were incubated at 40° C . for 14 , 24 and 48 hours . lytically active and hydrolyzes phosphate esters . The assay Samples were analyzed by SDS - PAGE , GP -HPLC , AEX , is performed at enzyme saturation to reach and maintain RP -HPLC , Peptide Mapping , and Potency as listed in Table Vmax for duration of the measurement. The pNPP is hydro 30 to determine comparability . The test results for the lyzed into a yellow colored product (maximal absorbance at temperature forced degradation study are listed in Table 31 405 nm ) . The rate of reaction is directly proportional to the enzyme activity . One unit ( U ) corresponds to 1 umol of for the three 20K batches and three 2K batches. PNPP formed per minute under the employed method con ditions . The Specific Activity by PNPP ( enzymatic activity TABLE 33 per mg protein ) was calculated from the enzymatic activity Batches Used for the Degradation Study and the Protein Concentration by Ango . The results are Concentration shown in Table 29 . Name mg/ mL Hydroxyapatite Binding # 35 90 . 9 # 36 96 . 4 # 38 40 . 0 [0279 ] The % hydroxyapatite binding determined by the # 40 107 . 6 HA Binding Assay of asfotase alfa 2K batches were com # 42 92 . 9 parable with the 20K batches with all values in the range of # 34 101. 6 85 - 97 % . The mean % HA binding is 91 % for both the 20K and 2K batches ( Table 29 ) . The HA binding assay measures the enzymatic activity of asfotase alfa complex for PNPP when bound to hydroxyapatite . A synthetic substrate , para SDS -PAGE (Non -Reduced and Reduced ) Nitrophenyl Phosphate (PNPP ), when hydrolyzed by asfo [0283 ] TO and T48 hour samples were analyzed by SDS tase alfa , produces a yellow colored product that can be PAGE ( non - reduced and reduced ) for purity . The main band quantifiable by absorbance at 405 nm . This assay was % by non - reduced SDS -PAGE was 100 % for all batches at performed at substrate saturation in order to reach and T = 0 and decreased to a mean of 97 . 3 % for 20K batches and maintain the Vmax for the duration of the reaction . 96 . 8 % for 2K batches as shown in Table 31 . The trend of US 2018 /0230445 A1 Aug . 16 , 2018 44 degradation is comparable for the 20K and 2K batches. High Peptide Mapping molecular weight species was detected in the T48 hour sample for all six batches. This species disappeared upon [0287 ] TO , T24 and T48 hour samples for 20K and 2K reduction . This result suggests the formation of disulfide batches were analyzed by peptide mapping for evaluation of bond -related aggregates is a degradation pathway for all potential chemical degradation . There was no observed batches. No extra bands were observed by reduced SDS significant difference between 20K and 2K at all the time PAGE for both the 20K and 2K batches at T = 0 and T = 48 points ( T = 0 hour and T = 48 hours ) . This method denatures , hours , indicating no degradation in the peptide bonds . This reduces and alkylates protein with guanidine hydrochloride, test separates the protein based on its molecular mass and dithiothreitol and iodoacetate , followed by digestion with provides analysis of the purity of the intact protein expressed the protease trypsin . The peptide fragments produced are as a percent main band . The protein sample is separated on separated by gradient reversed phase HPLC and detected at 210 nm . a gel , stained with Coomassie Blue Stain for visualization , Specific Activity (PNPP ) destained and analyzed by densitometry . Molecular weight [0288 ] Specific activity (PNPP ) of asfotase alfa 20K of asfotase alfa , degradation products , and process related batches and 2K batches was determined by enzymatic assay impurities are estimated by comparison with known molecu after 40° C . incubation at each time point ( TO , T14 , T24 , and lar weight standards . Quantitative measurements of sepa T48 ) . The activities of the 20K batches were comparable rated proteins are performed by scanning and analyzing with with the 2K batches at each time point . Decrease in the densitometric analysis . activity caused by thermal stress follows similar slopes for all batches over time. Protein phosphatase , enzyme controls GP -HPLC the removal of phosphate ( PO3- ) group from protein mol ecules . PNPP Phosphatase Assay was used to detect phos [0284 ] Degradation to form aggregates and fragments phatase activity and to compare 20K and 2K batches . This caused by thermal stress was monitored by GP -HPLC and method is used for the determination of asfotase alfa enzy the results are shown in Table 31. The peak profiles for all matic activity using PNPP as a substrate . Asfotase alfa is a batches at T = 0 were similar suggesting the presence of the recombinant protein that has one domain from the human same species. Thermal stress resulted in the appearance of a tissue non -specific alkaline phosphatase enzyme . This peak at approximately 6 minutes , corresponding to large domain is catalytically active and hydrolyzes phosphate aggregates, which increased over time. The peak profiles of esters . The assay is performed at enzyme saturation to reach all batches at T = 14 hours , T = 24 hours and T = 48 hours were and maintain Vmax for duration of the measurement. The similar, which indicates similar degradation pathway . The PNPP is hydrolyzed into a yellow colored product (maximal slopes of decrease of the main peak over time for all batches absorbance at 405 nm ). The rate of reaction is directly were similar taking into account the fact that the materials proportional to the enzyme activity. One unit ( U ) corre used were at different ages . Similar slope indicates similar sponds to 1 umol of PNPP formed per minute under the degradation kinetics . employed method conditions. The Specific Activity by PNPP (enzymatic activity per mg protein ) is calculated from Anion Exchange Chromatography ( AEX ) the enzymatic activity and the Protein Concentration by A280 [ 0285 ] Degradation of asfotase alfa was also monitored by [0289 ] Comparability of asfotase alfa manufactured at AEX , which separates proteins based on charge . The same 20K scale and asfotase alfa manufactured at 2K scale was peak profiles were observed for all batches at T = 0 , suggest established . Three batches from each scale was analyzed ing the same species in all batches . Thermal stress resulted side - by - side , if possible , using physicochemicalmethods to in the increase of peaks in the retention time window of evaluate asfotase alfa purity , impurities , charge variants , approximately 22 - 26 minutes . Comparable peaks profiles size , structure , glycosylation , disulfide bond , free thiols and for all batches were observed for all batches at T = 14 hours, activity . In addition , side -by -side forced degradation study T = 24 hours and at T = 48 hours , which indicates formation of was carried out to evaluate the degradation pathways and the same species by thermal degradation . Similar slopes of kinetics. The results demonstrate that the manufactured degradation ,monitored by decrease of the main peak , were products at 20K and 2K scales were comparable . observed for all batches taking into account the slight [0290 ] The purity of the batches was assessed by analyti difference at T = 0 (as disclosed previously ) and the age cal ultracentrifugation , SDS - PAGE / LOC , SDS - PAGE, GP difference of different batches. HPLC , and AEX . The results showed comparable purity for all the batches . The same types of aggregates at very low RP -HPLC comparable levels were detected in all batches by AUC , SDS -PAGE and GP -HPLC . [ 0286 ] When analyzed by RP -HPLC , a mean peak in the [0291 ] Charge variants , common for recombinant pro retention time window of approximately 21 . 2 - 21 . 3 minutes teins, were evaluated by cIEF . The same species at compa was observed for all batches , indicating the same species in rable levels was observed for all batches , suggesting con all batches . The main peak was the only major peak sistent level of posttranslational modifications . observed for all batches at T = 0 . Thermal stress resulted in an [0292 ] The molecular size of the batches was assessed by increase in a peak eluting immediately after the main peak . SEC -MALS , and intact MALDI- TOF molecular weight. The same peak from thermal degradation observed for all Identity was confirmed by deglycosylated /reduced & degly batches at T = 14 , T = 24 and T = 48 hours suggests the same cosylated MALDI- ToF molecular weight and deglycosy degradation pathway. Degradation kinetics as monitored by lated /reduced & deglycosylated ESI- ToF molecular weight . the decrease of the main peak was comparable for all batches Structure was also evaluated by CD , disulfide bonding and as evidenced by similar slopes of all batches . free thiols (LC /MS ) , total free thiol assay , metal content US 2018 / 0230445 Al Aug . 16 , 2018 45 analysis (ICP - MS/ ICP - AES ) , and phosphorylation site [0296 ] In summary , asfotase alfa 20K batches and 2K occupancy . Comparable molecular weights and comparable batches are comparable as demonstrated by extended char hydrodynamic size were obtained for all batches , indicating similar modifications and similar conformation of asfotase acterization using multiple orthogonal methods and side -by alfa of all batches. side forced degradation study. Thus, material from 20K [ 0293] Asfotase alfa glycosylation was assessed by batches should have comparable safety and efficiency as MALDI- TOF free glycan , 2AB labeled oligosaccharide material from the 2K batches . fHPLC , LC -MS glycopeptide analysis , and total sialic acid content. The same oligosaccharide species at comparable levels were obtained for all batches. Example 10 . Additional Comparability of Asfotase [0294 ] The activity of the drug substance was assessed by Alfa Manufactured at 2 , 000 L ( 2K ) and 20 ,000 L specific activity (PNPP ) , HA binding and PPi activity . The (20K ) Scales results showed that that asfotase alfa 20K batches were comparable to asfotase alfa 2K batches. [0297 ] Seven batches ofasfotase alfa were produced at the [ 0295] Lastly , the same degradation pathway and similar 20 ,000 L scale , and five additional batches of asfotase alfa degradation kinetics of the 20K and 2K batches was were produced using the 2 , 000 L scale . The products for observed by the side -by - side forced degradation study using both scales were analyzed and found to be comparable ( data thermal stress . not shown ) .

SEQUENCE LISTING

< 160 > NUMBER OF SEO ID NOS : 1 < 210 > SEQ ID NO 1 < 211 > LENGTH : 726 < 212 > TYPE : PRT 2222122 32 3 > ORGANISM : Artificial Sequence < 220 > FEATURE : AAAAA< 223 > OTHER INFORMATION : Synthetic polypeptide < 400 > SEQUENCE : 1 Leu Val Pro Glu Lys Glu Lys Asp Pro ?Lys Tyr Trp Arg Asp Gin Ala Gin Glu Thr Leu Lys Tyr Ala Leu Glu Leu EJGin Lys Leu Asn Thr Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val Ser 135 40 Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gin Leu His His Asn Pro 50 Ep.: Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala Leu am 80 Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly Thr 85 Ala Thr Ala Tyr 5mmLeu Cys Gly Val Lys Ala Asn Glu Gly Thr Val Gly

Val Ser Ala Ala Thr Glu Arq Ser Arg Cys Asn ?Thr Thr Gln Gly Asn 115 120 Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser Val mmmm 130 Gly Ile Val Thr Thr Thr Arg Val Asn His Ala ?Thr Pro Ser Ala Ala m?mm 160 Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met Pro 165

Pro Glu Ala Leu Ser Gin Gly Cys Lys Asp Ile Ala Tyr Gln Leu Met

His Asn Ile Arg Asp Ile Asp Val Ile Met Gly Gly Gly Arg mmmmmLys Tyr 195 200 mmmm | Met Tyr Pro Lys Asn Lys Thr Asp Val Glu Tyr Glu Ser Asp Glu Lys 210 mama mmmm mmmmm