physicochemical and biological characterization of the proposed...

Research ArticlePhysicochemical and Biological Characterization ofthe Proposed Biosimilar Tocilizumab

Shiwei Miao1 Li Fan1 Liang Zhao1 Ding Ding2 Xiaohui Liu2

HaibinWang2 andWen-Song Tan1

1State Key Laboratory of Bioreactor Engineering East China University of Science and Technology Shanghai 200237 China2Hisun Pharmaceutical (Hangzhou) Co Ltd Fuyang Hangzhou Zhejiang 311404 China

Correspondence should be addressed to Wen-Song Tan wstanecusteducn

Received 13 July 2016 Revised 18 October 2016 Accepted 27 October 2016 Published 2 March 2017

Academic Editor Yves Renaudineau

Copyright copy 2017 Shiwei Miao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

HS628 has been developed as a proposed biosimilar product of originator tocilizumab (Actemra) An extensive physicochemicaland biological characterization was conducted to assess similarity between HS628 and originator tocilizumab The aminoacid sequence was shown to be identical between HS628 and originator tocilizumab The higher order structure was foundto be indistinguishable from originator tocilizumab Concerning purity and heterogeneity HS628 was demonstrated to havesimilar posttranslational modifications charge heterogeneity size heterogeneity and glycosylation to originator tocilizumabMoreover HS628 exhibited highly similar binding affinity and antiproliferative activity as well as capability of inhibiting STAT3phosphorylation compared to originator tocilizumab Taken together HS628 can be considered as a highly similar molecule tooriginator tocilizumab in terms of physicochemical and biological properties

1 Introduction

Tocilizumab (Actemra) is a recombinant humanized IgG1monoclonal antibody which binds to human interleukin-6 (IL-6) receptors (IL-6R) By inhibiting IL-6 binding toboth soluble and membrane-bound IL-6R (sIL-6R and mIL-6R) tocilizumab blocks IL-6-mediated signal transduction[1] Actemra is approved by the US Food and Drug Admin-istration (FDA) for the treatment of rheumatoid arthritisand juvenile idiopathic arthritis patients It has also beendemonstrated that tocilizumab has anticancer potency viaapoptosis induction as an agonistic IL-6R regulator and maybe utilized as a new target molecule for non-small-cell lungcancer (NSCLC) [2]

According to the FDA the European Medicines Agency(EMA) and the World Health Organization (WHO) guide-lines biosimilar products must demonstrate similarity interms of quality safety and efficacy with the referenceproduct [3ndash5] On March 6 2015 the FDA approved SandozIncrsquos (Sandoz) Zarxio (filgrastim-sndz) as the first biosimilarproduct for use in the USA The EMA has already approved

a number of biosimilar products mainly cytokines CT-P13(Remsima Inflectra) a biosimilar product of referenceinfliximab (Remicade) is the first biosimilar monoclonalantibody approved by the EMA for use in all indications forwhich Remicade is approved [6]

In 2015 six of the ten top-selling drugs are antibody-based therapeutics With the increasing use of therapeu-tic monoclonal antibodies (mAbs) there has been a hugedemand for the development of biosimilar mAbs Regulatoryapproval for a biosimilar product relies on the demonstrationof comparability towards the reference product starting withan extensive physicochemical and biological characteriza-tion which will provide evidence to support the extent ofadditional clinical evaluation [7 8]

A biosimilar product will not be exactly like its referenceproduct but the critical quality attributes (CQAs) need tomatch so that the biosimilar product can have similar efficacysafety and immunogenicity to those of the reference product[9ndash11] A CQA is defined in the ICH Guideline Q8 [12]as a physical chemical biological or microbiological prop-erty or characteristic that should be within an appropriate

HindawiBioMed Research InternationalVolume 2017 Article ID 4926168 13 pageshttpsdoiorg10115520174926168

2 BioMed Research International

limit range or distribution to ensure the desired prod-uct quality safety and efficacy Potential CQAs of mAbsmay include charge-related variants size-related variantsoxidation-related variants glycosylation structural variantsprocess-related impurities and biological properties [13ndash15]In the case of adalimumab themost critical assays are TNF-120572binding and neutralization those that directly measured theprimary mechanism of action (MOA) of the product [16]

So far many biosimilarmAbs papers have been publishedon top-selling mAbs such as adalimumab rituximab inflix-imab bevacizumab and trastuzumab [17ndash21] but not yet ontocilizumab HS628 has been developed by Hisun Pharma-ceutical in China as a proposed biosimilar tocilizumab oforiginator tocilizumab In this study we describe a subset ofthe state-of-the-art methods of physicochemical and biolog-ical analysis that were performed to demonstrate similaritybetween HS628 and originator tocilizumab

2 Materials and Methods

21Materials Originatortocilizumab(Actemra 4mL 80mg10mL 200mg and 20mL 400mg) batches were purchasedfromRoche Pharma (Schweiz) Ltd (manufactured by ChugaiPharma Manufacturing Co Ltd) The proposed biosim-ilar product HS628 was produced by Zhejiang HisunPharmaceutical (Zhejiang China) HS628 drug product(4mL 80mg) was used for physicochemical and functionalcomparability study

22 Methods An array of state-of-the-art and orthogonalanalytical techniques were used to compare HS628 withtocilizumab All chromatographic analyses were performedon Agilent 1260 high performance liquid chromatography(HPLC) systems (Agilent Technologies Boblingen Ger-many) Primary sequences verified with tryptic peptidemapping and thewholemolecule exactmasses were analyzedusing reverse-phase ultraperformance liquid chromatogra-phy system coupled with a UV detector and a quadru-pole time-of-flight mass spectrometer (RP-UPLC-Q-TOF)Higher order structure was evaluated by circular dichroism(CD) and differential scanning calorimetry (DSC) Freeproteinogenic thiol groupwas quantified using Ellmanrsquos assayThe disulfide bridging pattern was assessed using RP-UPLC-Q-TOF system after peptide mapping under nonreducingconditions Posttranslational modifications were identifiedby RP-UPLC-Q-TOF system after tryptic peptide mappingunder reducing conditions Charge heterogeneity of pro-tein sample with or without carboxypeptidase digestionwas assessed by cation exchange chromatography (CEX-HPLC) and imaged capillary isoelectric focusing (icIEF)Size heterogeneity (purity) was determined by size exclu-sion chromatography (SEC) and capillary electrophoresis-sodium dodecylsulfate (CE-SDS) N-Glycosylation patternsof the products were assessed by LC-MS peptide mappingand normal-phase HPLC with fluorescence detection (NP-HPLC-FL) Functional properties were evaluated by surfaceplasmon resonance (SPR) antiproliferative assay and inhibi-tion of STAT3 phosphorylation

221 Nonreduced and Reduced PeptideMapping with Reverse-Phase (RP) HPLC with UV andMass Spectrometric DetectionReduced peptide mapping was performed to identify theprimary sequences of tested products Nonreduced peptidemapping was carried out to determine the disulfide bridgingpattern Protein sample was diluted in water at 2mgmL and400 120583L of sample solution was mixed with guanidine-HCl inTris buffer (01molL Tris pH 80) to a final concentrationof guanidine-HCl of 6molL The solution was incubated at37∘C for 2 hrs After incubation sample solution was reducedwith dithiothreitol (DTT) at 10mgmL and incubated at37∘C for 30min Alkylation was conducted by adding a20120583L aliquot of a 1molL iodoacetamide stock solution atroom temperature for 40min in the dark The buffer wasexchanged to 005molL ammonium bicarbonate at pH 78by ultrafiltration The protein sample was then digested withsequencing grade trypsin at a 1 40 enzyme-sample ratioat 37∘C overnight The enzymatic reaction was quenchedwith the addition of formic acid to a final concentrationof 01 For disulfide bridge identification the sample wasprepared with the same methods as described above withoutreduction and digested using both trypsin andGlu-C enzymeat a 1 1 40 ratio of trypsin Glu-C sample RP-UPLC-MSwas performed on a Waters ACQUITY UPLC H-Class Biosystem coupled with a UV detection and a Q-TOF massspectrometer Mobile phase A was water with 01 TFAand mobile phase B was acetonitrile with 01 TFA Peptidemapping was conducted using an ACQUITYUPLCBEH 300C18 column (17 120583m 21mm times 150mm Waters USA) with aflow rate of 02mLmin The gradient started at 5 phase Bfor 5min and then increased from 5 phase B to 45 phaseB in 45min followed by a gradient from 45 phase B to95 phase B in 10min followed by holding at 95 phaseB for another 10min as an elution step The UV detectionwavelength was set at 214 nmThemass spectrometer was setto run in positive ion mode with a sample cone voltage of60V capillary voltage of 3000V mass resolution of 30000and mz range of 50ndash2000 The mass spectrometer wascalibrated with NaI (2 120583g120583L) in waterisopropanol (5050)and tuned with leucine enkephalin solution (LE 1 ng120583L) inwateracetonitrile (5050) with 01 formic acid The ion ofLE at mz 5562771 was used to conduct mass correctionThe acquired raw spectra data were then deconvoluted andanalyzed using BiopharmaLynx software (version 133 Build7)

222MolecularMass DeterminationUsing RP-UPLC-Q-TOFSample solution was diluted to a final concentration of1mgmL in 005M Tris-HCl (pH 75) Analysis of intactprotein and reduced protein was run on a MassPrepmicro desalting column (Waters USA) with a flow rateof 02mLmin The gradient began at 10 phase B for1min and a linear gradient from 10 phase B to 60phase B in 10min was then performed followed by anincrease to 90 phase B in 2min followed by holdingat 90 for another 5min The Q-TOF mass spectrometerwas run in positive ion mode Sample cone voltage cap-illary voltage mass resolution and mz range were set at

BioMed Research International 3

60V 3000V 30000 and 250ndash4000 respectively The massspectrometer was calibrated with NaI solution at a finalconcentration of 2120583g120583L in waterisopropanol (5050) andwas tuned with leucine enkephalin solution (LE 1 ng120583L) inwateracetonitrile (5050) with 01 formic acid The masscorrection was conducted using ion of LE at mz 5562771The raw spectra data acquired were then deconvoluted andanalyzed using BiopharmaLynx software (version 133 Build7)

223 Ellmanrsquos Assay Free proteinogenic thiol group wasquantified by using Ellmanrsquos reagent (DTNB) L-Cysteinesolution (10mM) was formulated as 100 120583M 75 120583M 50 120583M25 120583M 20120583M 10 120583M and 5 120583M standard solutions with theassay buffer (100mM PBS 1mM EDTA pH 74) 150 120583L8M guanidine hydrochloride was added to each well of themicroplate Then 25120583L test solution or standard solutionwas added to each well and for each solution 3 replicateswere applied Finally 25 120583L Ellmanrsquos solution (4mgmL 551015840-dithiobis(2-nitrobenzoic acid)) was added to each well Theabsorbance was measured at 412 nm after 3 hrs of incubationThe content of free thiol was determined by external standardmethod This method is applicable to detect denaturedsample

224 Circular Dichroism (CD) CD experiments were per-formed on a Chirascan-Plus spectrometer (Applied Photo-physics UK) Near-UV CD spectra were recorded from 250to 320 nm wavelength step size was 1 nm bandwidth was1 nm and time per point was 15 s Far-UV CD spectra wererecorded within the range of 190ndash260 nm wavelength stepsize was 1 nm bandwidth was 07 nm and time per pointwas 07 s For each sample three scans were performedand baseline correction was applied Noise reduction andnormalization of the spectrum were performed

225 Differential Scanning Calorimetry (DSC) DSC mea-surements were carried out on aMicroCal VP-Capillary DSCSystem (GE Healthcare USA) Scans were recorded at a rateof 180∘Ch Samples were diluted to 05mgmLwith referencebuffer and scanned from 15 to 110∘C For each sample threescans were performed

226 Cation Exchange Chromatography (CEX) CEX sepa-ration was performed using a weak CEX column (ProPacWCX-10 4 times 250mm Dionex Germany) connected to anAgilent 1260 HPLC system Mobile phase A consisted of10mM sodium phosphate (pH 75) and mobile phase Bconsisted of 10mM sodium phosphate and 100mM sodiumchloride (pH 75) Samples were diluted using equilibrationbuffer consisting of mobile phases A and B at a ratio of85 15 (vv) to a final concentration of 2mgmL For C-terminal lysine cleavage 15120583L of 1mgmL carboxypeptidaseB (CPB Roche Germany) was added to 1mL of the samplesolution and the mixture was incubated at 37∘C for 30minThe separation gradient was set as follows 0 to 3min holdingat 15 B 3 to 6min 15 Bndash30 B 6 to 36min 30ndash70 Band 36 to 38min 70ndash100 BThe column was then washedwith 100 B for 5min followed by equilibration using 15 B

for 15min The column temperature was set at 30∘C and theabsorbance of the eluent was monitored at 214 nm

227 Imaged Capillary Isoelectric Focusing (icIEF) Isoelec-tric point and charge heterogeneity were determined usingiCE3 Capillary IEF System (ProteinSimple USA) The sam-ple was diluted using 8M urea to a final concentrationof 2mgmL 200120583L of sample mixture for icIEF analysisconsisted of 70 120583L 1 methyl cellulose 10 120583L Pharmalyte (pI3ndash10) 05 120583L pI marker (pI = 710) 05120583L pI marker (pI =1010) 50 120583L sample solution in 8M urea 50 120583L 8M ureaand 19 120583L water The sample mixture was injected into thecartridge and focused by applying a potential of 1500V for1min for the first focusing step and a potential of 3000Vfor 10min as the second focusing step The isoelectric point(pI) and charge variants were analyzed using Chrom PerfectAnalysis software

228 Size Exclusion Chromatography (SEC) SEC was con-ducted on aTSK-GelG3000SWXL column (TosohBioscienceJapan) connected to an Agilent 1260 HPLC system The SECseparation buffer contained 20mMPBS and 200mM sodiumchloride at pH 75 20 120583L of protein sample at a concentrationof 2mgmLwas injected onto the columnThe separationwasconducted at a flow rate of 05mLmin and was monitoredat 280 nm Molecular weight determination of monomersand polymers was tested by SEC-MALS-RI Multiangle staticLight Scattering (MALS) detector was DAWN HELEOS II(Wyatt Technology USA) and Refractive Index (RI) detectorwas Optilab T-rEX (Wyatt Technology USA)

229 Capillary Electrophoresis with Sodium Dodecyl Sul-fate (CE-SDS) CE-SDS was performed on 7100 CE System(Agilent USA) equipped with a diode array detector Fornonreduced CE-SDS analysis the antibody samples at afinal concentration of 1mgmL were mixed with SDS samplebuffer (10 vv) and 125mM iodoacetamide The mixturewas heated at 70∘C in a water bath for 10min cooled atroom temperature and centrifuged at 10000 rpm for 5minfor injection For reduced CE-SDS the samples were treatedas described above with 120573-mercaptoethanol (5 vv) insteadof iodoacetamide Before separation the capillary was rinsedwith 01molL NaOH for 2min and 01molL HCl for 1minfollowed bywater for 1minThen the capillary was filled withSDS sieving gel buffer for another 15min in forward directionThe samples were injected at the anode at minus10 kV for 30 s andseparated at minus15 kV with reverse polarity for 40min

2210 Glycan Analysis The N-glycan analysis was carriedout using normal-phase HPLC florescence (NP-HPLC-FL)approach The N-linked oligosaccharides of protein werereleased by incubation with PNGase F 02mg protein wasmixed with 01 U PNGase F in 20mM Tris-HCl buffer (pH74) and incubated at 37∘C overnight The deglycosylatedprotein was then precipitated by adding precooled ethanoland incubated at minus20∘C for a further 2 hrs The mixturecontaining released N-glycans was centrifuged at 10000 rpmat 4∘C for 05 hrs and the supernatant was completely driedusing vacuum centrifugation Dried N-glycan was mixed


with 20 120583L labeling reagent (04molL 2-aminobenzamide1molL NaCNBH3 in 3 7 (v v) acetic acid DMSO) andincubated at 65∘C for 4 hrs in the dark The mixture wasthen cleaned using Ludger Clean S-cartridges (Ludger UK)according to the manufacturersrsquo instructions The labeled N-glycans were separated on an ACQUITY UPLC BEH Glycancolumn at 02mLmin (17 120583m 21 times 150mm Waters) linkedto an Agilent 1290 UPLC system with fluorescence detector(excitation at 330 nm emission at 420 nm) Mobile phase Awas 125mmolL ammonium formate (pH 44) in water andmobile phase B was 100 acetonitrile The separation beganwith 20 phase A from 0 to 5min and a linear gradient ofmobile phase A from 20 to 45 was performed to separatethe labeled N-glycans followed by an elution step with 60mobile phase A for a further 15min

2211 Surface Plasmon Resonance (SPR) Binding The kineticinteractions of protein samples with IL-6R Fc120574RIIIa andFcRn were evaluated using a Biacore X100 plus biosensor ForIL-6R affinity analysis anti-Fc antibody was immobilized ona CM5 sensor chip surface according to the manufacturerrsquosrecommendation with a level of sim2500 response units (RU)reached The protein sample at a concentration of 10 120583gmLwas injected at a flow rate of 10 120583Lmin for 60 s and capturedby the anti-Fc antibody IL-6R in a series of concentrationsranging from 150 ngmL to 47 ngmL was injected into theflow cells at a flow rate of 30 120583Lmin for 180 s with adissociation time of 500 s usingHBS-EP+buffer For Fc120574RIIIaand FcRn binding determination anti-His antibody wasimmobilized on a CM5 sensor chip surface as recommendedby the manufacturerrsquos instructions with an RU of sim2500 Theprotein sample at 10 120583gmL was injected into the flow cellsat 10 120583Lmin for 60 s and captured by the anti-His antibodyFc120574RIIIa and FcRn in serially diluted concentrations from150 ngmL to 47 ngmL were injected at a flow rate of30 120583Lmin for 180 s The dissociation phase using HBS-EP+buffer was then set to 500 s The primary sensorgram datawas processed using Biacore evaluation software 10 for thedetermination of association rate constant (119896119886) dissociationrate constant (119896119889) and dissociation equilibrium constant(119870119863) data

2212 Antiproliferation Assay DS-1 cells (ATCC CRL-11102) were seeded in RPMI-1640 media with 10 FBS andincubated in 96-well plates at 37∘C A series of concentrationsranging from 800 120583gmL to 1221 ngmL of HS628 and origi-nator tocilizumabwere added and incubated for 72 hrs CCK-8 (Dojindo Japan) was added to stain the cells and incubatedat 37∘C for 3 hrsThe absorbancewasmeasured at 450 nmand650 nm Test results were expressed as the relative percentageof the EC50 from the dose-response curve of HS628 withrespect to originator tocilizumab

2213 Inhibition of STAT3 Phosphorylation U937 cells(ATCC CRL-15932) in the logarithmic growth phasewere starved in RPMI-1640 media with HS628 originatortocilizumab and an irrelevant antibody control (anti-CD20mab) at 37∘C for 3 hrs The cells were pulsed or not with rhu-IL6 (Prospec Israel) for 15minThe cells were then fixed (BD

Cytofix Fixation Buffer BD Biosciences USA) for 12minpermeabilized (Perm Buffer III BD Biosciences) for 30minand incubated with Alexa Fluor 647 Mouse Anti-Stat3 (BDBiosciences) in BD Pharmingen Stain Buffer for 1 hour atroom temperature Cells were analyzed on BD FACSCaliburinstrument (BD USA)

3 Results and Discussion

HS628 a biosimilar product of originator tocilizumab wasproduced in a genetically engineered Chinese hamster ovary(CHO) cell line The gene sequences of HS628 were reverse-engineered from the amino acid sequence of originatortocilizumab A company-internal selection of expressionvectors and transfection and selection methods were usedfor cell line development The upstream process was per-formed in fed-batch mode in a bioreactor with a proprietarychemically defined medium Feeding of various nutrientsincluding galactose appeared to be very critical to maintainthe glycan profiling of HS628 as similar as possible to thatof originator tocilizumab Following upstream productionHS628 was purified by industry standard chromatographicpurification and viral inactivation methods which consistof Protein A affinity anion exchange chromatography andcation exchange chromatography in combination with viralinactivation (low pH) virus clearance (nanofiltration) andultrafiltrationdiafiltration (UFDF) steps Downstream pro-cess efficiency was established adequately for the removal ofprocess-related impurities (eg host cell protein and DNA)and product-related impurities (eg aggregates)The residuallevels of process-related impurities comply with existingguidelines for biopharmaceuticals The capacity of viralinactivation and clearance procedures have been validatedby National Institutes for Food and Drug Control (China)The drug substance was formulated to the final drug productusing the same formulation as the originator tocilizumab

A candidate biosimilar product should be similar to theoriginator product in terms of physicochemical character-istics and functional properties [22 23] To demonstratethe biosimilarity the state-of-the-art robust methodologies(Table 1) including chromatographic electrophoretic andcell-based bioassay methods along with CD DSC MS andSPR technologies were employed to compare biosimilartocilizumab HS628 and originator tocilizumab The selectedmethods are widely used in similarity assessment of biosimi-lar products which are capable of detectingminor differencesin the protein structures as well as identifying and quantify-ing product-related variants [24ndash26]

31 Physicochemical Properties

Primary Structure The RP-HPLC-UV tryptic peptide mapof the biosimilar HS628 showed indistinguishable chro-matograms of the fragmented peptides from the originatortocilizumab (Figure 1) Primary amino acid sequence analysisof the peptide fragments ofHS628 and originator tocilizumabwas obtained by using reduced peptide mapping followed byRP-UPLC-MS sequencing which resulted in 100 sequencecoverage for the heavy chain and light chain of two products


Table 1 Characterization strategy for the proposed biosimilar HS628 and originator tocilizumab

Category Attributes Methods

Primary structure Amino acid sequence Reduced RP-UPLC-MS peptide mappingMolecular weight RP-UPLC-MS

Higher order structure

Disulfide bridging Nonreduced RP-UPLC-MS peptide mappingFree thiols Ellmanrsquos assay

Secondary and tertiary structure Circular dichroism (CD)Thermodynamic stability Differential scanning calorimetry (DSC)

Posttranslational modifications Deamidation oxidation Reduced RP-UPLC-MS peptide mapping

Charge heterogeneity Charge variants CEX-HPLCIsoelectric point Imaged capillary isoelectric focusing (icIEF)

Size heterogeneityHigh molecular weight impurities SEC-HPLCLow molecular weight impurities Nonreduced CE-SDS

Nonglycosylated heavy chain (NGHC) Reduced CE-SDSGlycosylation Glycan profile NP-HPLC-FLBinding activity Affinity to IL-6R Fc120574RIIIa and FcRn Surface plasmon resonance

Potency Antiproliferative potency Antiproliferation assaySignal transduction inhibition Inhibition of STAT3 phosphorylation

Originator tocilizumab

HS628minus600minus400minus200

0200

(mAU

)

400600

20 40 60 80 1000Time (min)

Figure 1 Mirror plot of peptide mapping chromatograms obtainedfrom RP-UPLC-UV for originator tocilizumab and HS628

(Figures 2 and 3) The sequences obtained of HS628 werefound to be identical to the sequences obtained with origi-nator tocilizumab which were matched with the theoreticalsequence ofActemra [27]Mass analysis of intact and reduced(heavy chain and light chain) forms of HS628 by RP-UPLC-MS was observed to show consistent molecular masses withthose of the originator tocilizumab (Table 2)

Higher Order Structure Nonreducing peptide maps showedthe expected disulfide bridging pattern in both originatorproduct and HS628 The following disulfide bridges wereidentified intrachain [Cys(L23)-Cys(L88) Cys(L134)-Cys(L194) Cys(H22)-Cys(H96) Cys (H146)-Cys(H202)Cys(H263)-Cys(H323) Cys(H369)-Cys(H427)] andinterchain [Cys(L214)-Cys(H222) Cys(H228)-Cys(H228)Cys(H231)-Cys(H231)] The results of Ellmanrsquos assay showedthat the number of moles of free SH groups per mole of IgGwas comparable in HS628 and originator tocilizumab and inthe range of 04 (free SH molIgG mol)

The secondary structure (120572-helix 120573-sheet and randomcoil) and tertiary structure can be determined by CD spec-troscopy in the far-UV region and near-UV region respec-tively Far-UV CD spectra (Figure 4(a)) and near-UV CDspectra (Figure 4(b)) of HS628 and originator tocilizumabwere shown to overlap with each other and the contents of120572-helix 120573-sheet and random coil of the two products werecomparable respectively

The thermodynamic stability of HS628 and originatortocilizumab was evaluated by differential scanning calorime-try (DSC) The DSC thermograms of HS628 and originatortocilizumab were found to be superimposable Transitiontemperatures (119879119898) of HS628 by DSC (Figure 5) were 744∘C886∘C and 987∘C whereas for the originator tocilizumabthey were 744∘C 886∘C and 986∘CThe three 119879119898 values areconsidered to be linked to the unfolding of the CH2 Fab andCH3 domain respectively [28]

Collectively disulfide bridging pattern free thiol contentCD and DSC analyses confirmed that the higher orderstructure of HS628 was comparable to that of originatortocilizumab

Posttranslational Modifications RP-HPLC-UVMS peptidemapping was used to detect posttranslational modificationssuch as deamidation oxidation and phosphorylation Sim-ilarity levels of deamidation and oxidation modificationswere detected in HS628 and originator tocilizumab (Table 3)Both the number and the locations of these modificationswere generally consistent between HS628 and originatortocilizumab

Deamidation of the heavy chain and light chain of HS628and originator tocilizumabwas observed at the positionsHC-Asn77 HC-Asn288 HC-Asn317 HC-Asn386 HC-Asn436 LC-Asn34 LC-Asn137 and LC-Asn138 and the levels of deamida-tion of the two products were comparable


HS628Control coverage () 1000 Combined coverage () 1000 Analyte coverage () 00

Control unique coverage () 1000 Common coverage () 00 Analyte unique coverage () 00

EVQLQESGPG LVRPSQTLSL TCTVSGYSIT SDHAWSWVRQ PPGRGLEWIG

YISYSGITTY NPSLKSRVTM LRDTSKNQFS LRLSSVTAAD TAVYYCARSL

ARTTAMDYWG QGSLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD

YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY

ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK

DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS

TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV

YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL

DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

DIQMTQSPSS LSASVGDRVT ITCRASQDIS SYLNWYQQKP GKAPKLLTYY

TSRLHSGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ GNTLPYTFGQ

GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV

DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG

LSSPVTKSFN RGEC

Heavy chain

Light chain

∙ ∙

∙

∙

∙∙

∙

∙

∙

∙∙

∙ ∙∙

∙∙

∙ ∙ ∙

∙

∙

∙

∙ ∙ ∙

∙∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙

∙

∙∙

∙ ∙

∙

∙

∙∙∙

∙

2 201 to 214

2 151 to 200

2 101 to 150

2 51 to 100

2 1 to 50

1 401 to 449

1 351 to 400

1 301 to 350

1 251 to 300

1 201 to 250

1 151 to 200

1 101 to 150

1 51 to 100

1 1 to 50

Figure 2 Sequence coverage of the heavy chain and light chain of HS628















LSSPVTKSFN RGEC

Heavy chain

Light chain

Control coverage () 1000 Combined coverage () 1000 Analyte coverage () 00

Control unique coverage () 1000 Common coverage () 00 Analyte unique coverage () 00∙

∙

∙ ∙

∙

∙ ∙

∙ ∙

∙∙ ∙

∙

∙∙ ∙

∙

∙

∙ ∙

∙

∙

∙

∙

∙

∙∙∙∙

∙ ∙

∙

∙ ∙

∙∙∙

∙

∙

∙

∙∙

∙

∙

∙

∙ ∙

2 201 to 214

2 151 to 200

2 101 to 150

2 51 to 100

2 1 to 50

1 401 to 449

1 351 to 400

1 301 to 350

1 251 to 300

1 201 to 250

1 151 to 200

1 101 to 150

1 51 to 100

1 1 to 50

Figure 3 Sequence coverage of the heavy chain and light chain of originator tocilizumab

Table 2 The whole molecule heavy chain and light chain exact masses of originator tocilizumab and HS628 by MS

TypeOriginator tocilizumab (4 bathes)

Mass (Da)HS628 (4 bathes)

Mass (Da)B2014B10 B2019B26 B1018B13 B2027B13 20130901 20130902 20130903 20130904

Wholemolecule

G0FG0F 14788519 14788444 14788581 14788561 14788578 14788700 14788239 14788777G0FG1F 14804600 14804588 14804681 14804661 14804627 14804814 14804420 14804841G1FG1F 14820706 14820664 14820781 14820763 14820834 14820823 14820453 14820959G1FG2F 14836519 14836481 14836566 14836553 14836600 14836911 14836452 14837130

Deglycosylatedmolecule 14499863 14499691 14499730 14499767 14499702 14499781 14499466 14499908

Heavychain

G0F 5044532 5044518 5044546 5044550 5044357 5044388 5044411 5044375G1F 5060694 5060687 5060697 5060701 5060544 5060591 5060571 5060555G2F 5076795 5076780 5076792 5076796 5077280 5077206 5077406 5077670


Light chain 2350097 2350096 2350097 2350096 2350075 2350075 2350077 2350076


HS628Originator tocilizumab

Mea

n re

sidue

ellip

ticity

Wavelength (nm)

4000

3000

2000

1000

0

minus1000

minus2000

minus3000

200 210 220 230 240 250 260

(a)

20

0

minus20

minus40

minus60

minus80

minus100


260 270 280 290 300 310 320250Wavelength (nm)

Mea

n re

sidue

ellip

ticity

(b)

Figure 4 Comparison of CD spectra of HS628 and originator tocilizumab (a) Far-UV spectra (b) near-UV spectra

20 40 60 80 100 120

Original tocilizumab HS628

Cp

(mca

lmin

)

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

Temperature (∘C)

Figure 5 Comparison of DSC spectra of HS628 and originatortocilizumab

No detectable Met oxidation was observed on the lightchain of the two products Similar low levels ofMet oxidationon the heavy chain of HS628 and originator tocilizumabwereobserved at the position of HC-Met254

No detectable phosphorylation was observed inHS628 ororiginator tocilizumab

Charge Heterogeneity Antibody charge variants can beformed by chemical and enzymatic modifications [29]

Charge heterogeneity may substantially affect stabilitybiological activity and pharmacokinetics of antibodies [30]Cation exchange chromatography (CEX) was used to assess

the profile of charge variants that may be more acidic orbasic relative to the main peak It was revealed that theaverage abundances of the main peak acidic variants andbasic variants were within the same order of magnitude forHS628 and originator tocilizumab (Figure 6(a)) with themean values of 688 252 and 60 (119899 = 4) forHS628 and633 250 and 117 (119899 = 4) for originator tocilizumabrespectively The sum of the HS628 acidic variants wascomparable to that of originator tocilizumab while the sumof the basic variants was found to be slightly lower thanthat of originator tocilizumab Treatment of the mAbs withCPB removes C-terminal lysine heterogeneity The resultsobtained after digestion with CPB (Figure 6(b)) showed alsoa comparable content of charge variants between the twoproducts with the average abundances of the main peakacidic variants and basic variants of 687 253 and 60(119899 = 4) for HS628 and 658 257 and 85 (119899 = 4) fororiginator tocilizumab respectively

An orthogonal analytical technique for the evaluation ofcharge heterogeneity is imaged capillary isoelectric focusing(icIEF) The isoelectric points (pI) for the main peak were924 for both HS628 and originator tocilizumab and thecontents of the main peak of the two products were 669and 647 respectively (Figure 6(c)) Consistent with theCEX results no significant differences were observed in thecIEF chromatograms of the biosimilar HS628 and originatortocilizumab indicating the comparable charge heterogeneitybetween the two products

Size Heterogeneity It is known that protein aggregates inimmunomodulatory biologic formulations can trigger anunwanted immunogenic response [31ndash33] Size exclusionchromatography (SEC) was performed to detect the levels ofaggregates monomers and fragments The retention timesof monomeric HS628 and originator tocilizumab were thesame while average purity was 994 for HS628 (119899 = 4)


Table3Deamidationandoxidationmod

ificatio

nsof

HS628

andoriginator

tocilizum

ab

Mod

ificatio

nsite

Mod

ificatio

ntype

Masssignalintensity(

)

HS628

(20130901)

HS628

(20130902)

HS628

(20130903)

HS628

(20130904)

Orig

inator

tocilizum

ab(B2014B10)

Heavy

chain

N(77)QFSLR

Deamidation

52

42

38

39

37

DTL

M(254) ISR

Oxidatio

n25

1519

1833

FNWYV

DGVEV

HN(288) A

KDeamidation

226

218

206

205

214

VVS

VLT

VLH

QDWL

N(317)G

KDeamidation

998

997

998

996

997

GFY

PSDIAVEW

ESN(386)G

QPE

NNYK

Deamidation

756

826

749

766

755

WQQGNVFS

CSVMHE

ALH

N(436)H

YTQK

Deamidation

67

7767

62

68

Lightchain

ASQ

DISSY

LN(34)

WYQ

QKP

GK

Deamidation

1925

1519

16

SGTA

SVVC

LLN(137) N

FYPR

Deamidation

444

378

342

354

415

SGTA

SVVC

LLN

N(138)FYP

RDeamidation

59

55

47

49

58


Acidic variants Basic variants

Main peak

Originator tocilizumab HS6280

100200300400500600

(mAU

)

10 20 30 40 500Time (min)

(a)


Main peak

Basic variantsAcidic variants

10 20 30 40 500Time (min)

0100200300400500600

(mAU

)

(b)

marker

Main peak


00

01

02

03

04

05

06

Abso

rban

ce

75 80 85 90 95 10070pI

pI = 924

pI = 1010pI = 765 marker

(c)

Figure 6 Charge heterogeneity comparison of HS628 and originator tocilizumab (a) CEX chromatograms of HS628 and originatortocilizumab (b) CEX chromatograms of HS628 and originator tocilizumab after CPB digest and (c) cIEF chromatograms of HS628 andoriginator tocilizumab

and 990 for originator tocilizumab (119899 = 4) No fragments(low molecular weight variants) were found in both HS628and originator tocilizumab and there was also no indicationof different types of high molecular weight variants SEC-MALS-RI results showed that the molecular weights ofthe monomer and the dimer were 152 KDa and 301 KDarespectively

Nonreducing CE-SDS was used to separate monomersfrom higher molecular weight variants and fragments [HHL(125 kDa) HH (100 kDa) HL (75 kDa) HC (50 kDa) and LC(25 kDa)] In the electropherograms of HS628 and originatortocilizumab a number of fragments were resolved anddetected The amounts of free light chain (LC) free heavychain (HC) HL HH and HHL of HS628 and originatortocilizumab were comparable and the average percentage ofmAb (monomer) was 953 for HS628 (119899 = 4) and 950 fororiginator tocilizumab (119899 = 4) (Figure 7(a))

Reducing CE-SDS was used to assess the levels oflight chain heavy chain and nonglycosylated heavy chain(NGHC) Similar chromatographic profiles and purities (HC+ LC) of 990 were found for HS628 and originatortocilizumab (Figure 7(b)) The average level of NGHC ofHS628 was 014 (119899 = 4) which was slightly lower than thatof originator tocilizumab (072 119899 = 4)

Overall the results from SEC and CE-SDS show thatHS628 has a similar purity and aggregate level to originatortocilizumab

Glycosylation Glycosylation has been identified as a CQAfor many antibody-based drugs [34ndash36] LC-MSMS peptidemapping was used to characterize the glycosylation of thetwo mAbs Peptide mapping confirmed the glycosylationsite of HS628 and originator tocilizumab at Asn299 whilereduced CE-SDS revealed that in both molecules gt99 ofAsn299 was glycosylated N-linked glycans were releasedfrom tocilizumab by enzymatic hydrolysis using PNGaseFand then were labeled with 2-aminobenzamide followedby normal-phase HPLC with fluorescence detection (NP-HPLC-FL) The glycan patterns of HS628 and originatortocilizumabwere comprised of the same principal glycoforms(Figure 8) It should be noted here that no potential immuno-genic glycoforms such as NGNA residues were observedThe glycosylation pattern of the major abundant glycanssuch as the G0 G0F G1F G1rsquoF and G2F isoforms was verysimilar between HS628 and originator tocilizumab Smalldifferences could be identified when looking at the lowabundant glycan structures HS628 was shown to containslightly lower amounts of mannose structures (Man5 Man6)of around 07 than those of the originator (15ndash25)


Nonreducing CE-SDS mAb

HHL

LC HC HL HH HMWOriginator

tocilizumab

HS628

0

5

10

(mAU

)

15

20

25

30

25 30 35 40 45 50 5520(min)

(a)

Reducing CE-SDS LC HC

Originatortocilizumab

HS628

NGHC

minus20

0

20

(mAU

)

40

60

225 25 275 30 325 35 37520(min)

(b)

Figure 7 Comparison of CE-SDS electropherograms between HS628 and originator tocilizumab (a) Nonreducing (b) reducing LC lightchain HC heavy chain HL heavy-light chain HH heavy-heavy chain HHL heavy-heavy-light chain HMW high molecular weightsubstance NGHC nonglycosylated heavy chain

G0-N G0F-N G0

G0F

Man5Man6 G1FS1G2F

A1F A2FG2


HS628

60 70 80 90 100 110 120 13050Time (min)

0100200300400500600700

EU

G1F + G1F㰀

G1 + G1㰀

Figure 8 Comparison of the glycosylation pattern of HS628 andoriginator tocilizumab

Together HS628 and the originator share similar patternand abundance of various glycan moieties

32 Functional Characterization A comprehensive set ofpotency bioassays including SPR binding assays antiprolif-eration assay and inhibition of STAT3 phosphorylation weredeveloped to provide a complete evaluation of HS628rsquos func-tional integrity and comparability to originator tocilizumab

SPR Binding Assays SPR technique was used to determinethe affinity constants of HS628 and originator tocilizumab tosIL-6R recombinant human Fc120574RIIIa and FcRn receptors inparallel Fc120574RIIIa is implicated in inducingADCC (antibody-dependent cell-mediated cytotoxicity) Although the mecha-nism of action of tocilizumab does not involve ADCC thebinding activity of tocilizumab to Fc120574RIIIa was also assessedThe results show that the affinities of HS628 towards sIL-6R Fc120574RIIIa and FcRn were very comparable to those ofthe originator product respectively (Table 4) the data shownwere performed head to head and reflect the minimum andmaximum value of four originator batches and four HS628batches

Table 4 Affinity constants (119870119863) for binding to sIL-6R Fc120574RIIIaand FcRn receptors as determined by Biacore SPR

Originator tocilizumab 119870119863 HS628 119870119863sIL-6R 108sim178 nM 092sim117 nMFc120574RIIIa 021sim038 120583M 024sim025 120583MFcRn 029sim031 120583M 025sim026 120583M

Antiproliferation Assay In the cell-based bioassay the cellgrowth inhibiting activity was evaluated by adding IL-6 andHS628originator tocilizumab to DS-1 cells such that theycompete for the IL-6R on the cells The results demonstratedthat both products have the same potency to deplete DS-1cells being themean relative potencies towards the originatortocilizumab of 98 102 94 and 105 for four batches ofHS628 One of these curves was shown in Figure 9 indicatingthatHS628 had a comparable biological potency to originatortocilizumab

Inhibition of STAT3 Phosphorylation STAT3 a member ofSTAT family has been described in mediating IL-6 signalingthrough interaction with the IL-6R and IL-6 can inducephosphorylation of STAT3 [37]

Fluorescence Activated Cell Sorting (FACS) was usedto assess the biological activity of HS628 and originatortocilizumab to inhibit IL-6-induced STAT3 phosphorylationin U937 cells An irrelevant antibody control (anti-CD20mAb) was used as negative control The results showed thatIL-6 could induce 4302 STAT3 phosphorylation in U937cells while the rate of STAT3 phosphorylation was only099 without IL-6 The anti-CD20 mAb did not block IL-6-induced STAT3 phosphorylation since the rate of STAT3phosphorylationwas still 4266 BothHS628 and originatortocilizumab could inhibit STAT3 phosphorylation in U937cells as shown in Figure 10 An overlapping sigmoidal dose-response curve was obtained for both HS628 and originatortocilizumab Half-maximal effective concentration (EC50)


15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis

A B C DInnovator tocilizumab (Group 01 concentrationversus mean value)

143 0626 587 0138 0999

HS628 (Group 02 concentration versus mean value) 138 0615 627 0156 0999

4-P Fit y = (A minus D)(1 + (xC)B) + D R2

Figure 9 DS-1 cell growth inhibition curves of HS628 and originator tocilizumab

HS628Innovator Tocilizumab

0

20

40

60

80

100

Inhi

bito

ry ra

te (

)

0 1 2minus2 minus1minus3log (휇gmL)

DoseResp Fit of ACTEMRADoseResp Fit of HS628

Equation

Adj R-Squ

ACTEMRAACTEMRAACTEMRAACTEMRAACTEMRA

HS628

HS628

HS628HS628HS628

y = A1 + (A2 minus A1)(1 + 10((logx0 minus x)lowastp))

099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0

Figure 10 Comparison of inhibition of STAT3 phosphorylation between HS628 and originator tocilizumab (Actemra) The dose-responsecurves of inhibition effects on STAT3 phosphorylation of HS628 and originator tocilizumab (Actemra) were generated using 4-parameter-curve equation y = A1 + (A2 minus A1)(1 + 10((log1199090minus119909)lowast119901)) by the software OriginPro (Version 860 Sr2) The EC50 values of STAT3phosphorylation inhibition of HS628 and originator were calculated from the logx0 values from the corresponding curves The resultsshowed that the dose-response curves of HS628 and tocilizumab were highly overlapped indicating that the inhibition effect on STAT3phosphorylation of HS628 was highly comparable with that of originator tocilizumab (Actemra)


value calculated for HS628 from the dose-response curve wasfound to be similar to that of originator tocilizumab

Functional similarity is a very critical component of thetotality of evidence required for demonstration of biosimi-larity These results collectively demonstrated the high levelof similarity in functional properties between HS628 andoriginator tocilizumab

4 Conclusions

In the present work the comprehensive physicochemicaland biological characterization including the verification ofprimary structure higher order structure posttranslationalmodifications charge heterogeneity size heterogeneity gly-cosylation binding affinity to IL-6R Fc120574RIIIa and FcRnantiproliferative activity and inhibition of STAT3 phospho-rylation provided solid evidence to prove the similaritybetween the proposed biosimilar HS628 and originatortocilizumab Based on this research HS628 can be consideredas a highly similar molecule to originator tocilizumab interms of physicochemical and biological properties

Subsequently a comparative animal toxicity study wasconducted to evaluate the safety of the product in accor-dance with Good Laboratory Practice (GLP) (China Foodand Drug Administration (CFDA) 2003) No significantdifferences between HS628 and originator tocilizumab wereobserved through the comparative toxicological studies inanimals In addition HS628 was observed to exhibit a similarpharmacokinetics profile compared to that of the originatortocilizumab following a single-dose injection in cynomolgusAs such it can be anticipated that the proposed biosimilarHS628 would show comparable potency and safety as thereference originator product in future clinical trials

Competing Interests

The authors declare that they have no financial and personalrelationships with other people or organizations that caninappropriately influence this work and there are no com-peting interests regarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (nos 21206040 and 21406066)

References

[1] T Tanaka M Narazaki and T Kishimoto ldquoAnti-interleukin-6 receptor antibody tocilizumab for the treatment of autoim-mune diseasesrdquo FEBS Letters vol 585 no 23 pp 3699ndash37092011

[2] N H Kim S K Kim D S Kim et al ldquoAnti-proliferative actionof IL-6r-targeted antibody tocilizumab for non-small cell lungcancer cellsrdquoOncology Letters vol 9 no 5 pp 2283ndash2288 2015

[3] Food and Drug Administration Guidance for Industry Scien-tific Considerations inDemonstrating Biosimilarity to aReferenceProduct FDA Rockville Md USA 2015

[4] European Medicines Agency Guideline on Similar BiologicalMedicinal Products Containing Biotechnology-derived Proteinsas Active Substance Quality Issues EMA London UK 2014

[5] World Health OrganizationGuidelines on Evaluation of SimilarBiotherapeutic Products (SBPs) WHO Geneva Switzerland2009

[6] J Braun and A Kudrin ldquoProgress in biosimilar monoclonalantibody development the infliximab biosimilar CT-P13 in thetreatment of rheumatic diseasesrdquo Immunotherapy vol 7 no 2pp 73ndash87 2015

[7] S K Jung K H Lee J W Jeon et al ldquoPhysicochemicalcharacterization of RemsimardquomAbs vol 6 no 5 pp 1163ndash11772014

[8] A Beck HDiemer D Ayoub et al ldquoAnalytical characterizationof biosimilar antibodies and Fc-fusion proteinsrdquo Trends inAnalytical Chemistry vol 48 pp 81ndash95 2013

[9] J J Z Liao and P F Darken ldquoComparability of critical qualityattributes for establishing biosimilarityrdquo Statistics in Medicinevol 32 no 3 pp 462ndash469 2013

[10] I H Cho N Lee D Song et al ldquoEvaluation of the struc-tural physicochemical and biological characteristics of SB4 abiosimilar of etanerceptrdquomAbs vol 8 no 6 pp 1136ndash1155 2016

[11] P J Declerck ldquoBiosimilar monoclonal antibodies a science-based regulatory challengerdquo Expert Opinion on Biological Ther-apy vol 13 no 2 pp 153ndash156 2013

[12] International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse ICH Harmonized Tripartite Guideline PharmaceuticalDevelopment Q8 R2 2009

[13] N Alt T Y Zhang PMotchnik et al ldquoDetermination of criticalquality attributes for monoclonal antibodies using quality bydesign principlesrdquo Biologicals vol 44 no 5 pp 291ndash305 2016

[14] A S Rathore A Weiskopf and A J Reason ldquoDefiningcritical quality attributes for monoclonal antibody therapeuticproductsrdquoBioPharm International vol 27 no 7 pp 34ndash43 2015

[15] L A Bui S Hurst G L Finch et al ldquoKey considerations in thepreclinical development of biosimilarsrdquo Drug Discovery Todayvol 20 supplement 1 pp 3ndash15 2015

[16] J Velayudhan Y-F Chen A Rohrbach et al ldquoDemonstrationof functional similarity of proposed biosimilar ABP 501 toadalimumabrdquo BioDrugs vol 30 no 4 pp 339ndash351 2016

[17] J Liu T Eris C Li S Cao and S Kuhns ldquoAssessing analyticalsimilarity of proposed amgen biosimilar ABP 501 to adali-mumabrdquo BioDrugs vol 30 no 4 pp 321ndash338 2016

[18] J Visser I Feuerstein T Stangler T Schmiederer C Fritschand M Schiestl ldquoPhysicochemical and functional compara-bility between the proposed biosimilar rituximab GP2013 andoriginator rituximabrdquo BioDrugs vol 27 no 5 pp 495ndash507 2013

[19] C A Lopez-Morales M P Miranda-Hernandez L C Juarez-Bayardo et al ldquoPhysicochemical and biological characteriza-tion of a biosimilar trastuzumabrdquo BioMed Research Interna-tional vol 2015 Article ID 427235 10 pages 2015

[20] K McKeage ldquoA review of CT-P13 an infliximab biosimilarrdquoBioDrugs vol 28 no 3 pp 313ndash321 2014

[21] S BandyopadhyayMMahajan TMehta et al ldquoPhysicochemi-cal and functional characterization of a biosimilar adalimumabZRC-3197rdquo Biosimilars vol 5 pp 1ndash18 2015

[22] S A Berkowitz J R Engen J R Mazzeo and G B JonesldquoAnalytical tools for characterizing biopharmaceuticals and theimplications for biosimilarsrdquo Nature Reviews Drug Discoveryvol 11 no 7 pp 527ndash540 2012


[23] B J Scott A V Klein and J Wang ldquoBiosimilar monoclonalantibodies a canadian regulatory perspective on the assessmentof clinically relevant differences and indication extrapolationrdquoJournal of Clinical Pharmacology vol 55 no 3 pp S123ndashS1322015

[24] W Li B Yang D Zhou J Xu Z Ke and W-C SuenldquoDiscovery and characterization of antibody variants usingmass spectrometry-based comparative analysis for biosimilarcandidates of monoclonal antibody drugsrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 1025 pp 57ndash67 2016

[25] S-C Chow F Song and H Bai ldquoAnalytical similarity assess-ment in biosimilar studiesrdquoAAPS Journal vol 18 no 3 pp 670ndash677 2016

[26] S Chow ldquoChallenging issues in assessing analytical similarityin biosimilar studiesrdquo Biosimilars vol 5 pp 33ndash39 2015

[27] M Tsuchiya K Sato M M Bendig S T Jones and JW Saldanha ldquoReconstituted human antibody against humaninterleukin 6 receptorrdquo EP patent 0628639 B1 1999

[28] C B Andersen M Manno C Rischel M Thorolfsson and VMartorana ldquoAggregation of a multidomain protein a coagula-tion mechanism governs aggregation of a model IgG1 antibodyunder weak thermal stressrdquo Protein Science vol 19 no 2 pp279ndash290 2010

[29] L A Khawli S Goswami RHutchinson et al ldquoCharge variantsin IgG1 isolation characterization in vitro binding propertiesand pharmacokinetics in ratsrdquomAbs vol 2 no 6 pp 613ndash6242010

[30] Y Du A Walsh R Ehrick W Xu K May and H LiuldquoChromatographic analysis of the acidic and basic species ofrecombinant monoclonal antibodiesrdquo mAbs vol 4 no 5 pp578ndash585 2012

[31] EMMoussa J P Panchal B SMoorthy et al ldquoImmunogenic-ity of therapeutic protein aggregatesrdquo Journal of PharmaceuticalSciences vol 105 no 2 pp 417ndash430 2016

[32] S Kumar S K Singh XWang B Rup andDGill ldquoCoupling ofaggregation and immunogenicity in biotherapeutics T- and B-cell immune epitopes may contain aggregation-prone regionsrdquoPharmaceutical Research vol 28 no 5 pp 949ndash961 2011

[33] W Wang S K Singh N Li M R Toler K R King and SNema ldquoImmunogenicity of protein aggregatesmdashconcerns andrealitiesrdquo International Journal of Pharmaceutics vol 431 no 1-2 pp 1ndash11 2012

[34] D Reusch andM L Tejada ldquoFc glycans of therapeutic antibod-ies as critical quality attributesrdquoGlycobiology vol 25 no 12 pp1325ndash1334 2015

[35] M M C Van Beers and M Bardor ldquoMinimizing immuno-genicity of biopharmaceuticals by controlling critical qualityattributes of proteinsrdquo Biotechnology Journal vol 7 no 12 pp1473ndash1484 2012

[36] L K Hmiel K A Brorson andM T Boyne ldquoPost-translationalstructural modifications of immunoglobulin G and their effecton biological activityrdquo Analytical and Bioanalytical Chemistryvol 407 no 1 pp 79ndash94 2015

[37] A C Bharti N Donato and B B Aggarwal ldquoCurcumin(diferuloylmethane) inhibits constitutive and IL-6-inducibleSTAT3 phosphorylation in human multiple myeloma cellsrdquoJournal of Immunology vol 171 no 7 pp 3863ndash3871 2003

Submit your manuscripts athttpswwwhindawicom

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Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


limit range or distribution to ensure the desired prod-uct quality safety and efficacy Potential CQAs of mAbsmay include charge-related variants size-related variantsoxidation-related variants glycosylation structural variantsprocess-related impurities and biological properties [13ndash15]In the case of adalimumab themost critical assays are TNF-120572binding and neutralization those that directly measured theprimary mechanism of action (MOA) of the product [16]

So far many biosimilarmAbs papers have been publishedon top-selling mAbs such as adalimumab rituximab inflix-imab bevacizumab and trastuzumab [17ndash21] but not yet ontocilizumab HS628 has been developed by Hisun Pharma-ceutical in China as a proposed biosimilar tocilizumab oforiginator tocilizumab In this study we describe a subset ofthe state-of-the-art methods of physicochemical and biolog-ical analysis that were performed to demonstrate similaritybetween HS628 and originator tocilizumab

2 Materials and Methods

21Materials Originatortocilizumab(Actemra 4mL 80mg10mL 200mg and 20mL 400mg) batches were purchasedfromRoche Pharma (Schweiz) Ltd (manufactured by ChugaiPharma Manufacturing Co Ltd) The proposed biosim-ilar product HS628 was produced by Zhejiang HisunPharmaceutical (Zhejiang China) HS628 drug product(4mL 80mg) was used for physicochemical and functionalcomparability study

22 Methods An array of state-of-the-art and orthogonalanalytical techniques were used to compare HS628 withtocilizumab All chromatographic analyses were performedon Agilent 1260 high performance liquid chromatography(HPLC) systems (Agilent Technologies Boblingen Ger-many) Primary sequences verified with tryptic peptidemapping and thewholemolecule exactmasses were analyzedusing reverse-phase ultraperformance liquid chromatogra-phy system coupled with a UV detector and a quadru-pole time-of-flight mass spectrometer (RP-UPLC-Q-TOF)Higher order structure was evaluated by circular dichroism(CD) and differential scanning calorimetry (DSC) Freeproteinogenic thiol groupwas quantified using Ellmanrsquos assayThe disulfide bridging pattern was assessed using RP-UPLC-Q-TOF system after peptide mapping under nonreducingconditions Posttranslational modifications were identifiedby RP-UPLC-Q-TOF system after tryptic peptide mappingunder reducing conditions Charge heterogeneity of pro-tein sample with or without carboxypeptidase digestionwas assessed by cation exchange chromatography (CEX-HPLC) and imaged capillary isoelectric focusing (icIEF)Size heterogeneity (purity) was determined by size exclu-sion chromatography (SEC) and capillary electrophoresis-sodium dodecylsulfate (CE-SDS) N-Glycosylation patternsof the products were assessed by LC-MS peptide mappingand normal-phase HPLC with fluorescence detection (NP-HPLC-FL) Functional properties were evaluated by surfaceplasmon resonance (SPR) antiproliferative assay and inhibi-tion of STAT3 phosphorylation

221 Nonreduced and Reduced PeptideMapping with Reverse-Phase (RP) HPLC with UV andMass Spectrometric DetectionReduced peptide mapping was performed to identify theprimary sequences of tested products Nonreduced peptidemapping was carried out to determine the disulfide bridgingpattern Protein sample was diluted in water at 2mgmL and400 120583L of sample solution was mixed with guanidine-HCl inTris buffer (01molL Tris pH 80) to a final concentrationof guanidine-HCl of 6molL The solution was incubated at37∘C for 2 hrs After incubation sample solution was reducedwith dithiothreitol (DTT) at 10mgmL and incubated at37∘C for 30min Alkylation was conducted by adding a20120583L aliquot of a 1molL iodoacetamide stock solution atroom temperature for 40min in the dark The buffer wasexchanged to 005molL ammonium bicarbonate at pH 78by ultrafiltration The protein sample was then digested withsequencing grade trypsin at a 1 40 enzyme-sample ratioat 37∘C overnight The enzymatic reaction was quenchedwith the addition of formic acid to a final concentrationof 01 For disulfide bridge identification the sample wasprepared with the same methods as described above withoutreduction and digested using both trypsin andGlu-C enzymeat a 1 1 40 ratio of trypsin Glu-C sample RP-UPLC-MSwas performed on a Waters ACQUITY UPLC H-Class Biosystem coupled with a UV detection and a Q-TOF massspectrometer Mobile phase A was water with 01 TFAand mobile phase B was acetonitrile with 01 TFA Peptidemapping was conducted using an ACQUITYUPLCBEH 300C18 column (17 120583m 21mm times 150mm Waters USA) with aflow rate of 02mLmin The gradient started at 5 phase Bfor 5min and then increased from 5 phase B to 45 phaseB in 45min followed by a gradient from 45 phase B to95 phase B in 10min followed by holding at 95 phaseB for another 10min as an elution step The UV detectionwavelength was set at 214 nmThemass spectrometer was setto run in positive ion mode with a sample cone voltage of60V capillary voltage of 3000V mass resolution of 30000and mz range of 50ndash2000 The mass spectrometer wascalibrated with NaI (2 120583g120583L) in waterisopropanol (5050)and tuned with leucine enkephalin solution (LE 1 ng120583L) inwateracetonitrile (5050) with 01 formic acid The ion ofLE at mz 5562771 was used to conduct mass correctionThe acquired raw spectra data were then deconvoluted andanalyzed using BiopharmaLynx software (version 133 Build7)

222MolecularMass DeterminationUsing RP-UPLC-Q-TOFSample solution was diluted to a final concentration of1mgmL in 005M Tris-HCl (pH 75) Analysis of intactprotein and reduced protein was run on a MassPrepmicro desalting column (Waters USA) with a flow rateof 02mLmin The gradient began at 10 phase B for1min and a linear gradient from 10 phase B to 60phase B in 10min was then performed followed by anincrease to 90 phase B in 2min followed by holdingat 90 for another 5min The Q-TOF mass spectrometerwas run in positive ion mode Sample cone voltage cap-illary voltage mass resolution and mz range were set at





































0200

(mAU

)

400600

20 40 60 80 1000Time (min)

























LSSPVTKSFN RGEC

Heavy chain

Light chain

∙ ∙

∙

∙

∙∙

∙

∙

∙

∙∙

∙ ∙∙

∙∙

∙ ∙ ∙

∙

∙

∙

∙ ∙ ∙

∙∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙

∙

∙∙

∙ ∙

∙

∙

∙∙∙

∙

2 201 to 214

2 151 to 200

2 101 to 150

2 51 to 100

2 1 to 50

1 401 to 449

1 351 to 400

1 301 to 350

1 251 to 300

1 201 to 250

1 151 to 200

1 101 to 150

1 51 to 100

1 1 to 50
















LSSPVTKSFN RGEC

Heavy chain

Light chain



∙

∙ ∙

∙

∙ ∙

∙ ∙

∙∙ ∙

∙

∙∙ ∙

∙

∙

∙ ∙

∙

∙

∙

∙

∙

∙∙∙∙

∙ ∙

∙

∙ ∙

∙∙∙

∙

∙

∙

∙∙

∙

∙

∙

∙ ∙

2 201 to 214

2 151 to 200

2 101 to 150

2 51 to 100

2 1 to 50

1 401 to 449

1 351 to 400

1 301 to 350

1 251 to 300

1 201 to 250

1 151 to 200

1 101 to 150

1 51 to 100

1 1 to 50





Mass (Da)B2014B10 B2019B26 B1018B13 B2027B13 20130901 20130902 20130903 20130904

Wholemolecule



Heavychain

G0F 5044532 5044518 5044546 5044550 5044357 5044388 5044411 5044375G1F 5060694 5060687 5060697 5060701 5060544 5060591 5060571 5060555G2F 5076795 5076780 5076792 5076796 5077280 5077206 5077406 5077670


Light chain 2350097 2350096 2350097 2350096 2350075 2350075 2350077 2350076



Mea

n re

sidue

ellip

ticity

Wavelength (nm)

4000

3000

2000

1000

0

minus1000

minus2000

minus3000

200 210 220 230 240 250 260

(a)

20

0

minus20

minus40

minus60

minus80

minus100


260 270 280 290 300 310 320250Wavelength (nm)

Mea

n re

sidue

ellip

ticity

(b)


20 40 60 80 100 120


Cp

(mca

lmin

)

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

Temperature (∘C)











ificatio

nsof

HS628

andoriginator

tocilizum

ab

Mod

ificatio

nsite

Mod

ificatio

ntype


)

HS628

(20130901)

HS628

(20130902)

HS628

(20130903)

HS628

(20130904)

Orig

inator

tocilizum

ab(B2014B10)

Heavy

chain

N(77)QFSLR

Deamidation

52

42

38

39

37

DTL

M(254) ISR

Oxidatio

n25

1519

1833

FNWYV

DGVEV

HN(288) A

KDeamidation

226

218

206

205

214

VVS

VLT

VLH

QDWL

N(317)G

KDeamidation

998

997

998

996

997

GFY

PSDIAVEW

ESN(386)G

QPE

NNYK

Deamidation

756

826

749

766

755

WQQGNVFS

CSVMHE

ALH

N(436)H

YTQK

Deamidation

67

7767

62

68

Lightchain

ASQ

DISSY

LN(34)

WYQ

QKP

GK

Deamidation

1925

1519

16

SGTA

SVVC

LLN(137) N

FYPR

Deamidation

444

378

342

354

415

SGTA

SVVC

LLN

N(138)FYP

RDeamidation

59

55

47

49

58



Main peak


100200300400500600

(mAU

)

10 20 30 40 500Time (min)

(a)


Main peak


10 20 30 40 500Time (min)

0100200300400500600

(mAU

)

(b)

marker

Main peak


00

01

02

03

04

05

06

Abso

rban

ce

75 80 85 90 95 10070pI

pI = 924


(c)









HHL


tocilizumab

HS628

0

5

10

(mAU

)

15

20

25

30

25 30 35 40 45 50 5520(min)

(a)



HS628

NGHC

minus20

0

20

(mAU

)

40

60

225 25 275 30 325 35 37520(min)

(b)


G0-N G0F-N G0

G0F

Man5Man6 G1FS1G2F

A1F A2FG2


HS628

60 70 80 90 100 110 120 13050Time (min)

0100200300400500600700

EU

G1F + G1F㰀

G1 + G1㰀











15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis


143 0626 587 0138 0999





0

20

40

60

80

100

Inhi

bito

ry ra

te (

)



Equation

Adj R-Squ


HS628

HS628

HS628HS628HS628


099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0





4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





































0200

(mAU

)

400600

20 40 60 80 1000Time (min)

























LSSPVTKSFN RGEC

Heavy chain

Light chain

∙ ∙

∙

∙

∙∙

∙

∙

∙

∙∙

∙ ∙∙

∙∙

∙ ∙ ∙

∙

∙

∙

∙ ∙ ∙

∙∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙

∙

∙∙

∙ ∙

∙

∙

∙∙∙

∙

2 201 to 214

2 151 to 200

2 101 to 150

2 51 to 100

2 1 to 50

1 401 to 449

1 351 to 400

1 301 to 350

1 251 to 300

1 201 to 250

1 151 to 200

1 101 to 150

1 51 to 100

1 1 to 50
















LSSPVTKSFN RGEC

Heavy chain

Light chain



∙

∙ ∙

∙

∙ ∙

∙ ∙

∙∙ ∙

∙

∙∙ ∙

∙

∙

∙ ∙

∙

∙

∙

∙

∙

∙∙∙∙

∙ ∙

∙

∙ ∙

∙∙∙

∙

∙

∙

∙∙

∙

∙

∙

∙ ∙

2 201 to 214

2 151 to 200

2 101 to 150

2 51 to 100

2 1 to 50

1 401 to 449

1 351 to 400

1 301 to 350

1 251 to 300

1 201 to 250

1 151 to 200

1 101 to 150

1 51 to 100

1 1 to 50





Mass (Da)B2014B10 B2019B26 B1018B13 B2027B13 20130901 20130902 20130903 20130904

Wholemolecule



Heavychain

G0F 5044532 5044518 5044546 5044550 5044357 5044388 5044411 5044375G1F 5060694 5060687 5060697 5060701 5060544 5060591 5060571 5060555G2F 5076795 5076780 5076792 5076796 5077280 5077206 5077406 5077670


Light chain 2350097 2350096 2350097 2350096 2350075 2350075 2350077 2350076



Mea

n re

sidue

ellip

ticity

Wavelength (nm)

4000

3000

2000

1000

0

minus1000

minus2000

minus3000

200 210 220 230 240 250 260

(a)

20

0

minus20

minus40

minus60

minus80

minus100


260 270 280 290 300 310 320250Wavelength (nm)

Mea

n re

sidue

ellip

ticity

(b)


20 40 60 80 100 120


Cp

(mca

lmin

)

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

Temperature (∘C)











ificatio

nsof

HS628

andoriginator

tocilizum

ab

Mod

ificatio

nsite

Mod

ificatio

ntype


)

HS628

(20130901)

HS628

(20130902)

HS628

(20130903)

HS628

(20130904)

Orig

inator

tocilizum

ab(B2014B10)

Heavy

chain

N(77)QFSLR

Deamidation

52

42

38

39

37

DTL

M(254) ISR

Oxidatio

n25

1519

1833

FNWYV

DGVEV

HN(288) A

KDeamidation

226

218

206

205

214

VVS

VLT

VLH

QDWL

N(317)G

KDeamidation

998

997

998

996

997

GFY

PSDIAVEW

ESN(386)G

QPE

NNYK

Deamidation

756

826

749

766

755

WQQGNVFS

CSVMHE

ALH

N(436)H

YTQK

Deamidation

67

7767

62

68

Lightchain

ASQ

DISSY

LN(34)

WYQ

QKP

GK

Deamidation

1925

1519

16

SGTA

SVVC

LLN(137) N

FYPR

Deamidation

444

378

342

354

415

SGTA

SVVC

LLN

N(138)FYP

RDeamidation

59

55

47

49

58



Main peak


100200300400500600

(mAU

)

10 20 30 40 500Time (min)

(a)


Main peak


10 20 30 40 500Time (min)

0100200300400500600

(mAU

)

(b)

marker

Main peak


00

01

02

03

04

05

06

Abso

rban

ce

75 80 85 90 95 10070pI

pI = 924


(c)









HHL


tocilizumab

HS628

0

5

10

(mAU

)

15

20

25

30

25 30 35 40 45 50 5520(min)

(a)



HS628

NGHC

minus20

0

20

(mAU

)

40

60

225 25 275 30 325 35 37520(min)

(b)


G0-N G0F-N G0

G0F

Man5Man6 G1FS1G2F

A1F A2FG2


HS628

60 70 80 90 100 110 120 13050Time (min)

0100200300400500600700

EU

G1F + G1F㰀

G1 + G1㰀











15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis


143 0626 587 0138 0999





0

20

40

60

80

100

Inhi

bito

ry ra

te (

)



Equation

Adj R-Squ


HS628

HS628

HS628HS628HS628


099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0





4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















LSSPVTKSFN RGEC

Heavy chain

Light chain

∙ ∙

∙

∙

∙∙

∙

∙

∙

∙∙

∙ ∙∙

∙∙

∙ ∙ ∙

∙

∙

∙

∙ ∙ ∙

∙∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙

∙

∙∙

∙ ∙

∙

∙

∙∙∙

∙

2 201 to 214

2 151 to 200

2 101 to 150

2 51 to 100

2 1 to 50

1 401 to 449

1 351 to 400

1 301 to 350

1 251 to 300

1 201 to 250

1 151 to 200

1 101 to 150

1 51 to 100

1 1 to 50
















LSSPVTKSFN RGEC

Heavy chain

Light chain



∙

∙ ∙

∙

∙ ∙

∙ ∙

∙∙ ∙

∙

∙∙ ∙

∙

∙

∙ ∙

∙

∙

∙

∙

∙

∙∙∙∙

∙ ∙

∙

∙ ∙

∙∙∙

∙

∙

∙

∙∙

∙

∙

∙

∙ ∙

2 201 to 214

2 151 to 200

2 101 to 150

2 51 to 100

2 1 to 50

1 401 to 449

1 351 to 400

1 301 to 350

1 251 to 300

1 201 to 250

1 151 to 200

1 101 to 150

1 51 to 100

1 1 to 50





Mass (Da)B2014B10 B2019B26 B1018B13 B2027B13 20130901 20130902 20130903 20130904

Wholemolecule



Heavychain

G0F 5044532 5044518 5044546 5044550 5044357 5044388 5044411 5044375G1F 5060694 5060687 5060697 5060701 5060544 5060591 5060571 5060555G2F 5076795 5076780 5076792 5076796 5077280 5077206 5077406 5077670


Light chain 2350097 2350096 2350097 2350096 2350075 2350075 2350077 2350076



Mea

n re

sidue

ellip

ticity

Wavelength (nm)

4000

3000

2000

1000

0

minus1000

minus2000

minus3000

200 210 220 230 240 250 260

(a)

20

0

minus20

minus40

minus60

minus80

minus100


260 270 280 290 300 310 320250Wavelength (nm)

Mea

n re

sidue

ellip

ticity

(b)


20 40 60 80 100 120


Cp

(mca

lmin

)

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

Temperature (∘C)











ificatio

nsof

HS628

andoriginator

tocilizum

ab

Mod

ificatio

nsite

Mod

ificatio

ntype


)

HS628

(20130901)

HS628

(20130902)

HS628

(20130903)

HS628

(20130904)

Orig

inator

tocilizum

ab(B2014B10)

Heavy

chain

N(77)QFSLR

Deamidation

52

42

38

39

37

DTL

M(254) ISR

Oxidatio

n25

1519

1833

FNWYV

DGVEV

HN(288) A

KDeamidation

226

218

206

205

214

VVS

VLT

VLH

QDWL

N(317)G

KDeamidation

998

997

998

996

997

GFY

PSDIAVEW

ESN(386)G

QPE

NNYK

Deamidation

756

826

749

766

755

WQQGNVFS

CSVMHE

ALH

N(436)H

YTQK

Deamidation

67

7767

62

68

Lightchain

ASQ

DISSY

LN(34)

WYQ

QKP

GK

Deamidation

1925

1519

16

SGTA

SVVC

LLN(137) N

FYPR

Deamidation

444

378

342

354

415

SGTA

SVVC

LLN

N(138)FYP

RDeamidation

59

55

47

49

58



Main peak


100200300400500600

(mAU

)

10 20 30 40 500Time (min)

(a)


Main peak


10 20 30 40 500Time (min)

0100200300400500600

(mAU

)

(b)

marker

Main peak


00

01

02

03

04

05

06

Abso

rban

ce

75 80 85 90 95 10070pI

pI = 924


(c)









HHL


tocilizumab

HS628

0

5

10

(mAU

)

15

20

25

30

25 30 35 40 45 50 5520(min)

(a)



HS628

NGHC

minus20

0

20

(mAU

)

40

60

225 25 275 30 325 35 37520(min)

(b)


G0-N G0F-N G0

G0F

Man5Man6 G1FS1G2F

A1F A2FG2


HS628

60 70 80 90 100 110 120 13050Time (min)

0100200300400500600700

EU

G1F + G1F㰀

G1 + G1㰀











15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis


143 0626 587 0138 0999





0

20

40

60

80

100

Inhi

bito

ry ra

te (

)



Equation

Adj R-Squ


HS628

HS628

HS628HS628HS628


099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0





4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Mea

n re

sidue

ellip

ticity

Wavelength (nm)

4000

3000

2000

1000

0

minus1000

minus2000

minus3000

200 210 220 230 240 250 260

(a)

20

0

minus20

minus40

minus60

minus80

minus100


260 270 280 290 300 310 320250Wavelength (nm)

Mea

n re

sidue

ellip

ticity

(b)


20 40 60 80 100 120


Cp

(mca

lmin

)

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

Temperature (∘C)











ificatio

nsof

HS628

andoriginator

tocilizum

ab

Mod

ificatio

nsite

Mod

ificatio

ntype


)

HS628

(20130901)

HS628

(20130902)

HS628

(20130903)

HS628

(20130904)

Orig

inator

tocilizum

ab(B2014B10)

Heavy

chain

N(77)QFSLR

Deamidation

52

42

38

39

37

DTL

M(254) ISR

Oxidatio

n25

1519

1833

FNWYV

DGVEV

HN(288) A

KDeamidation

226

218

206

205

214

VVS

VLT

VLH

QDWL

N(317)G

KDeamidation

998

997

998

996

997

GFY

PSDIAVEW

ESN(386)G

QPE

NNYK

Deamidation

756

826

749

766

755

WQQGNVFS

CSVMHE

ALH

N(436)H

YTQK

Deamidation

67

7767

62

68

Lightchain

ASQ

DISSY

LN(34)

WYQ

QKP

GK

Deamidation

1925

1519

16

SGTA

SVVC

LLN(137) N

FYPR

Deamidation

444

378

342

354

415

SGTA

SVVC

LLN

N(138)FYP

RDeamidation

59

55

47

49

58



Main peak


100200300400500600

(mAU

)

10 20 30 40 500Time (min)

(a)


Main peak


10 20 30 40 500Time (min)

0100200300400500600

(mAU

)

(b)

marker

Main peak


00

01

02

03

04

05

06

Abso

rban

ce

75 80 85 90 95 10070pI

pI = 924


(c)









HHL


tocilizumab

HS628

0

5

10

(mAU

)

15

20

25

30

25 30 35 40 45 50 5520(min)

(a)



HS628

NGHC

minus20

0

20

(mAU

)

40

60

225 25 275 30 325 35 37520(min)

(b)


G0-N G0F-N G0

G0F

Man5Man6 G1FS1G2F

A1F A2FG2


HS628

60 70 80 90 100 110 120 13050Time (min)

0100200300400500600700

EU

G1F + G1F㰀

G1 + G1㰀











15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis


143 0626 587 0138 0999





0

20

40

60

80

100

Inhi

bito

ry ra

te (

)



Equation

Adj R-Squ


HS628

HS628

HS628HS628HS628


099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0





4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



ificatio

nsof

HS628

andoriginator

tocilizum

ab

Mod

ificatio

nsite

Mod

ificatio

ntype


)

HS628

(20130901)

HS628

(20130902)

HS628

(20130903)

HS628

(20130904)

Orig

inator

tocilizum

ab(B2014B10)

Heavy

chain

N(77)QFSLR

Deamidation

52

42

38

39

37

DTL

M(254) ISR

Oxidatio

n25

1519

1833

FNWYV

DGVEV

HN(288) A

KDeamidation

226

218

206

205

214

VVS

VLT

VLH

QDWL

N(317)G

KDeamidation

998

997

998

996

997

GFY

PSDIAVEW

ESN(386)G

QPE

NNYK

Deamidation

756

826

749

766

755

WQQGNVFS

CSVMHE

ALH

N(436)H

YTQK

Deamidation

67

7767

62

68

Lightchain

ASQ

DISSY

LN(34)

WYQ

QKP

GK

Deamidation

1925

1519

16

SGTA

SVVC

LLN(137) N

FYPR

Deamidation

444

378

342

354

415

SGTA

SVVC

LLN

N(138)FYP

RDeamidation

59

55

47

49

58



Main peak


100200300400500600

(mAU

)

10 20 30 40 500Time (min)

(a)


Main peak


10 20 30 40 500Time (min)

0100200300400500600

(mAU

)

(b)

marker

Main peak


00

01

02

03

04

05

06

Abso

rban

ce

75 80 85 90 95 10070pI

pI = 924


(c)









HHL


tocilizumab

HS628

0

5

10

(mAU

)

15

20

25

30

25 30 35 40 45 50 5520(min)

(a)



HS628

NGHC

minus20

0

20

(mAU

)

40

60

225 25 275 30 325 35 37520(min)

(b)


G0-N G0F-N G0

G0F

Man5Man6 G1FS1G2F

A1F A2FG2


HS628

60 70 80 90 100 110 120 13050Time (min)

0100200300400500600700

EU

G1F + G1F㰀

G1 + G1㰀











15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis


143 0626 587 0138 0999





0

20

40

60

80

100

Inhi

bito

ry ra

te (

)



Equation

Adj R-Squ


HS628

HS628

HS628HS628HS628


099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0





4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Main peak


100200300400500600

(mAU

)

10 20 30 40 500Time (min)

(a)


Main peak


10 20 30 40 500Time (min)

0100200300400500600

(mAU

)

(b)

marker

Main peak


00

01

02

03

04

05

06

Abso

rban

ce

75 80 85 90 95 10070pI

pI = 924


(c)









HHL


tocilizumab

HS628

0

5

10

(mAU

)

15

20

25

30

25 30 35 40 45 50 5520(min)

(a)



HS628

NGHC

minus20

0

20

(mAU

)

40

60

225 25 275 30 325 35 37520(min)

(b)


G0-N G0F-N G0

G0F

Man5Man6 G1FS1G2F

A1F A2FG2


HS628

60 70 80 90 100 110 120 13050Time (min)

0100200300400500600700

EU

G1F + G1F㰀

G1 + G1㰀











15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis


143 0626 587 0138 0999





0

20

40

60

80

100

Inhi

bito

ry ra

te (

)



Equation

Adj R-Squ


HS628

HS628

HS628HS628HS628


099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0





4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



HHL


tocilizumab

HS628

0

5

10

(mAU

)

15

20

25

30

25 30 35 40 45 50 5520(min)

(a)



HS628

NGHC

minus20

0

20

(mAU

)

40

60

225 25 275 30 325 35 37520(min)

(b)


G0-N G0F-N G0

G0F

Man5Man6 G1FS1G2F

A1F A2FG2


HS628

60 70 80 90 100 110 120 13050Time (min)

0100200300400500600700

EU

G1F + G1F㰀

G1 + G1㰀











15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis


143 0626 587 0138 0999





0

20

40

60

80

100

Inhi

bito

ry ra

te (

)



Equation

Adj R-Squ


HS628

HS628

HS628HS628HS628


099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0





4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


15

13

11

09

07

05

03

010001 001 01 1 10 100 1000

x-axis

y-a

xis


143 0626 587 0138 0999





0

20

40

60

80

100

Inhi

bito

ry ra

te (

)



Equation

Adj R-Squ


HS628

HS628

HS628HS628HS628


099618 09980

ValueA1

A2

A1

A2

p

p

EC50

EC50

24379

84515

minus03774

09967

04193

32728

86384

minus03332

09739

04642

172273

237884

005376

011254

123336

178997

003919

007865

Standard er

logx0

logx0





4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




4 Conclusions



Competing Interests


Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

physicochemical and biological characterization of the proposed...

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