Characterization of recombinant monoclonal IgG2 antibodies using LC-MS and limited 英文文献原版

1、Accepted Manuscript CharacterizationofrecombinantmonoclonalIgG2 using LC-MS and limited ys-C digestion Christine Nowak, Rekha Patel, Hongcheng Liu PII:S1570-0232(18)30547-6 DOI:doi:10.1016/j.jchromb.2018.05.010 Reference:CHROMB 2174 o appear in: Received date:3 April 2018 Revised date:8 May 2018 Acc

2、epted date:10 May 2018 Pleasecitethisarticleas:ChristineNowak,RekhaPatel,HongchengLiu Characterization of recombinant monoclonal IgG2 antibodies using LC-MS and ys-C digestion. The address for the corresponding author was captured as a forallauthors.Pleasecheckifappropriate.Chromb(2017),doi: j.jchro

3、mb.2018.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. a service to our customers we are providing this early version of the manuscript. manuscript will undego copyediting, typesetting, and review of the resulting proof it is published in its final form.

4、Please note that during the production process errors be discovered which could afect the content, and all legal disclaimers that apply to journal pertain. Characterization of recombinant monoclonal G2 antibodies using C-MS and limited Ls-C diestion Christine Nowak, Rekha Patel and Honchen iu* Produ

5、ct characterization, Alexion Pharmaceuticals, 100 Collee Street, New Haven, CT06510 * Correspondin author Email: HYPERLINK mailto:LiuH iuH Telephone: 203-271-8354 Abstract Recombinant monoclonal antibodies have been routinel characterized at intact, subunit and peptide levels b C-MS. Papain and peps

6、in have been the enzmes commonl used to cleave gG into various framents to facilitate in-depth characterization. However, non- specific cleavage for both papain and pepsin and the need of a reducin reaent for papain has limited their usae. n contrast, IdeS has gained popularit due to its specificit

7、and independence of reducin reaent. Results presented in the current stud demonstrated that the readil available endoprotease Ls-C can cleave gG2 at a specific peptide bonds in CH2 domain to enerate a homogeneous F(ab)2 frament, and the Fc regions was digested to peptides under limited digestion con

8、dition. The enerated F(ab)2 frament is suitable for further analsis because of its homogeneit. The posttranslational modifications located in the Fc reion includin lcoslation, deamidation and C-terminal heteroeneit can be rapidl analzed b C-MS. Ke words: iquid chromatoraph-mass spectrometr; monoclon

9、al antibod; Endoprotease Ls-C 1. Introduction Recombinant monoclonal antibodies (mAbs) are heteroeneous due to various post- translational modifications (PTMs) and deradations that occurr durin cell culture or purification. Molecular variants differin in mass, chares and hdrophobicit have been routi

10、nel analzed b respective analytical methods such as mass spectrometr, ion-exchane chromatoraph and hdrophobic interaction chromatoraph. n addition to analsis at intact, and peptide levels, mAbs have been routinel analzed at subunit levels. Subunit level analsis has offered some unique advantaes, inc

11、ludin relativel hiher resolution compared to intact analsis, reasonable turnaround time compared to peptide level analsis and localization of modifications to either Fab or Fc reion, simplifin further analsis. For subunit analsis, mAbs are cleaved usin various proteases to enerate Fab, F(ab)2, or Fc

12、. Traditionall, pepsin and papain have been used to generate immunolobulin (gG) framents. Pepsin cleaves peptide bonds below the hinge reions to enerate F(ab)2 frament 1-3. The major cleavage site of pepsin is between the two leucine (eu) residues in the sequence of APELGG4. Papain cleaves an antibo

13、d in the upper hinge reion generatin Fab frament1, 5, 5-7 mainl between histidine (His) and threonine (Thr) in the sequence of DKTHTC to generate Fab and Fc5. However, non-specific cleavae from pepsin and papain resulting in non-homoeneous framents, which can potentiall complicate further analsis an

14、d thus limitstheir usage for mAb characterization. Additionall, reducing reagent required for papain activit ma cause partial reduction of gG disulfide bonds. imited Ls-C diestion preferentiall cleaves a specific peptide bond in the upper hine reion of IG1 between amino acids lsine (Ls) and Thr in t

15、he amino acid sequence of DKTHTC to generate Fab and Fc framents8, 9. n the past several ears, deS has ained popularit because it cleaves gG below the hinge reion with high specificit10-12. Several other enzmes have been used to enerate gG framents, includin Plasmin13, Cathepsin 14-16, matrilsin17 a

16、nd stromelsin17 but the have not been widel used for mAb characterization because of lack of specificit, low efficienc or the need of reducing reaents. Hereof, we report a novel cleavage site of gG2 b limited Ls-C digestion. gG2 has also been a subclass of choice for the development of recombinant m

17、onoclonal antibod therapeutics and antibod-dru conjuates because of the reduced effector functions 18-22. However, gG2 with heterogeneous inter chain disulfide bond structures poses unique challenges for in-depth characterization23, 2. We demonstrated that Ls-C under limited digestion condition clea

18、ved at a specific peptide bond in the upper CH2 domain of gG2. nterestinl, the cleavae site was localized to between a Ls and a Proline (Pro) residue, which is tpicall resistant to protease diestion. imited Ls-C diestion of gG2 enerated homogeneous F(ab)2 frament. Additionall, the Fc frament was dig

19、ested to peptides, which can be analzed b C-MS to determine lcoform profile, deamidation of the conserved susceptible asparaine (Asn) residue, and C-terminal heteroeneit. 2. Experimntal 2.1Materials and methods Recombinant monoclonal G2/4 hbrid antibod (mAb-1) and gG2 (mAb-2) were expressed in a Chi

20、nese hamster ovar (CHO) cell line and purified at Alexion (New Haven, CT). gG2 meloma protein, acetonitrile, dithiothreitol, uanidine hdrochloride, iodoacetic acid and trifluoroacetic acid (TFA) were purchased from Sigma (St,ouis, MO). Lys-C was purchased from Walko (Richmond, VA). 2.2 Size-exclusio

21、n chromatography A Waters Alliance HPLC sstem and a TSKgel G3000SWx column (7.8 mm .D. x 30 cm, Tosoh Bioscience, King of Prussia, PA) were used for size exclusion chromatograph. A mobile phase containin 20 mM sodium phosphate and 150 mM sodium chloride, pH 7.0, was used isocrasticall to analze vari

22、ous samples at a flow-rate of 1 m/minute for 15 minutes. The proteins eluting off the column were monitored usin UV280nm. Fractions correspondin to the main peak from limited Ls-C diestion were collected manuall. The fractions were concentrated usin Amicon ultra centrifugal device with a molecular w

23、eiht cutoff of 30 kDa (Millipore, Burlinton, MA). 2.3 Limited Lys-C digestion MAb-1 was diluted to 2 mg/m usin phosphate buffered saline (pH 7.4). Ls-C was added to aliquots of the samples at 1:10, 1:20 and 1:100 Ls-C: protein ratios (w:w) and incubated at 37 C for 1 hour. Digestion usin 1:10 Ls-C t

24、o mAb-1 was repeated with longer incubation time points of 2, 3 and 4 hours. Mab-2 and the meloma protein were diested using 1:10 Ls-C to protein ratio at 37 C for 2 hours. 2.4 Complete Lys-C digestion MAb-1 and the collected SEC fraction after limited Ls-C diestion were denatured and reduced usin 6

25、 M uanidine hdrochloride in 20 mM Tris, pH 7.8 and 10 mM dithiothreitol at 37 C for 30 minutes. The reduced samples were then alklated b the addition of idodoacetic acid to a final concentration of 35 mM at 37 C for an additional 30 minutes. The sample pH was adjusted to approximatel 7.8 usin sodium

26、 hdroxide immediatel after the addition of iodoacetic acid. The samples were then buffer exchaned into 20 mM Tris, pH 7.8, usin eba desalting columns (Millipore). The samples were digested usin Ls-C at a ratio of 1:10 (w:w) at 37C for 4 hours. 2.5 LC-MS analysis A Maxis 4 G mass spectrometer (Bruker

27、, illerica, MA ) and a Mass Prep MicroDesalting column (Waters, Milford, MA) were used for molecular weight measurement. Approximatel 10 of each sample was injected into the column at 95% mobile phase A (0.1% TFA, 5% acetonitrile in water) and 5% mobile phase B (0.1% TFA in water). Proteins were elu

28、ted off the column b increasin the percentae of mobile phase B to 90% and remained at 90% for 1.5 minutes. The column was washed usin 90% mobile phase B and then equilibrated with 5% mobile phase B before the next injection. The flow-rate was set at 0.5 m/min and the column temperature was set at 60

29、 C. The mass spectrometer was run in positive mode with the followin settins: a scan rane of m/z 900-5500, as temperature of 220C, drin as of 10 /min, nebulizer at 2 psig and voltage at 120eV. The same mass spectrometer, UPC and a Proto 200 C18 column (1.0 x 20 mm, Higins Analtical) were used for pe

30、ptide analsis. Peptides of mAb-1 and its Ls-C frament collected from SEC were injected at 98% mobile phase A (0.1% TFA in water) and 2% mobile phase B (0.1% TFA in acetonitrile) and then eluted b increasin mobile phase B to 35% within 135 minutes. The column was then washed usin 90% mobile phase B f

31、or 5 minutes and then equilibrated usin 2% mobile phase B for 15 minutes. The flow-rate was set at 50 /minute. The column temperature was set at 60 C. The mass spectrometer was run in positive mode with the followin settins: a scan range of m/z 900-5500 for molecular weiht and m/z of 150-3000 for pe

32、ptide analsis. 3. Results and discussion Mab-1 was diested usin Ls-C at 1:100, 1:20 and 1:10 (w: w) Ls-C to mAb-1 ratios at 37 C for 1 hour. An aliquot of the sample without Ls-C was also incubated at 37 C for 1 hour and used as control. Deconvolutaed mass spectra from C-MS analsis of those samples

33、are shown in Figure 1. n the control sample, onl one major peak correspondin to intact mAb-1 with the major lcoform of G0F was observed (Figure 1, A). However, additional peaks with lower molecular weihts were observed when the samples were digested with Ls-C. The intensities of the additional peaks

34、 increased with hiher amount of Ls-C, while the intensit of the intact mAb peak decreased concurrentl (iure 1, B-D). The peak, which became the onl major peak in the samples diested with hiher amount of Ls-C corresponded to mAb-1 F(ab)2 frament due to cleavage between amino acid Ls and Pro in the se

35、quence of VFFPPKPKDTM in the CH2 domain. The peak with the molecular weight of 100580 Da shown in Figure 1B indicated a different cleavae site between amino acids Ls and Asp in the same amino acid sequence of one heav chain. The peak with the molecular weiht of 100804 Da corresponds to cleavae betwe

36、en Ls and Asp of both heav chains. The peak with the molecular weiht of 124708 Da in Fiure 1B was due to cleavae between Ls and Asp of one heav chain, while the other heav chain remained intact. The peak with the molecular weiht of 124482 Da was caused due to cleavae between amino acid Ls and Pro of

37、 one heav chain, while the other heav chain remained intact. To optimize the diestion condition, mAb-1 aliquots were further diested using 1:10 Ls-C to mAb-1 ratio at 37 C for 2, 3 and 4 hours. As shown in Figure 1, E-G, onl one peak was observed with longer incubation time. These results indicated

38、that a homogeneous F(ab)2 frament was obtained using limited Ls-C diestion. The cleavae between amino acids Ls and Pro was unexpected because it is generall believed that peptide bond with Pro on the C-terminal side is resistant to protease diestion, e., trpsin, Ls-C25. To confirm the cleavae site,

39、mAb-1 and the F(ab)2 frament obtained from Ls-C digestion and SEC separation were full digested usin Ls-C after denaturation, reduction and alklation. Analsis of the peptides demonstrated that onl those generated from cleavage between amino acids Ls and Pro were observed (Data not shown), supportin

40、cleavage between the Ls and Pro residues under the limited and complete diestion conditions. Because mAb-1 is an gG2/4 molecule with Fab, hinge and upper CH2 from G2 and lower CH2 and CH3 from G4, althouh, the amino acid sequence around this newl identified cleavage site are the same as a tpical gG2

41、 molecule, mAb-2, a recombinant monoclonal G2 molecule was also diested to determine whether or not the same procedure can be applied to enerate homogeneous F(ab)2 frament. The deconvoluated mass spectra of mAb-2 after limited Ls-C digestion using 1:10 Ls-C to mAb-2 ratio at 37 C for 2 hours are sho

42、wn in Figure 2. MAb-2 was completel diested, generatin a homogeneous F(ab)2 frament. Based on the molecular weight, the cleavae site was also localized to the same cleavage site. t is worthwhile to mention that an gG2 meloma protein was also diested usin the limited Ls-C digestion condition. Because

43、 of molecular weight heteroeneit of the gG2 myeloma protein, SEC was used to analze the samples before and after diestion. imited Ls-C diestion resulted in the eneration of a peak with lower molecular weight similar to the peaks enerated from digestion of mAb-1 and mAb-2, confirmin the occurrence of

44、 cleavae (Supplementar Figure 1). Taken toether, the newl identified limited diestion condition can be generall applied to gG2 molecules to generate homogeneous F(ab)2 frament. We saw several potential application of this limitd Ls-C diestion procedure to mAb characterization. First, generation of h

45、omogeneous F(ab)2 enables further in-depth characterization of modifications resided in this frament without the complicatin factors from non-specific cleavae as in the cases of pepsin and papain. Thus, it provides an alternative option in addition to deS for gG2. Secondl, the lcan profile on the co

46、nserved N-linked lcoslation site can be evaluated from analzing lcopeptides obtained from this limited digestion procedure. A mass spectrum showin various lcoforms is shown in Figure 3. The peaks were assined based on the known amino acid sequence and the common oligosaccharide structures of mAbs. T

47、he major lcoform of mAb-1 is G0F, which is followed b G1F. Hih mannose tpes of oligosaccharides with five (M5) and six (M6) mannose residues were also observed. Third, the level of deamidation at the conserved Asn deamidation hot-spot 2630 was analzed (iure 4). The major peak corresponds to the pept

48、ide with the original Asn, while the peak after corresponds to the peptide with a sinle site deamidation because of the molecular weight increase of 1 Da. The site of deamidation was assined to the second Asn residue in this peptide and the deamidation product was assined as Asp31. Tpicall, a peak p

49、rior to the main peak corresponding to the peptide containing an isoaspartate residue from deamidation of the susceptible Asn followed b a lcine residue was tpicall observed as a result of pre- existing deamidation product and procedure-induced artifact31. The lack of peak prior to the main peak ind

50、icated that this short limited diestion procedure can eliminate procedure-induced artifact. astl, this limited Ls-C digestion procedure was applied to determine the relativel levels of the three commonl observed mAb C-terminal variants, includin removal of C- terminal Ls, with the oriinal C-terminal

51、 Ls and C-terminal amidation3. The extracted ion chromatorams corresponding to each peptide is shown in Figure 5. nteration of the EC peak areas indicates that mAb-1 contains 95.7%, 3.7% and 0.6% of C-terminal peptides without C- terminal Ls, with C-terminal Ls or C-terminal amidation respectivel. A

52、ll the expected peptides from the Fc portion that can be retained b the reversed-phase column were identified (Supplement data), including peptides containin the two disulfide bonds. 4. Conclusion A new procedure of limited digestion of gG2 molecules usin Ls-C was established in the current stud. Un

53、der appropriate condition, homoeneous F(ab)2 was obtained from Ls-C digestion. The generated F(ab)2 should allow a straightforward analsis b other techniques such as ion exchane chromatoraph without the issue of complicated chromatorams from non-homogeneous diestion. Analsis of the peptides from the

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