16 Supplement to BioPharm International August 2010 www.biopharminternational.com

Advances in Protein Characterization Biosimilars

Rapid Assessment of Molecular Similarity between a Candidate Biosimilar and

an Innovator Monoclonal Antibody Using Complementary LC–MS Methods

Intact protein LC–MS detected a mass variance of 62 Da and peptide mapping located a difference of two amino acids.

Microheterogeneities in glycosylation were confirmed using LC–FLR.

Martin Gilar, HonGwei Xie, asisH CHakraborty, JooMi aHn, yinG QinG yu, deepalaksHMi p. daksHinaMoortHy, weibin CHen, st. JoHn skilton, Jeffery r. Mazzeo

AbStrActthere is an emerging interest in developing biosimilar monoclonal antibodies (MAbs). to avoid expensive clinical trials and shorten time to market, the biosimilar industry must establish that a developing product is, as much as pos-sible, similar to a marketed innovator product through com-prehensive analysis. Here, we demonstrate that ultra high pressure liquid chromatography (UHPLc) and mass spec-

Martin Gilar, pHd, is a principal researcher, HonGwei Xie, pHd, is a senior research scientist, asisH CHakraborty, pHd, is a senior chemist, JooMi aHn is a senior research chemist, yinG QinG yu, pHd, is a principal chemist, weibin CHen, pHd, is a principal chemist, st. JoHn skilton, pHd, is the senior marketing manager, and Jeffery r. Mazzeo, pHd, is the biopharmaceutical business director, all at Waters Corporation, Milford, MA, 508.482.3119, martin_gilar@waters.com. deepalaksHMi p. daksHinaMoortHy is a senior applications specialist at Waters India Pvt Ltd, Bangalore, India.

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Advances in Protein Characterization Biosimilars

trometry (MS) can be used routinely to characterize minor differences between a candidate biosimilar and an innovator IgG1 MAb. A two amino acid residue variance in the heavy chain sequence was detected by Lc–MS intact protein mass measurement and located by tryptic peptide map-ping with data independent acqui-sition Lc–MS. Microheterogeneities due to N-linked glycosylation and chemical degradation were compre-hensively catalogued and compared. the results show that complementary Lc–MS methods can be used as a set of routine tools for rapid compari-son of molecular similarity between a candidate biosimilar and a commer-cially available MAb.

Recombinant monoclo-nal antibodies (MAbs) represent a c lass of eff icient, but expen-sive, biotherapeutics.

Developing less costly generic “biosimilar” MAbs is of great inter-est to both drug companies and consumers. Biosimilar drugs can be defined as compounds that are as similar as possible, structurally and functionally, to an innova-tor drug. The driving force for the interest in biosimilars for generic drug manufacturers is the upcom-ing patent expiration for marketed products.1 However, developing biosimilar products is challenging. Currently, there are no registered biosimilars in the US, and only recently has a mechanism for their approval been created.2 Biosimilar manufacturers will be under pres-sure to ensure that their products conform as closely as possible to existing products if they wish to avoid repeating expensive clini-cal trials and thus reach the mar-ket faster. Therefore they have a vested interest in comprehensive product analysis at all stages of development and manufacturing.

The key criteria for approval of biosimilars include quality, effi-

cacy, and safety. Therefore, the generic biopharmaceuticals indus-try must try to demonstrate con-sistency of a biosimilar to the innovator reference product in every aspect. A number of physi-cochemical and biological tests are required by regulatory author-ities for the characterization of MAbs.3,4 European Union regula-tions specify that state-of-the art characterization studies should be applied to the “similar biological product” (Section 5.2)5 and that physicochemical characterization should include a determination of the primary structure (section 5.2.2) while post-translational modifications (PTMs) such as gly-cosylation exhibit only minor lev-els of microheterogeneity.

In the work presented here, we demonstrate the application of state-of-the-art liquid chromatog-raphy (LC) and mass spectrometry (MS) to perform a comprehen-sive comparison of innovator and biosimilar MAbs. LC–MS analy-sis was carried out at the intact protein level, providing the infor-mation about molecular weight and glycan heterogeneity. Middle-down analysis of the MAb after reduction provided mass infor-mation on the antibody light chain and heavy chain separately, whereas the bottom-up approach (analysis of the MAb trypt ic digest) allowed us to locate and identify the differences in primary MAb sequence and PTMs.

For peptide mapping, we used a novel, data-independent acquisition method that allows for simultaneous collection of both MS and fragment ion data for every peptide in the data set independent of their mass, inten-sity, and retention time.6,7 The data-independent MS acquisition method permits reliable acquisition of both quantitative (peptide MS signal) and qualitative (peptide sequence) data in a single LC–MS analysis.

The goal of this manuscript is to demonstrate the utility of a compre-hensive set of advanced UHPLC–MS methods and their suitability for comprehensive comparison of inno-vator products and biosimilar MAb drug candidates, using a biosimilar of trastuzumab as an example.

Materials and MethodsSeparations were performed on an Acquity UHPLC system (Waters Corporation) equipped with tun-able ultraviolet (TUV; for peptide mapping) or fluorescence (FLR, for released glycan profiling) detec-tors. All MS measurements were made on a Synapt HDMS system (Waters). The systems were con-trolled by MassLynx 4.1 software (Waters); data processing was per-formed with BiopharmaLynx v. 1.2 (Waters). Simglycan (v. 2.75) (Premier Biosoft International) was used for glycan identification from a MALDI MS–MS experiment. The conditions of intact protein analy-sis and peptide mapping were the same as described in recently pub-lished papers.8,9 The conditions for hydrophilic interaction chroma-tography-fluorescence (HILIC–FLR) analysis were described elsewhere.10

Results and Discussion Intact Protein LC–MS Analysis The intact MS analysis of large proteins such as MAbs requires appropriate preparation but can be performed routinely. The sam-ples must be thoroughly desalted before MS analysis to obtain a

A mass shift of ~32 Da was observed for all glycoforms, suggesting a modification of the heavy chain.

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Advances in Protein Characterization Biosimilars

sufficient signal. In our experi-ence, the best results are obtained with on-line desalting.11

The reversed-phase desalting col-umn was used in an on-line setup to obtain UHPLC–MS data shown

in Figure 1. Deconvoluted data for the innovator and biosimilar MAbs are presented as a mirror plots; the glycoform annotation is based on the deconvoluted MS signals. One can clearly observe protein mass heterogeneity resulting from the presence of several main protein glycoforms. Several dominant peaks can be distinguished for each MAb sample; the difference in mass of ~162 Da corresponds most likely to loss of galactose, G, and ~146 to fucose, F, from the N-linked gly-can core structure. The molecu-lar weight of the innovator MAb is consistent with its expected pri-mary sequence, i.e., its mass cor-responds to the sum of the mass of two G0F, G0F/G1F, two G1F, G1F/G2F, or two G2F glycans (Figure 1, upper trace). Interestingly, the biosimilar candidate shows differ-ent glycoform heterogeneity and all main glycoforms show a mass shift of ~64 Da compared with the inno-vator drug. This shift is putatively caused by the presence of unknown PTMs or by differences in the pri-mary amino acid sequence.

Further investigation of intact protein mass data suggests the presence of minor glycoforms such as G0/G0F in each MAb (a mass difference of 146 Da, due to incom-plete occupancy of a fucosylation site on one of the two G0F core glycan structures). In addition, a low-level Man5/Man5 form was detected in both MAbs.

Although the intact protein analysis is fast and useful, it is difficult to quantitatively mea-sure the relative glycoform ratios, especially for minor glycoform species (Figure 1). Therefore, we performed a partial reduction of MAb samples, followed by on-line desalting and MS analysis. The deconvoluted masses of light and heavy chains are shown in Figures 2A and 2B, respectively. Whereas the light chains are identical, the

Figure 1. Intact MAb MS analysis. Multiply charged spectra were deconvoluted into protein molecular mass and compared using BiopharmaLynx software. Heterogeneity is caused by the MAb’s glycoforms. Notice the mass shift of biosimilar drug candidate compared to the reference innovator sample.

Figure 2. UHPLC–MS analysis of reduced MAb samples. Data were processed by BiopharmaLynx software. A) Light chain of innovator and biosimilar MAb are identical. B) Heavy chain of biosimilar candidate MAb shows consistent mass shift of ~32 Da for all protein glycoforms compared to the reference sample. Glycosylation pattern differences were detected.

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heavy chain data for the inno-vator and biosimilar MAbs show a distinct mass shift for all gly-coforms. Analyzing the 50-kDa heavy chain alone allows for bet-ter assessment of glycosylation heterogeneity (whereas the infor-mation about heterogeneity on the intact MAb molecular level is now lost). Man5, G0, G0F, G1, G1F, G2, and G2F glycoforms were clearly distinguishable in deconvoluted MS spectra (Figure 2B).

When comparing the innova-tor to the biosimilar MAb, the consistent mass shift of ~32 Da is observed for all glycoforms, sug-gesting a possible modification of the biosimilar MAb candidate heavy chain. This is consistent with the previously detected ~64 Da difference in the intact pro-tein mass (i.e., the combined con-tribution of two heavy chains). In the next section, we describe a peptide mapping experiment used to pinpoint the origins of the mass shift.

Application of Peptide Mapping to Locate Sequence VariantsTryptic digests of both samples were analyzed using the data-inde-pendent acquisition UHPLC–MS approach (“UPLC–MSE”). It has been shown that data-independent acquisition LC–MS with alternate low and elevated collision energy scanning provides signif icant benefits in terms of completeness and speed of peptide mapping.8 In contrast to data-dependent acqui-sition (DDA) LC–MS–MS, which requires a list of precursors (tar-geted DDA) or relies on intensity-biased precursor selection on the fly (often resulting in omission of minor peptides), data-independent acquisition MS allows for sequenc-ing of all peptides above the limit of detection and for accurate quantification by MS signals in a single analysis.

Figure 3 shows a mirror plot of UHPLC–MS peptide maps of the biosimilar and innovator MAbs generated by BiopharmaLynx software. The detected tryptic

peptide masses were matched against the theoretical values using a published trastuzumab sequence.12 Although the total ion chromatogram traces do not show

Figure 3. UHPLC–MS tryptic peptide maps of innovator and biosimilar MAbs presented as total ion chromatograms.

Figure 4. UHPLC–MS data of peptide maps shown as deisotoped and charge reduced chromatograms (all charge states and isotopes were deconvoluted into singly charged ion “stick”). Selected part of chromatogram between 3.5 and 7.5 min shows one unique peptide detected in innovator and biosimilar MAb digests.

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significant differences between samples (Figure 3), the compare function in the software visual-ized chromatogram regions for each of the MAbs that differed. Besides differences in glycosyl-at ion, the sof tware indicated addit ional d i f ferences, made apparent by displaying charge-reduced and isotope-deconvo-luted MS “stick chromatogram” plots, as shown in Figure 4. The mirror plot of the selected region of the chromatogram highlights that the innovator and biosimi-lar MAbs each contain a unique peptide without a correspond-ing signal in the other sample. The peptide HT35 (heavy chain, tryptic peak T35) with the amino acid sequence EEMTK found in the innovator product was not present in the biosimilar candi-date MAb. Instead, a peak with a mass of 605.32 Da was observed. The calculated difference in mass between this unknown peptide and EEMTK is ~32 Da, which cor-relates with our previous results for the differences in mass in the heavy chain (~32 Da) and intact MAb (~64 Da).

The MS data suggest that the innovator and biosimilar MAbs have a local inconsistency in the primary amino acid sequence located in the HT35 peptide. Because the UHPLC–MS data con-tain fragmentation data for all peptides (including the unknown and unidentified ones) it was pos-sible to investigate the unknown peak in BiopharmaLynx, as well as in the PepSeq programs, with the latter capable of assisting de novo sequencing. This task was simpli-fied by the availability of infor-mation about alternative MAb allotypes in the DrugBank.13,14 An alternative sequence matched the HT35 peptide mass and the frag-mentation pattern matched the DELTK sequence, with a differ-

Figure 5. Spectra of unique peptides, obtained using data-independent acquisition MS, confirm the primary sequence of HCT35 peptides for innovator antibody. As expected, it is EEMTK. Biosimilar drug candidate sequence of the HCT35 peptide is DELTK, which is a known MAb allotype.

Figure 6. LC-FLR profiling of released and 2-AB labeled glycans. Peak assignment was verified by reference standards and by MALDI–MS analysis. Relative quantitation values shown are based on fluorescence signal.

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Advances in Protein Characterization Biosimilars

ence of two amino acids from the innovator MAb EEMTK peptide. Both sequences were confirmed using data-independent acquisi-tion MS (Figure 5).

Analysis of Glycans Intact protein UHPLC–MS and peptide mapping provided infor-mation about the heterogeneity of MAb glycosylation and indi-cated that there are differences in the glycosylation patterns of the investigated samples. To gather more detailed and quantitative information about the glycosyl-at ion patterns, we performed LC analysis using a hydrophilic inte rac t ion chromatog raphy (HILIC) column of released gly-cans using f luorescent detec-tion (FLR). Glycans were cleaved from MAbs by PNGase F enzyme, labeled with 2-AB fluorescent dye, and analyzed using an Acquity UHPLC HILIC glycan column. Chromatograms featuring well resolved glycans including iso-mers of G1 a/b and G1F a/b are shown in Figure 6.

The HILIC method quantified differences in the glycan profiles of the innovator and biosimi-lar MAbs. Whereas G1F was the most abundant glycan structure in the innovator product, G0F was the most abundant in the biosimilar sample. The sensitivity of fluorescent detection permits the relative quantitation of minor glycans. Glycan identity was con-firmed by LC–MS–MS.

ConclusionsComprehensive investigation of an innovator MAb (trastuzumab) and a biosimilar candidate MAb with LC–MS and LC–FLR tech-n iques revea led unexpec ted d i f ferences in thei r pr imary sequence. Intact protein analy-sis showed mass differences and heterogeneity in the glycosyl-

ation patterns. To further elu-cidate whether the differences result from sequence variations or post-t ranslat ion modif ica-tions, a peptide mapping experi-ment was performed, revealing an unexpected peptide sequence. The UHPLC–MS data analysis of the peptides in BiopharmaLynx and PepSeq software, in conjunc-tion with information about pos-sible allotype sequences f rom DrugBank, allowed the identifi-cation of the biosimilar candi-date drug sequence in a single UHPLC–MS experiment. Clear differences in the levels of gly-cosylation observed in LC–MS experiment of intact and dena-tured MAbs were confirmed by an LC –FLR exper iment w ith released labeled glycans detected by f luorescence. The proposed set of experiments is robust and suitable for characterization and quality monitoring of biophar-maceutical proteins at all stages of drug development. ◆

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The MS data suggest that the innovator and biosimilar MAbs have a local inconsistency in the primary amino acid sequence located in the HT35 peptide.