Abstract

One of the significant analytical challenges for the biopharmaceutical industry includes comprehensivecharacterization of protein therapeutics over thecourse of drug development. Developing a methodto monitor and detect process- and product-relatedimpurities is challenging due to their large molecularweight, inherent complexity, heterogeneity and theirsusceptibility to undergo degradation during the development processes. In this report, we describe analytical platforms using liquid chromatography coupled with electrospray ultra-high resolution quadrupole timeof-flight mass spectrometry (LC-ESI UHR QTOF/MS)with enhanced dynamic range, high speed MS MSacquisition and high mass accuracy to characterizeaglycosylated trastuzumab derived from Xpress CF™technology. The LC-MS method developed here couldbe used as a generic assay for the determination ofintact and monoisotopic subunits masses, as well asfor the identification of sites of modifications, sequencevariants and low-level host cell proteins.

Authors

Lieza M. Danan-Leon, Tyler HeibeckSutro Biopharma, South San Francisco, CA, USA

Guillaume Tremintin, Milady R. NiñonuevoBruker Daltonics, Fremont, CA, USA

Bru

ker

Dal

toni

cs is

con

tinua

lly im

prov

ing

its p

rodu

cts

and

rese

rves

the

rig

ht

to c

hang

e sp

ecifi

catio

ns w

ithou

t no

tice.

© B

ruke

r D

alto

nics

02-

2015

, LC

MS

-101

, 18

34

919

Application Note LCMS-101

Enabling Characterization of Protein Therapeutics Derivedfrom Xpress CF™ Technology with LC-ESI UHR QTOF/MS

Keywords Instrumentation and Software

antibodies impact HD

protein therapeutics maXis 4G

ProteinScape™ ProteinScape™

trastuzumab SNAP algorithm

Herceptin

TOF-MS

intact mass

peptide mapping

modifications

subunit analysis

sequence variant

host cell proteins

In this work, we have developed and employed LC-ESI/UHR QTOF MS-based platforms to characterize aglysosylated trastuzumab derived from Xpress CF™ technology. Trastuzumab, marketed as Herceptin® for breast cancer treatment, was utilized as a model monoclonal antibody to present the capabilities of Xpress CF™ technology in producing quality drug products with similar quality attributes as its mammalian expressed counterpart. We have analyzed process- and product-related impurities through intact mass and sub-unit analysis in addition to peptide mapping. Additionally, information on sites and levels of modifications, sequence variants, and low-level host cell proteins in aglycosylated trastuzumab manufactured using different bioreactor conditions were obtained.

Experimental

Intact and Sub-units Mass Analysis

Mass analysis of intact trastuzumab was performed usingthe sample as is, without further purification. Approximately,~1-2.5 ug of antibody was loaded, on-column,desalted and separated onto a reversed phase column.For sub-units mapping, trastuzumab was digested withIdes (Immunoglobulin-degrading enzyme of Streptococcuspyogenes, FabRICATOR, Genovis), and reduced with6M guanidine-HCl and 50 mM TCEP. The reaction wasquenched by adding 10% trifluoroacetic acid (TFA). Thisprocedure yielded Fd, Fc/2 and LC fragments of trastuzumab. LC-MS analysis of intact and IdeS fragmentswere accomplished using a Dionex UltiMate 3000 systemcoupled with a Bruker UHR QTOF MS (maxis 4G). DetailedLC-MS settings are shown in Table 2.

Introduction

With the increasing demand to rapidly and systematicallyexplore various protein variants to identify drug candidatesat quantities adequate for exploratory and pre-clinicaldevelopment, Xpress CF™ technology – Figure 1 wasestablished and applied to manufacture novel protein therapeutics such as monoclonal antibodies and antibody drug conjugates (ADC) [1]. Monoclonal antibodies (mAb) and their derivatives (ADC, bi and tri-specific mAbs, mAb fragments, and conjugated mAbs) are a rapidly growing class of biotherapeutics for the treatment of life threatening diseases such as cancer, inflammatory, infectious and autoimmune diseases [2]. However, due to their large molecular weight, inherent complexity and heterogeneity these biomolecules present significant analytical challenges to the biopharmaceutical industry. In addition their susceptibility to modifications and residue substitutions requires in-depth characterization to ensure a controlled and reproducible production process.

nanoLC

System NanoAdvance LC

Analytical column Dionex PepMap Acclaim C18, 250 x 0.075 mm, 2 µm

Trap Column Dionex PepMap Acclaim C18, 20 x 0.10 mm, 5 µm

Column temp 60 ºC

Sample amount ~ 1 µg on-column

Flow rate 400 nL/min

Gradient 5-50% - 90 min gradient, B (Acetonitrile) Total run time: 120 min

nanoLC

System impact HD™

Source Captive Spray™ with nanobooster (ACN)

MS conditions 4 Hz spectra rate

Active Exclusion after 1 spectra

RT2 = 3x

MS/MS conditions IDAS 4 -20 Hz

Cycle time 7 s

Scan range 150 – 2200 m/z

Cell-free Protein Synthesis

Figure 1: Overview of Cell-free Protein SynthesisSystem for Protein Production

Table 1: LC-MS Parameters for Peptide Mapping

Peptide Mapping

Aglycosylated trastuzumab cell free (CF) were producedin three batches of cell free reactions in 100 mL bioreactorsfollowed by a three-step purification process usingMabSelect SuRe™, Capto™ adhere, and Capto™ MMC chromatography. Purified proteins were characterized usinga Bruker UHR QTOF mass spectrometer (impact HD) withenhanced dynamic range and high speed MS/MS acquisition. After purification, the proteins were digested with trypsin to obtain appropriate peptide lengths suitable for C18 separation (90 min gradient). Eluted peptides were ionized, isolated and fragmented using collision-induced dissociation and mass analyzed. Shown in Table 1 are the detailed LC-MS parameters used in this report for peptide mapping.

For detecting sites of modifications, sequence variant analysis and host cell proteins, the MS/MS data weresearched against a customized database in ProteinScape™.Levels of modifications and unintended amino acid substitutions were also quantified.

Results and Discussion

Intact and Sub-unit Mass

For the intact and sub-units mass analysis, trastuzumabsamples were desalted, separated using a reversed-phasecolumn and analyzed by UHR QTOF MS (maxis 4G). The Intact mass spectrum (Figure 2) displays multiple peaks from ~m/z 2000 - 4000 with charge states distribution of 35+ to 72+. The multiply-charged envelope was deconvoluted using MaxEnt algorithm in Bruker Data Analysis software to obtain the neutral average molecular weight (MW) of aglycosylated trastuzumab, 145 946.75 Da (major). This experimental average MW is ~0.1 Da different from the calculated average MW of 145 946.67 that corresponds to ~0.56 ppm mass error. In addition to the expected major protein isoform, an average MW of 145 974.27 Da, roughly 30% of the main peak and ~ 27 Da mass differences from the expected MW was also observed. Upon close examination of the expanded intact mass spectrum, the mass spectrum shows a slightly wider peak width along with the presence of shoulder peaks. These data indicate the presence of heterogeneities that can be elucidated by additional experiments. The observed mass difference arises from various modifications on the light and heavy chain of the antibody.

To further characterize the findings in the intact mass analysis, the trastuzumab was cleaved into sub-units (Fd, LC and Fc/2) by FabRICATOR digestion followed by reduction with guanidine-HCl/TCEP. Leveraging the ultrahigh resolving power of the QTOF instrument, the sub-units are isotopically resolved and consequently, enabled the determination of their monoisotopic masses using the SNAP algorithm.

Shown in Figure 3 are the measured deconvoluted monoisotopic MW for the Fd, LC and Fc/2 fragments. Themasses 25 498.5516 Da (Fd), 23 904.0246 Da (Fc/2) and23 559.5772 Da (LC) correspond to ~0.3 ppm (on average)mass error, consistent with the results obtained from intactmass analysis. Aside from the expected primary sub-units,additional fragments with higher MW are detected. Thisdata suggests variants/modified forms of the antibody arepresent. Nevertheless, the high mass accuracy (sub ppmmass error) data and the capability to determine the monoisotopic masses verify the sequence fidelity of the antibody.

HPLC

System Dionex Ultimate 3000

Analytical column Phenomenex Aeris Widepore 3.6 µ C4, 150 x 2.1 mm

Column temp 70 ºC (intact); 80 ºC (sub-unit)

Sample amount ~ 1.5 µg (intact); 2.5 µg (sub-unit) on-column

Flow rate 400 µL/min

Gradient Intact: 5-95% - 10 min gradient, B (Acetonitrile, 0.1% FA) Sub-units: 15-30% -17 min gradient B

TOF/MS

System maXis 4G ETD

Source Bruker Apollo II ESI source

MS conditions ESI voltage: 4 kV, 8 L/min, 250 ºC dry gas, 2.5 bar nebulizing gas

Intact: 150 eV ISCID

Scan range 500 – 4200 m/z

Table 2: LC-MS Parameters for Intact and Sub-Unit Mass Analysis

Sites and Levels of Modifications

Protein therapeutics are prone to degradation during theentire drug manufacturing processes - from fermentationto purification, formulation, filling and upon storage.The degradation should be controlled specifically if themodification alters the protein’s biological activity or elicitsimmunogenicity. In order to detect such modifications, anLC-MS method (peptide mapping) was developed. Fromthe results, the trastuzumab’s identity was confirmed with>90 % sequence coverage. The MS/MS data searchedagainst a customized IgG1 trastuzumab database identifiedcommon modifications such as oxidation and deamidation.For example, deamidation of asparagine (Asn)residues were detected in the sequences LSCAASGFNIK(21-31), and in NTAYLQMNSLR (78-88), both in the heavychain of trastuzumab. Typically, deamidation occurs in theAsn residue as was indicated earlier. A not so commonglutamine (Gln) deamidation was also detected in thesame peptide. Shown in Figure 4 are the MS/MS spectraof the native and deamidated peptides. The fragmentsy6, y7, y8 and y9 ions (all shifted +~0.98 Da) suggest thepossible sites of deamidation to either be in the Gln or the

second Asn residues. However, since y5 and b3 fragmentsremained unchanged, both Asn residues can be ruled outand therefore, confirmed the deamidation site to be in theGln residue.

Furthermore, sites of methionine oxidation include theN-terminal methionine in the heavy chain with residues,MEVQLVESGGGLVQPGGSLR (1-20). The mass error forthis oxidized peptide was -1.77 ppm, indicating a highlyconfident identification. The site of oxidation was furtherconfirmed by the high quality MS/MS data, showing a consecutive y-ion series from residues 6-18 and a few b-ions. Oxidation levels were also calculated using the peak areas of the high resolution extracted ion chromatograms (hrEIC) of the native and modified peptides. Figure 5 displays the extracted ion chromatograms for both the native and oxidized peptides manufactured at 25 oC and 30 oC. As shown, there was no significant difference in the % methionine oxidation for this peptide, DTLMISR. In the tested conditions, the slight increase in the bioreactor temperature does not affect the level of Met256 oxidation.

0

1000

2000

3000

Intens.

1500 2000 2500 3000 3500 4000 m/z

Figure 2: Averaged mass spectrum (full scale – top, expanded - bottom) and deconvoluted mass spectrum (upper right) of aglycosylated trastuzumab derived from Cell-Free technology.

145946.75

0

200

400

600

800

Intens.

145750 145800 145850 145900 145950 146000 146050 146100 146150 146200 146250 m/z

Deconvoluted Mass (Average)

Calc (Ave) Expt'l (Ave)145946.67 145946.75

224765+ 2282

64+ 231863+

235562+

239461+

243460+

247559+

251858+

256257+

260856+

265553+

270454+ 2755

53+ 280852+

286351+

292150+

298049+

304248+

310747+

317446+

324545+

331944+

339644+

2200 2400 2600 2800 3000 3200 3400 m/z0

1000

2000

3000

Intens.

∆M/M = 0.56 ppm

Sequence Variants

Sequence variants are unintended amino acid substitution, which may have a pertinent and impactful influence on the safety and efficacy of biotherapeutics. Thus, detection and identification of these substitutions is critical in the course of bioprocess development. One of the current approaches is by employing LC-UV and LC-MS/MS in combination with Mascot error tolerant search (ETS) [2]. This workflow has been proven to be effective to detect low-level sequence variants. However, one of the caveats of such approach is the fairly high number of false positives, requiring a tedious manual inspection of every MS/MS spectra and chromatograms to validate the results. This leads to a very long turn-around time to obtain the results. Herein, we use a targeted search approach where we specifically look fora particular sequence variant in the sample. Following thedatabase search is a manual validation of the tandem MS.Since the search is well-defined, the number of hits to bemanually validated is of reasonable amount. As an example,

Figure 6 shows the annotated MS/MS from a 1% levelasparagine to serine (Asn -> Ser) substitution, corresponding to a mass shift of ~-27Da for a peptide sequence SGTASVVCLLNNFYPR. From the peptide sequence, the substitution could either be on the first asparagine (Asn11) or on the second asparagine (Asn12). As shown, the MS/MS displays a series of y-ions ranging from y2 to y12 and a few b-ions with adequate signal intensity which is sufficient to locate the site of substitution. From the inspection of the MS/MS spectrum we find that the y5 is shifted by -27 Da which proves that the Asn at position 12 is substituted by the Ser. If Asn at position 11 would be substituted, the y5 ion would be observed at the expected position. Additionally, y6 ions up to y12 ions are all -27 Da off pointing further to the exact location of the amino acid substitution. Likewise, the retention time of the variant (~3 min later) further reinforces the substitution of a hydrophilic moiety (Asn) by a serine residue.

Figure 3: Sub-unit mapping of aglycosylated Trastuzumab

25498.5516

23904.0246

23559.5772

0

1

2

3

4x10Intens.

0

1

2

3

44x10

0

1

2

3

4x10

20000 21000 22000 23000 24000 25000 26000 27000 28000 29000 m/z

Fd

Fc/2

LC

0

1

2

3

4x10Intens.

23900 23910 23920 23930 23940 23950 m/z

0

1

2

3

4x10Intens.

25490 25500 25510 25520 25530 25540 25550 m/z

0.0

0.5

1.0

1.5

2.0

2.5

3.04x10

Intens.

23550 23560 23570 23580 23590 23600 m/z

Mcalc : 25498.558

∆M/M: - 0.25 ppm

Mmono

Mmono

Mmono

Mcalc : 23904.024

∆M/M: - 0.02 ppm

Mcalc : 23559.564

∆M/M: - 0.56 ppm

Low-Level Host Cell Proteins

Aglycosylated trastuzumab CF was also analyzed for thepresence of low-level host cell proteins. From previousELISA-based analysis of the three batches of samples takenat two different bioreactors conditions (i.e. 25 ºC and 30 ºC),~ < 2 ppm host cell proteins were quantified. Without theneed for 2D-LC-MS/MS and further sample processing, atleast three E.coli proteins including FKBP-type peptidyl-prolyl cis-trans isomerase, thios:disulfide interchange protein DsbC and chaperone protein DnaK were detected in trastuzumab CF. These residual proteins are somewhat expected as components of the Xpress CF™ system to facilitate proper protein assembly.

Figure 4: Annotated MS/MS of glutamine deamidation

Deamidation (N, Q) – + 0.9848 Da Potential sites of deamidation

Conclusions

Extensive characterization of aglycosylated trastuzumabderived from Xpress CF™ Technology was enabled byutilizing LC-ESI UHR QTOF/MS systems with enhanceddynamic range , high speed MS/MS acquisition and highmass accuracy. Characterization partly involves intact andsub-units mass mapping to determine the average andmonoisotopic MW (with sub ppm mass error) that allowsus to verify sequence integrity and to profile product heterogeneity.

Orthogonal to MW determination, detectionand identification of modifications, sequence variants andlow-level host cell proteins was accomplished throughpeptide mapping. Taken together, combining effectivesoftware packages with high performance and high massaccuracy instrumentation provides a complete solution tocomprehensively characterize protein therapeutics.

native – RT 31.85 min

Deamidated – RT 32.88 min

Figure 5: Relative quantitation of Met256 oxidation, a known degradation hot spots on the heavy chain from triplicate injections . Example of extracted ion chromatogram (EIC) of trastuzumab manufactured at 25 oC – A and 30 oC – B for the native unaltered peptide (m/z 418.2207) and oxidized peptide (m/z 426.2182) used for quantitation.

15 20 25 30 35 40Time [min]

A – 25 oC

B – 30 oC

oxidized peptide

native unaltered peptide

DTLMISR

Sample % oxidation

25 oC 14.7 ± 0.8

30 oC 14.7 ± 1.8

Figure 5: Annotated MS/MS of asparagine to serine substitution.

native – RT 50.5 min

variant – RT 53.4 min

Bru

ker

Dal

toni

cs is

con

tinua

lly im

prov

ing

its p

rodu

cts

and

rese

rves

the

rig

ht

to c

hang

e sp

ecifi

catio

ns w

ithou

t no

tice.

© B

ruke

r D

alto

nics

02-

2015

, LC

MS

-101

, 18

34

919

For research use only. Not for use in diagnostic procedures.

Acknowledgements

The authors thank Wolfgang Jabs, Julian Phillips, Sandy

Yates and Sunil Bajad for their insightful suggestions and

technical guidance.

References

- Ying G, Garces E, Yang J, Zhang J, Tran C, et al. (2012)

Aglycosylated antibodies and antibody fragments

produced in a scalable in vitro transcription-translation

system. MAbs 4: 217–225.

- Reichert JM (2011) Antibody-based therapeutics to

watch in 2011. MAbs 3: 76–99.

- Yang Y, Strahan A, Li C, Shen A, Liu H, et al. (2010)

Detecting low level sequence variants in recombinant

monoclonal antibodies. MAbs 2: 285–298.

Bruker Daltonik GmbH

Bremen · GermanyPhone +49 (0)421-2205-0Fax +49 (0)421-2205-103

Bruker Daltonics Inc.

Billerica, MA · USA Phone +1 (978) 663-3660 Fax +1 (978) 667-5993

ms.sales.bdal@bruker.com - www.bruker.com