Accepted Manuscript

Quantification of a recombinant antigen in an immuno-stimulatory whole yeast cell-

based therapeutic vaccine

Jenny Wang, Daniel Stenzel, Ally Liu, Dengfeng Liu, Darren Brown, Alexandre
Ambrogelly

PII: S0003-2697(18)30007-1
DOI: 10.1016/j.ab.2018.01.006
Reference: YABIO 12897

To appear in: Analytical Biochemistry

Received Date: 12 October 2017

Revised Date: 25 December 2017
Accepted Date: 11 January 2018

Please cite this article as: J. Wang, D. Stenzel, A. Liu, D. Liu, D. Brown, A. Ambrogelly, Quantification of
a recombinant antigen in an immuno-stimulatory whole yeast cell-based therapeutic vaccine, Analytical
Biochemistry (2018), doi: 10.1016/j.ab.2018.01.006.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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 ACCEPTED MANUSCRIPT

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Quantification of a Recombinant Antigen in an Immuno-Stimulatory
Whole Yeast Cell-based Therapeutic Vaccine

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Jenny Wang1, Daniel Stenzel1, Ally Liu1, Dengfeng Liu1, Darren Brown1, and Alexandre
Ambrogelly1,*

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Biologics Analytical Operations, Pharmaceutical & Biologics Development, Gilead
Sciences, 4010 Ocean Ranch Blvd, Oceanside, California, 92056, United States
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* To whom correspondence should be addressed

alexandre.ambrogelly@gilead.com
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Keywords: whole cell therapeutic vaccine, Saccharomyces cerevisiae, Tarmogen®, ELISA,

MRM, Antigen, HBV, S Antigen, X Antigen, Core Antigen, immuno modulatory
 ACCEPTED MANUSCRIPT

Abstract
Therapeutic vaccines represent an emerging class of immune-modulatory treatments for
cancer, infections, and chronic diseases. One such vaccine was designed as an immune

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stimulator of the T cell response against HBV antigens to eliminate HBV infected cells and
offer a therapeutic avenue to treat patients suffering from chronic hepatitis B infection.
Whole deactivated Saccharomyces cerevisiae cells expressing a recombinant fusion of HBV X,

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S and Core antigens elicit T cell responses in mice and activate human T cells linked with viral
clearance. As the therapeutic efficacy of the yeast-based vaccine relies on the production of

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the recombinant antigen, analytical methods designed to accurately and precisely quantitate
the fusion protein in the midst of all the yeast proteins are necessary. We report the
development and characterization of western blot, quantitative ELISA and mass

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spectrometry based orthogonal methods to support the assessment of manufacturing
consistency.
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Introduction
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Therapeutic vaccines constitute an emerging class of immuno-modulatory therapeutic

solutions for the treatment of a number of cancers and other ailments such as viral infectious
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diseases and chronic diseases [1-3]. Although only few have yet received approval from the
health authorities, many therapeutic vaccine candidates show promises in clinical studies
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alone or in combination with other immune-modulatory molecules such as an Anti-PD1

therapeutic monoclonal antibody [1, 4-6].
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In a manner similar to prophylactic vaccines, therapeutic vaccines are subdivided into

different categories depending on the nature of the antigen used. The active component of
the therapeutic vaccine may be polyvalent or antigen specific. Polyvalent antigens can be a
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mix of well characterized recombinant proteins or constituted from autologous or allogenic

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preparations of tumor cells for instance [4]. Antigen specific therapeutic vaccines may be
constituted of recombinant proteins, peptides known to include a defined immunogenic
sequence, or even nucleic acids in the form of DNA or mRNA coding for the sequence of the
desired antigen.

Regardless of the nature of the antigen selected, the mode of action rests on the same basic
immunological principles [4]. The active component of the therapeutic vaccine is presented
by dendritic cells via the MHC class I and II to CD4+ and CD8+ T cells, and in the presence of
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suitable activation signals, T cell expansion and migration occur leading eventually to the
eradication of the malignant or infected cells. Unlike drugs directly targeted at one or more
critical pathways responsible for tumor expansion, therapeutic vaccines aim at training T cells
to eliminate the diseased cells.

The Tarmogens® are a class of such vaccines. They consist of intact, heat-inactivated

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Saccharomyces cerevisiae yeast cells expressing a recombinant antigenic target protein
under the control of a strong promoter. Immunization with a Tarmogen® results in antigen-
specific cellular immune responses against the target protein, with the whole inactivated

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yeast cells mediating the phagocytosis by macrophages and dendritic cells and serving as
adjuvant [7-9].

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Two of these whole Yeast cell-based therapeutic vaccines are currently in phase 2 clinical
development for the treatment of medullary thyroid cancer and a variety of metastatic
cancers. These candidates express a modified recombinant version of brachyury protein and

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carcinoembryonic antigen. A third vaccine candidate was designed to target HBV antigens to
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improve HBsAg seroconverstion rates and offer a therapeutic avenue to treat patients
suffering from chronic hepatitis infection. Whole deactivated Saccharomyces cerevisiae cells
containing a recombinant fusion of HBV X, S and Core specific T cell antigens were shown to
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elicit T cells in mice and activate human T cells in vitro that are linked with viral clearance [7].

As the expected biological activity of the vaccine depends on the presence of the
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recombinant antigen in the yeast cells, analytical methods able to quantify the mass of
recombinant antigen present in a defined number of yeast cells are necessary. We report in
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this paper, a combination of orthogonal analytical methods used to quantify the

recombinant fusion of HBV X, S and Core specific T cell antigens expressed in the HBV
Tarmogen®.
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Results
Antigen structure and properties

The antigen expressed by the yeast cells is composed of the fusion of portions of three of the
four HBV gene products encoded in the viral DNA genome: X, S and Core; the S antigen being

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flanked by the X antigen at the amino terminus and by the Core antigen on the carboxy
terminus (Fig. 1) [7]. A 6x Histidine tag is placed at the carboxy terminus of the fusion protein

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for immuno-detection. Owing to the presence of a relatively high number of hydrophobic
amino acids in the primary sequence, the recombinant antigen is only soluble in solutions
containing sodium dodecyl sulfate (SDS) or other detergents. Typical workflows for the

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qualitative or quantitative assessment of the recombinant antigen begin with yeast cell
counting and cell lysis.

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Yeast cells counting
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Antigen content is expressed in mass of antigen per unit of yeast cell, one yeast unit (YU)
amounting to 1 x 107 cells. Accurate and precise determination of the number of yeast units
per unit of volume is therefore important to ensure the consistency of the cell lysis step and in
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fine the adequate measurement of antigen content. The number of yeast cells per unit of
volume is measured by micro flow imaging (MFI), an analytical method that uses flow
microscopy for the characterization of subvisible particulates ranging from 1 µm to above 50
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µm. The method is suitable as the typical diameter of a yeast cell is about 4 µm, and offered a
precise and accurate measurement of the number of cells per volume unit over a 0.025 to
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0.075 YU/mL range. The method allows also measurement of the average diameter
distribution of the yeast cell population. The yeast cell diameter was found to typically range
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from 1 µm to 10 µm, with an average at 4.5 µm and a distribution mode between 4.5 and 4.8
µm (Fig. 2).

Preparation of yeast cell lysates

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The preparation of the cell lysate is of critical importance for the accurate quantitation of the
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antigen. Prior to lysis, a fixed number of yeast cell units are washed with cold 1x PBS to
remove any remnant cell media and suspended in a buffer containing SDS and 2-
mercaptoethanol. Cell lysis is performed using silica beads in a shaker and recovered soluble
total protein content after sedimentation of the cell debris is determined by a BCA type assay.
FACS based experiments using an imaging flow cytometer suggest that the bulk of the antigen
is localized in the periplasmic space or associated with the cell wall (Fig. S1). The antigen being
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insoluble and possibly membrane bound, the concentration of detergent and 2-

mercaptoethanol was optimized to maximize fusion antigen recovery.

Antigen Identification

The presence of the intended recombinant fusion of HBV X, S and Core antigens in the yeast
cell lysate was demonstrated using 1D and 2D LC-MS after digest with various combinations of

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Trypsin, LysC, GluC, and Chymotrypsin (Table 1). The sample analyzed contained a mixture of
proteins including the recombinant fusion of HBV antigens and yeast proteins. The sequence

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of all peptides identified as belonging to the antigen was confirmed by MS/MS. Sequence
coverage of the recombinant fusion was 72% when all digests were considered. Both amino-
and carboxy- termini peptides were detected and identified (Table 1). The part of the

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recombinant fusion antigen not detected by peptide mapping covered a hydrophobic
sequence region ranging from amino acids 317 to 475 (Fig. S2). The antigen was found to be
present with and without its amino terminal methionine processed. This observation is

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consistent with the documented presence of a methionine aminopeptidase involved with the
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maturation of proteins in a number of organisms including yeast [10]. The relative amount of
recombinant fusion antigen was found to be less than 10% of the total proteins but was the
most abundant protein in the samples. Over 1800 yeast proteins were identified. The summary
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of the protein identification result is in Table 1.

Antigen Integrity
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The mass spectrometry results are consistent with the presence of the recombinant fusion
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HBV antigen in the yeast cells. However, it did not demonstrate that the antigen is present in
its intended full length. Because of the high content in hydrophobic amino acids, the
recombinant fusion protein is soluble only in solutions containing a significant amount of
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detergent. As detergents tend to be incompatible with mass spectrometry, attempts at

collecting intact mass data after antibody mediated pull down were unsuccessful. A different
approach, relying on western blot was therefore used to demonstrate the integrity of the
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recombinant antigen. A series of western blots was performed, using anti-S antigen, anti-X
antigen and anti-6xHistidine tag antibodies (Fig. 3 and 4). For each of the three antibodies, the
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band in the gel that showed the strongest immunoreactivity was indicative of a protein with an
electrophoretic mobility consistent with a mass of about 65 to 68 kDa (Fig. 3 and 4). As the
predicted mass of the recombinant antigen is 73.3 kDa and the observed mass is reasonably
close to that value, we concluded that the antigen is predominantly present it its full-length
form.

This conclusion was further supported by in-gel digest of the protein included in a small piece
of polyacrylamide gel excised from the region where the immune-reactive protein was found.
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The digest was performed with a combination of trypsin and chymotrypsin proteases (Table 1).
The resulting peptides were analyzed by 2D nano LC-MS/MS. The sequence of all the peptides
identified as part of the recombinant antigen was confirmed by MS/MS. Although the
sequence coverage was lower than when the digest was performed in solution, peptides
covering both amino and carboxy extremities of the predicted fusion antigen polypeptide were
detected with high confidence. In line with results obtained upon in-solution digest,

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approximately half of the recombinant antigen was missing the amino terminal methionine in
the in-gel digested sample. Immunoreactivity towards smaller proteins were also detected,

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regardless of which of the three antibodies was used (Fig. 3 and 4). The electrophoretic
mobility of these bands was consistent with potential fragments of the X-S-Core HBV antigen
with molecular weights of 45 kDa and 55 kDa (Fig. 4). However, we cannot rule out that the

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immunoreactivity towards these bands might be caused by unspecific binding of the primary
or secondary antibodies (Fig. 4).

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Antigen quantitation by Western blot
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Based on the mass spectrometry and western blot results, we developed a semi-quantitative
Western blot using an anti-6xHistidine tag antibody for the quantitation of the recombinant
antigen. Due to its lack of solubility, the recombinant antigen could not be purified from yeast
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and therefore could not be used as standard. A commercially available highly purified
6xHistidine tagged polypeptide was thus used as standard and 0.25 to 3.5 pmol per lane were
loaded over five gel lanes (Fig. 3). The Yeast lysate was loaded on the gel based on the total
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protein concentration determined by the BCA assay, such that the expected response would
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fall within the linear range established for the standard. Total yeast load ranged from 3 to 9 µg
over four gel lanes, for a nominal load target of 6 µg in typical assay conditions. Based on the
linearity of the response as a function of mass loaded on the gel, the immunoreactivity of the
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standard and the recombinant antigen was found to be comparable. However, intermediate
precision over multiple days and analysts was overall poor, and quantitation of the western
blot signal based on densitometry greatly depended on exposure time and blot background
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(%CV of mean antigen concentration was 18% for 6 repeats). The seven biological replicates
tested by this method yielded antigen content ranging from about 700 ng/YU to 1100 ng/YU.
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Alternatively, HBV specific detection antibodies such as an anti- S antigen and anti- X antigen
monoclonal antibodies were also explored in the western blot format (Fig. 4). The Western
blot methods based on these two different antibodies yielded a similar range of antigen
content as when the anti-6xHistidine tag antibody was used. While specificity towards the
fusion protein was improved when the anti- S antigen and anti- X antigen monoclonal
antibodies were used, the approach remained semi-quantitative.
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Antigen quantitation by ELISA

A more quantitative immune-absorbent assay relying on an ELISA format was developed. In

this assay, the recombinant fusion protein is captured using the anti- S antigen antibody and
detected using the anti-6xHistidine antibody conjugated to HRP. The signal is normalized to a

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calibration standard consisting of a recombinant HBV-S antigen protein carrying a 6xHistidine
tag on its carboxy terminus and spiked into a 20 µg protein per mL lysate of yeast transformed

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with an empty vector. The sample preparation was optimized such that the SDS content,
critical for efficient cell lysis and protein solubilization, was reduced by a 20x dilution in an
assay buffer containing 1% Triton-X, a detergent less detrimental to antibody reagents used in

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ELISA. This assay format greatly improved the precision and accuracy of the X-S-Core HBV
antigen concentration determination. The %RSD of the mean antigen concentration for
repeatability was 3%, and for intermediate precision was 6 % for twelves independent

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measurements over multiple days and analysts. The 6xHistidine tagged recombinant HBV-S
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antigen standard concentration mean accuracy was 96 to 100% within the assay linearity
range of 1.0 to 6.6 nM (equivalent to 233 to 11,611 ng per YU), greatly expending the dynamic
range of the assay compared to the western blot format. The ELISA was specific to the X-S-
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Core antigen in yeast lysate; no interference was found in the lysate prepared from vector
control yeast, placebo, and formulation buffer. As for the Western blot, antigen concentration
results for unknown samples were only accepted provided a number of system suitability
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acceptance criteria were met for standards, reference standard and assay controls.
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The ELISA method was also found to be stability indicating. Antigen concentration was found
to decrease in deactivated intact yeast samples stored at 40°C for up to three months. In
contrast, antigen content in samples stored at 2-8°C remained stable for at least 5 months (Fig.
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5).

Antigen quantitation by LC-MS/ MRM

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Multiple reactions monitoring (MRM) relies on the use of synthetic peptides containing at least
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one heavy stable isotope labelled amino acid that has identical sequence to a matching
peptide in the endogenous protein to be quantified [11]. The heavy peptide is spiked into a
tryptic digest preparation of a sample and used as internal standard for quantitation. Being
chemically equivalent, each pair of standard and endogenous peptides coelute during
chromatography, have identical ionization efficiency and produce identical fragmentation
patterns. However, in the mass spectrometer, owing to their mass differences, endogenous
peptide and fragmentation products from the endogenous and matching standard can easily
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be separated. The chemical identity and mass difference of the endogenous and standard
peptides are the basis for the MRM quantitation methodology.

Four tryptic peptides of the recombinant fusion of HBV X, S and Core specific T cell antigens
were selected for the development of the MRM based quantitation method based on a series
of in silico criteria (Table 2 and S1). These criteria include peptide length (7-25 amino acids),

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the absence of sequence homology to yeast proteins, and stability (the absence of amino acids
known to undergo degradation such as deamidation/isomerization or oxidation). The heavy
peptides were synthesized to achieve both the highest possible purity and absolute

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concentration determination, and spiked into different yeast lysates. MRM method
development included the optimization of the digest time (Fig. 6), the selection and

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optimization of the specific transitions (a given precursor and fragment ions pair) used for
quantitation, verification of the absence of matrix interference and optimization of the
multiplexing as more than one peptide could be used at the time. A calibration curve was

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constructed for every peptide based on serial dilution of the synthetic peptides spiked in the
yeast lysate. Of the four peptides investigated, we selected DLLDTASALYR, based on signal
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strength, linearity, and overall method performance. This peptide was used to further qualify
the method. In addition to linearity, LOQ and LOD, and reproducibility were assessed (Table 2).
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The method was used to analyze antigen content in six independent biological replicates.
Antigen content ranged from about 700 ng/mL to 1000 ng/mL. Values were systematically
lower than those determined by ELISA, possibly reflecting the impact of the absence of SDS on
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antigen extraction efficiency. However, both methods showed a high level of orthogonality as
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the antigen content trends were highly similar (Fig. 6). While the two methods were found to
be highly complementary for freshly prepared samples, the correlation ceased to be evident
when forced degraded material were used, highlighting different stability indicating
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properties. Indeed, the ELISA requires the integrity of both the 6xHistidine tag and S antigen
epitopes on a same polypeptide to generate a positive signal, while the concentration
determined using MRM only requires a 11 amino acid fragment and thus do not reflect the
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integrity of the X-S-Core HBV antigen. The impact of fragmentation X-S-Core HBV antigen after
yeast inactivation on potency has not been determined. Based on the mode of action, it is
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assumed that fragmentation post yeast cell in activation may not constitute an impediment to
activity.
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Discussion and conclusion

Vaccines whether therapeutic or prophylactic are regulated like other biologics by FDA's
Center for Biologics Evaluation and Research (CBER) and are subject to similar Chemistry,

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Manufacturing, and Controls (CMC) rules and regulations [12,13]. Establishing fit for purpose
and validated analytical methods as part of the control strategy and for extended
characterization is critical for assuring quality and safety of the vaccine [12]. To align with such

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expectations, we developed and qualified two analytical methods for the identification and
quantification of the active component of a yeast-based therapeutic vaccine designed to treat

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chronic hepatitis B patients. These orthogonal methods rely on proteomics approaches
commonly used for complex matrices and are designed to detect the X-S-Core HBV antigen
fusion protein in the midst of a large number of yeast proteins. Both methods can potentially

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be validated and are amenable to Quality control testing environment. However, the stability
indicating nature as well as the ease of execution and lower equipment requirement make of
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the ELISA format a more likely choice for lot release and stability testing. The accurate
quantitation of the recombinant HBV antigen directly affects the manufacturing controls
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strategy, shelf life, comparability between processes and manufacturing scales, but also allows
a better understanding of the correlation between dosing and clinical efficacy.
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Competing interests
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Authors declare no competing financial interests

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Acknowledgements
We would like to thank JadeBio for conducting the LC-MS work. Our Gratitude goes to Dick Ill,
Andy Snowden, Ashraf Amanullah, Rachel Bright, Dell Farnan, and Yas Saotome, for their
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support. We would like to thank GlobeImmune leadership, for granting permission to publish
this work and the many interesting conversations on the biology supporting the mode of
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action of the HBV Tarmogen®. Our gratitude also goes to Leo Ambrogelly for is help in making
the figures ready for publication.
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Material and Methods

Yeast cell number quantitation by MFI
An estimated amount of 20 YU worth of yeast cells suspended in 1.2 mL were first sonicated
for 20 s at 3 to 5 watts. Cells were then diluted to approximately 0.05 YU/ mL with water

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(500,000 cells per mL). MFI instrument readiness was verified prior each run by verifying
particle size and concentration using NIST-traceable polystyrene beads standard (5 µm); The
acceptable range for the polystyrene beads equivalent circular diameter was set to 4.75-5.25
µm, and particle concentration is ± 15% of the polystyrene beads nominal concentration used.

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The diluted samples were analyzed on the MFI in triplicate as per the manufacturer
recommendations (DPA4200 Micro-flow Imager, Brightwell). Baseline runs should not contain
more than 500 particles/mL, and diluted samples should not contain more than 750,000 cells

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per mL.
Cell lysis and total yeast protein concentration in cell lysate
20 YU of yeast cells were pelleted in a centrifuge (16000 x g for 5 min) and resuspended by

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vortexing at 1400 rpm for 10 min in lysis buffer (phosphate, 4% (w/v) SDS, 2% (v/v) 2-
mercaptoethanol, EDTA, protease inhibitor) and 0.5 mm glass beads (Sigma). Each lysate was
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incubated at 95 °C ± 3 °C for 10 minutes. Glass beads and cells debris were decanted, and the
supernatant was recovered. The supernatant was clarified by centrifugation at 16000 x g for 5
min. Total protein concentration of the lysate was determined using Pierce 660 colorimetric
protein assay as per the manufacturer insert. Protein concentration in the lysate was
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determined against a BSA calibration curve obtained by diluting a stock solution in lysis buffer
and ranging from 0 to 2 mg/mL over 6 concentrations in triplicates. The assay was considered
valid if %RSD of the standard triplicates and if linearity of the standard curve met a predefined
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acceptance criteria. Samples were also tested in triplicates; sample results were considered
valid only of %RSD of the sample triplicates met a predefined acceptance criteria.
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Western Blot
Yeast Cell lysates of samples and reference standard were diluted in 2X loading SDS sample
preparation buffer (125 mM Tris, 20% v/v glycerol, 4% SDS,0.005% w/v bromophenol blue, 2%
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v/v 2-mercaptoethanol, EDTA, protease inhibitor cocktail from Roche) and a volume of 10 µL
(6 µg of total yeast protein) per lane was loaded in triplicate. 6xHistidine tagged standard
polypeptide, Marker (Precision plus protein standard) and Magic Mark XP standard were also
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loaded to the same volume. The SDS PAGE was a NuPAGE 10% Bis-Tris gel run in 1X NuPAGE
MES SDS Running Buffer for 40 min at 200 V. Prior to setting up the transfer, the gel and
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nitrocellulose membrane were extensively washed in DPBS plus 0.05% Tween-20. Transfer was
done using standard methodology in a wet transfer apparatus for 2hr at 25V. Western blot
was conducted following standard methodologies. Membranes were blocked in 2% BSA-DPBS-
T for up to 17 hours. The primary antibody was a mouse anti-6xHistidine tag (Qiagen, 1:1000
dilution) and secondary was HRP-couple goat anti-mouse (Jackson ImmunoResearch, 1:1000
dilution). Blots were revealed using ECL reagent, pictures of the blot were taken at different
exposure times. The picture used for data analysis was the one that presented the best signal
intensity without signal saturation as indicated by Image Lab 5.1 software. Data collection and
densitometry analyses were conducted using a BioRad imager. Results for samples were
considered valid only if the reference standard lysate generated an antigen concentration
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value within a predefined range considered as accurate. Similar methodology and conditions
were used with anti-X (Millipore) and anti-S (International Immuno Diagnostics) primary
antibodies.
Quantitative ELISA
Yeast cell lysate was diluted 20 folds in a 1% Triton-X containing assay buffer (corresponding to
20 ug/mL total yeast proteins), reducing the SDS and 2-mercaptoethanl concentrations to 0.2%

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w/v and 0.1% v/v, respectively. The target antigen was captured by anti-HBsAg immobilized
on ELISA plate, then detected by HRP labeled 6x Histidine tag antibody. The calibration
standard was a recombinant HBsAg 6x Histidine tagged protein (ProSpec), spiked into 20

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µg/mL of the vector control yeast lysate at 0.13 – 65.95 nM. Three levels of Assay control
samples were prepared at 100X concentration in 20 µg/mL of the vector control yeast lysate at
1.06, 2.64, 6.60 nM, and stored at -80°C. Reference lysate and sample lysate were tested at 20,
10, 5, and 2.5 µg/mL of yeast protein concentration. The antigen content in a lysate, expressed

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as ng/YU, was determined using interpolation against the standard curve. Results for samples
were considered valid only if the calibration standards, reference lysate, and assay control
lysates generated an antigen concentration value within a predefined range considered as

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accurate.
Identification and in solution Coverage Assessment by LC-MS
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The yeast cell lysate was digested with a set of different enzymes combinations including LysC,
LysC-Trypsin, LysC-Trypsin-Chymotrypsin, LysC-Trypsin-GluC. Each sample was analyzed by 1D
and/or 2D nano LC-MS/MS. Yeast cell lysates were diluted in Hepes buffer containing 1 mM
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TCEP. Digest enzymes were added together or sequencially and each digest was allowed to
proceed for up to 17 hours at 37°C. Free thiols were capped using a solution of iodoacetamide
for 30 min at 37°C in the dark. 1D LC analysis was performed on a 5 µm Zorbax SB-C18
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(Agilent) packed into a fused silica capillary tubing (200 µm ID x 20 cm length).

Chromatography was performed on an Agilent 1200 HPLC system. The capillary was developed
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in a 0-80% gradient of acetonitrile for 120 min. A fresh capillary was used for each sample
tested. 2D LC analysis was performed by coupling the RP-HPLC first dimension with a SCX
column for the second dimension. Peptides were first eluted from the RP1 to the SCX column
using a 0–80% acetonitrile gradient for 60 min. The peptides were then fractionated by the
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SCX column using a series of five salt gradients (30 mM, 50 mM, 70 mM, 100 mM, and 1 M
ammonium acetate) followed by high resolution reverse phase separation using an acetonitrile
gradient of 0–80% for 120 min. Spectra were acquired using an LTQ linear ion trap tandem
mass spectrometer (Thermo Electron Corp., San Jose, CA) using automated, data-dependent
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acquisition. The mass spectrometer was operated in positive ion mode with a source
temperature of 250 °C. The collected MS and MS/MS spectra were analyzed against UniProt
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yeast database, the fusion of HBV X, S and Core specific T cell antigens sequence, and decoy
sequence databases using Spectrum Mill (Agilent).
In gel Digest and LC-MC
Gel pieces excised from the SDS PAGE were washed in 10 mM Ammonium Bicarbonate (pH
8.0) and in 50% acetonitrile in 10 mM Ammonium Bicarbonate (pH8.0) until completely
destained. Gel pieces were further washed with 100 % acetronitrile and dry down in a
SpeedVac at room temperature for 5 min after removal of supernatant. LysC, Trypsin and
Chymotrypsin were added in one pot or sequencially. Digests were performed in 50 mM Hepes
(pH7.0) and in the presence of 1 mM TCEP at 37°C for 4 to 16 hr depending on the enzyme.
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Acetylation was performed with iodoacetamide in the dark at 37°C for 30 minutes. Each
sample was analyzed by 2D nano LC-MS/MS as indicated above.

Multiple reaction monitoring

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20 YU of yeast cells were pelleted and washed 6 times with cold PBS buffer. The cells were
lysed in 8 M urea and 10 mM DTT using a glass beads and a blender. The cell lysate was heated
at 65°C prior to the addition of LysC enzyme and incubation at 37°C for 30 min. Digestion was

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followed by alkylation with iodoacetamide and dilution into an ammonium bicarbonate and
urea solution. The synthetic peptide standards (Thermo) were then spiked into the samples at
different levels. Samples were further digested with trypsin and LysC at 37°C for 2 hours.

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Digests were cleaned using a MCX column and injected on the LC-MS. Separation by LC was
achieved using a BEH130 (Waters) RP-UPLC column developed in a gradient of water/0.1%
formic acid and Acetronitrile/0.1% formic acid on an Agilent 1290 system. Mass spectrometry
was performed on a triple quadruple mass spectrometer (Agilent 6490). A total of 34

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transitions were monitored. Data analysis was performed using Mass Hunter (Agilent) and
skyline (University of Washington). Each sample was injected a minimum of three times. For
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each sample injection, the synthetic to endogenous peptides ratio was reported.
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Figure Legends
Fig. 1. Cartoon representation of the recombinant HBV X-S-Core fusion Antigen. M (MADEAP
metabolic stability tag), X = 60 amino acids, Large S (envelope) = 399 amino acids, Core 182
amino acids, His6 = 6xHistidine tag.
Fig. 2. Determination of the Yeast cell population distribution by MFI. ECD stands for
Equivalent Circular Diameter.

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Fig. 3. HBV X-S-Core fusion antigen integrity and content determination by Western Blot using
an anti-6xHistidine tag antibody. (A) Blot showing molecular weight markers (Mw) with the
molecular mass of each of the proteins indicated in kDa, the 6xHistidine tagged polypeptide

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used as standard, the analysis in triplicates of a sample lysate and of the reference standard
lysate, and a blank line to demonstrate specificity (blk). (B) Blot showing four biological
replicate of lots analyzed in duplicate. The Molecular weight marker (MMXP) with the

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molecular mass of each of the proteins indicated in kDa, and the empty yeast control for
specificity (Yvec Control) are shown. The estimated molecular weight of the main
immunoreactive species in each lane is shown. (C) Plot showing the correlation between the
HBV X-S-Core fusion antigen content (ng/YU) determined by ELISA (Y axis) and by anti-

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6xHistidine tag antibody Western blot (X axis).
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Fig. 4. HBV X-S-Core fusion antigen integrity by Western Blot using anti-S and anti-X
Antibodies. The plot of HBV X-S-Core fusion antigen degradation kinetics followed by Western
Blot when incubated at 60°C for up to 24 hours by anti-His antibody detection. The blot is
showing immunoreactivity towards a yeast lysate containing the HBV X-S-Core fusion antigen,
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using Anti-X, Anti-S and anti-6xHistidine tag (Anti-HIS) antibodies, just after sample
preparation (T0) and after incubation for 24 hours of the yeast cells at 60°C (T24).
Fig. 5. Stability of antigen content in samples stored at 2-8°C (solid squares) and for up to 5
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months at 40°C (solid triangles) for up to 3 months by ELISA. Relative antigen content is
expressed as a percentage of the antigen content determined at 1 month for each storage
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condition.
Fig. 6. HBV X-S-Core fusion antigen concentration determined by MRM LC-MS/MS. (A) Plot
showing the linearity of the MRM method expressed as ratio of detected heavy to light
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DLLDTASALYR peptides as a function of the heavy DLLDTASALYR peptide spiked in (B) Plot of
kinetic of trypsin/LysC digest at 37°C expressed as the ratio of the peptide DLLDTASALYR
concentrations after 45 min of digest time. (C) Plot showing the correlation between the HBV
X-S-Core fusion antigen content (ng/YU) determined by MRM LC-MS/MS (Y axis) and by ELISA
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(X axis).
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Table 1: Recombinant HBV fusion antigen coverage assessment after in solution and in gel
digests with combination of different proteses

Technology Fusion HBV Antigen

Digest Enzymes Unique Proteins identified
Platform % sequence coverage

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In solution digest
LysC 2D MS/MS 252 3%
LysC-Try 1D MS/MS 426 52%
LysC-Try 2D MS/MS 1637 54%

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LysC-Try-Chymotrypsin 1D MS/MS 268 58%
LysC-Try-Chymotrypsin 2D MS/MS 1370 52%

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LysC-Try-GluC 1D MS/MS 381 49%
LysC-Try-GluC 2D MS/MS 1144 49%
Total 1878 72%
In gel digest

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LysC-tryp-Chymo Nano LC MS/MS 591 58%
LysC-Try Nano LC MS/MS 767 42%
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Total 817 62%
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Table 2: Summary of MRM method performance using selected peptide

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Average CV% CV%

LOD LOQ
Concentration (n=6) (n=3)
Peptide DR Linearity
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µg/YU LC-SRM assay µg/YU µg/YU

DWEELGEELEGQWSSKPR 0.699 5.20 1.72 0.01014 0.01014 >2 0.99768

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DLLDTASALYR 0.5964 5.87 6.83 0.00304 0.01014 >2 0.99774

TPPAYRPPNAPILSTLPETTVVR 0.178 9.00 1.89 0.00304 0.01014 >2 0.99899

ANSANPDWDFNPNK 0.3932 11.04 4.11 0.01014 0.01014 >2 0.99666

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