RESEARCH ARTICLE

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The Degree and Length of O-Glycosylation of Recombinant

Proteins Produced in Pichia pastoris Depends on the Nature
of the Protein and the Process Type
Bojana Radoman, Clemens Grünwald-Gruber, Bernhard Schmelzer, Domen Zavec,
Brigitte Gasser, Friedrich Altmann, and Diethard Mattanovich*

1. Introduction
The methylotrophic yeast Pichia pastoris is known as an efficient host for the
production of heterologous proteins. While N-linked protein glycosylation is Glycosylation is an essential protein mod-
ification playing a role in protein stability,
well characterized in P. pastoris there is less knowledge of the patterns of
structure and activity, as well as in protein
O-glycosylation. O-glycans produced by P. pastoris consist of short linear quality control in the endoplasmic reticu-
mannose chains, which in the case of recombinant biopharmaceuticals can lum (ER).[1] It also has an impact on cell
trigger an immune response in humans. This study aims to reveal the growth and development. Only properly gly-
influence of different cultivation strategies on O-mannosylation profiles in P. cosylated and folded proteins can success-
fully pass from the ER to the Golgi and
pastoris. Sixteen different model proteins, produced by different P. pastoris
further to their final location.[2] Besides its
strains, are analyzed for their O-glycosylation profile. Based on the obtained importance for native protein folding and
data, human serum albumin (HSA) is chosen to be produced in fast and slow maturation, glycosylation is also a cru-
growth fed batch fermentations by using common promoters, PGAP and PAOX1 . cial posttranslational modification (PTM)
After purification and protein digestion, glycopeptides are analyzed by of biopharmaceuticals, as it can influence
LC/ESI-MS. In the samples expressed with PGAP it is found that the degree of their in vivo half-life, solubility, immuno-
genicity, and bioactivity.[3,4]
glycosylation is slightly higher when a slow growth rate is used, regardless of
There are two main types of glyco-
the efficiency of the producing strain. The highest glycosylation intensity is sylation, which are evolutionarily con-
observed in HSA produced with PAOX1 . The results indicate that the served in animals and fungi, N- and
O-glycosylation level is markedly higher when the protein is produced in a O-glycosylation.[2] In eukaryotic cells, N-
methanol-based expression system. linked glycans are attached to the amide
group of an asparagine residue within the
consensus sequence Asn-Xxx-Ser/Thr. O-
linked glycans are attached to the hydroxyl
B. Radoman, B. Schmelzer, Prof. B. Gasser, Prof. F. Altmann, group of the amino acids serine (Ser) or threonine (Thr), but
Prof. D. Mattanovich there is no known consensus sequence. There are several differ-
Austrian Centre of Industrial Biotechnology (ACIB) ent types of O-glycan structures present in mammalian cells, with
Vienna 1190, Austria the only O-glycosylation type conserved among fungi, animals,
E-mail: diethard.mattanovich@boku.ac.at
and humans being O-mannosylation.[5] This modification was
B. Radoman, B. Schmelzer, Dr. D. Zavec, Prof. B. Gasser,
Prof. D. Mattanovich
first identified in baker’s yeast.[1] It starts in the ER, where, simi-
Department of Biotechnology larly to N-glycans, it uses a dolichol-phosphate for the transfer of
BOKU—University of Natural Resources and Life Sciences the first sugar to Ser or Thr. Different from N-linked glycans, this
Vienna 1190 Austria sugar is a single mannose residue (Man), which is transferred by
Dr. C. Grünwald-Gruber, Prof. F. Altmann enzymes of the protein–mannosyl–transferase (PMT) family.[6]
Department of Chemistry Yeasts have a long-term history as hosts for biopharmaceu-
BOKU—University of Natural Resources and Life Sciences
Vienna 1190 Austria ticals with several approved products on the market. The first
yeast employed for heterologous protein production was Saccha-
The ORCID identification number(s) for the author(s) of this article romyces cerevisiae which is still routinely used for the production
can be found under https://doi.org/10.1002/biot.202000266 of many different biopharmaceuticals including insulin and vac-
© 2020 The Authors. Biotechnology Journal published by Wiley-VCH cines against hepatitis B and human papillomavirus. Besides S.
GmbH. This is an open access article under the terms of the Creative cerevisiae there are other yeasts which were proven to be very use-
Commons Attribution-NonCommercial-NoDerivs License, which permits
use and distribution in any medium, provided the original work is
ful for the production of heterologous proteins, among them the
properly cited, the use is non-commercial and no modifications or methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii). It
adaptations are made. grows to very high cell densities in minimal media and can effi-
DOI: 10.1002/biot.202000266 ciently secrete proteins to the culture broth.[7,8]

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However, yeast glycosylation patterns are not optimal for
proteins which have a medical application. While the structure
of N-glycans differs between yeasts and humans, the major
problem regarding O-glycans is their unwanted occurrence
in recombinant proteins at positions where they would not
natively be found. Thus, several attempts to engineer either
N- or O-glycosylation in yeasts have been carried out (recently
summarized by Van Landuyt et al.[9] ). In recent years, several
studies were published concerning the glycoproteome of S.
cerevisiae, which tried to resolve the main mechanistic steps of
the O-glycosylation machinery.[1,5,10–12] Compared to the detailed
knowledge of S. cerevisiae, protein glycosylation in P. pastoris
has not been fully understood yet. N-glycosylation in P. pastoris
has been analyzed in more detail and through tremendous
glycoengineering humanization of N-glycosylation has been
achieved.[13,14]
Concerning O-glycosylation in P. pastoris, there are still many
questions that need to be answered. According to one previous
study, P. pastoris produces short O-glycans with up to 3-Man. O-
glycans produced by Candida albicans and S. cerevisiae are much
longer, with up to 5- and 13-Man, respectively.[15] Other studies,
however, suggest that P. pastoris can produce proteins with O-
glycans consisting of 6-Man.[16,17] These mannose residues are
mostly 𝛼-1,2 or 𝛽-linked, and they can also be phosphorylated.[4]
𝛽-linkages on recombinant glycoproteins could cause an im-
munogenic response in humans due to interaction with the
mannose receptor, or dendritic cell-specific intercellular adhe-
sion molecule-3-grabbing non-integrin (DC-SIGN).[18] However,
it has been shown that proteins produced in P. pastoris with a sin-
gle mannose residue do not induce an immunogenic reaction.[19]
In the case of some recombinant proteins, no significant differ-
ence in immunogenicity between a glycosylated protein and its
non-glycosylated form was observed. The authors suggested that
this could be either due to the absence of highly immunogenic
terminal 𝛼-1,3-mannosyl groups, or due to lower complexity of
mannose type O-glycans in P. pastoris compared to more com-
plex ones in mammalian cells.[17]
As there is no clear picture on the lengths and structure of
O-glycans on recombinant proteins produced in P. pastoris and
only a little knowledge to which degree they are decorated with
an O-glycan, we decided to study O-glycosylation in P. pastoris
more comprehensively. Due to the lack of a consensus sequence,
analysis of O-glycans is quite demanding. Previous studies on
O-glycans in P. pastoris were mostly focused on a single particu-
lar protein produced in this yeast.[15,17,20,21] Hence, we decided to
analyze several different recombinant proteins produced in our
laboratory. Considering glycosylation as an important attribute of
recombinant protein quality, it is of great interest to investigate
the impact of culture conditions on this PTM. Therefore, we fur-
ther aimed to understand the impact of cultivation strategies on
the degree and compositions of O-glycans in proteins produced
by P. pastoris. In particular, we compared the impact of culture
conditions that are usually employed with two most commonly
used promoters, the constitutive glyceraldehyde-3-phosphate
dehydrogenase promoter (PGAP ) and the inducible alcohol oxi-
dase 1 promoter (PAOX1 ), on protein O-glycosylation. Based on
this overview on O-glycan profiles of different model proteins
with detailed information about their lengths, positions and site
occupancy, human serum albumin (HSA) was chosen as the
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model protein for studying the impact of cultivation conditions
and bioprocess strategies on O-glycosylation.
2. Experimental Section
2.1. Strains and Model Proteins
Sixteen model proteins (listed in Table 1) were produced in dif-
ferent batch or fed-batch fermentations. The expression strain
used for the production of most of the proteins was P. pastoris
CBS7435, except for HSA and human trypsinogen which were
produced by P. pastoris CBS2612, while antigen-binding frag-
ment 1 and invertase were produced by P. pastoris X-33. Accord-
ing to Braun-Galleani et al.,[33] these three strains have nearly
identical genome sequences. Proteins were produced either in
bioreactor cultivation or in a shake flask, under the control of
PGAP or PAOX1 .
2.2. Analysis of Glycosylation in the Model Proteins
For this study, model proteins were either purified or used di-
rectly from the culture supernatants from bioreactor or shake
flask cultivations. All non-purified samples were applied to
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS
PAGE) and after Coomassie Blue staining, bands were cut out
before in-gel digestion. Model proteins were digested by trypsin
(Promega) in-gel or in-solution. The MS analysis is described be-
low in the glycopeptide analyses section. Purified model proteins
were further investigated with intact mass analysis, which is de-
scribed below.
2.3. Strains and Vectors Used for the Production of Human
Serum Albumin under the Different Promoters
The expression strain used in this study was P. pastoris CBS7435
wild-type strain. Two pPuzzle vectors with the HSA gene were
constructed previously in the laboratory. One of the vectors
contains PAOX1 [34] and the other contains PGAP . Both vectors were
linearized before the transformation of P. pastoris wild-type. The
selection was based on Zeocin resistance.
2.4. Strain Transformation and Screening
P. pastoris transformation was done as previously described by
Ata et al.[35] 3 µg of each linearized and purified (by Wizard SV
Gel and PCR Cleanup System, Promega Corp) plasmid was used
for electro-transformation. After recovering, cells were plated on
YPD-agar plates (10 g L−1 yeast extract, 20 g L−1 soy peptone,
20 g L−1 glucose, 20 g L−1 agar-agar) with 50, 100, or 500 mg
mL−1 Zeocin. 24 randomly selected clones were grown at 25 °C
at 280 rpm in selective YPD medium (10 g L−1 yeast extract, 20 g
L−1 soy peptone, 20 g L−1 glucose). After 24 h the cells were cen-
trifuged, and re-suspended in minimal ASM medium (6.3 g L−1
(NH4)2 HPO4 , 0.8 g L−1 (NH4)2 SO4 , 0.49 g L−1 MgSO4 × 7H2 O,
2.64 g L−1 KCl, 0.054 g L−1 CaCl2 × 2H2 O, 22 g L−1 citric acid
monohydrate, 1.47 mL L−1 PTM0 trace metals, 0.8 mg L−1 biotin,
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Table 1. List of analyzed recombinant proteins. The name of the strains and promoters used for the production are shown. The maximum number of the
detected mannose residues (column "maximum glycosylation") and the site occupancy percentage (column "% of glycosylation") are given. Sequence
coverage (SC) is the percentage of the total protein sequence that was found in MS/MS analysis. Underlined values are observed from the intact mass
analysis. Sequence coverage for these three samples was very low and glycopeptide analysis showed that these proteins were not glycosylated. However,
the intact mass analysis provided information on the existence of glycosylation in these proteins.

# Trivial name Promoter Strain Production Maximum % of SC [%] Reference for

process glycosyla- glycosy- protein sequence
tion lation

1 Single domain antibody PAOX1 CBS7435 Bioreactor No 0 83.6 Abbeele et al.[ 22 ]

2 Single domain antibody PGAP CBS7435 Bioreactor No 0 83.6 Abbeele et al.[ 22 ]
3 Single chain variable fragment 1 PAOX1 CBS7435 Bioreactor 3 Man 13.4 40.6 Rader et al.[ 23 ]
4 Single chain variable fragment 2 PAOX1 CBS7435 Shake flask 2 Man 1.8 54.9 Lin et al.[ 24 ]
5 Single chain variable fragment 3 PAOX1 CBS7435 Shake flask 1 Man 3.3 76.7 Dall’Acqua et al.[ 25 ]
6 Single chain variable fragment 3 PGAP CBS7435 Shake flask 1 Man 2.2 76.7 Dall’Acqua et al.[ 25 ]
7 Antigen binding fragment Fab 1 PGAP X-33 Bioreactor 4 Man 44.9 33.1 Kunert et al.[ 26 ]
8 Antigen binding fragment Fab 2 PAOX1 CBS7435 Bioreactor 4 Man 8.0 47.5 Gasser et al.[ 27 ]
9 Antigen binding fragment Fab 2 PGAP CBS7435 Bioreactor 4 Man 50.8 47.5 Gasser et al.[ 27 ]
10 Antigen binding fragment Fab 3 PAOX1 CBS7435 Bioreactor 2 Man 8.8 66.4 Gasser et al.[ 27 ]
11 Human serum albumin PAOX1 CBS2612 Bioreactor 5 Man 42.9 88.3 Meloun et al.[ 28 ]
12 Trypsinogen (human) PGAP CBS2612 Shake flask 3 Man 10.7 51 Gaboriaud et al.[ 29 ]
13 Invertase PGAP X-33 Shake flask N-glyc N-glyc 36.1 Reddy et al.[ 30 ]
14 Carboxylesterase PGAP CBS7435 Bioreactor 3 Man 14.9 69.1 Heinl et al.[ 31 ]
15 Carboxypeptidase B PGAP (glucose) CBS7435 Bioreactor 1 Man 1.6 76.1 Burgos et al.[ 32 ]
16 Carboxypeptidase B PGAP (methanol) CBS7435 Bioreactor 1 Man 2.3 76.1 Burgos et al.[ 32 ]

20 mL L−1 NH4 OH (25%); pH set to 6.5 with KOH) and polysac-
charide solution with enzyme (PSE). PSE provides glucose lim-
iting conditions with a slow glucose release system with 12.5 g
L−1 polysaccharide and 0.4% enzyme (for PAOX1 screenings) and
25 g L−1 polysaccharide and 0.75% enzyme (for PGAP screenings)
(m2p-labs GmbH, Germany). For screening of the clones with
the inducible promoter PAOX1 cells were fed with methanol (once
with 0.5% (v/v) and three times with 1% (v/v)). Cells were har-
vested at t = 48 h of the cultivation by centrifugation at 13 000 rpm
for 5 min.
2.5. Relative Gene Copy Number
The relative gene copy number was determined by quantitative
real-time PCR as already described by Prielhofer et al.[36] All sig-
nals were normalized to the housekeeping gene ACT1.
2.6. Bioreactor Cultivation
The fed batch fermentations were done in a DASGIP Parallel
Bioreactor System (Eppendorf AG, Germany) with a final work-
ing volume of 0.7 L. The four high and four low producer strains
with the HSA gene under the control of PGAP and four high pro-
ducer strains with the HSA gene under the control of PAOX1 were
cultivated. Strains with the GAP promoter were cultivated at the
fast growth rate of 𝜇 = 0.12 h−1 and slow growth rate of 𝜇 =
0.05 h−1 , while strains with AOX1 promoter were cultivated at
the same slow growth rate of 𝜇 = 0.05 h−1 .
In the PGAP fermentations, a ≈18h batch phase (300 mL batch
volume) was followed by a glucose fed batch phase (550 g glucose,
10.4 mL PTM0 trace salts stock solution and 10.4 mL of 0.2 g L−1
biotin stock solution per liter). Feed was exponential to the equa-
tion y = 2.897 × e0.12x for the fast growth rate or y = 0.994 × e0.05x
for the slow growth rate fermentations. In the PAOX1 fermenta-
tion, ≈18 h batch phase was followed by an exponential methanol
fed batch phase (100% methanol), according to the equation y =
1.2761 × e0.05x , where y presents a volumetric feed (mL h−1 ) at the
time x (h).
Fermentations were terminated when the maximum volume
(limited by the reactor volume) was reached. This was achieved
after ≈14 h of the feed phase in the fast growth rate PGAP fermen-
tation, after around 51 h of the feed phase in the slow growth
rate PGAP fermentation and after 55 h of the feed phase in the
slow growth rate PAOX1 fermentation. All the fermentations were
performed at 25 °C, the dissolved oxygen (DO) was controlled at
DO = 30% by adjustment of stirrer speed and air flow, and the
pH 5.0 was controlled with NH4 OH (25%).
The media used for cultivation was BSM described by Mel-
litzer et al.[37] consisting of 11.48 g L−1 H3 PO4 , 0.5 g L−1 CaCl2
× 2H2 O, 7.5 g L−1 MgSO4 × 7H2 O, 9 g L−1 K2 SO4 , 2 g L−1 KOH,
40 g L−1 glycerol, 0.25 g L−1 NaCl, 4.35 mL L−1 PTM0 , 0.87 mg
L−1 biotin, 0.1 mL L−1 Glanapon 2000, pH set to 5.5 with 25%
NH4 OH. PTM0 consisted of 6.0 g L−1 CuSO4 × 5H2 O, 0.08 g L−1
NaI, 3.36 g L−1 MnSO4 × H2 O, 0.2 g L−1 Na2 MoO4 × 2H2 O, 0.02 g
L−1 H3 BO3 , 0.82 g L−1 CoCl2 , 20.0 g L−1 ZnCl2 , 65.0 g L−1 FeSO4
× 7H2 O, 5 mL L−1 H2 SO4 (95%–98%).
2.7. Protein Purification
The fermentation broth was centrifuged for 30 min at 10 000
rcf and 4 °C (Beckman Coulter Avanti Centrifuge, J-20 XP).

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The supernatant was ultrafiltrated. HSA was further purified
on a HiTrap Blue HP column (GE Healthcare). Following the
manufacturer’s instructions, samples were diluted to the compo-
sition of the equilibrating buffer (EB), 20 mm sodium phosphate,
pH 7.0, before the purification. The column was equilibrated
with five volumes of EB. The samples were loaded at a flow
rate of 2 mL min−1 on an ÄKTA lab-scale protein purification
systems (GE Healthcare). The unbound fraction was washed
off with five volumes of EB. HSA was eluted from the column
with five volumes of elution buffer, 20 mm sodium phosphate,
2 m NaCl, pH 7.0 in 1.5 mL fractions. Fractions were checked
by SDS-PAGE. The purest fractions were pooled together. Five
volumes of phosphate-buffered saline (0.24 g L−1 KH2 PO4 , 1.8 g
L−1 Na2 HPO4 × 2H2 O, 0.2 g L−1 KCl, 8 g L−1 NaCl) were used
for buffer exchange to decrease the salt concentration.
2.8. SDS PAGE
Samples were analyzed by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE
12% Bis/Tris Gel 1.0 mm (Life Technologies) and MOPS buffer
according to the manufacturer’s instructions. After electrophore-
sis, the proteins were visualized by Coomassie Blue staining.
2.9. Glycopeptide Analysis
Digested model protein samples were applied to LC/ESI-MS for
glycopeptide analyses. After purification, HSA samples from the
bioreactor cultivations were digested in solution first by trypsin
and then with GluC (Promega). Additional single digestion was
done by LysC (Promega).
Mass spectrometry analysis was performed as already de-
scribed by Hennicke et al.[38] Briefly, carbamidomethylated and
trypsin digested samples were loaded on a BioBasic C18 column
(BioBasic-18, 150 × 0.32 mm, 5 µm, Thermo Scientific) equili-
brated with 80 mm ammonium formate buffer as the aqueous
solvent. A flow rate of 6 µL min−1 was performed with a 35 min
gradient of 5% to 40% acetonitrile, followed by a 7 min gradient
from 40% to 95% delivered by a Dionex Ultimate 3000 LC. Detec-
tion was performed in a positive ion DDA mode with Q-TOF MS
(Bruker maXis 4G, Bruker) with the standard electrospray ion-
ization (ESI) source. Manual glycopeptide searches were made
using DataAnalysis 4.0 (Bruker).
2.10. Intact Mass Analysis
Purified model proteins were injected to a LC/ESI-MS system
via a Thermo ProSwift RP-4H column (0.2 × 250 mm). A gra-
dient from 10% to 80% acetonitrile in 0.05% trifluoroacetic acid
(using a Thermo ProSwift RP-4H column (0.2 × 250 mm)) at
a flow rate of 8 µL min−1 was applied over 30-min. Detection
was performed with a Q-TOF instrument (Bruker maXis 4G)
equipped with the standard ESI source in positive ion, MS mode
(range: 400–3000 Da). Instrument calibration was performed us-
ing ESI calibration mixture (Agilent). Data were processed using
Data Analysis 4.0 (Bruker) and the spectrum was deconvoluted
by MaxEnt.
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2.11. Statistical Analyses
Statistical analyses were performed by GraphPad Prism (Graph-
Pad, La Jolla, CA, USA). To compare the total glycosylation
amount of the two most glycosylated peptides in PGAP fermen-
tations the differences between sample means were statistically
examined by using two-way analysis of variance (ANOVA), with
Sidak post-test to adjust the p-value for multiple comparisons. To
compare the total glycosylation amount of the two most glyco-
sylated peptides from PAOX1 versus PGAP fermentations the dif-
ferences between sample means were statistically examined by
using unpaired Student’s t-test. Differences were considered sig-
nificant for p < 0.05. The results are presented as mean ± stan-
dard deviation (SD). To assess the significance of the instance
of glycosylation of all peptides in HSA fermentations data were
subjected to arcsine square root transformation and evaluated by
one-sample t-test.
3. Results
3.1. Frequency and Length of O-Glycans Differs Markedly
between Different Model Proteins
The degree of O-glycosylation in sixteen different industrially rel-
evant recombinant secretory proteins produced by P. pastoris was
analyzed by LC-ESI-MS. The proteins (Table 1), were digested by
trypsin to obtain glycopeptides for site-specific analysis. Depend-
ing on their purity the protein samples were either digested in
solution or, after SDS PAGE, in gel. Purified proteins were also
analyzed by intact mass analysis.
The analysis of these 16 model proteins provided a broad
overview of the O-glycan profiles present in P. pastoris-derived
proteins with information about their positions (Table S1, Sup-
porting Information) and site occupancy. It was interesting to see
that most of the proteins had only short chain glycans with a very
low site occupancy in most of the proteins (Table 1). Exceptions
were a single domain antibody that was not O-glycosylated at all,
independent of the used promoter and cultivation strategy and
invertase which was only N-glycosylated. A higher site occupancy
was found in HSA, ≈43% and two Fab fragments, #7 (44.9%) and
#9 (50.8%). However, it is important to mention that HSA glyco-
sylation percentage is the glycosylation of one peptide (Figure 1)
and it is coming from glycopeptide analysis while the glycosyla-
tion amount of Fab #7 and #9 was calculated from the intact mass
analysis. This fact is particularly important in the case of Fab 2,
which was produced under PAOX1 (#8 in Table 1) and PGAP (#9
in Table 1). A significantly higher fraction of glycosylation was
observed when measured by intact mass analysis (#9). Sequence
coverage of sample #8 was low. Unfortunately, intact mass anal-
ysis for this sample was not successful. Sequence coverage of
sample #9 was low as well, and no glycosylation was observed
in peptide analysis. Nevertheless, in intact mass analysis, 4-Man
residues were detected for this sample, with a glycosylation frac-
tion of 50.8%. Thus, the fraction of glycosylated protein cannot
be directly compared between peptide and intact mass analysis.
According to the literature P. pastoris produces glycans with up
to 6-Man residues. However, we observed only mannose chains
with a maximum of 5-Man residues. A single mannose was found
in 4 out of the 16 analyzed proteins. In two proteins we found
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Intens.
6
x10
Line style glycoform %
not glyc 57.1
2.0
1 Man 16.7
2 Man 23.9
1.5
3 Man 2.0
4 Man 0.2
1.0
5 Man 0.2

0.5

0.0
20 21 22 23 24 25 26 Time [min]
[M+2H]2+
not glyc
956.47
rel Int %

1000.61

[M+2H]
2+ [M+2H]2+
1 Man 2 Man 1222.99

1037.50 1118.52
[M+2H]2+ [M+2H]2+
[M+2H]
2+ 4 Man 5 Man
1017.54
3 Man
969.50
986.47 1084.02 1141.07 1199.55 1274.96 1354.65

950 1000 1050 1100 1150 1200 1250 1300 1350 m/z
Figure 1. Mass spectrometry analysis of HSA peptide RPCFSALEVDETYVPK (position: 485–500), found in the sample digested by trypsin. The possible
glycosylation sites are indicated in bold. A) Base peak chromatograms of the not glycosylated peptide and its glycoforms. The not glycosylated form
(marked with a full line) is the largest fraction of this peptide with 57.1% of the peptide pool. The most abundant glycoform was 2-Man with 23.9%.
Chromatograms of the glycoforms with 4- and 5-Man residues cannot be seen as each of them makes only 0.2% of the peptide pool. However, they are
presented in the summed MS spectrum of the same peptide (B).

a glycoform with 2-Man. Three proteins had glycans with up to Table 2. List of HSA peptides that are glycosylated in either or both PGAP
3-Man, while three others had a glycan chain with 4-Man. Only and PAOX1 based processes, with their positions in the HSA sequence.
Possible glycosylation sites are indicated in bold.
HSA showed a single glycosylated tryptic peptide carrying five
mannose residues (Figure 1). The total site occupancy of this pep-
Peptide Sequence Glycosylated in: Position
tide was 42.9% and the majority was the glycoform with two man-
noses. The glycoforms with 4- and 5-Man occurred in amounts of 1 TPVSDR PAOX1 467–472
about 0.2% only. Since HSA showed a more diverse glycan pro- 2 CCTESLVNR PGAP and PAOX1 476–484
file, we decided to use it as a model for further analysis. 3 RPCFSALEVDETYVPK PGAP and PAOX1 485–500
4 MPCAEDYLSVVLNQLCVLHEK PAOX1 446–466
5 LVRPEVDVMCTAFHDNEETFLK PAOX1 115–136
3.2. Abundance of O-Glycosylation of HSA Is Higher on 6 SHCIAEVENDEMPADLPSLAADFVESK PGAP and PAOX1 287–313
Methanol than on Glucose

3.2.1. Analysis of O-Glycosylation of HSA Produced in the Glucose

Fed-Batch
First, four high (H) and four low-producing (L) P. pastoris
CBS7435 strains with the HSA gene under the control of the
PGAP promoter were selected. The selection was based on the
determination of biomass-specific product yields in correlation
to the gene copy number. HSA gene copy numbers are summa-
rized in Figure S1, Supporting Information. The chosen strains
were used for the production of HSA at two different growth
rates, 𝜇 = 0.12 h−1 and 𝜇 = 0.05 h−1 . After purification, HSA
was first digested by trypsin and then additionally by GluC. The
samples were analyzed by LC/ESI-MS. The use of two proteases
helped to cover all peptides with all possible glycosylation sites,
except the peptide ATK (position: 539–541). To detect this pep-
tide, samples were digested by the enzyme LysC. The missing
peptide was detected as ATKEQLK. As there is no sequence
motif for O-glycosylation, all Thr and Ser positions had to be
checked for modification. This was achieved by manual glycopep-
tide searches in tryptic, trypsin/GluC and LysC digests.
In all investigated HSA samples, both high and low-producing
strains, three glycosylated peptides were identified (Table 2). The
peptide RPCFSALEVDETYVPK showed the highest degree of
glycosylation and up to three mannose residues, independent of
the growth rate. In some of the high producer replicates, from

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the fast growth rate, a glycan with 5-Man was observed as well, A) Pepde 2
while, in the slow growth rate samples, some of the replicates

Total amount of glycosylation (%)

had glycans with up to 4-Man. However, these glycoforms were 30
Fast growth rate
present in a very low amount, even less than 0.5% (data not Slow growth rate
shown). Peptides 2 and 6 carried only 1-Man. However, as we will
20
describe later, in the PAOX1 fermentation, peptide 2 was glycosy-
lated with more than one Man with almost 30% site occupancy.
Peptide 6, on the other hand, turned out to be glycosylated with 10
only one Man in all processes and all samples, and always with
≈2% site occupancy or less. Hence, peptide 6 was not analyzed
in more detail. 0

n
The degree of O-glycosylation in PGAP based samples was

s io

sio

s io

sio
es

es
es

es
pr

pr

pr

pr
slightly higher when the slow growth rate was applied. However,

Ex

Ex

Ex

Ex
A

A
A

A
according to the statistical analysis, done by ANOVA with Sidak

HS

HS

HS

HS
h

h
w

w
post-test, this difference was not significant (Figure 2).

g
Lo

Lo
Hi

Hi
3.2.2. Analysis of HSA O-Glycosylation in the Samples from a PAOX1 B) Pepde 3
Based Process Compared to PGAP

Total amount of glycosylation (%)

40
Fast growth rate
Methanol fed batch fermentation was designed in a way to ac- Slow growth rate
30
complish the same slow growth rate as it was used in the glucose
fed batch fermentation. Four high-producing P. pastoris CBS7435
20
strains with the HSA gene under the control of PAOX1 , were cul-
tivated at the same slow growth rate of 𝜇 = 0.05 h−1 . HSA was
10
purified, digested and analyzed in the same way as it had been
done with previous samples.
0
The results showed that the number of glycosylated peptides
n

n
s io

sio

s io

sio
and the degree of peptide glycosylation depends on the type of
es

es
es

es
pr

pr

pr
pr

process applied. Besides peptides 2, 3, and 6, which were carrying

Ex

Ex

Ex

Ex
A

A
A

A
HS

HS
HS

HS
O-glycans in both the PGAP and PAOX1 based processes, the anal-
h

h
w

w
g

g
Lo

Lo
Hi

Hi

ysis of the samples obtained by PAOX1 fermentation showed the

presence of three other glycosylated peptides (1, 4, and 5) listed
in Table 2. Also, it was observed that the number of Man units
in the glycosylated peptides 2 and 3 in the PAOX1 samples was
higher than in the samples produced on glycose. In the PAOX1
samples, each of these peptides had glycans with up to 8-Man.
However, like in the PGAP samples, the glycoforms with 1, 2, and
3 mannoses were the most abundant, while the abundances of all
longer glycoforms were <1%, except in case of peptide 2, where
3.8% of the peptide was found as the 5-Man glycoform. Regard-
ing peptide number 6, it also had only 1-Man unit, with the gly-
cosylation abundance <1.5% (data not shown). The three other
peptides found to be glycosylated only in the PAOX1 samples car-
ried either one Man (peptides 1 and 5) or 4-Man (peptide 4).
The glycosylation levels of peptides 2 and 3 obtained from
the PGAP and PAOX1 produced samples, were statistically com-
pared by using an unpaired student’s t-test. The differences were
considered significant for p < 0.05 (Figure 3). According to this
analysis, the abundance of O-glycosylation of HSA was signif-
icantly higher when it was produced under PAOX1 , than under
PGAP . Finally, it should be mentioned that peptide 3 was always
the most highly glycosylated peptide, with a site occupancy up
to 32.8% (if produced by PGAP process) or 42.7% (PAOX1 process)
(Table 3).
4. Discussion
To understand O-glycosylation of recombinant proteins pro-
duced by P. pastoris in more detail, we organized this study in
Figure 2. Comparison of the total glycosylation amount of the two most
highly glycosylated HSA peptides. A) peptide 2, B) peptide 3, obtained in
fast (blue) and slow (red) PGAP fermentations. The results are presented
as mean ± SD. The number of samples is n = 4 for all except the high
producers from the slow growth rate, where n = 3. The reason for this is
that the protein was degraded during the production process in one of the
replicates. Statistical analysis was performed by GraphPad Prism (Graph-
Pad, La Jolla, CA, USA). To compare the total glycosylation amount of the
two most glycosylated peptides in PGAP fermentations, the differences be-
tween sample means were statistically examined by ANOVA, with Sidak
post-test to adjust the p-value for multiple comparisons. Differences were
considered significant for p < 0.05. No significant difference in the glycosy-
lation amount was found neither between producers nor between different
processes.
two different parts. First, we analyzed sixteen pharmaceutically
relevant proteins produced in our laboratory, to get an overview
of the O-glycan positions and their lengths. Then, based on
these data, we selected the most glycosylated protein as a model
to examine the impact of different cultivation strategies on the
glycan composition.
Our analysis of the different model proteins showed that the
longest O-glycans produced by P. pastoris were oligosaccharides
of five mannose residues. However, when human serum albu-
min was produced in the CBS7435 strain in different bioreactor
cultivations, glycans were ranging from 1–8 mannose residues,
which showed that P. pastoris produces longer glycans as well.
Previous studies[39,40] suggested that P. pastoris strains produce
proteins with up to 6 Man residues. Reports on O-glycans longer

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Peptide 2 Peptide 2
*
%)

%)
40 40
p = 0.1140 GAP - slow growth rate p = 0.0447 GAP - fast growth rate
a mount of glycosylation

a mount of glycosylation
AOX - slow growth rate
(

AOX - slow growth rate

(
30 30

20 20

10 10
Total

Total
0 0
GAP AOX GAP AOX

Peptide 3 Peptide 3
** *
%)

%)
50 p = 0.0071 50 p = 0.0312
GAP - slow growth rate GAP - fast growth rate
a mount of glycosylation

a mount of glycosylation
AOX - slow growth rate AOX - slow growth rate
(

(
40 40

30 30

20 20

10 10
Total

Total
0 0
GAP AOX GAP AOX

Figure 3. Comparison of the total glycosylation amount of two most highly glycosylated HSA peptides. A) peptide 2, B) peptide 3, from PAOX1 versus
PGAP fermentations. The results are presented as mean ± SD. The number of samples for PGAP fast growth rate is n = 4, while for the slow growth rate
PGAP fermentation and the samples from PAOX1 fermentation n = 3, due to protein degradation in one of the replicates. To compare the total glycosylation
amount of the two most glycosylated peptides from PAOX1 versus PGAP fermentations the differences between sample means were statistically examined
by an unpaired Student’s t-test. Differences were considered significant for p < 0.05. The total glycosylation amount is significantly higher in the samples
produced under the PAOX1 .

Table 3. Percentage of a non-glycosylated fraction of the HSA peptides obtained from different processes, with the standard deviations. GAP F-H and
GAP F-L—samples from the PGAP fast growth rate fermentation, high producer strain (H) and low producer strain (L); GAP S-H and GAP S-L—sample
from the PGAP slow growth rate fermentation, high producer strain (H) and slow producer strain (L), AOX S-H—samples from PAOX1 slow growth rate
fermentation of the high producer strain. Values of peptides that were never glycosylated in the given conditions are indicated in italics, while those
significantly glycosylated (p-value > 0.05) are marked in bold. It should be noted however that also for the other conditions glycosylation of peptide 6
was identified in some samples.

Peptide GAP F-H GAP F-L GAP S-H GAP S-L AOX S-H

1 100% 100% 100% 100% 99.4% ± 0.00

2 89.7% ± 0.11 89.4% ± 0.09 78.7% ± 0.05 78.1% ± 0.04 71.4% ± 0.02
3 74.5% ± 0.09 78.7% ± 0.10 67.2% ± 0.02 72.6% ± 0.05 57.3% ± 0.01
4 100% 100% 100% 100% 93.6% ± 0.03
5 100% 100% 100% 100% 99.7% ± 0.00
6 99.3% ± 0.01 98.7% ± 0.02 99.4% ± 0.01 99.0% ±0.01 98.6% ± 0.00

than six mannoses are rare. To our best knowledge, glycans with
11 mannoses have been observed solely by O’Learya et al.[41]
Duman et al.[42] and Gomathinayagam et al.[43] claimed
that the major form of P. pastoris O-glycans are dimeric and
trimeric mannoses. In our case, the glycoforms with one and two
mannoses were the most abundant. Apte-Deshpande et al.[44]
observed that protein produced by P. pastoris can appear simul-
taneously in both forms, glycosylated and non-glycosylated. We
observed the same, as illustrated in Figure 1, where the largest
fraction of the shown HSA peptide appears unglycosylated,
while the glycosylated part is a mix of different glycoforms.
As the O-glycosylation amount and the glycan length is dif-
ferent in different recombinant proteins, the question arises
whether the cultivation process has an impact on it as well. It
has been shown in mammalian cells that different process pa-
rameters, such as are growth rate, pH, dissolved oxygen and
temperature, can influence N-glycosylation by influencing cell
metabolism.[45–47] In the second part of the present study, we thus
investigated the impact of different cultivation strategies on the
O-glycosylation profile in P. pastoris. The impact of the two most
commonly used cultivation conditions, methanol, and glucose
feed were assessed. PAOX1 and PGAP are typically used promoters
for the expression of recombinant proteins in P. pastoris. PAOX1 is
widely used as it allows the control of expression of recombinant
proteins. However, the requirement of methanol in PAOX1 pro-
cesses is its biggest disadvantage.[48] It was also shown that the

Biotechnol. J. 2021, 16, 2000266 2000266 (7 of 9) © 2020 The Authors. Biotechnology Journal published by Wiley-VCH GmbH
www.advancedsciencenews.com
overall efficiency of protein production with these two promoters
depends on the type of expressed protein.[49] Rebnegger et al.[50]
demonstrated a positive growth-related product formation when
HSA was produced under the PGAP , while Kobayashi et al.[51]
observed negative growth-related product formation when the
same protein was produced under PAOX1 . We compared the in-
fluence of fast and slow growth rates on O-glycosylation of re-
combinant HSA produced in glucose fed batch fermentations.
Then we compared the obtained results with those acquired in
methanol fed fermentation. Consistently to Rebnegger et al.[50]
we observed higher specific productivity (qP ; a product formed
per biomass and time) in the fast growth rate fermentations and
lower qP in the slow growth rate glucose fed fermentation (Ta-
ble S2, Supporting Information). Regarding the glycosylation, we
did not observe statistically significant differences in the total O-
glycosylation amount among all conditions, but we did observe
a small increase in the glycosylation level at slow growth. Nev-
ertheless, we detected glycans up to 5-Man long at fast growth,
and 4-Man long at slow growth. However, these long glycoforms
were found in some of the biological replicates only. The most
commonly found glycoform was only 3-Man long in both growth
rates. In contrast, the glycosylation level of HSA was significantly
higher when it was produced with PAOX1 . The glycans from the
PAOX1 samples were longer, with up to 8-Man residues, and the
total glycosylation amount was significantly higher. It is maybe
interesting to note that the qP in slow methanol fed fermenta-
tion was higher than that in slow glucose fed fermentation (Sup-
porting information). O-glycosylation in S. cerevisiae was found
to render proteins more hydrophilic[52,53] and may therefore help
to prevent aggregation at higher production rates. It is tempt-
ing to conclude the potential benefit of a higher degree of O-
glycosylation at high qP .
Taken together, our work presents the impact of two most
commonly used cultivation conditions on the O-glycosylation
level of recombinant proteins produced in P. pastoris. Our results
can be a starting point for further progress in this field. It was
clearly shown that the glycosylation level is higher in a methanol
fed process. As methanol-based processes are frequently used
for protein production, it would be very important to investigate
the impact of methanol on O-glycosylation in more detail. This
may help to understand O-glycosylation machinery of P. pastoris
better, and to develop a strategy to avoid or adapt this type of
glycosylation for biopharmaceutical production.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
The authors want to thank Roland Prielhofer and Corinna Rebnegger
for making the PGAP background construct used in this study and also
Jonas Burgard, Martin Dragosits, Markus Buchetics, Gabriele Wilt, Fabian
Schneider, Artur Schuller, Martin Nagl, and Marion Rothmüller who either
constructed P. pastoris strains secreting the model proteins or provided
samples of model proteins for glycan analysis. The authors are also grate-
ful to Gordana Wozniak-Knopp for providing the ÄKTA lab-scale protein
purification systems, where all the samples were purified. Helena Her-
rmann kindly advised on statistical analysis. This work has been supported
Biotechnol. J. 2021, 16, 2000266 2000266 (8 of 9)
www.biotechnology-journal.com
by the Austrian BMWD, BMVIT, SFG, Standortagentur Tirol, Government
of Lower Austria, and ZIT through the Austrian FFG-COMET-Funding Pro-
gram. EQ-VIBT is acknowledged for providing mass spectrometry and fer-
mentation equipment.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available from the cor-
responding author upon reasonable request.
Keywords
fed batch fermentation, human serum albumin, O-glycosylation, specific
productivity, yeast
Received: June 3, 2020
Revised: September 9, 2020
Published online: October 8, 2020
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