Development of Simple, Rapid & High-Throughput
Glycoanalytics for Biopharmaceuticals
Introduction

MethodGlycosylation is a post-translational modification that
can influence the safety and efficacy of biologic drugs,
making it a Critical Quality Attribute for biotherapeutic
manufacturing. It is typically characterised using lectins(bioaffinity proteins that recognize and bind specificglycan structures) for HPLC and MS-based analysis.
However, these methods have limited throughput andrely on plant-based lectins that lack specificity andare difficult to produce recombinantly. GlycoSeLect’sRecombinant Prokaryotic Lectins (RPLs) offer superior
specificity, consistency, and scalability over plantbased lectins to streamline the analysis of intact glycosylated biomolecules. By integrating RPLs into the Sartorius Octet system for glycoanalysis of biotherapeutics
provided by Allergan Biologics, and assay validation byCPI, this project aimed to develop a novel platform forsimpler, faster glycoprofiling.

Two biopharmaceutical samples, follicle stimulating hormone (FSH) and the Fc fusion protein Eylea, wereselected for glycoanalysis using the Sartorius Octet, a label-free detection system based on biolayer interferometry. Briefly, RPL-Gal1 and RPL-Sia1 were immobilised on biosensors via the biotin-streptavidin interaction
and used to monitor terminal β1-4 galactose and sialic acid structures, respectively. Assay robustness wasassessed by determining the linearity, repeatability, and percentage spike recovery of each assay.

Figure 1 (A) – Octet red 384; (B) – Octet red 384 stage; (C) –over time graph; (F)Binding rate over concentration graph

AMethod Overview(A) The Octet biosensor tipFabBHeavy chain
Light chain

Fc

Octet biosensor (SAX)

Recombinant Prokaryotic
Lectin (RPL)mAbGlycans
mAbdisplaying
displaying
Glycans(B) RPL immobilisation protocol
Step

BufferDuration

Agitation

Baseline

TBST

60sec

1000rpm

Loading

RPLs (at20µ/ml)

600sec1000rpm

Wash

TBST

180sec1000rpm

C(C) Concentration ranges evaluated for each RPL-Biopharmaceutical pairLinearity range(µ/ml)

Spike recovery
Concentration (µ/ml)

Repeatability
Concentration (µ/ml)

Gal1-FSH

500-7.850, 25, 12.5200

Gal1-Eylea

500-15.6(≥15.6)

N/A

200

Sia1-FSH

150-1.2

15, 7.5, 1.950

Sia1-Eylea

250-7.8

N/A

100

RPL- Biopharmaceutical pairanalysis and isolation of intact glycoproteins.
RPLs are much
more specific,
profile must be characterised
and monitored
throughoutconsistentproduct developm
manufacturing
processes.
Lectinsareto
bioaffinity
proteins
that can recognize
and scalable than their plan counterparts. This
projectaims
developa novelResults
to specific glycan structures on intact glycosylated biomolecules and can theanalytical platform to advance therapid analysis of the glycosylation profiles ofused for the analysis of these molecules. Currently available lectins are predB
biotherapeutics. Using GlycoSeLect
intolack
ForteBio’sbiosensor
plantRPLs
based.integrated
These, however,specificity, arestructurallyplatform
complex (glycoSpike
RecoveryLinearity
Repeatability
and difficultby
to produce
recombinantly.
we will analyse biotherapeutics
providedAllergan
Biologics.GlycoSelect’s Recombinant Prokaryo
Concentration

et red 384 stage; (C) – Octet biosensor tip; (D) 96 well sample plate; (E)Binding
concentration graph

The linearity of FSH (β1-4 galactose) analysis
was determined by measuring the binding rate
two replicates (n=2) at 8 concentrations andCofdemonstrated an acceptable range for theassay of 7.8 to 500 µg/ml (%CV <20%).
Data is shown in Figure 1Methods

A(RPLs) are superior glycoselective molecules that facilitate simple and effectanalysis and isolation of intact glycoproteins. RPLs are much more specific, cand scalable than their plan counter parts. This project aims to develop a noanalytical platform to advance the rapid analysis of the glycosylation profiles
Conclusion/Summary
biotherapeutics. Using GlycoSeLect RPLs integrated into ForteBio’s biosenso
Known
Well
Well 2 Biologics.
Avg.
will ofanalyse
providedby 1Allergan
Octetweanalysis
FSH (β1-4biotherapeuticsgalactose)% Spike

Percentage spike recovery tests to investigate
Assay repeatability and precision was assessed
matrixinterference
duringquantitation
by measuringsix6 replicatesof FSH
at 200µg/showing the standard
Figure– Analysis of FSH
and Eylea
by Octet
curve obtainedwith with
Gal1 andSia1 RPLs-SAX of FSH(β1-4galactose)
showedrecoveries
ml. Data aresensors
shownin dots=standard
Figure 1, withconcentra(bluecurveand red dots= 6 replicates of middle of standard curve + experimental points).of 80-120%
with a %CV of <10%, indicating that glycan detion results in Figure 2. The assay showed good
tection is not compromised by the presence ofrepeatability with a %CV of <10% for FSH quanthe sample buffer (Figure 3).
titation.

Conc. Avg.

%CVThesuitability of a bynovelAllergan
high-throughput
methodfor thedetectionof glycans
Conc.
Conc.
Conc.
Conc.
(µ/ml)
Two biopharmaceuticalsamples
provided
wereanalysed
using
thein%CV
Octet
Recovery(µg/ml)(µg/ml)(µg/ml)(µg/ml)Repetition
FSH
at200
µg/ml
purifiedbiopharmaceuticalsampleshas
been
assessed.
The
method,which
usesthe500.0
500.2
3.65
12.5 to detect
13.8
14.1 β1-4Samples
14.0
1.52
112%
FSH (follicle
stimulatinghormone)
Eylea
(Fc
fusionprotein).
1190.4 Sia1
Octet
system with Gal1andand
RPLs-SAX
sensors
terminalgalactose
250.0platform:
250.0
2.55
25.0
27.5
27.0 and27.3
1.30for 109%
andsialic acid structures,197.7
demonstrates high intra-assay
repeatability
linearity
2 Methods
125.0125.1
2.44
were
testedforterminal
β1-4galactoseandsialicacid
structures
using
RPL-Gal1
50.0
52.1
52.0
52.1 indicate
0.14 and104%
thespike recoveriesof 80-120%
3 supplied FSH and Eylea
198.4samples. Percentage
62.5
62.4
3.00
that
glycan detection
not compromisedby the presenceof the sample
buffer.
4200.931.3 RPL-Sia1,31.3
0.53
respectively.
robustnessof theis Octet
quantitation
assay
developed
at using thFigure 3. Percentage
spikerecovery for FSH (β1-4galactose) analysis
was determinedby the binding rateTheofTwobiopharmaceuticalsamples
providedbyAllergan
wereanalysed
5205.8galactose) from a matrix comprising a mix of
1.71
oncentration 15.6
levels:500 µg/ml,15.7
250 µg/ml,125µg/ml,
FSH
formulation
andbuffer.
GlycoSeLect
foreach
RPL-BiopharmaceuticalpairwasassessedatCPI
byassay
verifying
the SaThis
project consortium
brought
togethertheexpertise
andbuffer
resourcesof
CPI,
platform:
FSH(folliclestimulating
hormone)andEylea(Fc
fusion protein).
6
197.6
7.8 The acceptable1.07 range of
5.6 µg/ml, 7.87.8µg/ml and 0 µg/ml.
GlycoSeLect
UK Ltd,Scienceand Allergan
Ltd, toaddress
Conc.
Avg.
198.5 Pall Life
were tested
forForteBio
terminal
β1-4galactose
andBiologics
sialic acidstructuresusing RPL-G
0 linearity, 0.0
0.00
g/ml (%CV <20%).
repeatability
and percentage
spikerecoveryof
eachassay.
theneedfornew
glycoanalytical
approaches
tomeettheneedsof
thegrowing%CV
2.53
FSH (µg/ml)

B) – Biotinylated RPL’s on-line immobilisation method; (C) – Concentration ranges
utical pair.

BB

RPL-Sia1, respectively.
The robustness of the Octet quantitation assay develo
biotherapeutics
market.

AFigure 2. FSH (β1-4 galactose) concentration results.

GlycoSeLect for each RPL-Biopharmaceutical pair was assessed at CPI by ver

C spike
B Acknowledgements
linearity, repeatability and percentage
recovery of each assay.
Contact
A

Cristina Blood
The Centre for Process Innovation (CPI)
1 Union Square, Central Park
Darlington
County Durham
DL1 1GL,UKC

B

Email:

www.uk-cpi.com

Figure1. Linearity
determination
for FSH (β1-4galactose).
for FSH (β1-4galactose);(B) – Analysisof FSH (β1-4 galactose)
by Octetshowingd curve andred dots= 6 replicatesof FSH at data
200µg/ml+ experimentalpoints ).
(A) Average
concentration
and%CV (B) Standardcurve (blue dots) and experimental points / six replicatesof FSH at 200µg/ml (red dots).

Dcristina.blood@uk-cpi.com

Octet red 384

ED

Octet biosensor tipEFF

www.glycoselct.com

Conclusion/Summary
Conclusion/SummaryCB

Furthermore, the Octet method demonstrated percentage spike r
120%, high intra-assay linearity and repeatability for both the sup
Results/Conclusion
BAEylea samples with Gal1 and Sia1 RPLs-SAX sensors. These sensor
suitabilityof a novel
methodtheTheOctet
system to detectterminalhigh-throughput
β1-4 galactose and sialic
acid’s on-line immobilisation method; (C) – Concentration ranges
FSHand suitability
Eyleabiopharmaceuticalsamples.
6 shows
the standard
obtainepurifiedsamples
hascurve
beenassTheofFigurea novelhigh-throughput
metho
L’s on-line
method;(C)FSH– Concentration
rangesSimilarimmobilisation
spike recoveries wereseen for(sialic acid), and forboth Eylea assays, which all demonstrated high intra-assay linearity and repeatability. Figure 6 shows thesamples.
standard curves obtained for these samples.
Figure 6 – andAnalysis ofFSH and Eyleaby Octet
the standardOctet
systemwith Gal1
Sia1
RPLs-SAX
sensorpurifiedbiopharmaceuticalsampleshasshowing
been
ass
sensors (blue dots=standard curve and red dots= 6 replicates of mC
FE
Figure 1 (A) – Octet red 384; (B) – Octet red 384 stage; (C) – Octet biosensor tip; (D) 96 well sample plate; (E)Binding
over time graph; (F)Binding rate over concentration graphandsialic
acidwith
structures,
demonstrates
highintra
Octet
systemGal1 andSia1 RPLs-SAX
sensortheand Eyleademonstrates
samples. Percentage
andsuppliedsialic acidFSH
structures,
high intrs
thatglycan detection
not compromisedby thethe supplied
FSHConclusion/Summaryand isEylea
samples. Percentage
alysis was determined by the binding rate ofthat glycan detection
is notof compromised
bymethotheThe suitability
a novel high-throughput
Figure 2 (A) – Method Overview; (B) – Biotinylated RPL’s on-line immobilisation method; (C) – Concentration ranges
levels:500determinedµg/ml,µg/ml,125 µg/ml,
tested for each250
RPL- Biopharmaceuticalpair.
purified biopharmaceuticalsamples has
been
ass
alysis wasbythe binding
rate ofThis project consortium
brought
togethertheexpOctet
systemcurve.
with Gal1 and Sia1 RPLs-SAX sensor
FigureFigure
6andAnalysis
6 –0
Analysisof
FSHofandFSH
Eylea
andEylea
by Octetby FSH
Octet
showing
showing
the standard
thestandard
curve
curve
obtained
obtainedwith withwithGal1
withpoints
andGal1Sia1andRPLs-SAX
Sia1
RPLs-SAXat the middleFigure
4.– Standard
curves(bluedots)
forandEylea.
Reddots
represent
experimental/ six
replicatesof standardµg/mlµg/ml.
The
acceptable
range
oflevels:
500
250
125
µg/ml,
ctet
red 384 stage;(C)µg/ml,– Octetbiosensor
tip;µg/ml,(D) 96
well sample
plate;(E)Binding
GlycoSeLect
UK Ltd,ForteBio
Pall together
LifeScience
sensors
sensors(blue (bluedots=standard
dots=standard
curvecurveand redanddots=
red dots=
6 replicates
6 replicatesof middleof middleof standardof standard
curvecurve+ experimental+ experimentalpoints).
points).
and sialic
acid
structures,demonstrates
highandintr
This
project
consortium
brought
theexper concentration graphResults20%).
the supplied FSH and Eylea samples. Percentage
8 µg/ml and 0 µg/ml. The acceptable range oftheneed for new
glycoanalyticalm
GlycoSeLect
UK Ltd,ForteBio Pallapproaches
Life SciencetoandB
that glycan detection is not compromised by the20%).
The linearity of FSH (β1-4 galactose) analysis was determined by the binding
rate of for new
biotherapeutics
market.
the needglycoanalytical approaches to mtworeplicates(n=2)
at8concentrationlevels:500µg/ml,250µg/ml,
125µg/ml,
Conclusion/Summary
Acknowlegments
This project consortium brought together the exp
Conclusion/Summary
Conclusion/Summary
biotherapeutics
market.
62.5 µg/ml, 31.3 µg/ml, 15.6 µg/ml, 7.8 µg/ml and 0 µg/ml. The acceptable
range of
GlycoSeLect UK Ltd, ForteBio Pall Life Science andConcentration

Acknowledgements
Acknowledgements

The integrationof RPLs
Sartorius
Octet
systemrepresents a novel,
This project consortium brought together the expertise and resources of
the assay
was into7.8 theto 500µg/ml(%CV<20%).
the byneedfor
new glycoanalytical
approaches
to mFigure
6 – Analysis ofUK
FSHandForteBio
Eylea
Octet
showingthe(nowstandard
obtained with withhigh-throughput method for detecting glycans in purified biopharmaceutical
CPI,
GlycoSeLect
Ltd,
PallLifeSciencepart curveof Sartorius
TheThesuitability
suitabilityof aofnovel
a novelhigh-throughput
high-throughput
methodmethodfor thefor thedetection
detectionof glycans
ofsensors
glycansininABbiotherapeutics
market.
curve
andtored
dots= 6thereplicatesof newmiddleof standard curve + esamples. Assay robustness is validated by high linearity, repeatability, andGroup)(blueand dots=standard
Allergan Biologics
Ltd
addressneed forglycoanapurified
purifiedbiopharmaceuticalbiopharmaceuticalsamplessamples
has
has
been
been
assessed.
assessed.
TheThemethod,method,
which
which
uses
uses
thethepercentage spike recovery, and confirms the viability of this approach forlytical methods within the growing biotherapeutics market.
simpler,
fasterglycoprofiling.
Octet
Octet
systemsystemwithwith
Gal1Gal1andandSia1Sia1
RPLs-SAXRPLs-SAX
sensors
sensors
to detect
to detectterminal
terminal
β1-4β1-4
galactosegalactoseC

Acknowledgements

andandsialicsialic
acidacid
structures,
structures,
demonstrates
demonstrates
highhigh
intra-assay
intra-assay
repeatability
repeatability
andandlinearity
linearity
for forthe thesuppliedsupplied
FSHFSHandandEylea
Eylea
samples.
samples.
Percentage
Percentage
spike
spike
recoveries
recoveriesof 80-120%
of 80-120%
indicate
indicate
GlycoSeLect
thatthatglycanglycandetection
detection
is notis notcompromised
compromisedby theby thepresence
presenceof theof thesample
samplebuffer.
buffer.
The suitability of a novel high-throughput method for the detection(B) – Biotinylated RPL’s on-line immobilisation method; (C) – Concentration ranges
This(B)
Thisprojectproject
consortium
broughtbrought
together
togetherthetheexpertise
expertiseandandresources
ofby Octet
CPI,of showing
CPI,
euticalpair.
purifiedbiopharmaceutical samples has been assessed. The metho
Figure3consortium(A)– Linearity
determination
for FSH(β1-4galactose);(B)– Analysisof FSH(β1-4resourcesgalactose)ose);– Analysis
ofFSH(β1-4galactose)
byOctetshowing
the standard curve (blue dots=standard curve and red dots= 6 replicates of FSH at 200µg/ml + experimental points ).
GlycoSeLect
UKForteBio
ForteBio
PallLife LifeScienceScienceand).and
AllerganAllergan
Biologics
Biologics
Ltd,Ltd,
to address
to address
Octetsystemwith Gal1 and Sia1 RPLs-SAX sensors to detect termins=GlycoSeLect
6 replicatesofUKFSHLtd,at Ltd,200µg/ml+Pall
experimental
pointsose); (B) – Analysis of FSH (β1-4 galactose) by Octet showing
GlycoSeLect
the theneedneedfor newfor new
glycoanalyticalglycoanalytical
approaches
approaches
to meetto meetthe theneeds
needsof theofand
thegrowing
growingsialicacid structures, demonstrates high intra-assay repeatabil
ts= 6 replicates of FSH at 200µg/ml + experimental points ).
biotherapeutics
biotherapeutics
market.
market.
the supplied FSH and Eylea samples. Percentage spike recoveries othat glycan detection is not compromised by
the presence of the sa
www.glycoselct.com
4 galactose) analysis was determined by the binding rate ofConclusion/Summary