IPDO INTERNAL JOUNRAL

FLUORESCENCE CORRELATION SPECTROSCOPY: A SENSITIVE TOOL TOWARDS

IDENTIFICATION OF EARLY STAGE INSTABILITIES IN PEPTIDE AND PROTEIN BASED
FORMULATIONS

Sounik Saha, Bankanidhi Sahoo, Archan Dey & Atanu Sengupta

ACT Lab

ABSTRACT:
Protein/peptide-based formulations are susceptible to degradation in various ways including misfolding and
aggregation. This becomes a major challenge in the formulation of peptide or protein-based drugs. So, it is
desirable to study such instability of the above-mentioned formulations earlier in the life cycle of the
formulation. In this work, we have evaluated the scope of a single molecule technique to detect early
instabilities in a peptide formulation. The single molecule fluorescence technique, Fluorescence Correlation
Spectroscopy (FCS), has been used in various applications including protein aggregation. This work evaluates
the applicability of (FCS) to detect early aggregation and kinetics of evolution of aggregates for
peptide/protein based drug molecules. This work establishes FCS as a more sensitive tool over traditional
fluorimeter based detection of peptide aggregation. It also shows that early detected aggregates by FCS can
be correlated to in vitro biological activity to a certain degree.

INTRODUCTION:
Peptide and protein based formulations are inherently unstable due to the susceptibility of the
peptide/protein backbone to misfold and aggregate. These undesirable aggregation is one of the major
concerns in formulating peptide/protein based formulations. This impacts efficacy of the drug and can elicit
severe immune response which presents a safety concern as well. Hence detection of such aggregation at a
very early stage in the life cycle of a peptide/protein based formulation is of paramount importance.
Traditional methods to detect protein aggregation employ techniques such as DLS, Thioflavin-T based
fluorimeter assay, Multi-angle light scattering, Analytical ultracentrifugation etc. However, all these
techniques are either limited by their selectivity and/or sensitivity to detect aggregation at an early stage of
product life cycle. For e.g., light scattering is not very sensitive for the low molecular weight aggregates
particularly formed early in the aggregation pathway and present in sparse population. Fluorescence
techniques, however, can have sensitivity at single molecule level and can be used to detect these sparsely
populated species, since the sensitivity can go down to sub-nanomolar concentration. One such technique
is Fluorescence Correlation Spectroscopy (FCS) which is currently finding use to detect and derive kinetics of
evolution of protein aggregates. FCS is a highly sensitive analytical technique which provides information on
diffusion times (useful for particle size calculation with no lower limit), conformation and concentration of
fluorescent molecules (Methods and Protocols, Methods in Molecular Biology, vol. 1345). The high
sensitivity of FCS helps to monitor phenomenon such as self-association at concentration regimes of sub-
nanomolar thus enabling it to monitor aggregation at the single molecule level.
An important molecule of interest is Liraglutide, which is a Glucagon-like peptide analogue (GLP-1)
(Fig. 1). GLP-1 is an aggregation prone peptide and has been thoroughly studied using various
physicochemical techniques (J. Am. Chem. Soc., 2016, 138 (50), 16259–16265; FEBS Lett. 2002, 515, 165-70).
Liraglutide poses similar challenges as well in terms of aggregation behavior as the peptide backbone is not
significantly modified from GLP-1. Thus, process control at API stages, limiting impurities and adopting
formulation strategies to circumvent aggregation induced potency loss and immunotoxicity purports
methods to detect aggregated states at sufficiently early in the lifecycle of product development. Here we
show the applicability of a novel FCS setup known as cuvette FCS (Ref: Biophys J. 2018; 115(3), 455-466) (Fig.
2) which is a leap forward for detection of early fibrils/aggregates of liraglutide and determine any instability
of the formulated product.

Figure 1: Mechanism of Action of Liraglutide (Note: Formation of Fibrills is undesirable in this whole cycle)

Figure 2: Novel cuvette FCS set up used in this work

Results and Discussion:

Instrument & methodology: The novel cuvette FCS setup at Tata Institute of Fundamental Research (TIFR)-
Hyderabad (Biophys J. 2018; 115(3), 455-466.) addresses all sensitivity issues associated with fluorimeter-
based experiments to detect aggregation. Further all experiments possible in traditional fluorimeter can be
performed in cuvette FCS setup with much higher sensitivity and at single molecule level. Besides these,
this instrument has the unique ability to study protein aggregation in a label free condition i.e. there is no
need to attach a fluorophore to the protein/peptide in certain cases. This FCS uses lasers line suitable for
Thioflavin T excitation which undergoes fluorescence enhancement upon binding to protein aggregates,
thereby makes it possible to detect only the aggregates. The nonbound Thioflavin T is very weakly
fluorescent, so does not interfere with the measurements. In simple words, this can detect only the
aggregates selectively.
FCS vs fluorimeter based assay: To determine if FCS has the ability to detect peptide aggregation at a
much earlier stage than traditional fluorimeter based Thio-T assay, Victoza (innovator) was studied using
the methodology as stated at the end in the procedure section. In parallel, Thio-T fluorescence was also
recorded for 324 hours using a fluorimeter. The results show that over the period of 92 hours of
accelerating conditions insignificant increase in Thio-T fluorescence takes place in the innovator sample as
measured using fluorimeter (Fig. 3, A). A very slight increase in Thio-T fluorescence signal happens at 162
hrs (~7 days) but at 324 hours (~14 days) notable changes in Thio-T fluorescence is observed under
accelerated conditions.

Figure 3: Comparative Thio-T fluorescence monitoring in innovator sample as monitored through

fluorimeter and FCS in parallel to compare aggregate detection ability of FCS and fluorimeter.

Contrastingly, the innovator batch sample which appear to be free of any significant aggregation (confirmed
from sporadic Thio-T spikes in FCS data as shown in Fig. 3, B-F) up to 44 hours in FCS demonstrated notable
increase in Thio-T positive structures at the 92 hour time point indicating that aggregation started post 48

of the sparsely population of the aggregates which does not contribute significantly to the total signal.
However, FCS being a single molecule sensitive technique is able to detect these aggregates as early as
possible. This data indicates FCS it is possible to detect any aggregation events at a much earlier stage than
traditional fluorimeter based Thioflavin-T assay.
Comparative Assessment of innovator and a trial in-house formulation

Figure 5: Dynamics of evolution of peptide aggregates of USRLD and inhouse F-068 batch as
monitored through thio-T staining and subsequent FCS kinetics monitoring for 44 hours.

Fig. 5 shows comparative FCS profiles of innovator batch and a trial inhouse batch. The unfiltered inhouse
batch sample shows the presence of a large number of preformed aggregates (data not shown). So, the
inhouse batch sample was
of aggregation using Thio-T. Nearly similar time points were chosen for monitoring the dynamics of
aggregation for the innovator sample as well. As compared to RLD, the trial inhouse batch (developmental
sample) possesses more Thio-T positive structures which are of relative much larger length scale
considering the relative intensity of the spikes. Over a period of 18 hour no change was observed in the
number of Thio-T positive structures in the trial inhouse batch, however, the next time point of 44 hours
demonstrated large increase in number of Thio-T positive structures indicative of fibrillation. This
fibrillation was further confirmed by imaging using Total Internal Reflection Fluorescence (TIRF) microscopy
(Fig. 6). In contrast, RLD sample showed no Thio-T positive structures at initial time point and there was
insignificant increase in number of spikes up to 44 hour time point indicating peptide aggregation might
not have been induced to a significant degree till 44 hours’ time point under the accelerated conditions.
 Fig. 6: Three representative images of Thio-T stained fibrils formed from inhouse product upon
incubation at 37 C with 300 rpm stirring for 72 hours. Scale bar is 5000 nm.

Batch Sample Analysis and correlation with cAMP assay:

Next we explored if detecting early instability and hence peptide aggregation has any possible correlation
with biological activity measured through cyclic AMP assay (Note: Performed by IPDO bioassay team). To
determine that FCS analysis was performed on three formulations of a single inhouse API batch which were
formulated in three different ways. Table 1 provides the batch number and the formulation strategy for
the three samples.
Table 1: Formulation batches studied
Sl. No. Batch Number Formulation Batches cAMP assay
01 Sample A Heated 80 C for 3 mins 8.4  0.6 M
02 Sample B Heated 50-58 C for 3 mins 14.2  0.8 M
03 Sample C Heated 40-50 C for 3 mins 26.7 ± 2.5 M

Results for the three samples are presented in Fig. 7. As can be seen from Fig. 7, Sample A had the lowest
number of spikes (indicative of preformed aggregates) at t = 0 hr amongst the three samples followed by
Sample B and Sample C. All the samples under accelerating conditions demonstrated increase in number of
spikes up to 92 hrs. The cAMP IC50 data also shows an apparent correlation with the amount of
preformed fibril and instability of the formulations with the Sample A batch with the lowest IC50 being the
most stable of the three formulations and Sample C batch with the highest IC50 being most unstable with
respect to fibrillation amongst the three batches. This indicates a certain degree of correlation of
biological activity as measured by cAMP assay and this gives positive indications that we can use
quantitation of aggregates as an alternate measurement in correlation to cAMP assay.
 Fig. 7: Dynamics of evolution of peptide aggregates in three formulation batches as monitored
through Thio-T staining and subsequent FCS kinetics monitoring for 92 hours.
CONCLUSION:
The FCS setup employed here is able to detect early aggregation in Peptide and protein based formulations
which traditional Thioflavin-T assay fails to capture. This work also further showed correlation between
bioactivity and aggregation to a certain degree. Overall this work demonstrated the usefulness of single
molecule based fluorescence technique towards development of stable peptide/protein based
formulations.

Materials and Methods

 As such RLD/inhouse sample was allowed to equilibrate to room temp.
 Approximately 2 ml of equilibrated sample was transferred to a quartz cuvette in the FCS setup.
 Initial FCS data for the samples was recorded immediately after addition of Thio-T to a final
concentration of 2 µM under stirring.
 In case, aggregates are observed in the initial samples (in case of inhouse sample, preformed
aggregates are very common), the samples are filtered using 0.2 um syringe filters and FCS
measurement done immediately which is considered as t=0 measurement.
 The sample was recovered and transferred to a clean glass tube containing a magnetic bead and
stirred at 37 ºC with 300 rpm speed.
 At various time points sample was withdrawn for measurement by FCS.

ACKNOWLEDGEMENTS
We are thankful to Prof. Kanchan Garai from Tata Institute of Fundamental Research, Hyderabad for his
support with the FCS setup. We are also grateful to all the CFTs in Liraglutide project for their support.

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