This document describes two projects undertaken by the author to develop novel analytical methods. Project One involved developing a cation/chromatofocusing HPLC method to analyze charge variants in StreptAvax vaccine proteins. Testing showed the method could monitor changes in protein variants due to thermal stress. Project Two aimed to quantify residual guanidine HCl and DTT in purified protein solutions, which is important for protein refolding and meeting safety requirements. The author developed these methods to address challenges in analyzing pharmaceutical proteins.

1. Dennis L.Thireault,MS
Senior RA (cGMP) Protein
Biochemistry
GSK Biologicals Background

2. Novel Method Development
(cGMP)
• Project One: Cation/Chromatofocusing
(CFX) of StreptAvax Proteins
• Project Two: Methods to quantify residual
guanidine HCl (GnHCl) and 1, 4-
dithiothreitol (DTT) in purified protein
solutions
• These were just some of the many novel
new method development I did here

3. Project One: CFX Analysis of
Streptavax Proteins
• Background
– Vaccine and protein drug formulations are between pH 4.5 – 7.5
– In this pH range you generate charge variants
• Deamidation-N,Q
• cyclic imide formation-N, D
• iso-Asp formation-N, D
• oxidation-N
• The StreptAvax proteins have a number of putative hot spots
– Asn-Gly, Asp-Gly, Asn-Ser, Asn-Thr and Asn-Asp (Pearlman
and Wang)
• Some of these sites have been identified by previous peptide
mapping LC/MS experiments on the StreptAvax proteins.
• StreptAvax drug substances has a large number of total Asn and
Asp

4. Project One: CFX Analysis of
StreptAvax Proteins
• The purpose of this project will be to develop a
routine HPLC assay to isolate and quantitate
charge variants in the StreptAvax proteins.
• Previous analysis of StreptAvax drug substance
lots by IEF-PAGE has revealed multiple pI forms
of StreptAvax protein at time zero of storage for
multiple lots (Table 1). Thermal stress induces
an overall significant shift on these gels to a
more acidic pI (Table 2).

5. Project One: CFX Analysis of
StreptAvax Proteins
Table 1: Calculated pI on Broad and Narrow pH range gels with single 10 µg Load
Sample Theoretical pI
Observed pI
pH 3–7 Conditions
Observed pI
pH 3–10 Conditions
Hexavalent A
Lot 20200173
6.0 6.2
average from doublet bands
6.1
average from doublet bands
Septavalent B
Lot 20200236
5.8 6.2
unfocused band
5.8
unfocused band
Septavalent C
Lot 20200213
5.3 5.7
unfocused band
5.1
Septavalent D
Lot 20200214
5.1 5.3 5.0
BSA 4.7 5.0
unfocused band
5.0
unfocused band

6. Project One: CFX Analysis of
StreptAvax Proteins
Table 2: Proposed Reference Material lots Control versus Thermal Stress
Relative pI from One Analysis
StreptAvax™
Protein
-20o
C
(Control)
40o
C for 6 Days
(Thermal Stress)
Hexavalent A
Lot No. 20200396
pI = 5.9
pI = 5.8
(2 distinct bands)
pI = ~5.3
More diffuse and acidic
2 – 3 bands
Septavalent B
Lot No. 20300077
pI = 6.2
Diffuse band
pI = 5.3
Much more focused and acidic band
Septavalent C
Lot No. 20200470
pI = 5.3
Basic shadow band at constant intensity
(artifact?)
pI = 5.2
Slightly more diffuse but more acidic band
Septavalent D
Lot No. 20300070
pI = 5.3 pI = 5.2
Slightly diffuse

7. Project One: CFX Analysis of
StreptAvax Proteins
• The StreptAvax proteins all have a low pI, and formulated in 1X PBS
• The protein pI<formulation pH
– Cation exchange mode was selected since we are interested in the charge variants
– [WCX] Carboxylmethyl (CM) functional groups could not be used due to low pI
• A Research Associate III previously spent months on this project
– SCX columns (Sulfonic Acid and other functional groups) with different pore sizes
– pH Ranges and Eluting Salt Conditions (Ca2+ > Mg2+ > Na+ > K+ >NH4+) and molarities
• The best result that was achieved is seen above in the chromatogram
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
Hexavalent A CEX

8. Project One: CFX Analysis of
StreptAvax Proteins
• First project when I started at ID Biomedical
– Effort, money and time were spent on it with little success
• I examined all the previous work and concluded
– There was nothing I would have done differently
– Previous efforts had failed even with outstanding work
– Time to think outside of the box – pH Gradient
(Chromatofocusing)
– Only 2 basic functional groups to work with, CM and S
• Never been performed at analytical scale (prep only)
– NPR HPLC Column Technology (Dionex and Tosoh Columns)
• Natural eluting properties of the protein variants and the functional
group resins of column utilizing weak buffer conditons
• NPR unlike Porous columns should generate sharp peaks with
these conditions
• Everything else had failed so why not give it a shot?

9. Project One: CFX Analysis of
StreptAvax Proteins
• HPLC parameters:
• Column: Dionex WCX-10 cation exchange column, 2.1 x 250 mm (10 μm particles, non-porous)
• Amount of sample injected (μl):
• A: 2 (~10 µg)
• Mobile phase A: 10mM Acetate-Phosphate, 5 mM Borate Buffer pH 7.00
• Mobile phase B: 10mM Acetate-Phosphate, 5 mM Borate Buffer pH 8.55
• Flow rate: 0.2 ml/min
• Gradient: 0% to 100% mobile phase B in 100 minutes
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• Hexavalent A.3 L/N 20200396 P/N 40.10324 Acetate-Phosphate-Borate Buffer
• Dionex WCX-10 λ218nm
• -20°C > 24 months Duplicate injections

10. Summary Table of Charge Variant Component Content of Multiple
Hexavalent A lots (stored at -20°C)
Hexavalent A L/N 20200396 (-20C) L/N 20200173 (-20C) L/N 2040288 (-20C)
Charge Variant Name Avg % Area Avg % Area Avg % Area
Component 6' 1.25
Component 5' 2.37 1.21
Component 4' 4.44 3.27 1.16
Component 3' 5.46 4.53 2.08
Component 2 23.79 19.42 17.17
Component 1 62.72 71.58 79.60

11. Project One: CFX Analysis of
StreptAvax Proteins
• The composition of charge variant components
is similar across multiple lots of drug substance
stored at the recommended storage condition of
-20°C
• The major component for the three lots analyzed
averages 72 ± 9% of the total and the secondary
component averages 20 ± 4%
• These numbers were later confirmed by LC/MS
studies, but would this be useful for stress
studies?

12. CFX Hexavalent A.3 L/N 20400288 P/N 40.10324 Acetate-Phosphate-Borate
Buffer Dionex WCX-10 λ218nm
Stressed at +40°C – 0, 48, 72, 96 Hours used to monitor thermal stress
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• Overlay plot of effect of
increasing thermal stress to Hexa
A on CFX-HPLC profile

13. Septavalent B.6 L/N 2040764 P/N 40.10325 Phosphate Buffer - Acetonitrile
Dionex WCX-10 λ218nm -20°C ~ 1 Month
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• Septavalent B.6 L/N 2040764 P/N 40.10325 Phosphate Buffer - Acetonitrile
• Dionex WCX-10 λ218nm -20°C ~ 1 Month
• Septavalent B was already
thermally degraded

14. Septavalent C.2 L/N 20200470 P/N 40.10326 Acetate-Phosphate Buffer
Tosoh SP-NPR λ218nm Stressed at +40°C – 0, 48, 72, 96 Hours
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• Overlay plot of effect of increasing
thermal stress to Septa C on CFX-
HPLC profile
Septa C at time
zero

15. Septavalent D.3 L/N 20300070 P/N 40.10327 Acetate-Phosphate Buffer
Tosoh SP-NPR λ218nm Stressed at +40°C – 0, 24, 48, 72, 96,120
and 144 Hours
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• Overlay plot of effect of
increasing thermal stress to
Septa D on CFX-HPLC profile
Septa D at time
zero

16. Project One Conclusion: CFX
Analysis of StreptAvax Proteins
• Storage or stress of the drug substance at elevated temperatures
induces a dramatic shift in charge variant component content as
illustrated in the chromatographs and table 2 above of material
stored at -20°C or stressed at 40°C. The shift is directly observed
into the minor components with newer components also appearing
as early eluting peaks as observed in the stressed chromatograms.
• The molecular identity of each of the charge variant components
resolved on CFX-HPLC is not yet known. Collection of component
peaks for further analysis will be required to identify the molecular
events that give rise to each of the charge variants observed.
Likewise, the potency or activity of each of the charge variant
components is not yet known. Isolation of sufficient quantity of each
component for animal immunization will be required to assess the
potency of these components.
• A novel high resolution cation/chromatofocusing (CFX) - HPLC
method has been developed and applied to the analyses of
streptAvax drug substances.

17. Project Two: Methods to quantify residual guanidine HCl (GnHCl) and
1, 4-dithiothreitol (DTT) in purified protein solutions
• Guanidine hydrochloride (GnHCl) is a strong chaotropic
agent
– Causes protein structures to be disrupted
– This ability is useful for the denaturizing and subsequent
refolding of proteins
– Used as the first step in refolding proteins or enzymes into their
active form
• In addition to the solubilizing chaotropic agent, the
presence of low molecular weight thiol reagents such as
1, 4-dithiothreitol (DTT) or 2-mercaptoethanol is
generally required.
– reduce inter- and intra-molecular disulfide bonds possibly
formed by air oxidation during cell disruption
– maintain cysteines in their reduced state

18. Project Two: Methods to quantify residual guanidine HCl (GnHCl) and
1, 4-dithiothreitol (DTT) in purified protein solutions
• However, removal of the chaotropic agent and
the reducing agent from the solution containing
the solubilized protein must occur prior to
formation of the native structure in the
physiological conditions needed for refolding.
• Accurate and sensitive monitoring of the removal of
GnHCl and DTT from a therapeutic recombinant
protein drug is necessary to monitor its clearance in
the purification process as well as to meet the safety
and regulatory requirements for human or animal use.

19. Project Two: Methods to quantify residual guanidine HCl (GnHCl) and
1, 4-dithiothreitol (DTT) in purified protein solutions
• Several methods have been developed previously for the
determination of GnHCl.
– Detection of guanidine compounds by the use of paper
chromatography was published by Jones and Thompson in 1963
– GC/ECD or GC/FID procedure is developed in 1977 that
measured the hexafluoroacetylacetonate derivatives of methyl-
guanidine, guanidine and agmatine.
– An ion-exchange chromatographic method measured the o-
phthalaldehyde (OPA) derivatives of amines, guanidines and
hydroxylcinnamic acid amines by fluorescence detection.
– A cation-exchange method with UV detection that measures
GnHCl levels in high salt and protein matrices. However, this
method still requires significant sample cleanup with centrifugal
filters to remove the protein interference at 195nm and also
requires a UV detector capable of accurately monitoring at
195nm to achieve an LOQ of 250 ng/mL.

20. Project Two: Methods to quantify residual guanidine HCl (GnHCl) and
1, 4-dithiothreitol (DTT) in purified protein solutions
• Develop novel HPLC methods for the
determination of GnHCl and DTT analyses
– Rapid and Accurate measurement
– Ability to detect < 1ppm (ug/mL)
– Sample matrices containing high salt and
protein
– Utilize direct injection with no sample cleanup
– I examined what had been done previously,
and decided to use HPAEC-PAD.

21. Project Two: Methods to quantify residual guanidine HCl (GnHCl) and
1, 4-dithiothreitol (DTT) in purified protein solutions
• The development of these methods on HPAEC-PAD
grew out of my personal experience with carbohydrate
and amino acid analysis which suggested this
chromatography system would provide excellent
resolution and sensitivity for GnHCl and DTT.
• Outside of amino acid analysis, carbohydrates analysis,
and oligosaccharide mapping, HPAEC is under utilized
• Electrochemical detection under anionic conditions is
specific for functional groups that are oxidized at the
detection voltage. Current from amine or thiol oxidation
is measured at the first potential E1. The second, E2,
cleans the gold electrode. The third potential, E3,
reduces the gold oxide back to gold.

22. Project Two: Methods to quantify residual guanidine HCl (GnHCl) and
1, 4-dithiothreitol (DTT) in purified protein solutions
• Because electrochemical settings specific for the detection of DTT
or GnHCl were unavailable, voltammetry waveforms were
generated. These waveforms were used to develop a pulse
amperometry (PAD) detection method. The optimized PAD
parameters for the detection of both DTT and GnHCl are E1 at
+0.15V (420ms), E2 at +0.80V (180ms), and E3 at -0.15V (360ms).
• The signal-to-noise ratios and resolution of both molecules are very
reasonable using the described methods, as illustrated by the
baseline and peak shapes of GnHCl and DTT shown in
representative chromatographs of a BSA solution with and without
spiked GnHCl and an F1V solution with and without spiked DTT.
• A simple change in the mobile phase was all that was required for
analysis – the column (Dionex PA-1) remains the same (MA-1, PA-
10, PA-100, and PA-200 were also evaluated)
– 100 mM NaOH Isocratic for GnHCl analysis
– 10 mM NaOH Isocratic for DTT analysis

23. Voltammetric response for (A) GnHCl, 100 μg/mL in 100 mM NaOH, (B) DTT, 10 μg/mL
in 10 mM NaOH eluent with 3 mm Au Working Electrode, Hydrogen Reference Electrode
and 50 μm Spacer with forward scan from + 0 mV to + 1200 mV. The dashed line
represents the corresponding NaOH eluent blank.

24. Chromatograms of optimized HPLC separation of (B) GnHCl and (D) DTT spiked into respective
protein solutions. Conditions: Dionex CarboPac PA-1 column 250mm x 4.6mm, mobile phase NaOH
buffer (100 mM for Gn HCl, 10 mM for DTT); flow rate 1.0 mL/min, temperature 40ºC, 20μL injection
volume, optimized PAD triple potential waveform. Concentrations: GnHCl 195 ng/mL (ppb) in 2.0
mg/mL BSA and DTT 100 ng/mL (100 ppb) in 1.1 mg/mL F1V.

25. Project Two Conclusion: Methods to quantify residual guanidine HCl
(GnHCl) and 1, 4-dithiothreitol (DTT) in purified protein solutions
• The amounts of GnHCl and DTT in purified protein solutions can be
accurately and conveniently measured by the novel method.
• The advantages of these methods over previously described
methods includes:
– no sample preparation is required, other than removal of any
particulates
– The methods are easily automated to provide data in support of process
development, protein folding, or residual release testing of purified
proteins in clinical development
– The LOQs are equivalent or lower than those of other reported
methods-GnHCl (155 ng/mL) and DTT (100 ng/mL)
– The methods were developed using an older, but very functional,
autosampler and LC system. We expect better performance of the
methods on state-of-the art LC systems.
– Thireault, D.L. and Stroop, S.D. Determination of residual Gn HCl and
DTT in purified protein solutions using HPAEC-PAD Journal of
Chromatography A (Submitted, not published)

26. Professional Background
• MS in Regulatory Affairs from UW School of Pharmacy (GPA
3.9/4.0) while working full time
• BS in Chemistry-Bemidji State University
• Graduate research in Bioanalytical and Biophysical Chemistry at
Montana State-Bozeman- Characterization of GPCRs
• Certificates in Project Mgmt., Program Mgmt., and Clinical Trials
from UW Extension
• Active Member of PMI, Toastmasters, TU
• Non-Profit Executive Officer for WA largest conservation group for
cold-water fisheries >10yrs. Active volunteer in Fremont CC.
• PMP Exam will be taken soon, RAC will than follow
• High success rate at every organization I have worked at no matter
what capacity, area, or field. Considered a strong team player.
• Owned my own consulting business since 2006.
• Many of the IVD tests I developed were the first commercial CLIA
assays for these analytes and are now FDA approved.

27. Protein Chemistry Background
• Graduate Student Montana State University 1989-1994
– Graduate research emphasis was on characterization of G
Protein-Coupled Receptors (GPCRs) utilizing bioanalytical and
biophysical methods.
• Rhodopsin (Bovine and Human)
• Bacteriorhodopsin (aka Purple Membrane or BR) from halobacteria
– At this time, it was thought that any bacterial life found on Mars would
exhibit a similar proton gradient into chemical energy system.
• N-formyl Peptide Receptor (FPR)
– GPCR involved in stimulating a variety of differential responses in
neutrophils including chemotaxis, degranulation, superoxide production,
transcriptional activation and actin reorganization.
– The predominant emphasis was modeling the interaction of the
GPCR transmembrane “Loops” with G-Protein by utilizing
peptides in conjunction with 2D NMR and Molecular Modeling.

28. Protein Biochemistry Background
• Methods utilized in my graduate research included.
– Tangential Flow Filtration (Harvesting from ROS)
– Purification Techniques (Affinity, Gels, HPLC, Dialysis, etc.)
– Formulations
– NMR-Predominately Tr-NOESY, TOCSY and Solid State
– Mass Spectrometry
– X Ray Crystallography
– Ruthenium Dye Laser
– Fluorescent and Radioisotope labeling
– UV Kinetics, Biacore, Peptide Synthesis, etc.
– Molecular Modeling with Silicon Graphics Workstations
– I also grew all my Halobacteria, and scaled it up myself

29. Protein Biochemistry Background
• Worker Protection Chemist for Montana
Department of Agriculture
– Best equipped state analytical laboratory in country, we had every
conceivable GC and HPLC detector configuration including SFE.
– You were expected to not only learn how to run everything, but also how
to perform your own service engineering in the event of equip. failure.