METHOD DEVELOPMENT AND VALIDATION FOR DETERMINATION OF HUMAN SERUM ALBUMIN MONOMER, DIMER AND OTHER AGGREGATES USING SIZE EXCLUSION CHROMATOGRAPHY 

METHOD DEVELOPMENT AND VALIDATION FOR DETERMINATION OF HUMAN
SERUM ALBUMIN MONOMER, DIMER AND OTHER AGGREGATES USING SIZE
EXCLUSION CHROMATOGRAPHY
NORTHEASTERN ILLINOIS UNIVERSITY
DEPARTMENT OF CHEMISTRY
NOVEMBER 25, 2019

INTRODUCTIONS
 MATERIALS AND EQUIPMENT
 HUMAN SERUM ALBUMIN
 SIZE EXCLUSION CHROMATOGRAPHY
 METHOD DEVELOPMENT
• Effect of Ionic Strength
• Effect of pH
• Effect of Organic Solvent
• Effect of Flow Rate
• Effect of Injection Volume
• Effect of Column Temperature
 FORCE DEGRADATION
• Acidic Hydrolysis
• Basic Hydrolysis
• Oxidative Hydrolysis
• UV Degradation
 METHOD VALIDATION
• LINEARITY
• REPEATABILITY
• PRECISION
• ROBUSTNESS
1. Flow Rate
2. Column Temperature
3. Injection Volume
4. Auto-sampler Temperature
5. Concentration and pH of Mobile Phase
6. Wavelength
7. Stability
• SYSTEM SUITABILITY TEST
• SPECIFICITY
 CONCLUSIONS

Materials and Equipment
Agilent AdvanceBio SEC 300A, 2.7 um (300 mm x 4.6 mm i.d), polymer
coated silica stationary phase.
Agilent 1100 HPLC

CHEMICAL AND REAGENT
• Human Serum Albumin MW = 66.5 kDa; Lot #: 21K7600
• Deionized Water
• Methanol HPLC Grade
• Monobasic Sodium Phosphate; MW = 119.98 g/mol
• Dibasic Sodium Phosphate; MW = 141.96 g/mol
• Sodium Chloride; MW = 58.44 g/mol
• Acetone HPLC Grade
• Sodium Hydroxide 12M
• Hydrochloride Acid 12M
• Hydrogen Peroxide 27%
• Phosphoric Acid Conc.
Mobile Phase (200ml)
1. Weighting sodium phosphate mono
and di basic
2. Adjusting pH
3. Filtering in vacuum
4. Degassing
Henderson-Hasselbach equation
pH = pKa + log ([A−]/[HA])

BLOOD
Human Serum Albumin (HSA)
Encyclopædia Britannica, Inc
1. Transport
• Gases(O2, CO2)
• Nutrient
• Waste
• Hormones
• Heat
2. Protection
• Leukocytes, or white blood
cells
• Antibodies and other
proteins
• Platelet factors
3. Regulation
• pH
• Water balance

HUMAN SERUM ALBUMIN
Synthesized in the liver. Blood pH 7,4. (HSA negatively charged)
585 amino acids; 66,500 Da; 80x80x80Å ; 4mg/ml; 60%
Glomerular membrane; 3 Domains
Transports fatty acids, amino acids, drugs, metals.
Replace of albumin.
Contains 11 binding sites.

SIZE EXCLUSION CHROMATOGRAPHY
AdvanceBio SEC 300A, 2.7 um, (300 mm x 4.6 mm i.d)
Stored in sodium azide 0.02%
100% deionized water, ≥ 2 column volumes
Stored in mobile phase to three days if no salt
Stored in 20% methanol or 20% ethanol (if not used for more than 1 week).
Temperature < 80oC
pH 2 - 8.5
Flow rate 0.1-0.7 ml/min

•Solubility of sample
•Avoid interaction with column packing
Solvent
Deionized water
• Salt addition
• Organic solvent addition
Buffer
150 mM sodium phospate pH 7
METHOD DEVELOPMENT
Vinj = 5 𝞵l
F = 0.1ml/min
T = 30oC
UV = 214 nm
150 mM Sodium Phosphate pH 7
AdvanceBio SEC 300A, 2.7 um,
(300 mm x 4.6 mm i.d)
Vinj = 1 𝞵l
F = 0.2 ml/min
T = 30oC
UV = 214 nm
150 mM Sodium Phosphate
pH 7
HSA 10 mg/ml

Monomer
NaCl
(mM)
Area
(mAU)
N Height
Tailing
factor
Resolution
Selectivi
ty
Retention
time (min)
Sample
Control
2368 11126 112.5 1.4 2.37 2.24 13.892
50 1949 10363 93.63 1.5 2.31 2.12 12.970
100 1980 10155 95.08 1.5 2.33 2.13 12.987
150 1779 9574 69.95 2.1 1.95 2.07 13.026
Effect of ionic strength
Sample
Control
379.4 3692 12.2 1.05 1.05 - 12.306
50 272.6 3989 9.64 1.03 1.34 1.45 11.538
100 286.5 4050 10.21 1.08 1.37 1.43 11.536
150 425 2277 9.96 - - 1.21 11.557
NaCl
(mM)
Area
(mAU)
Nr. of
Theoretic
al
plates
Heigh
t
Tailin
g
facto
r
Selectivit
y
Retentio
n time
(min)
Sample
Control
52.38 3591 1.9 - - 11.258
50 34.3 4021 1.41 - - 10.597
100 39.6 3911 1.60 1.14 - 10.569
150 47.1 - 3.53 - 1.98 10.629Dimer
Trimer
• Secondary interactions between packing material and
proteins
• Sample control: 150 mM Na3PO4, pH 7
• Vinj = 0.2 𝞵l
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm
? NaCl + 50 mM Na3PO4, pH 7

50 mM NaCl + 50 mM Na3PO4 pH 7
150 mM Na3PO4 pH 7
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm
AdvanceBio
SEC 300A, 2.7 um,
(300 mm x 4.6 mm i.d)

min5 10 15 20
mAU
0
20
40
60
80
100
*DAD1 A, Sig=214,4 Ref=360,100 (SECINA00041.D)
150 mM Na3PO4 pH 7

Effect of pH
pH
Area
(mAU)
Nr. of
theoretical
plates
Heigh
t
Tailin
g
factor
Resoluti
on
Selectivit
y
Retention
time (min)
6.57 2132 10887 105.27 1.34 2.33 2.26 13.447
7 2368 11126 112.5 1.4 2.37 2.24 13.892
7.54 2352 11136 115.99 1.32 2.33 2.26 13.450
6.57 327.57 3786 11.06 0.998 1.37 1.46 11.938
7 379.47 3692 12.2 1.05 1.05 - 12.306
7.54 365.86 3731 12.31 0.93 1.26 1.43 11.937
pH
Area
(mAU)
Nr. of
theoretical
plates
Height
Tailing
factor
Retention
time (min)
6.57 47.47 3702 1.8 - 10.915
7 52.38 3591 1.9 - 11.258
7.54 79.91 2362 2.5 0.93 10.876
Monomer
Dimer
Trimer
Vinj = 0.2 ul
F = 0.2 ml/min
Tcolumn = 30oC
Wavelength = 214 nm
Mobile phase = 150 mM Na3PO4
AdvanceBio SEC 300A, 2.7 um, (300 mm x 4.6 mm i.d)
150 mM Na3PO4, pH ?

min0 2 4 6 8 10 12 14 16
mAU
0
20
40
60
80
100
Effect of pH
pH 7
pH
6.57
pH 7.54

Monomer
% ACN
Area
(mAU)
Nr. of
theoretical
plates
Height
Tailing
factor
Resoluti
on
Selectivity
Retention
time (min)
Sample
control
2368 11126 112.5 1.4 2.37 2.24 13.892
3 1854.2 10736 88.4 1.5 2.34 2 13.058
9 1263 10766 62.46 1.57 2.386 2.24 13.078
Effect of organic solvent
% ACN
Area
(mAU)
Nr. of
theoretica
l plates
Height
Tailing
factor
Resoluti
on
Selectivit
y
Retention
time (min)
Sample
control
379.47 3692 12.2 1.05 1.05 - 12.306
3 296.57 3842 9.9 1 1.2 1.4 11.588
9 169.9 4171 6.29 0.919 1.218 1.396 11.616
% ACN
Area
(mAU)
Nr. of
theoretical
plates
Height
Tailin
g
factor
Retention
time (min)
Sample
control
52.38 3591 1.9 - 11.258
3 63.22 2571 2 - 10.638
9 17.7 2590 0.76 1.374 10.666
Dimer
Trimer
Vinj = 0.2 ul
F = 0.2 ml/min
Tcolumn = 30oC
Wavelength = 214 nm
Mobile phase = 50 mM Na3PO4 + ACN
1) 3% ACN + 50 mM Na3PO4 pH 7
2) 9% ACN + 50 mM Na3PO4 pH 7
3) Sample control :150 mM Na3PO4 pH 7

0 5 10 15 20
mAU
0
20
40
60
80
100
DAD1 A, Sig=214,4 Ref=360,100 (SECINA00041.D)
11.258
12.306
13.892
0 5 10 15 20
mAU
0
10
20
30
40
50
60
70
80
10.638
11.588
13.058
19.748
0 5 10 15 20
mAU
0
10
20
30
40
50
11.616
13.078
19.780
Sample control :150 mM Na3PO4 pH 7
3% ACN + 50 mM Na3PO4 pH 7

min5 10 15 20 25
mAU
0
20
40
60
80
100
Sample control :150 mM Na3PO4 pH 7

Monomer
Flow rate
(ml/min)
Area
mAU
Nr. of
theoretic
al plates
Height
Tailing
factor
Resoluti
on
Selectivity
Retenti
on time
(min)
0.1 98140.6 7557 2045.47 1.588 2.1 2.146 27.085
0.15 67549.6 7339 2058.97 1.512 2.054 2.102 17.956
0.2 50814.5 7062 2038.3 1.396 2.049 2.081 13.448
Effect of flow rate
0.1 18536.9 3639 305.49 - 1.25 1.43 24.092
0.15 13481.8 3376 316.49 - 1.204 1.432 15.963
0.2 10223.8 3365 315.62 - 1.183 1.435 11.943
Flow
rate
ml/min
Area
(mAU)
Nr. of
theoretic
al plates
Heig
ht
Resol
ution
Selecti
vity
Retenti
on time
(min)
0.1 3701 2705 58.2 0.86 1.41 22.024
0.15 2904.9 2429 65.09 0.822 1.417 14.387
0.2 2274.7 2301 62.27 0.786 1.434 10.914Dimer
Trimer
Vinj = 0.2 𝞵l,
T column = 30oC,
Wavelength = 214 nm
Mobile phase 50 mM Na3PO4 pH 7
Higher flow rate  lower run time
Lower flow rate  higher resolution
Compromise between resolution and run time
F = ?,

min0 5 10 15 20 25 30
mAU
0
250
500
750
1000
1250
1500
1750
2000
Monomer
Dimer
Trimer0
20000
40000
60000
80000
100000
120000
0 0.05 0.1 0.15 0.2 0.25
PeakAreamAU
Flow Rate ul/min
Effect of Flow Rate
F =
0.1
ml/mi
n
F =
0.2
ml/mi
n
F =
0.15
ml/mi
n
Effect of flow rate

Injection
volume
(µl)
Area
(mAU)
Nr. of
theore
tical
plates
Height
Tailing
factor
Resol
ution
Select
ivity
Retent
ion
time
(min)
0.2 2368 11126 112.5 1.4 2.37 2.24 13.892
0.5 5906 11868 288.69 1.4 2.42 2.26 13.885
1 11860 10669 553.62 1.41 2.31 2.20 13.896
2 23259 10234 1058.9 1.41 2.26 2.16 13.897
0.2 379.47 3692 12.2 1.05 1.05 - 12.306
0.5 901.8 3910 29.82 0.99 1.36 1.45 12.309
1 1886.7 3652 59.44 0.99 1.25 1.19 12.325
2 3909.2 3507 120.01 0.99 1.2 1.44 12.328
Injection
volume
(µl)
Area
(mAU)
Nr. of
theore
tical
plates
Height
Tailing
factor
Resol
ution
Select
ivity
Retent
ion
time
(min)
0.2 52.38 3591 1.9 - - - 11.258
0.5 143.8 3479 5.1 - - - 11.249
1 434.2 2666 11.51 - - - 11.262
2 859.1 2352 24.92 - - - 11.263
Effect of injection volume
Dimer
Monomer
Trimer
Flow rate = 0.2 ml/min
Tcolumn = 30oC
Wavelength = 214 nm
0.5% - 4% of total column volume

Monomer
Dimer
Trimer0
5000
10000
15000
20000
25000
0 0.5 1 1.5 2 2.5
PeakAreamAU
Injection Volume ul

Column
Temp.
oC
Area
(mAU)
Nr. of
theoret
ical
plates
Height
Tailing
factor
Resolu
tion
Selecti
vity
Retenti
on time
(min)
20 2151.5 9679 98.54 1.428 2.253 2.14 13.376
28 1647.4 10846 79.65 1.317 2.25 2.102 13.569
30 2368 11126 112.5 1.4 2.37 2.24 13.892
50 2077 11142 104.86 1.356 2.476 2.376 13.304
Column
Temp.
oC
Area
(mAU)
Nr. of
theoret
ical
plates
Height
Tailing
factor
Resolu
tion
Selecti
vity
Retenti
on time
(min)
20 37.1 3167 1.37 1.156 - - 10.840
28 35.1 3269 1.26 1.133 - - 11.011
30 52.38 3591 1.9 - - - 11.258
50 28.1 3645 1.07 1.035 - - 10.773Column
Temp.
oC
Area
(mAU)
Nr. of
theoret
ical
plates
Height
Tailing
factor
Resolu
tion
Selecti
vity
Retenti
on time
(min)
20 285.9 3528 9.42 1.036 1.3 1.401 11.861
28 232.6 3739 7.81 0.97 - - 12.036
30 379.47 3692 12.2 1.05 1.05 - 12.306
50 220.8 4153 7.9 1.059 1.382 1.505 11.773
Effect of Column temperature
Monomer
Dimer
Trimer
20oC  91oC
50oC  60oC
Flow Rate = 0.2 ml/min
Vinj = 0.2 𝞵l
Wavelength = 214 nm
Temp. Pressure

'3D' Signal Overlay
min0 2.5 5 7.5 10 12.5 15 17.5
mAU
0
20
40
60
80
100
120
Print of window 47: '3D' Signal Overlay
JMS LC-01 8/27/2019 2:25:23 PM Malvina Page 1 of 1
Effect of Column temperature
30 oC
28 oC
20 oC
50 oC

Effect wavelength
Wavelength
(nm)
Area
(mAU)
Nr. of
theoretical
plates
Height
Tailing
factor
Resoluti
on
Selectivity
Retention
time (min)
214 2368 11126 112.5 1.4 2.37 2.24 13.892
215 988.8 11066 48.40 1.32 2.36 2.33 13.557
218 1783.3 11042 87.06 1.35 2.39 2.27 13.542 Wavelength
(nm)
Area
(mAU
)
Nr. of
theoretic
al plates
Heig
ht
Tailin
g
factor
Resolu
tion
Selectivit
y
Retentio
n time
(min)
214 52.38 3591 1.9 - - - 11.258
215 18.3 3758 0.7 0.998 - - 11.026
218 26.8 4217 1.13 1.161 - - 10.974
Wavelength
(nm)
Area
(mAU)
Nr. of
theoretical
plates
Heigh
t
Tailing
factor
Resoluti
on
Selectivity
Retention
time (min)
214 379.4 3692 12.2 1.05 1.05 - 12.306
215 135.7 3841 4.67 0.977 - - 12.025
218 249 3945 8.54 1 - - 12.009
Monomer
Dimer
Trimer
0.3 sec
Flow rate = 0.2 ml/min
Tcolumn = 30oC
Vinj = 0.2 ul

min0 2 4 6 8 10 12 14 16
mAU
0
20
40
60
80
100
Effect wavelength
215
nm
218
nm
214
nm

FORCED DEGRADATION STUDIES
Acidic Hydrolysis
Basic Hydrolysis
Oxidative Hydrolysis
UV Degradation
Conversion of amide side chain of asparagine to
either aspartic acid or isoaspartic acid.
Methionine side chains near the surface of
protein to methionine sulfoxide
Upon absorption, they can transfer an electron to nearby disulfide bonds formed between cysteine (Cys) amino acids,
causing them to break. By breaking these key bonds, the protein begins to unravel, and very reactive Cys radicals are
created which can promote side-reactions and protein aggregation or clumping

Acidic Hydrolysis
Time
(Hour)
Area
(mAU)
Height Width Retention Time % Degradation
Sample control 379.47 12.2 0.48 12.306 0
0 240 8.17 0.45 12.088 36.8
1 216.3 7.19 0.46 12.079 43
24 386.1 - 0.50 11.986
Peak area higher than
sample control
Time
(Hour)
Area
(mAU)
Height Width Retention Time % Degradation
Sample control 2368 112.5 0.31 13.892 0
0 1717.5 82.89 0.31 13.623 27.5
1 1515.5 72.71 0.31 13.625 36
24 2483 86.12 0.32 13.568
Peak area higher
than sample control
Time
(Hour)
Area
(mAU)
Height Width
Retention
Time
% Degradation
Sample
control
52.38 1.9
0.44
11.258
0
0 27.7 1.09 0.42 11.021 47
1 29.13 0.92 0.42 11.038 44.4
24 190.3 2.33 0.54 10.948
Peak area higher
than sample
control
5 mg/ml HSA + 0.1M HCl
37 oC
• Mobile phase: 150 mM Na3PO4, pH 7
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm

Current Chromatogram(s)
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
20
40
60
80
100
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
10
20
30
40
50
60
70
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
10
20
30
40
50
60
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
10
20
30
40
50
60
70
80
Acidic HydrolysisWithout HCl
T = 0 hour without
heated
T = 24 hours
T = 1 hour
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm

Basic hydrolysis
min0 5 10 15 20 25 30
mAU
0
20
40
60
80
100
5 mg/ml HSA + 0.1M NaOH
T = 0 min. 95.5 %
T = 60
min
100% T = 1440
min
Sample control
Without NaOH
37 oC
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
20
40
60
80
100
11.258
12.306
13.892
min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
12.007
13.571
20.577
min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
50
100
150
200
250
300
350
400
11.963
13.580
20.589
Sample control
Without H2O2
T = 0
hour
T =
1hour
T = 24
hours
5 mg/ml HSA + 3%
H2O2
Heated at 37oC
Monomer 16%
Dimer 14.4%
Monomer. 16%
Dimer 46%
Trimer 100%
2.5%
After 10 min at room temperature
24 Hours incubated at 37 oC
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
Sample control Without H2O2
T = 0 hour
T = 1 hour

min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
20
40
60
80
11.258
12.306
13.892
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
20
40
60
80
100
12.098
13.621
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
10
20
30
40
50
12.099
13.633
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
-50
0
50
100
7.572
7.908
10.366
Sample control No UV
T = 30 min
T = 24 hours
T = 1 hour
UV degradation
254 nm
Monomer. 16%
Dimer 4.9%
Trimer 100%
Monomer. 46.3%
Dimer 8.5%
Trimer 100%

min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
20
40
60
80
100
120
140
160
180
UV degradation
254 nm
Sample control No UV
T = 30 min
T = 60 min
T = 24 hours
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm

METHOD VALIDATION
 Linearity
 Repeatability
 Precision
 Robustness
1. Flow Rate
2. Column Temperature
3. Injection Volume
4. Auto-sampler Temperature
5. Concentration and pH of Mobile Phase
6. Wavelength
7. Stability
8. System suitability test
 Specificity
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm

LINEARITY
y = 1101.7x - 472.77
R² = 0.99914
y = 167.39x - 83.468
R² = 0.99896
y = 27.913x - 21.735
R² = 0.98939
0
1000
2000
3000
4000
5000
6000
7000
0 1000 2000 3000 4000 5000 6000 7000
PeakAreamAU
Concentration (ppm)
Linearity
Monomer
Trimer
Dimer
LOD
(mg/ml)
Monomer 1.4
Dimer 1.6
Trimer 2.6
LOQ
(mg/ml)
Monomer 4.3
Dimer 5
Trimer 7.
ICH: 80 -120%

REPEATABILITY
Sample Injection Nr. Area Std. Dev Average RSD%
Monomer
1 5832.9
52.66 5795.31 0.91
2 5702.2
3 5792.2
4 5806.4
5 5783.6
6 5854.6
Dimer
1 873.7
10.31 869.7 1.19
2 850.1
3 872.7
4 876.6
5 867.2
6 878
Sample Injection Nr. Area Std. Dev Average RSD%
Trimer
1 134.3
2.66 134.4 1.98
2 129.3
3 135
4 135.8
5 135
6 137
RSD < 1%

METHOD PRECISION
Peak Area
Sample
Nr.
Area SD Mean RSD%
1
Monomer
5703.6
107.4 5616.4 1.9
2 5620.9
3 5657
4 5625.6
5 5684.1
6 5407.2
1
Dimer
909.2
31.4 869.3 3.6
2 884.1
3 889
4 855.3
5 857.8
Sample
Nr.
Area SD Mean RSD%
1
Trimer
149.8
6.9 141.4 4.9
2 144.5
3 144.3
4 133.3
5 135.2
1 2 3 4 5
6
Same concentration 5mg/ml
RSD < 2%

Sample Nr.
(5mg/ml)
Retention
Time (min)
SD Mean RSD%
Prep-1
Monomer
13.497
0.005 13.5 0.04
Prep-2 13.492
Prep-3 13.508
Prep-4 13.497
Prep-5 13.497
Prep-6 13.496
Prep-1
Dimer
11.961
0.008 11.9 0.07
Prep-2 11.952
Prep-3 11.968
Prep-4 11.976
Prep-5 11.966
Prep-6 11.972
Sample
Nr.
(5mg/ml
)
Retentio
n Time
(min)
SD Mean RSD%
Prep-1
Trimer
10.936
0.007 10.9 0.07
Prep-2 10.935
Prep-3 10.933
Prep-4 10.948
Prep-5 10.943
Prep-6 10.952
METHOD PRECISION
Retention time

FLOW RATE
Flow Rate ±0.01 Peak Area Tailing factor Retention
0.19 Monomer 6209 1.54 14.309
Dimer 910.5 1.07 12.683
Trimer 139.4 1.04 11.605
0.2 Monomer 5805.9 1.51 13.552
Dimer 852.4 1.08 12.007
Trimer 132 1.03 10.98
0.21 Monomer 5498.3 1.5 12.96
Dimer 806.19 1.07 11.429
Trimer 123.29 1.04 10.455
ROBUSTNESS
Monomer. 6.5 %
Dimer 5.3%
Trimer 6.6%
Monomer.
Dimer 5.3%
Trimer
Monomer. 5.3 %
Dimer 5.4%
Trimer 6.6%
Monomer. 4.4 %
Dimer 4.8%
Trimer
Retention time
Peak Area

COLUMN TEMPERATURE
Column
Temperature
Peak Area Tailing factor Retention
29 Monomer 5862.7 1.51 13.481
Dimer 860.18 1.08 11.93
Trimer 132.54 10.912
30 Monomer 5869.9 1.516 13.472
Dimer 858.83 1.059 11.945
Trimer 130.09 N/A 10.927
31 Monomer 5794.2 1.51 13.449
Dimer 850.37 1.07 11.91
Trimer 129.84 1.05 10.887
1.2%

INJECTION VOLUME
Injection Volume Peak Area Tailing factor Retention
0.4
Monomer 4635.8 1.391 13.368
Dimer 676.8 1.055 11.843
Trimer 101.7 1.061 10.844
0.5 Monomer 5671 1.366 13.369
Dimer 828.6 1.048 11.840
Trimer 124.2 N/A 10.832
0.6 Monomer 7154.8 1.384 13.365
Dimer 1061 1.045 11.842
Trimer 160.3 1.06 10.834
Monomer. 23.5 %
Dimer 21.9%
Trimer 22.5%
Monomer.
Dimer 18%
Trimer
Peak
Area

AUTO-SAMPLER TEMPERATURE
Auto-sampler
Temperature
4
Monomer 5413.7 1.438 13.597
Dimer 816.01 1.033 12.055
Trimer 128.8 1.057 11.022
5 Monomer 5777.1 1.389 13.365
Dimer 803.5 1.043 11.842
Trimer 116.3 1.058 10.822
6 Monomer 5653.2 1.395 13.363
Dimer 784.5 1.037 11.839
Trimer 112.6 1.045 10.828
Monomer. 6.3 %.
higher
Dimer 1.5%
lower
Trimer 9.7%
lower
Monomer. 2.1 %
Dimer 2.4%.
Trimer 3.2%
Lower

CONCENTRATION OF BUFFER
Concentration of
Buffer
140
Monomer 5968.4 1.523 13.456
Dimer 879.94 1.081 11.916
Trimer 134.45 1.051 10.895
150
Monomer 5805 1.510 13.552
Dimer 852.4 1.08 12.007
Trimer 132 1.03 10.98
160
Monomer 5895.6 1.516 13.479
Dimer 863.46 1.087 11.936
Trimer 130.93 1.068 10.913

pH OF BUFFER
pH of Buffer Peak Area Tailing factor Retention
6.9 Monomer 5837 1.517 13.475
Dimer 921.03 1.046 11.941
Trimer 263.17 0.736 10.912
7 Monomer 5805 1.51 13.552
Dimer 852.4 1.08 12.007
Trimer 132 1.03 10.98
7.1 Monomer 5801.4 1.52 13.469
Dimer 853.1 1.07 11.934
Trimer 130 1.05 10.915
Higher

WAVELENGTH
Wavelength Peak Area Tailing factor Retention
212
Monomer 6520 1.514 13.459
Dimer 963.11 1.077 11.922
Trimer 149.61 1.074 10.906
214
Monomer 5805 1.51 13.552
Dimer 852.4 1.08 12.007
Trimer 132 1.03 10.98
216
Monomer 5145.9 1.509 13.453
Dimer 755.4 1.079 11.916
Trimer 115.4 1.081 10.892
Monomer 10.9 %. lower
Dimer 11.5% lower
Trimer 11.8% lower
Monomer 11.4 %. lower
Dimer 12.6% lower
Trimer 12.6% lower
Peak Area

STABILITY
Time (hours) Area (mAU)
Nr. of
Theoretical Plates
Height Resolution
Retention
time
0
Monomer 5393 10353 258 2.3 13.4
Dimer 814 3752 27 1.3 11.9
Trimer 127 3383 4.6 - 10.9
24
Monomer 5387 10554 259.3 2.3 13.4
Dimer 800 3741 27 1.3 11.9
Trimer 121 3439 4.5 - 10.9
48
Monomer 5672 10303 271.3 2.4 13.4
Dimer 829 3951 28.4 1.3 11.8
Trimer 124 3426 4.6 - 10.8
72
Monomer 6010 10287 286.4 2.4 13.4
Dimer 887 3942 30.43 1.4 11.8
Trimer 133 3446 5 - 10.8
10%
5%

SYSTEM SUITABILITY TEST
Number of theoretical plates > 2000
Tailing factor 0.9< Tf > 2
Resolution monomer-dimer > 2
Resolution dimer-trimer 1
RSD% monomer < 1%
RSD% dimer < 2%
RSD% trimer < 2%

SPECIFICITY
• Vinj = 0.2 𝞵l HSA
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm
• 150 mM Sodium Phosphate pH 7
• Vinj = 0.2 𝞵l Deionized Water
• F = 0.2 ml/min
• T =30 oC
• 𝞴 =214nm
• 150 mM Sodium Phosphate pH 7

CONCLUSION
• Unstable under basic conditions even without heating
• More stable under acidic conditions.
• 150 mM sodium phosphate ph 7.
• Very small changes in all chromatographic parameters do not have a considerable affect in
resolution and separation.

REFERENCES
• KAZUAKI TAGUCHI, VICTOR TUAN GIAM CHUANG, TORU MARUYAMA, MASAKI OTAGIRI, (2012). PHARMACEUTICAL ASPECTS OF THE RECOMBINANT HUMAN SERUM ALBUMIN DIMER:
STRUCTURAL CHARACTERISTICS, BIOLOGICAL PROPERTIES, AND MEDICAL APPLICATIONS. DEPARTMENT OF BIOPHARMACEUTICS, GRADUATE SCHOOL OF PHARMACEUTICAL SCIENCES,
KUMAMOTO UNIVERSITY, KUMAMOTO 862-0973, JAPAN; SCHOOL OF PHARMACY, FACULTY OF HEALTH SCIENCES, CURTIN HEALTH INNOVATION RESEARCH INSTITUTE, CURTIN UNIVERSITY,
PERTH 6845,WESTERN AUSTRALIA, AUSTRALIA; CENTER FOR CLINICAL PHARMACEUTICAL SCIENCES, KUMAMOTO UNIVERSITY, KUMAMOTO 862-0973, JAPAN; FACULTY OF
PHARMACEUTICAL SCIENCES, SOJO UNIVERSITY, KUMAMOTO 860-0082, JAPAN; DDS RESEARCH INSTITUTE, SOJO UNIVERSITY, KUMAMOTO 860-0082, JAPAN. PAGE 3033-3046
• MICHAEL DOCKAL, DANIEL C. CARTER, AND FLORIAN RUKER. (1999). THE THREE RECOMBINANT DOMAINS OF HUMAN SERUM ALBUMIN PRINTED IN U.S.A. STRUCTURAL
CHARACTERIZATION AND LIGAND BINDING PROPERTIES., UNIVERSITY OF AGRICULTURAL SCIENCES, MUTHGASSE 18, VIENNA, AUSTRIA AND NEW CENTURY PHARMACEUTICALS INC.,
HUNTSVILLE, ALABAMA. PAGE 29303–2931
• JUDITH VAJDA & REGINA RÖMLING, TOSOH BIOSCIENCE GMBH, STUTTGART, D. (2012). NEW APPROACHES TO HPLC ANALYSIS OF ANTIBODY AGGREGATES AND FRAGMENTS.
CHROMATOGRAPHY TODAY. PAGE 44-45
•
• IRA KRULL, ANURAG S. RATHORE. (2015). CHALLENGES IN THE DETERMINATION OF PROTEIN AGGREGATES. VOLUME 33, ISSUE 1, PAGE 42–49.
• JOHN DEN ENGELSMAN, PATRICK GARIDEL, RONALD SMULDERS, HANS KOLL, BRYAN SMITH, STEFAN BASSARAB, ANDREAS SEIDL, OTMAR HAINZL, WIM JISKOOT. (2010). STRATEGIES FOR
THE ASSESSMENT OF PROTEIN AGGREGATES IN PHARMACEUTICAL BIOTECH PRODUCT DEVELOPMENT. DIVISION OF DRUG DELIVERY TECHNOLOGY LEIDEN/AMSTERDAM CENTER FOR
DRUG RESEARCH LEIDEN UNIVERSITY, THE NETHERLANDS
• HANNS-CHRISTIAN MAHLER, WOLFGANG FRIESS, ULLA GRAUSCHOPF, SYLVIA KIESE. (2008). PROTEIN AGGREGATION: PATHWAYS, INDUCTION FACTORS AND ANALYSIS. FORMULATION R&D
BIOLOGICS, PHARMACEUTICAL AND ANALYTICAL R&D, F. HOFFMANN-LA ROCHE LTD., BASEL, SWITZERLAND; DEPARTMENT OF PHARMACY, PHARMACEUTICAL TECHNOLOGY AND
BIOPHARMACEUTICS, LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN, MUNICH, GERMANY. PP.2909-2934
• ANDERS SØBYE ASGER KOLDING MARIE-LOUISE KNOP LUND MIA DALGAARD JENSEN. (2015). PROTEIN STABILITY: A STUDY OF THE STABILITY OF HEN EGG-WHITE LYSOZYME EXPOSED TO
CHEMICAL AND THERMAL DENATURATION AT PH 4, PH 7, AND PH 10. SCHOOL OF ENGINEERING AND SCIENCE AALBORG UNIVERSITY, SKJERNVEJ. PAGE 5-95.
• DAVID CARR. A GUIDE TO THE ANALYSIS AND PURIFICATION OF PROTEINS AND PEPTIDES BY REVERSED-PHASE HPLC. ADVANCED CHROMATOGRAPHY TECHNOLOGIES, 1 BERRY STREET,
ABERDEEN, SCOTLAND. PAGE 1-60

METHOD DEVELOPMENT AND VALIDATION FOR DETERMINATION OF HUMAN SERUM ALBUMIN MONOMER, DIMER AND OTHER AGGREGATES USING SIZE EXCLUSION CHROMATOGRAPHY

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