Peptide Analytics
Peptide Analytics Applying Lessons Learned to Small Molecules Lisa Caralli –Director of Pharmaceutics
Small Molecules Commonly… Peptides Commonly…
Hydrolyze Oxidize/reduce Isomerize/racemize Can be light sensitive Lack a UV chromophore Have polymorphs
Hydrolyze Oxidize/reduce Isomerize/racemize Deamidate Can be light sensitive Have spectral doppelgangers Stick Take up water Contain a lot of extra stuff per mg of bulk API Aggregate
Oxidation/Reduction/Hydrolysis/Light Small Molecules
Reversed-Phase
Peptides
Usually C18 Usually UV Usually low pH
< pKa Hydrophobic interactions Ion-pairing?
Reversed-Phase C3 – C18 UV Wider pH range
Multiple pKa’s
Specific Concerns Crosslinking Oxidation
Methionine (S) Tryptophan Cysteine (S) Histidine Tyrosine
Isomerization/Racemization Small Molecules
Regulatory Guidelines Chiral methods Normal Phase
Peptides
No Guideline for peptides D and L amino acids – controlled at synthesis Look for key isomers by RP Asn or Asp to Iso-Asp
Isomerization
Demond W, et al. Orthogonal HPLC Methods for Quantitating Related Substances and Degradation Products of Pramlintide. AAPS PharmSciTech 2000; 1 (1) article 6.
Resolution of Iso-Asp 0.26 0.24 0.22 0.20 0.18
TFA < pH2
0.16 0.14
AU
0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 -0.04 -0.06 14.00
14.50
15.00
15.50
16.00
16.50
17.00
17.50
18.00
18.50
19.00
19.50
20.00 Minutes
20.50
21.00
21.50
22.00
22.50
23.00
23.50
24.00
24.50
25.00
25.50
0.14 0.12
Phosphate pH 2
0.10
AU
0.08 0.06 0.04 0.02 0.00 -0.02 8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
12.50 Minutes
13.00
13.50
14.00
14.50
15.00
15.50
16.00
16.50
17.00
0.30 0.28 0.26 0.24 0.22 0.20 0.18 0.16 0.14
AU
Phosphate pH 4
0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
12.50 Minutes
13.00
13.50
14.00
14.50
15.00
15.50
16.00
16.50
17.00
0.060 0.055 0.050 0.045
0.035 0.030 AU
Phosphate pH 6.5
0.040
0.025 0.020 0.015 0.010 0.005 0.000 -0.005 -0.010 14.00
14.50
15.00
15.50
16.00
16.50
17.00
17.50
18.00
18.50
19.00
19.50
20.00
20.50 21.00 Minutes
21.50
22.00
22.50
23.00
23.50
24.00
24.50
25.00
25.50
26.00
26.50
27.00
27.50
28.00
Deamidation Small Molecules
Not a term usually associated with SM
Peptides
Concern for peptides containing Asn and to a lesser extent Gln Asparagine/Aspartate change difficult to separate by RP May necessitate ionexchange
“Orthogonal method”
Deamidation
Demond W, et al. Orthogonal HPLC Methods for Quantitating Related Substances and Degradation Products of Pramlintide. AAPS PharmSciTech 2000; 1 (1) article 6.
pKa ~ 3.7
8
Ion-Exchange Ion-Exchange
More column to column variability High salts hard on system System salting out risk if interchanging RP with IE
No UV Chromophore
ELSD CAD RI Fluorescence MS
15.033 Peak 1 0.80 0.70 0.60 0.50 AU
Peptides
0.40 0.30 0.20 0.10 0.00 210.00
220.00
230.00
240.00
250.00
260.00
270.00
280.00
290.00
300.00
310.00
320.00
nm
1.00
7.400 Peak 1
0.90 0.80 0.70 0.60 AU
Small Molecules
0.50 0.40 0.30 0.20 0.10 0.00 210.00
220.00
230.00
240.00
250.00
260.00
270.00
nm
280.00
290.00
300.00
310.00
320.00
No UV Chromophore
ELSD CAD RI Fluorescence MS
15.033 Peak 1 0.80 0.70 0.60 0.50 AU
Peptides
0.40 0.30 0.20 0.10 0.00 210.00
220.00
230.00
240.00
250.00
260.00
270.00
280.00
290.00
300.00
310.00
320.00
nm
1.00
7.400 Peak 1
0.90 0.80 0.70 0.60 AU
Small Molecules
0.50 0.40 0.30 0.20 0.10 0.00 210.00
220.00
230.00
240.00
250.00
260.00
270.00
nm
280.00
290.00
300.00
310.00
320.00
When UV Chromophore is Present Small Molecules
Select wavelength for mass balance (potency
Peptides
loss = purity loss) Response Factor (RF) may be considered
Ensure good signal to noise Using TFA try to use a higher wavelength Target LOQ ≤ 0.05%
Use amide bond (210220 nm) No RF’s needed
Don’t use wavelength of aromatic residues Ensure good signal to noise TFA produces poor signal to noise at 220 nm Target LOQ ≤ 0.05% Low dose: LOQ ≤ 0.1% may be acceptable* *ICH Q3B(R2) Impurities in New Drug Products
Sensitivity 0.010
0.009
0.008
TFA 220 nm LOQ ~ 0.1%
AU
0.007
0.006
0.005
0.004
0.003 16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
42.00
Minutes
0.0020 0.0018 0.0016 0.0014
Perchlorate 220 nm LOQ < 0.01%
AU
0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
21.00 Minutes
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
Sensitivity 0.010
0.009
0.008
TFA 220 nm LOQ ~ 0.1%
AU
0.007
0.006
0.005
0.004
0.003 16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
42.00
Minutes
0.0020 0.0018 0.0016 0.0014
Perchlorate 220 nm LOQ < 0.01%
AU
0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
21.00 Minutes
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
Similar Spectra
Similar Spectra
Similar Spectra Small Molecules
Use peak purity assessment to determine if related substances are separated
Peptides
Need orthogonal detection to determine if peaks are fully separated Mass spec?
Leads companies to go into Phase 1 with two separation mechanisms
Adsorption Small Molecules
Rarely an issue
Peptides
Type of filter Type of HPLC vial How to prepare LOQ sensitivity samples? How to test dosing solutions? Carryover “Bio-inert” plumbing (no stainless)
LOQ Determination
Small Molecule 0.0140
LOQ Sample Response
0.0135 0.0130
30000
0.0125 0.0120
0.0110
20000 Area
AU
0.0105 0.0100 0.0095 0.0090
15000 10000
0.0085
5000
0.0080 0.0075
0
0.0070 0.0065
0
0.0060 0.0055
y = 75507x + 175.41 R² = 1
25000
0.0115
6.50
7.00
7.50
8.00
8.50
9.00 9.50 Minutes
10.00
10.50
11.00
11.50
12.00
0.05
0.1 0.15 0.2 0.25 0.3 % of Method Nominal Conc.
0.35
0.4
ICH QB2: Validation of Analytical Procedures – Methodology; 1996
19
LOQ Determination - Peptide 0.024
Sample = 0.2% of Method Nominal
0.022 0.020 0.018
AU
0.016 0.014
Plastic
0.012 0.010 0.008
Glass
0.006 0.004 7.00
8.00
9.00
10.00
11.00
12.00
13.00 Minutes
14.00
15.00
16.00
17.00
18.00
19.00
LOQ Determination - Peptide 0.024
Sample = 0.2% of Method Nominal
0.022 0.020 0.018
0.014
Plastic
0.012 0.010 0.008
Glass
0.006 0.004 7.00
8.00
9.00
10.00
11.00
12.00
13.00 Minutes
14.00
15.00
16.00
17.00
Area Counts per mg/mL 80000000 70000000
Response Factor
AU
0.016
60000000 50000000 40000000 30000000 20000000 10000000 0 0
0.1
0.2 0.3 0.4 Sample Concentration (mg/mL)
0.5
0.6
18.00
19.00
LOQ Determination - Peptide 0.024
Sample = 0.2% of Method Nominal
0.022 0.020 0.018
0.014
Plastic
0.012 0.010 0.008
Glass
0.006 0.004 7.00
8.00
9.00
10.00
11.00
12.00
13.00 Minutes
14.00
15.00
16.00
17.00
18.00
19.00
Area Counts per mg/mL 80000000
Method Nominal
70000000
Response Factor
AU
0.016
60000000 50000000 40000000 30000000 20000000
0.2% of Nominal
10000000 0 0
0.1
0.2 0.3 0.4 Sample Concentration (mg/mL)
0.5
0.6
Hygroscopicity Small Molecules
Not usually an issue for free forms More an issue for salts
Peptides
Usually an issue Karl Fischer titration Adsorption to apparatus – fouling
Reference Standard Program Primary Standard (solid) Vialed Working Standard
The Other Stuff Accurate Peptide Content Small Molecules
Assay Correction
Peptides
HPLC purity Salt counterion (?) Water ↓ Residual solvents ↓
Assay Correction HPLC purity Peptide Content
Salt counterion Water Residual Solvents Residue on Ignition
Peptide Content AAA Nitrogen Analysis Accurate water at time of analysis? What is peptide content if water is changing?
Aggregation Small Molecules
Normally not an issue
Peptides
Often an issue RP difficult to resolve due to high retention Agency may ask for size-exclusion (SEC) if known pathway DLS/MALS as research tool
Aggregation Reversed Phase Control
0.012 0.010
AU
0.008 0.006 0.004 0.002 0.000
Light
0.012 0.010
AU
0.008 0.006 0.004 0.002 0.000 8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00 21.00 Minutes
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
Aggregation 0.160 0.150
Reversed Phase
0.140 0.130 0.120 0.110
~27%
0.100 0.090
AU
0.080 0.070 0.060 0.050 0.040 0.030 0.020 0.010 0.000 -0.010 -0.020 6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00 20.00 Minutes
21.00
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
33.00
1.00 0.80
SEC
AU
0.60 0.40
~30%
0.20 0.00 -0.20 0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00 Minutes
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
THANK YOU! Lisa Caralli Director of Pharmaceutics, Pharmatek 2292 Carroll Road Suite 200 San Diego, CA 92121 858-805-6383
Small Molecules Commonly… Peptides Commonly…
Hydrolyze Oxidize/reduce Isomerize/racemize Can be light sensitive Lack a UV chromophore Have polymorphs
Hydrolyze Oxidize/reduce Isomerize/racemize Deamidate Can be light sensitive Have spectral doppelgangers Stick Take up water Contain a lot of extra stuff per mg of bulk API Aggregate
Oxidation/Reduction/Hydrolysis/Light Small Molecules
Reversed-Phase
Peptides
Usually C18 Usually UV Usually low pH
< pKa Hydrophobic interactions Ion-pairing?
Reversed-Phase C3 – C18 UV Wider pH range
Multiple pKa’s
Specific Concerns Crosslinking Oxidation
Methionine (S) Tryptophan Cysteine (S) Histidine Tyrosine
Isomerization/Racemization Small Molecules
Regulatory Guidelines Chiral methods Normal Phase
Peptides
No Guideline for peptides D and L amino acids – controlled at synthesis Look for key isomers by RP Asn or Asp to Iso-Asp
Isomerization
Demond W, et al. Orthogonal HPLC Methods for Quantitating Related Substances and Degradation Products of Pramlintide. AAPS PharmSciTech 2000; 1 (1) article 6.
Resolution of Iso-Asp 0.26 0.24 0.22 0.20 0.18
TFA < pH2
0.16 0.14
AU
0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 -0.04 -0.06 14.00
14.50
15.00
15.50
16.00
16.50
17.00
17.50
18.00
18.50
19.00
19.50
20.00 Minutes
20.50
21.00
21.50
22.00
22.50
23.00
23.50
24.00
24.50
25.00
25.50
0.14 0.12
Phosphate pH 2
0.10
AU
0.08 0.06 0.04 0.02 0.00 -0.02 8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
12.50 Minutes
13.00
13.50
14.00
14.50
15.00
15.50
16.00
16.50
17.00
0.30 0.28 0.26 0.24 0.22 0.20 0.18 0.16 0.14
AU
Phosphate pH 4
0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
12.50 Minutes
13.00
13.50
14.00
14.50
15.00
15.50
16.00
16.50
17.00
0.060 0.055 0.050 0.045
0.035 0.030 AU
Phosphate pH 6.5
0.040
0.025 0.020 0.015 0.010 0.005 0.000 -0.005 -0.010 14.00
14.50
15.00
15.50
16.00
16.50
17.00
17.50
18.00
18.50
19.00
19.50
20.00
20.50 21.00 Minutes
21.50
22.00
22.50
23.00
23.50
24.00
24.50
25.00
25.50
26.00
26.50
27.00
27.50
28.00
Deamidation Small Molecules
Not a term usually associated with SM
Peptides
Concern for peptides containing Asn and to a lesser extent Gln Asparagine/Aspartate change difficult to separate by RP May necessitate ionexchange
“Orthogonal method”
Deamidation
Demond W, et al. Orthogonal HPLC Methods for Quantitating Related Substances and Degradation Products of Pramlintide. AAPS PharmSciTech 2000; 1 (1) article 6.
pKa ~ 3.7
8
Ion-Exchange Ion-Exchange
More column to column variability High salts hard on system System salting out risk if interchanging RP with IE
No UV Chromophore
ELSD CAD RI Fluorescence MS
15.033 Peak 1 0.80 0.70 0.60 0.50 AU
Peptides
0.40 0.30 0.20 0.10 0.00 210.00
220.00
230.00
240.00
250.00
260.00
270.00
280.00
290.00
300.00
310.00
320.00
nm
1.00
7.400 Peak 1
0.90 0.80 0.70 0.60 AU
Small Molecules
0.50 0.40 0.30 0.20 0.10 0.00 210.00
220.00
230.00
240.00
250.00
260.00
270.00
nm
280.00
290.00
300.00
310.00
320.00
No UV Chromophore
ELSD CAD RI Fluorescence MS
15.033 Peak 1 0.80 0.70 0.60 0.50 AU
Peptides
0.40 0.30 0.20 0.10 0.00 210.00
220.00
230.00
240.00
250.00
260.00
270.00
280.00
290.00
300.00
310.00
320.00
nm
1.00
7.400 Peak 1
0.90 0.80 0.70 0.60 AU
Small Molecules
0.50 0.40 0.30 0.20 0.10 0.00 210.00
220.00
230.00
240.00
250.00
260.00
270.00
nm
280.00
290.00
300.00
310.00
320.00
When UV Chromophore is Present Small Molecules
Select wavelength for mass balance (potency
Peptides
loss = purity loss) Response Factor (RF) may be considered
Ensure good signal to noise Using TFA try to use a higher wavelength Target LOQ ≤ 0.05%
Use amide bond (210220 nm) No RF’s needed
Don’t use wavelength of aromatic residues Ensure good signal to noise TFA produces poor signal to noise at 220 nm Target LOQ ≤ 0.05% Low dose: LOQ ≤ 0.1% may be acceptable* *ICH Q3B(R2) Impurities in New Drug Products
Sensitivity 0.010
0.009
0.008
TFA 220 nm LOQ ~ 0.1%
AU
0.007
0.006
0.005
0.004
0.003 16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
42.00
Minutes
0.0020 0.0018 0.0016 0.0014
Perchlorate 220 nm LOQ < 0.01%
AU
0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
21.00 Minutes
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
Sensitivity 0.010
0.009
0.008
TFA 220 nm LOQ ~ 0.1%
AU
0.007
0.006
0.005
0.004
0.003 16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
42.00
Minutes
0.0020 0.0018 0.0016 0.0014
Perchlorate 220 nm LOQ < 0.01%
AU
0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
21.00 Minutes
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
Similar Spectra
Similar Spectra
Similar Spectra Small Molecules
Use peak purity assessment to determine if related substances are separated
Peptides
Need orthogonal detection to determine if peaks are fully separated Mass spec?
Leads companies to go into Phase 1 with two separation mechanisms
Adsorption Small Molecules
Rarely an issue
Peptides
Type of filter Type of HPLC vial How to prepare LOQ sensitivity samples? How to test dosing solutions? Carryover “Bio-inert” plumbing (no stainless)
LOQ Determination
Small Molecule 0.0140
LOQ Sample Response
0.0135 0.0130
30000
0.0125 0.0120
0.0110
20000 Area
AU
0.0105 0.0100 0.0095 0.0090
15000 10000
0.0085
5000
0.0080 0.0075
0
0.0070 0.0065
0
0.0060 0.0055
y = 75507x + 175.41 R² = 1
25000
0.0115
6.50
7.00
7.50
8.00
8.50
9.00 9.50 Minutes
10.00
10.50
11.00
11.50
12.00
0.05
0.1 0.15 0.2 0.25 0.3 % of Method Nominal Conc.
0.35
0.4
ICH QB2: Validation of Analytical Procedures – Methodology; 1996
19
LOQ Determination - Peptide 0.024
Sample = 0.2% of Method Nominal
0.022 0.020 0.018
AU
0.016 0.014
Plastic
0.012 0.010 0.008
Glass
0.006 0.004 7.00
8.00
9.00
10.00
11.00
12.00
13.00 Minutes
14.00
15.00
16.00
17.00
18.00
19.00
LOQ Determination - Peptide 0.024
Sample = 0.2% of Method Nominal
0.022 0.020 0.018
0.014
Plastic
0.012 0.010 0.008
Glass
0.006 0.004 7.00
8.00
9.00
10.00
11.00
12.00
13.00 Minutes
14.00
15.00
16.00
17.00
Area Counts per mg/mL 80000000 70000000
Response Factor
AU
0.016
60000000 50000000 40000000 30000000 20000000 10000000 0 0
0.1
0.2 0.3 0.4 Sample Concentration (mg/mL)
0.5
0.6
18.00
19.00
LOQ Determination - Peptide 0.024
Sample = 0.2% of Method Nominal
0.022 0.020 0.018
0.014
Plastic
0.012 0.010 0.008
Glass
0.006 0.004 7.00
8.00
9.00
10.00
11.00
12.00
13.00 Minutes
14.00
15.00
16.00
17.00
18.00
19.00
Area Counts per mg/mL 80000000
Method Nominal
70000000
Response Factor
AU
0.016
60000000 50000000 40000000 30000000 20000000
0.2% of Nominal
10000000 0 0
0.1
0.2 0.3 0.4 Sample Concentration (mg/mL)
0.5
0.6
Hygroscopicity Small Molecules
Not usually an issue for free forms More an issue for salts
Peptides
Usually an issue Karl Fischer titration Adsorption to apparatus – fouling
Reference Standard Program Primary Standard (solid) Vialed Working Standard
The Other Stuff Accurate Peptide Content Small Molecules
Assay Correction
Peptides
HPLC purity Salt counterion (?) Water ↓ Residual solvents ↓
Assay Correction HPLC purity Peptide Content
Salt counterion Water Residual Solvents Residue on Ignition
Peptide Content AAA Nitrogen Analysis Accurate water at time of analysis? What is peptide content if water is changing?
Aggregation Small Molecules
Normally not an issue
Peptides
Often an issue RP difficult to resolve due to high retention Agency may ask for size-exclusion (SEC) if known pathway DLS/MALS as research tool
Aggregation Reversed Phase Control
0.012 0.010
AU
0.008 0.006 0.004 0.002 0.000
Light
0.012 0.010
AU
0.008 0.006 0.004 0.002 0.000 8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00 21.00 Minutes
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
Aggregation 0.160 0.150
Reversed Phase
0.140 0.130 0.120 0.110
~27%
0.100 0.090
AU
0.080 0.070 0.060 0.050 0.040 0.030 0.020 0.010 0.000 -0.010 -0.020 6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00 20.00 Minutes
21.00
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
33.00
1.00 0.80
SEC
AU
0.60 0.40
~30%
0.20 0.00 -0.20 0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00 Minutes
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
THANK YOU! Lisa Caralli Director of Pharmaceutics, Pharmatek 2292 Carroll Road Suite 200 San Diego, CA 92121 858-805-6383
