5. Introduction
• Importance of Bio therapeutics drugs have increased enormously during
recent years owing to their high specificity and low toxicity.
• They vary from small peptides to large proteins to fusion proteins.
• Market valued at around US$199 billion in 2013 projected to grow by
13.5% by 2020
• More than 600 biological drugs are currently approved and now account
for 30% of all drugs in development.
• For Bioanalysis we segregate peptides from proteins based on size.
Molecular weight above 6000 Da classified as proteins.
• Many of therapeutic drugs are below 6000 Da so classified as peptides.
5
7. Solubility
• General rules in predicting Solubility
• Peptides shorter than 5 amino acids are generally aqueous soluble
• Peptides containing <25% of hydrophobic amino acids are aqueous soluble.
• Hydrophobic peptides containing 50% or more of hydrophobic amino acids are
insoluble or partially aqueous soluble. These peptides needs to be dissolved in
DMSO, DMF or acetonitrile before making aqueous dilutions.
• Peptides containing >75% of hydrophobic amino acids (DEHKNQRSTY) capable of
forming gels these peptides are solubilized in organic solvents or pH of the buffer
to be adjusted.
7
8. Stability
• Plasma contains preoteases which degrades protein or peptide by acting at amide
linkage.
• DEP(Diisopropylfluorophosphate), Cocktails (Sodium fluoride, potassium oxalate,
Trichloro acetic acid), pefabloc (better than DEP)
• Choice of stabilizer is compound dependant.
• Even acidifying with formic acid or TFA will inhibit proteases.
• Storage conditions like -20, -800C needs to be evaluated
• Use ice baths where ever possible to slow down the reaction.
• Plasma to be separated within half hour of blood collection and stored in
conditions specified.
8
9. Non Specific Binding
• Peptides often exhibit adsorption to vials and tubes at low
concentration.
• Responsible for inconsistent results.
• Use of low adsoption polypropylene tubes, silica deactivated glass are
suggested for making dilutions.
• To minimize the adsorption serial dilutions can be prepared in plasma
• For matrix free solutions solubility may be enhanced by aqueous-
organic mixtures, adding BSA or surfactants to block adsorption sites
• For extracted samples organic content should be kept around 20% to
minimize adsorption.
9
11. Major Techniques (LCMS, LBA)
• Advantages of LCMS over LBA
• Selectivity between similar peptides and proteins (quality information)
• No antibodies required (less chances of cross reactivity)
• Higher throughput (less time to develop methods and easy method transfer)
• Simultaneous measurement of multiple analytes
• High dynamic range (typically greater than 4 orders of magnitude)
• Internal standard
• Use of AC/LC-MSMS sensitivity can be greatly enhanced.
• No reagent development.
11
14. Quantification through intact protein analysis.
• This approach is the true measurement of whole protein.
• MS uses SRM by Quadrapole and HRMS by Q-Tof or Orbitrap
• HRMS provides high level of selectivity owing to the mass accuracy and sensitivity
for some proteins which fragment poorly.
• HRMS may also have advantage of quantifying proteins and their derivatives after
biotransformation and post translation modifications.
• Currently this approach is used for peptides <10kDa molecular weight.
14
Protein ------- Sample prep-------LCMS
15. Surrogate peptide approach
• Commonly employed method for quantification of proteins.
• Usually signature peptides which are unique to the protein are selected.
• Selection Criteria:
1. Avoid peptides containing methionine, Cysteine or tryptophan
2. Avoid RR and KK in the sequence to minimize inconsistent digestion
3. Select peptides with length 5-15 amino acids.(too small too large may cause
specificity and selectivity issues)
• 4. Signature peptide can be generated by performing insilico digestion and can
be searched against all proteins in the biological matrices.
• https://www.expasy.org/proteomics
• http://www.uniprot.org/
15
Protein----digest-----peptides-----sample prep------LCMS
16. Protein digestion:
• Different enzymes used for digestion (trypsin, Glu-C, Lys-C, chymotrypsin, Asp-N)
• Trypsin is commonly employed owing to its availability, efficiency, low cost and
generates smaller fragments which are MS friendly.
• Conditions like incubation time, temperature, protein to enzyme ratio, organic
content around 10-30% ACN to speed up the time can be explored for efficient
and complete digestion.
• Pretreatment with Urea, rapigest and guanidine HCl will enhance the digestion
completeness.
• Denaturation, reduction and alkylation are required for complete sequence
coverage.
• For quantitative bioanalysis direct digestion will also give suroogate peptides
desired.
16
17. Sample clean up.
• Plasma contains more than 60% of Albumin and 30—35% of immunoiglobulins.
• Achieving analyte separation is essential to remove matrix effect or interfering
peaks caused by wide isotopic abundance caused by endogenous proteins and
peptides.
• Most of the peptides and proteins contains mixture of aminoacids. Achieving
sample clean up may be challenging.
• Molecules with high polar aminoacids display weak solubility and with high %
arginine residues will have a strong basic character.
• SPE followed by PPT is a good approach to start with.
17
18. Sample clean Up Cont…
• Generally larger protein precipitate around 40% of organic and smaller peptides
may need higher % of organic solvent.
• Aqueous acid preparations like 1% FA in ACN or 10% TFA in ACN can be tried if
recovery is not consistent.
• SPE avoids harsh organic conditions that can compromise solubility.
• Lower limits of quantitation can be achieved.
• Can be very selective with high recovery.
• Size exclusion can also be used prior to SPE to remove larger proteins.
• SPE needs expertise and with out SIL-IS chances of showing discrepancy in
recovery is more.
18
19. Chromatography
• For most LC seperations traditional C18 columns are good enough
• Recent advances in column technology of sub 2µ particles with pore size of 300
A0 will provide increased resolution, sensitivity and speed.
• Charge surface columns which will reduce secondary interactions are growing in
popularity.
• Decreasing flow rates to 200-400 µl/min, increasing column oven temperatures to
50 – 60 0C and performing shallower gradients will enhance sensitivity and peak
shape.
• Organic modifiers are always employed and acidic modifiers like formic acid and
TFA can also be tried to neutralize carboxyl.
• For very acidic and basic peptides or proteins ion exchange can also be tried.
19
20. Mass spectrometry.
• Peptide and proteins generally multiply charged in ES source
• Ratio of formation one charge state to other depends on flow rate and solvent
composition.
• So tuning to be performed with LC conditions.
• Peptides and proteins demonstrate wider isotopic distributions limiting the
sensitivity.
• Monitoring several charge states and isotopes can help in increasing the
sensitivity.
• Digestion of peptides and monitoring surrogate peptides will also help
• Peptides tend to fragment poorly. Increasing the collisional energy will
compromise the selectivity as it leads to individual amino acids.
• Avoid choosing immonium ions, water losses and adducts.
• Good practice is to choose b or y ions for specificity.
• With new technologies performing ETD will also generate more specific
fragments like a and z ions.
20
21. Challenges using LCMS
• Multiple charging and wide isotopic distribution restricts sensitivity
• Flow rate and solvent will effect charge state and ionization
• Poor fragmentation
• m/z value may exceed instruments mass range.
• Expertise
• Lack of regulatory guidance.
21
23. Method Validation cont….
• For LBS 4-6-20 rule applies(4 out of 6 should be within 20% of nominal value)
• For LCMS 4-6-15 rule applies
• Stability testing of digested proteins (false negatives if the peptide shows Non
specific binding to wells, false positives if the peptide used is more stable than
whole protein )
• Challenges during immunocapture purification with respect to IS.
• All experiments needs to be performed as for small molecules like selectivity,
precision and accuracy, stability studies, robustness and ruggedness.
23
34. Blank and LLOQ (1 ng/ml) of two unique peptides
RT: 0.00 - 8.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
3.85
3.93
3.94
4.01
1.29
NL: 2.07E4
m/z= 446.2736-446.2780 F:
FTMS + c ESI Full ms2
305.1734@cid25.00
[100.0000-1200.0000] MS
080117_ext_TRIAL_029
NL: 3.21E4
m/z= 650.3630-650.3696 F:
FTMS + c ESI Full ms2
463.2154@cid25.00
[100.0000-1200.0000] MS
080117_ext_TRIAL_029
NL: 8.83E2
m/z= 446.2736-446.2780 F:
FTMS + c ESI Full ms2
305.1734@cid25.00
[100.0000-1200.0000] MS
080117_ext_trial_001
NL: 0
m/z= 305.1719-305.1749 F:
FTMS + c ESI Full ms2
463.2154@cid25.00
[100.0000-1200.0000] MS
080117_ext_trial_001
Lloq- 1 ng/ml
Plasma BLK
34
35. Chromatograms of different concentrations for
peptide YKLW
RT: 0.00 - 8.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
1.29
3.85
3.93
3.83
3.93
3.87
3.95
3.83
3.91 4.02
NL: 8.83E2
m/z= 446.2736-446.2780 F:
FTMS + c ESI Full ms2
305.1734@cid25.00
[100.0000-1200.0000] MS
080117_ext_trial_001
NL: 2.07E4
m/z= 446.2736-446.2780 F:
FTMS + c ESI Full ms2
305.1734@cid25.00
[100.0000-1200.0000] MS
080117_ext_TRIAL_029
NL: 5.27E4
m/z= 446.2736-446.2780 F:
FTMS + c ESI Full ms2
305.1734@cid25.00
[100.0000-1200.0000] MS
080117_ext_trial_030
NL: 1.17E5
m/z= 446.2736-446.2780 F:
FTMS + c ESI Full ms2
305.1734@cid25.00
[100.0000-1200.0000] MS
080117_ext_trial_031
NL: 1.89E5
m/z= 446.2736-446.2780 F:
FTMS + c ESI Full ms2
305.1734@cid25.00
[100.0000-1200.0000] MS
080117_ext_TRIAL_032
1 ng/ml
2.5 ng/ml
blk
5ng/ml
10 ng/ml
35
36. Points to consider
• pH to be maintained at around 8 for Activity of DTT and Trypsin
• Percentage of organic modifier can be tried for enhanced digestion
• Sample were stored in -200c and were found to be stable for 14 days
• Ice bath was used during processing and temperature was maintained around 4
in centrifugal SPE.
• Oasis MAX was used as sorbent for sample clean up
• Protein heterogenicity to be considered (PTMs) and modifications during
processing.
• Calculation of net charge will also help in deciding SPE chemistry.
36
37. Conclusion
• Quantification using HRMS for proteins and large molecules gives better
selectivity and sensitivity.
• HRMS gives qualitative and quantitative data.
• Modifications can be detected.
• Intact protein bioanalysis
• Less time for development of analytical method.
• Depleting high abundance proteins can increase the sensitivity.
37
38. References
• http://www.ionsource.com/
• https://www.expasy.org/proteomics
• Therapeutic peptide bioanalysis_Sciex application note.
• Peptide and protein bioanalysis_Waters application note.
• Quantitative bioanalysis of proteins by mass spectrometry.
Long Yuan et al.
• Approaches to analyzing therapeutic peptides and proteins by LC-MS/MS. Matthew
Ewles
• LC-MSMS of large molecule in regulated bioanalytical environment-which acceptance
criteria to apply. Magnus Knotssun.
• Protein and peptide drug analysis by MS: Challenges and oppurtunities for the discovery
environment. J.larry CAmpbell
• LC-MS-based bioanalysis in support of protein biotherapeutics development:
• current challenges & emerging opportunities. Rand Jenkins 38
