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INTRODUCTION

Purifying synthetic peptides is necessary since impurities can

potentially affect therapeutic safety. Here, we show a mass-

directed peptide isolation method that significantly improves

the purity of the targeted peptide. It is also necessary to

analyze the purified peptide for process related impurities and

post-purification degradation products.

Selecting optimal column chemistry is an important step in

developing a synthetic peptide impurity analysis. The use of

LC/MS as an effective tool for analytical RP column screening

will be demonstrated. By specifically interrogating a RP

chromatographic separation of synthetic peptides for

predicted impurities such as deamidation, misincorporation,

and fragmentation, a high degree of confidence can be

achieved in the final analytical method even when performed

with only a UV absorbance detector.

SYNTHETIC PEPTIDE IMPURITY ANALYSIS AND PURIFICATION

METHODS

For Purification:

Fraction collection system: Waters AutoPurification System (shown below)

Detection: UV: 2998 Photodiode Array Detector, 214 nm

Mass Spectrometer: Acquity QDa Mass Detector

RESULTS

CONCLUSIONS

Peptides are gaining more and more attention as potential biotherapeutics. Currently, more than 100 peptides are marketed worldwide. The synthetic peptide approach has the advantage of being generated in a quick and well-controlled way using solid phase peptide synthesis (SPPS). However, impurities do exist inevitably. They are originated from raw material, manufacturing process and storage conditions.

Hua Yang, Jo-Ann M. Jablonski, Stephan M. Koza, Joe Fredette and Weibin Chen

Waters Corporation, Milford, MA, USA

Figure 4a. Separation of bivalirudin with formic acid mobile phases. Gradient: 18-

38%B in 20 min. Orange traces: 15-35%B in 20 min, 10-30%B in 20 min. Orange

ovals indicate unique peaks observed on a particular column.

II. Column Selection for Synthetic Peptide Impurity Analysis

A. Bivalirudin (Brand name: Angiomax, Angiox) is a specific and re-

versible direct thrombin inhibitor. It is used to decrease the clotting abil-

ity of the blood and to help prevent harmful clots from forming in the

blood vessels.

Bivalirudin (Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu

-Ile-Pro-Glu-Glu-Tyr-Leu) is a 20 amino acid peptide, with a monoiso-

topic mass of 2178.986 Da. It has an asparagine (Asn) residue, which

could undergo deamidation. In addition, the poly-glycine motif in the se-

quence is of great interest.

Figure 4b. Separation of bivalirudin with TFA mobile phases. Gradient: 22-42%B

in 20 min. Orange traces: 14-34%B in 20 min. Orange ovals indicate the unique

peaks that were observed on BEH C18 300 Å with FA mobile phases were now

observed on all columns with TFA mobile phases.

Figure 4c. Separation of bivalirudin with ammonium formate (pH 10) mobile

phases. Gradient: 5-25%B in 20 min.

B. Ceruletide (Brand name: Takus, Tymtran) stimulates gastric, biliary,

and pancreatic secretion and certain smooth muscle. It is used as diag-

nostic aid in pancreatic malfunction. Ceruletide is also known as ce-

rulein or caerulein.

Ceruletide (Pyr-Gln-Asp-Tyr(SO3H)-Thr-Gly-Trp-Met-Asp-Phe-NH2) is a

10 amino acid peptide, with a monoisotopic mass of 1351.449 Da. It

has a chemical modification – sulfonic acid, which is predicted to make

the molecule extremely acidic. In addition, it has a pyroglutamic acid

residue at the N-terminus, a methionine (Met) residue that could be

prone to oxidation, and an amidated C-terminus.

Figure 5a. Separation of ceruletide with formic acid mobile phases. Gradient: 18-

38%B in 20 min. Orange traces: 28-48%B in 20min for CSH Phenyl Hexyl; 35-

55%B in 20 min for CSH Fluoro Phenyl

Figure 5b. Separation of ceruletide with TFA mobile phases. Gradient: 18-38%B in

20 min.

Figure 5c. Separation of ceruletide with ammonium formate (pH 10) mobile phases.

Gradient: 10-30%B in 20 min.

Impurity Mass Mass Putative identity

Peak 1 2180.00 1.01 deamidation

Peak 2a 2161.96 -17.03 2a = 2b + deamidation

Peak 2b 2160.99 -18.00 H2O loss

Peak 3 1167.54 -1011.45 Fragment [12-20]

Impurity Mass Mass Putative identity

Peak 1 1271.503 -79.960 SO3 loss

Peak 2 1074.417 -277.046 (-262-15): fragment [1-8] - 15Da

Peak 5 917.381 -434.082 (-354-80): fragment [4-10] and

SO3 loss

Table 1. Impurities of bivalirudin, identified by HRMS.

Table 2. Impurities of ceruletide, identified by HRMS.

TFA mobile phases

Formic acid mobile phases

Ammonium Bicarbonate pH 10 mobile phases

m/z = 595.30, [M+2H]2+

Figure 1. For purification, separation of eledoisin was optimized on a 4.6 x 100 mm

XSelect Peptide CSH C18 Peptide column, using various mobile phases with fo-

cused gradients (0.36% B change/column volume). The pH 10 mobile phases

gave the best separation, so it was used for the fraction collection in large scale.

Figure 2. Crude eledoisin was purified on a XSelect®

Peptide CSH™ C18 OBD™

Prep Column, 130 Å, 5 µm, 19 x 100 mm, using Ammonium Bicarbonate pH 10

mobile phases. Upper panel: UV trace; Lower panel: Mass detection by QDa.

QDa data was used to confirm that the fraction collected was indeed the main

peak of eledoisin.

For Analysis:

LC system: ACQUITY UPLC H-Class Bio

Detection: UV: Acquity TUV, 214 nm

Mass Spectrometer: HRMS

Mobile phases and columns: please see figures for details

I. Synthetic Peptide Purification

Eledoisin belongs to the tachykinin family of neuropeptide, which is be-

lieved to exhibit a wide spectrum of pharmacological and physiological

activities such as vasodilation, hypertension,and stimulation of extravas-

cular smooth muscle.

Eledoisin (pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2) is an 11

amino acid peptide, with a molar mass of 1188.4 Da.

Figure 3. A complementary reversed phase method was used to estimate the pu-

rity of the fractions (55 and 56) of eledoisin: a Peptide BEH C18 130 Å, 1.7 µm, 2.1

x 150 mm column with TFA mobile phases. The purity of eledoisin increased sig-

nificantly from 49% (crude) to >96% (purified fractions)

In this study, optimization of purification conditions of the synthetic peptide was first carried out using a small-scale sample. After the optimization, a large amount of sample (>35 mg) was purified on a Prep column.

Mass-directed purification was used to confirm the identity of the purified peaks. This method can be used to significantly increase the purity of the sample in a single step purification.

A variety of synthetic peptides were separated on several reversed-phase columns under different mobile phase conditions. Column screening revealed unique characteristics of different column chemistry. Below are some of the observations:

1. Selectivity

Selectivity changes not only among columns, but also under different mobile phase conditions.

2. Retentivity

For most of the synthetic peptides, CSH columns have the least retentivity, while the HSS T3 column has the most retentivity.

For peptides that have extremely low pKa (e.g. Ceruletide), CSH columns have the most retentivity, likely because the net charge on the peptide is negative even with acidic mobile phases and it interacts with the positive surface of the CSH particles, resulting in longer retention.

3. Loadability

CSH type columns generally have the highest peak capacity. Therefore, more sample can be loaded onto these columns without sacrificing the resolution.

Based on the chromatographic results, it is clear that there is no single column that is the most effective for all of the synthetic peptide impurity analyses attempted. As a result, screening columns for this application can be beneficial. As a starting point, below are some of the significant characteristics of the “Peptide” columns that Waters offer:

- Peptide BEH C18 columns (130 Å and 300 Å):

> For general use

> For more retention in FA mobile phases, use 300 Å column for higher molecular weight peptides (> 2100 Da) or bulky peptides (e. g. PEGylated peptide); use 130 Å column for lower molecular weight peptides (< 1400 Da)

- Peptide CSH C18 130 Å column:

> Significant selectivity differences compared to BEH C18 phases

> Narrowest peak width in both FA and TFA (highest loadability)

> Lower retention than HSS T3 and BEH C18 phases (with exceptions, see ceruletide)

- Peptide HSS T3 100 Å column:

> Highest peptide retention (with exceptions, see ceruletide)

> Highest retention for early eluting (short, hydrophilic) peptides

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