High performance liquid chromatography (HPLC) Technique

High performance liquid chromatography, namely HPLC, is a well-known and efficient separation and analysis technology for free PEG and its PEGylated conjugate. Moreover, HPLC is also a powerful technique that can be used for the quantitative measurement of PEG, PEGylated small drugs, PEGylated liposomes and PEGylated micelles. Some features such as conjugate molecular weight, polymer mass distribution, or the degree and sites of PEGylation can be measured.

Fig. 1. Schematic diagram of high-performance liquid chromatography (HPLC). (British Journal of Hospital Medicine. 2014 75(2):C18-21.)

What is HPLC and Its Principle?

In the chromatographic column, the separation process is realized through the interaction among components of the mixture, the stationary phase and the mobile phase:

Fig. 2. The migration illustration of molecules in an HPLC column. (Diagnostic Molecular Biology 2019: 187-214)

The stationary phase is an inert tube (column) packed with a very small porous powder, and the mobile phase is a solvent or mixed solvent that passes through the chromatographic column under high pressure. The sample is injected into the eluent before the column. When the sample is mixed with the mobile phase and passes through the chromatographic column at different rates, each component will flow out of the chromatographic column in turn due to the different adsorption capacity between the components and the stationary phase. Once separated, the component concentration could be converted into an electrical signal and transmitted to the computer software through a suitable detector, and the information you want can be obtained by analyzing the chromatogram curves.

PEG Analysis Services from BOC Sciences

BOC Sciences offers a comprehensive suite of PEG analysis services tailored to meet the diverse needs of researchers and industry professionals. These services include characterization, quantification, and validation of PEG molecules in a variety of formulations and matrices. By utilizing advanced analytical techniques such as chromatography, mass spectrometry and spectroscopy, BOC Sciences provides precise and reliable data on PEG purity, molecular weight distribution and structural integrity. In addition, BOC Sciences' expert team of chemists and analysts specializes in customizing analytical protocols to meet specific client needs. Whether assessing the stability of PEG-modified compounds or elucidating the mechanisms of PEG-drug interactions, BOC Sciences' PEG analysis services are designed to provide actionable insights to enhance product development and quality control processes.

In addition to PEG analysis, BOC Sciences offers specialized PEGylation analysis services designed to optimize the design and performance of PEGylated products. Through a combination of chemical, biological, and biophysical assays, BOC Sciences evaluates the conjugation efficiency, site specificity, and functional impact of PEGylation on target molecules. This in-depth analysis enables customers to fine-tune the PEGylation process to improve pharmacokinetics, bioavailability and therapeutic outcomes.

HPLC for PEGylation Quantification

Because that PEGs do not contain active chromophores to be detected by UV light, the single HPLC cannot be able to assess the localization of PEG within the conjugates, but can be successfully utilized when coupled with other analytical tools, for example, refractive index detection (RID), mass spectrometry (MS), and the increasingly popular evaporative light scattering detection (ELSD). The grafting thickness can be estimated as half of the enlarged particle size compared to that of the non-PEGylated particles.

RID-HPLC

PEG molecules separated by HPLC can be directly measured with a RID, which has been commonly employed to measure PEG concentrations in urine samples during studies examining intestinal permeability. Size exclusion HPLC with RID can also be used to measure the content of residual PEGs in PEGylated protein samples.

UV-based HPLC

Although the sensitivity of PEG when detected by UV absorption is not comparable to that detected by RID, it still can be improved by some modification methods, such as link of the benzoate group. Moreover, UV-based HPLC can also be useful to measure incorporated drug concentrations in PEGylated liposomes or micelles. In comparison with fluorescent detection, UV-vis detection can help avoid potential interference and improve the accuracy.

HPLC-MS/MS

High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) allows sensitive quantitation of free PEG in biological fluid samples.

HPLC-ELSD

The only requirement of ELSD is that the conjugates or molecules should not evaporate under the set conditions, while the solvent evaporates. HPLC-ELSD is commonly used for analysis of compounds that do not efficiently absorb UV radiation.

LC-MS/MS

Utilizing some pretreatment steps before analysis, such as denaturation, alkylation, precipitation or solid phase extraction, can reduce the complexity of samples, thereby allowing LC-MS/MS become a more versatile tool for detection and identification of the positional isomers formed upon PEGylation.

HPLC is an excellent choice for the quantification of PEGylated drugs and small molecules. Although generally useful, the HPLC conditions and detection method must be optimized for each compound based on the chromatographic and spectral properties of the conjugated drug or small molecule.

Featured PEG Products

In addition to analytical services, BOC Sciences offers a variety of standard PEG products, including PEG polymers of various molecular weights, PEG derivatives with different functional groups, and PEG conjugates for specific applications. These products are manufactured using high-quality raw materials and state-of-the-art synthesis techniques to ensure consistency and purity. Whether you need custom PEG polymers for a specific research project or standard PEG derivatives for commercial applications, BOC Sciences has the expertise and capabilities to meet your needs. With a focus on innovation, quality and customer service, BOC Sciences is the first choice for PEG synthesis services and products.

Strengths & Weaknesses of HPLC

High-performance liquid chromatography (HPLC) analysis of polyethylene glycol (PEG) has several advantages and disadvantages:

Strengths

• Sensitivity: HPLC is a highly sensitive technique that can detect low concentrations of PEG.
• Selectivity: HPLC can separate PEGs of different molecular weights, providing detailed information about the components.
• Speed: HPLC analysis of PEG is relatively fast and samples can be analyzed quickly.
• Accuracy: HPLC can accurately quantify PEG, making it suitable for quality control purposes.
• Reproducibility: If optimized properly, HPLC analysis can be highly reproducible, ensuring consistent results.

Fig. 3. Advantages and disadvantages of HPLC analysis of PEG.

Weaknesses

• Cost: HPLC equipment and consumables can be expensive, making them difficult to use for some laboratories.
• Sample preparation: Sample preparation for PEG HPLC analysis can be time-consuming and complex, especially for complex matrices.
• Limited information: While HPLC can provide detailed information about the composition of PEG, it may not provide information about other impurities or contaminants present in the sample.
• Interference: HPLC analysis of PEG may be susceptible to interference from other compounds present in the sample, affecting the accuracy of the results.

Column Screening in HPLC Analysis of PEG

Protein drugs modified with PEG can prolong the residence time in the blood, inhibit the renal clearance rate, improve the solubility and stability of the drug, reduce immunogenicity and other beneficial effects. In particular, the effectiveness of drugs that incorporate branched PEG will be further improved. However, PEG-modified drugs will produce isomers due to different reasons such as the number or molecular weight of bound PEG and different binding sites. Therefore, in the development, production and quality control of such drugs, the separation and analysis of isomers by HPLC method is a very important part. Next, we list some application examples of TSKgel columns in the analysis of PEG-modified biopharmaceuticals.

Case Study 1

SEC analysis of PEG modified lysozyme

Fig 4 is a chromatogram of using size exclusion chromatography column TSKgel G3000SWXL (5μm, 7.8mm I.D.×30cm) to separate PEG-modified lysozyme of different molecular weights. In PEG-modified lysozyme, up to 3 molecules of PEG can be bound, and they are eluted in order from the most to the least number of bound molecules. The greater the molecular weight of the bound PEG, the faster the PEG-modified lysozyme will elute from the column.

Fig. 4. Size exclusion chromatography (SEC) analysis of PEG modified lysozyme.

Case Study 2

IEC analysis of PEG recombinant human growth hormone

Somavert® is Pfizer's PEG-human growth hormone receptor blocker for the treatment of acromegaly. Fig 5 shows the separation using cation exchange chromatography column TSKgel SP-STAT (7μm, 4.6mm I.D.×10cm). Since PEG modification binds PEG to the amino groups of proteins, the greater the number of bound molecules, the positive charge will be reduced accordingly, and the faster it will elute from the ion exchange column.

Fig. 5. IEC analysis of PEG recombinant human growth hormone.

References

1. Cheng T L, Chuang K H, et al. Analytical measurement of PEGylated molecules. Bioconjugate chemistry. 2012, 23(5): 881-899.
2. Blum F. High performance liquid chromatography. British Journal of Hospital Medicine. 2014 75(2):C18-21.
3. Shen, C. H. Quantification and Analysis of Proteins. Diagnostic Molecular Biology. 2019: 187-214.