Strategies for improving protein therapeutic half-life using multi-detection SEC

Introduction

In the field of protein therapeutics, a protein-polymer conjugation strategy can improve the protein’s clinical potential by overcoming some of the challenges, such as physiochemical instability, immunogenicity and suboptimal circulation half-life.[1][2] This strategy is the covalent modification of a therapeutic protein with a polymer of biological or synthetic origin. A commonly used polymer is polyethylene glycol (PEG), and the process is known as PEGylation.[3]

In this study the resulting crude product of a PEGylation reaction performed on L-asparaginase was analyzed using the Malvern Panalytical OMNISEC multi-detection SEC system and the compositional analysis, with the following aims:

• to understand the sample’s constituents at the molecular level
• to assess the success of the PEGylation reaction and allow the scale-up of the reaction, while avoiding laborious and time-consuming purification of protein PEGylation trials 

This is to save time: analyze crude reaction mixture to make decisions on large scale synthesis.

Knowing the molecular characteristics of a product is necessary to assess the success of a reaction and to optimize both the production process and the final product properties. Size exclusion chromatography (SEC) is a particularly useful tool to assess protein PEGylation, the size difference between the native protein and PEGylated variant facilitates separation and thus molecular weight characterization of the different species. In addition, multi-detection SEC allows for the direct calculation of the composition of the conjugated sample – an important step to speed up workflows. 

The SEC system from Malvern Panalytical, the multi-detection OMNISEC system (Figure 1), can characterize macromolecules in terms of absolute molecular weight and molecular weight distribution, molecular size and structure. This multi-detection SEC also enables compositional analysis, which exploits the combination of the two concentration detectors, refractive index (RI) and UV-Vis, to determine the concentration of the two components in a sample, such as a PEGylated protein. This, combined with the data from the right angle or low angle light scattering (RALS/LALS) or multi-angle light scattering (MALS) detectors, and the online differential viscometer detector, allows for a full and accurate characterization of a conjugated sample.[4]

Figure 1: Malvern Panalytical multi-detection OMNISEC system, comprising OMNISEC RESOLVE (left), OMNISEC REVEAL (right) and OMNISEC software.

The synthesis of PEGylated L-Asparaginase

The chosen protein of this study, L-asparaginase, is a biopharmaceutical used in treatment of acute lymphoblastic leukemia (ALL) thanks to its ability to cleave L-asparagine to ammonia and L-aspartic acid leading to the death of the cancer cells.[5] In its native form this enzyme can however cause immune response, in addition to having a short half-life and low solubility. PEGylation renders this enzyme more suitable, and the approved medicine Oncaspar is one example of the benefits of the PEGylation of L-asparaginase. 

The N-terminal site-specific PEGylation via reductive alkylation at low pH (Figure 2)[6] is the methodology used in this study to produce homogeneous PEGylated L-asparaginase, with a single chain conjugated to the amine terminus of L-asparaginase.

Figure 2: Schematic of PEGylation reaction of L-asparaginase with PEG-aldehyde.

For this study a PEGylation reaction was carried out and the resulting product, reported in Table 1 along with the chosen conditions, has been characterized and described here. For this synthesis, the chosen PEG-aldehyde has a nominal molecular weight of 10 kDa and the buffer used is 100 mM NaOAc with 20 mM NaB(CN)H₃ at ambient temperature.  

Table 1: Synthesized sample and respective reaction conditions. Samples name: A-PEGy stands for PEGylated L-asparaginase, 10k is the nominal Mw of the PEG used.   

Compositional analysis of PEGylated L-Asparaginase

The OMNISEC^TM software compositional analysis method is designed to determine the real concentration profile of the two components constituting a heterogeneous sample. In general, the method is used when working not only with PEGylated proteins, like in this study, but also with copolymers, polymer blends, membrane proteins, protein and DNA complexes, AAVs[7] and many others. To know more about compositional analysis check this video: Compositional analysis with OMNISEC.[8] 

Results and discussion

In order to facilitate compositional analysis of the PEGylated sample and have full knowledge of the starting materials, the first samples analyzed were the pure protein L-asparaginase and PEG 10k. The chromatograms produced for these two samples are shown below in Figure 3. The SEC quantitative results for the two pure samples are shown in Table 2.

Figure 3: L-asparaginase and PEG 10k multi-detector chromatograms. UV-Vis detector chromatogram reported at 220 nm. a) L-asparaginase, which is a homotetramer in its biologically active form, elutes with multiple peaks. The main peak at 8-8.5 mL represents the tetrameric protein, while the small peak eluting at 7-7.4 mL is the octamer form of the protein. b) PEG elutes as a single peak at 8-9 mL, and to be noted is the absence of the positive sample’s peak in the UV-Vis detector chromatogram (220 nm), confirming the absence of UV absorbance for PEG.

Table 2: Quantitative results from the analysis of the pure samples L-asparaginase and PEG 10k. In the table: weight and number-average molecular weight (Mw and Mn), dispersity (Đ), intrinsic viscosity (IV), hydrodynamic radius (Rh).

The crude product of the PEGylation reaction was then analyzed: A-PEGy 10k (Table 1). The multi-detector chromatogram of this sample is shown in Figure 4. 

Figure 4: OMNISEC multi-detector chromatogram of sample A-PEGy 10k.

The PEGylated L-asparaginase A-PEGy 10k sample, as a crude product, shows a complex chromatogram with multiple peaks that at a first sight are difficult to understand. In Figure 5 the comparison of the A-PEGy 10k RI chromatogram with the ones obtained for the pure PEG and protein (shown above in Figure 3) helps to identify the peaks of unreacted PEG and protein within the PEGylated sample.  

Figure 5: Overlaid RI chromatograms of the conjugate samples A-PEGy 10k (green), PEG 10k (red), and L-asparaginase (purple). 

The two peaks of the A-PEGy 10k sample’s RI chromatogram that correspond to the peaks of the PEG and protein chromatograms, between 8 and 9 mL of retention volume, are the unreacted starting materials. The third peak at 7-8 mL, eluting before the unreacted reagents, corresponds to a reaction product with increased size in comparison to the unreacted protein and PEG. To assess whether this third peak is the desired PEGylated L-asparaginase product or a reaction side product, the A-PEGy 10k multi-detection chromatogram has been analyzed using the compositional analysis method in the OMNISEC^TM software. The complex chromatogram was analyzed by setting multiple limits as shown in Figure 6, and for each peak the weight fractions of PEG and protein have been calculated, see Table 3. 

Figure 6: Multi-detector chromatogram of A-PEGy 10k, showing limits and baselines set for four different peaks within the sample’s chromatogram: from left to right Peak 1-4.

Table 3: Quantitative results for Peak 1-4 for sample A-PEGy 10k. In the table: retention volume (RV), weight fraction of PEG and protein, and concentration of protein calculated for duplicate injections. 

The weight fractions in Table 3 confirm that the two peaks identified from the overlay in Figure 5 as PEG and protein (peaks 2 and 3), are indeed the unreacted reagents of the reaction. The two peaks both contain more than 91% of protein and PEG respectively. The reason why the weight fraction is not 100% PEG or protein, is due to the partial overlapping of the two peaks in the chromatogram, meaning that the two peaks cross-contaminate each other. 

Peak 1, at low retention volume (7.5 mL), contains 12.6% of PEG and 87.4% of protein proving that the peak corresponds to a PEGylated form of L-asparaginase. Plotting the relative concentrations of each component along with the RI chromatogram (Figure 7) shows how the components are distributed throughout the sample. It is evident that the protein is the main component of peak 1, the PEGylated protein peak, but the peak comprises PEG as well. Peaks 2 and 3, which partially overlap, comprise of predominately protein and PEG respectively. 

Figure 7: Overlay of RI chromatogram and concentration profiles of PEG 10k and L-asparaginase (protein) for sample A-PEGy 10k. OMNISEC compositional analysis. 

Conclusions

In this study multi-detection SEC analysis has proven to be a key tool for the development and optimization of PEGylation reactions.  The OMNISEC system from Malvern Panalytical was used to successfully characterize a crude product derived from PEGylation reaction of L-asparaginase. The powerful compositional analysis method allows for a full characterization of the PEGylated protein, ensuring a high level of understanding of the successfulness of the PEGylation reaction and the composition of the PEGylated protein product. The fast turnaround of the process, including reaction and straightforward SEC characterization, without the need for lengthy purification steps, empowers scientists that can make fast decisions when optimizing the production of a product, such as a PEGylated protein.   

References

1. J.E. Murray, N. Laurieri, R. Delgoda, “Chapter-24 – Proteins”, Pharmacognosy, 2017, 477-494
2. Sahar Awwad, Claire Ginn, Steve Brocchini, “The case for protein PEGylation”, Engineering of Biomaterials for Drug Delivery Systems, 2018, 27-49
3. “FDA Approved PEGylated Drugs” https://www.biochempeg.com/article/58.html
4. https://www.materials-talks.com/compositional-analysis-now-with-omnisec/ 
5. Saleh A. M. et al., "Purification and Characterization of Asparaginase from Phaseolus vulgaris Seeds", Evidence-Based Complementary and Alternative Medicine, vol. 2015, Article ID 309214, 2015
6. O. Kinstler et al., “Mono-N-terminal poly(ethylene glycol)-protein conjugates”, Adv. Drug Del. Rev., 2002, 54, 477-485
7. “The importance of Multi-Detection SEC in gene therapy” https://www.malvernpanalytical.com/en/learn/knowledge-center/application-notes/AN200930GeneTherapySEC.html
8. “Compositional analysis with OMNISEC” https://www.youtube.com/watch?v=jybjnIqNWB8&list=PL2wIBTZfZRjd7tyhnBRy2xLhToO3CCcY9&index=6