Effect of Excipients on Recombinant Interleukin-2 Stability in Aqueous Buffers

In order to retain structural and functional integrity, protein medicines are frequently stabilized with excipients in aqueous solutions. The goal of this investigation was to see how stable IL-2 is with excipients that are acceptable for cell therapy. We investigated the time-dependent stability of commercially available recombinant IL-2 in aqueous solutions (CTS, RPMI, PBS, and water) at different temperatures [2 ̊C 8 ̊C, room temperature (20 ̊C ± 2 ̊C) and 37 ̊C] in the presence of excipients (EDTA, methionine, histidine, and glycine) over a period of up to 30 days. To detect and quantify IL-2, reversed phase high performance liquid chromatography was employed. Electrophoresis on a sodium dodecyl sulfate polyacrylamide gel was used to assess conformational stability. We discovered that IL-2 stability was improved in aqueous solutions including excipients, and that it may have retained its biological activity and sterility in these conditions.


IL-2 Compatibility with Glass Vials
Surface adsorption is a concern for hydrophobic proteins like IL-2 since they are frequently employed in small amounts, resulting in lower protein concentration in the final product. The most commonly used processing and storage materials, glass and plastics, are well-known for adsorbing large amounts of protein [15]. This can result in protein loss, particularly in low-concentrated protein solutions where the percentage loss is high. IL-2 was diluted to 0.018 mg/mL in a solution of 10 mM sodium citrate and 0.1 N acetic acid, pH 3.0, and placed in glass screw neck vials to study adsorption. Each vial was sealed with a LectraBond cap and kept sealed for 0, 24, and 96 hours at 2˚C -8˚C. To see if IL-2 could be utilized in glass vials, the solutions were injected individually and the peak areas of the freshly created IL-2 solutions were compared.

Selection of Aqueous Solutions for IL-2 Stability
IL-2, a key cytokine for T cell activation, is frequently incorporated in or re- Therapy Systems (CTS) OpTimizer are becoming more popular [16]. Phosphate buffer saline (PBS) is a balanced pH salt solution that reduces protein adsorption [17] and is often used in cell culture for washing and diluting cells. Water is another of them, and it's widely employed for dilution or temporary storage of biomolecules or reagents.

Chromatographic Conditions and Instrumentation
The reverse phase high-performance liquid chromatography (RP-HPLC) was performed [18] with a Agilent 1100 series, comprised of a quaternary pump solvent delivery module, online degasser, thermostated column compartment, auto sampler, auto injector with 100 µL injection loop, and a Variable Wavelength Detector. Samples were maintained at 5˚C in the autosampler prior to analysis. The system was used in a room temperature (RT) HPLC laboratory (20˚C ± 2˚C). The analysis was performed on a ZORBAX 300SB-CN, 150 × 4.6 mm, 3.5 μm column, and column temperature was maintained at 30˚C during analysis. The mobile phase A contained 0.1% trifluoroacetic acid (TFA) in water and B 0.1% TFA in acetonitrile. Gradient elution was used, and the program was set as follows: 30% -80% B from 0 to 10 min; 80% -100% B from 10 to 12 min and 30% B from 13 -20 min. Injection volume was kept constant 50 μL. The flow rate of the mobile phase was set at 0.8 mL/min and the eluate was monitored at an UV wavelength of 214 nm. Chromatogram output, integration of peaks, calculation of peak areas and retention times were obtained using the Empower software, version 3.

Effect of Excipients on IL-2 Stability in Aqueous Solutions
To test the effect of antioxidants and amino acids on IL-2 stability, IL-2 was diluted to approximately 0.018 mg/mL in CTS, RPMI, PBS, and water in the presence of 1 mg/mL EDTA, 5 mg/mL methionine (Met), 5 mg/mL histidine (His), and 5 mg/mL glycine (Gly) in separate glass vials. For short-term stability, each sample vial was incubated at 37˚C for 0, 1, 2, 3, 4, and 5 days. For up to 30 days, solutions were incubated at 2˚C -8˚C, RT (20˚C ± 2˚C), and 37˚C to test their long-term stability. The percent recovery of IL-2 was calculated using linear regression of the analytical standard curve after each solution was evaluated individually using RP-HPLC. The results of the IL-2 assay were compared to samples that had been newly prepared and were free of excipients.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis
To learn more, 1 µg of IL-2 was prepared in CTS, RPMI, PBS, and water in separate vials with 1 mg/mL EDTA, 5 mg/mL Met, 5 mg/mL His, and 5 mg/mL Gly.
To ensure short-term stability, all vials were incubated at 37˚C for 0, 1, 2, 3, 4, and 5 days. For up to 30 days, solutions were incubated at 2˚C -8˚C, RT (20˚C ± 2˚C) and 37˚C to determine long-term stability. Periodically, the solutions were examined by SDS-PAGE under reduced conditions using 4% -20% tris-glycine SDS gradient gels, as described by Laemmli [19]. Before being put onto the gel for examination, samples of IL-2 stability were first treated in the sample buffer at 95˚C for 10 minutes. SeeBlue Plus2 pre-Stained Standard was used as the protein standard, and it was not further processed. Gels were run at RT in tris-glycine SDS buffer at a constant voltage (200V) until the dye front reached the end of the gel. SDS-PAGE gels were dyed using SimpllyBlueTM Safe Stain, washed twice in Milli-Q water, and then destained twice in Milli-Q water. The gels were imaged using a densitometer.

Results
The goal of these tests was to find an excipient that could keep IL-2 stable in a wide range of aqueous solutions utilized in cell therapy. The influence of antioxidants and amino acids on the aqueous stability of IL-2 was examined in order to select an appropriate excipient for stability studies.

Optimization of RP-HPLC Analytical Conditions
The RP-HPLC analytical conditions for separating and quantifying IL-2 in aqueous solutions (CTS, RPMI, PBS, and water) were investigated for their stability. The physicochemical properties and chromatographic behaviors of IL-2 were studied in the literature. Additionally, as per our previous analytical conditions [18], the impacts of a specific combination of column type, mobile phase composition, and detection system were studied. The maximum injection volume for future applications in biological sample analysis was set at 50 µL. The analysis was limited to 20 minutes, and the flow rate was set to 0.8 mL/min to sharpen the peak, resulting in an IL-2 retention time of 7.2 minutes. This flow rate was found to be the best for reducing total run time while maintaining acceptable column backpressure. To ensure that all of the components in the sample solution were sufficiently separated, the column temperature was kept at 30˚C The detection wavelength of 214 nm was investigated for IL-2 while determining the detection wavelength for the analytical procedure, and it was found to yield peaks that were extremely sensitive and reproducible. IL-2 separated well in 10 minutes in this final optimized RP-HPLC condition, followed by a re-equilibration to the original condition.
The selectivity of a chromatographic device refers to its capability to chemically distinguish between pattern components. Chromatograms from 0.1 N acetic acid, CTS, RPMI, PBS, and water were compared to those with IL-2 present to better understand the aqueous solutions matrix impact. For this study, 50 µL of each aqueous solution (without and with IL-2) were introduced into the HPLC apparatus individually. IL-2 eluted as a single peak under the specified chromatographic conditions. As shown by peak purity analysis, there seem to be no co-eluting peaks during the IL-2 retention time that could interfere with the peak of interest, implying that the IL-2 peak is pure. The chromatogram results are shown in Figure 1.  The result of the chromatograms shows that the peak of analyte was pure, and there are no co-eluting peaks at the retention time of the IL-2 to interfere with the peak of interest.

Linearity Study
According to our earlier studies [18], the linearity was explored. The capacity of an analytical method to produce test findings that are directly proportional to the analyte concentration within a certain range is known as linearity. The linearity of IL-2 was investigated throughout a range of concentrations from 0.00458 mg/mL to 0.0495 mg/mL. The calibration graph was created by plotting

IL-2 Compatibility with Glass Vials
IL-2 and other proteins can bind to a various surfaces, and changing the pH can change the degree of adsorption by raising protein charge density, which reduces the hydrophobic effect that causes molecules to congregate or adsorb [13]. This mechanism can significantly alter the secondary structure of IL-2, causing it to lose biological function or become unstable. Adsorption can also be minimized by optimizing the protein solution, adding excipients such as surfactants [15], and adjusting the pH and ionic strength. The pace of protein adsorption varies depending on the surface material and the protein, although it is normally a relatively fast process [20] [21]. In contrast, we investigated the compatibility of glass vials with IL-2 in aqueous solutions and found that the IL-2 peak area did not significantly differ much. This clearly proved that, in our testing conditions, irreversible adsorption to glass vials does not provide a major risk of losing significant amounts of IL-2. Surfactants, on the other hand, were not necessary to prevent IL-2 from adsorbing in glass vials under the conditions we examined. Table 1 summarizes the recovery of IL-2 at the start, after 24, and after 96 hours of storage at 2˚C -8˚C.

Effect of Excipients on the Stability of IL-2 in Aqueous Solutions
The pH, temperature, and components of an aqueous solution can have a considerable impact on the structure of a protein medicine, resulting in poor stability and therapeutic inactivity. To investigate the effect of antioxidants and amino acids on IL-2 stability in aqueous solutions, IL-2 was prepared in CTS, RPMI, PBS, and water in separate glass vials in the presence of antioxidants and amino acids. Sample vials were incubated at 37˚C for 0, 1, 2, 3, and 5 days for short-term stability, while IL-2 sample solutions were incubated for 30 days at different temperatures [2˚C -8˚C, RT (20˚C ± 2˚C) and 37˚C] for long-term stability. Following the completion of the incubation time, all of the stability samples were evaluated by RP-HPLC in a single batch. Table 2, Table 3 and

Effect of Antioxidants on IL-2 Stability in Aqueous Solutions
To minimize or reduce the increase in oxidized product or any other possible oxidized forms of IL-2, we evaluated two antioxidants, EDTA and Met. Because the antioxidant activity of these excipients varies depending on the protein to be stabilized, they were chosen on a basis in evidence [10] [22]. The results indicated that EDTA appreciably elevated the stability of IL-2 in CTS, RPMI, PBS, and water for up to 5 days at 37˚C. The findings of short-and long-term stability tests in aqueous solutions in the presence of EDTA at various temperatures are summarized in Table 2, Table 3 and     are presented in Table 2, Met had no effect on IL-2 stability in PBS (Table 3 and Figure 5). Met oxidation may be accelerated by the presence of high ionic strength, oxygen, and oxygen radicals in PBS. Met is a sulfur-containing amino acid that is found in proteins.
The primary chemical breakdown product of IL-2 has been identified as met-104 sulfoxide IL-2 [14] [23]. Met is a significant oxidant scavenger because it can react with a wide range of oxidants to produce methionine sulfoxide [24]. In We discovered that the stability of IL-2 did not improve (data not shown).

Effect of Amino Acids on IL-2 Stability in Aqueous Solutions
The protein stability of therapeutic IL-2 is influenced by its storage and distribution. Amino acid additions can be made at any point during the manufacturing and storage processes to help stabilize proteins. Proteins have been shown to be stabilized through an exclusion process in which specific amino acids hydrate the protein in solution preferentially [13]. The purpose was to see how the bulking components His and Gly, which are routinely employed in pharmaceutical protein formulations, affected IL-2 stability in aqueous solutions. In CTS, RPMI, PBS, and water, His and Gly were found to act as IL-2 buffering agents. The effect of His and Gly in stabilizing IL-2 in aqueous solutions was investigated using RP-HPLC and SDS-PAGE, as previously mentioned. At any temperature, Gly had no effect on CTS or RPMI, but His improved the stability of IL-2 in CTS and RPMI for up to 5 days at 37˚C. However, in PBS and water, both amino ac-  (Table 3 and Figure 5, Figure 6). The findings of short-and long-term stability tests in aqueous solutions in the presence of His and Gly at various temperatures are shown in Table   2, Table 3 and Figures 3-6. Amino acids have been studied extensively to see if they can aid in protein stabilization. A number of amino acids, including aromatic, basic, and acidic amino acids, as well as Met, have been shown to help in the stabilization of recombinant human interferon alpha-2b [25], while glutamic and aspartic acids have been shown to extend the half-life of recombinant human keratinocyte growth factor [26]. Although the features of the protein that has to be stabilized are likely to have an impact on the stabilizing effect of amino acids. In contrast to Gly in CTS and RPMI during cell treatment conditions, our data showed that His was the most effective excipient in retaining the stability of IL-2.
More investigation was performed to see whether the N-acetylcysteine (NAC), N,  [28]. Triton X-100 cannot be utilized to keep IL-2 stable in aqueous solutions because peroxides have a significant impact on protein oxidation and aggregation [29]. Excipients such as EDTA, Met, His, and Gly may affect IL-2 bioactivity, implying that increased stability does not always imply increased bioactivity.
Because the bioactivity of IL-2 in the presence of excipients was not investigated, the potential of IL-2 to boost the survival and proliferation of an IL-2 dependent cell line was not examined in this study.

SDS-PAGE Analysis
In order to understand the RP-HPLC findings, we performed an additional sta-     process used too much reducing agent, resulting in complete reduction. When the amount of reducing agent in the sample buffer was decreased (data not shown), no precipitates or dimer bands were seen, suggesting that non-disulfide cross-linking interactions did not occur. Because no protein aggregation bands exist in this condition, it's likely that precipitates formed physically and then disintegrated entirely in SDS sample buffer under sample conditions. Soluble or insoluble aggregates, reversible or irreversible aggregates, covalent or noncovalent aggregates are all possibilities [30].

Discussion
While IL-2 is an effective anticancer therapeutic in humans and a growth factor for T cells, its unstability makes it more difficult to use in liquid form. Quantitative measurement of IL-2 is widely used in the pharmaceutical industry and research institutes because its ability to directly affect human health can be dem- Stability tests reveal the possibility of IL-2 loss following the treatment of cells with supplement medium. However, we looked at how two antioxidants, EDTA and Met, affected IL-2 stability in four different aqueous solutions at different temperatures. The IL-2 stability in all four aqueous solutions increased up to 5 days at 37˚C in the presence of EDTA, but beyond 5 days, it dropped (data not shown). After 30 days of incubation at different temperatures [2˚C -8˚C, RT (20˚C ± 2˚C) and 37˚C], the stability of IL-2 is shown to be decreasing. Furthermore, the highest dose of EDTA tested (4 mg/mL) had no effect on IL-2 stability in aqueous solutions. EDTA has been employed as a food and medical additive in a variety of applications, including protease inhibition [31], but it is most commonly used as a stabilizer to prevent metal-induced adverse effects. Metal ions can have a major impact on thiol group stability. When EDTA was added to a solution that included no other metals, it greatly reduced oxidative damage and enhanced thiol group stability [32].
In CTS, RPMI, and water, IL-2 became more stable in the presence of Met. In aqueous solutions, Met, on the other hand, can act as a competitive antioxidant, preventing IL-2 oxidation. In PBS with Met, the stability of Il-2 was not improved, suggesting that Phosphate buffer speeds up Met degradation more than other buffers [33], resulting in methionine sulfoxide and methionine sulfone production. Protein folding and structural stability are affected because both species are larger and more polar than nonoxidized Met [34]. After 30 days of incubation at various temperatures, the stability of IL-2 was shown to be reduced (data was not shown). The highest concentration of Met tested (7.5 mg/mL) had no effect on the IL-2 stability in aqueous solutions. Overall, these findings show that EDTA and Met can stabilize IL-2 in CTS and RPMI for up to 5 days under cell treatment conditions.
In aqueous solutions, His and Gly were found as pharmacological buffering agents for IL-2, and each amino acid was examined separately in CTS, RPMI, PBS, and water solutions. In CTS and RPMI, the amino acid His stabilized IL-2 better than the other amino acid Gly. The finding that IL-2 was unstable in aqueous solutions containing Gly was surprising. It's possible that the presence of Gly in aqueous solutions, the formation of disodium salt crystals, and pH might also additionally stabilize a protein by preferential exclusion [35]. His has been shown in studies to be useful not just as a buffering agent, but also as a protein stabilizer in aqueous environments [36]. The most critical element [37] affecting IL-2 stability in aqueous solutions appears to be the positive charge on the sidechain of His. It's likely that the mechanism for IL-2 stabilization is the same for all basic amino acids, and that it binds transiently to IL-2 side chains, causing water populations in the solvation shell to change [37], making unfolding and aggregation less energetically beneficial. This example demonstrated that His can be used as a specific stabilizer and buffering agent for IL-2 in its aqueous form.

Conclusion
In order to analyze protein medicines in liquid form, many methods are available. Excipients including EDTA, Met, and His were shown to be very effective at stabilizing IL-2 in different buffers, pH, and ionic strength in this study, which used an analytical approach called RP-HPLC analysis. Our findings suggest that adding EDTA, Met, and His in medical formulations and cell treatment medium can help stabilize and reduce IL-2 aggregation, and that they could be a solution to any aggregation-related manufacturing or formulation difficulties.

Authors Contributions
A.S.P.G. conceived, designed, performed the experiments and wrote manuscript.
A.S. and T.S. reviewed the manuscript.  with Gly for 30 days at RT (20˚C ± 2˚C), (X) water with Gly for 5 days at 37˚Cand (Y) water with Gly for 30days at 37˚C. Periodically the solutions were analyzed separately by RP-HPLC and the percent recovery of IL-2 was determined.