IgG4 monoclonal antibody pharmaceutical preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an IgG4 monoclonal antibody pharmaceutical preparation as well as a preparation method and application thereof. The invention provides an IgG4 monoclonal antibody pharmaceutical preparation. The IgG4 monoclonal antibody pharmaceutical preparation comprises an IgG4 monoclonal antibody, poloxamer 188 and a pharmaceutically acceptable buffer solution. The research system evaluates the feasibility of replacing the traditional polysorbate by the poloxamer 188 for stabilizing the IgG4 monoclonal antibody. By combining thermodynamic analysis, interfacial property determination and multi-stress model experiments, the stress dependency difference of a surfactant stabilization mechanism is researched and revealed, and an important theoretical basis is provided for reasonable selection of a stabilizer in a protein preparation.

Description

An IgG4 monoclonal antibody drug formulation, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an IgG4 monoclonal antibody drug formulation, its preparation method, and its application. Background Technology

[0002] Monoclonal antibodies (mAbs) are key biopharmaceuticals for treating a variety of diseases, but their inherent physical instability limits their therapeutic potential. Protein aggregation is the most significant degradation pathway during manufacturing, storage, and transportation. Agitation stress is particularly challenging during processing and dispensing, primarily inducing aggregation through the adsorption, unfolding, and interaction of protein molecules at dynamically formed gas-liquid interfaces. Furthermore, freeze-thaw cycles, a common stress during storage and transportation, can also lead to protein instability through mechanisms such as ice-liquid interface adsorption, low-temperature concentration, and pH drift. These processes can form visible or invisible particles, thus affecting drug safety and efficacy.

[0003] Although stress studies are extremely important in formulation development, standardized research guidelines are still lacking. The degree of aggregation is highly dependent on specific stress parameters and the type of stress applied. For example, studies have shown that shaking and agitation induce aggregates of different properties and quantities in IgG1 formulations, with agitation having a more significant effect than shaking. Other factors, such as the container's filling level, type, size, and shaking intensity, also significantly affect the degree of particle aggregation. Similarly, repeated freeze-thaw cycles have been shown to impair the stability of some monoclonal antibodies, leading to oxidation, aggregation, and charge variation, with the oxidation effect being particularly pronounced in certain buffer systems. These findings collectively indicate that the type and intensity of stress are key factors determining protein aggregation, thus necessitating the evaluation of excipient protective effects under physiologically relevant conditions. This also provides a theoretical basis for our investigation of the protective mechanisms of polysorbate and poloxamer 188 (P188) under different stress conditions and for verifying whether P188 can serve as a substitute for polysorbate in IgG4 formulations.

[0004] Adding nonionic surfactants is a common strategy to mitigate interfacial-induced protein aggregation. Polysorbates (especially Tween 20 and Tween 80) have been widely used in commercial formulations. In contrast, poloxamer surfactants, especially P188, are increasingly being considered promising alternatives and are subject to extensive research. The stabilizing effects of these surfactants depend primarily on their molecular structure. For example, both Tween 20 and Tween 80 contain a dehydrated sorbitol ring linked to a polyoxyethylene chain and a fatty acid tail chain, with Tween 20 having a lauric acid chain and Tween 80 having an oleic acid chain. The long, unsaturated fatty acid chain of Tween 80 enhances its hydrophobicity and molecular flexibility. P188, on the other hand, is a linear triblock copolymer with a polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) structure. Compared to polysorbates, its molecular structure is much larger, leading to fundamentally different interfacial behaviors and interactions with proteins.

[0005] Despite the wide application of polysorbates, some inherent limitations remain, such as susceptibility to oxidative degradation and the potential formation of harmful impurities, raising concerns about their long-term stability and safety. In contrast, P188, with its saturated PEO-PPO-PEO backbone, exhibits excellent chemical stability and eliminates the risk of fatty acid chain-related degradation, thus becoming a promising alternative. This difference in structure and performance underscores the necessity of systematically studying the stability of P188 relative to polysorbates under various stress conditions to validate its potential as a reliable alternative in IgG4 formulations.

[0006] Surfactants provide stability primarily through three mechanisms: interfacial competition, direct binding, and micellar encapsulation. While interfacial competition is often considered the main pathway, the synergistic effects among these mechanisms and the influence of stress are not fully understood. A gap exists between academia and industry on this issue: some studies suggest that surfactants below the critical micelle concentration (CMC) can provide complete protection under certain stresses; however, industrial practice commonly uses concentrations far exceeding the CMC as a safety measure. This discrepancy reflects the inadequacy of current empirical methods. Without a clear understanding of how stress intensity affects the transition between monomer-driven and micellar-assisted mechanisms, the rational selection and dosage of surfactants will remain challenging.

[0007] To assess the feasibility of replacing polysorbate (P188) with IgG4 for stabilization, we employed an integrated mechanism-driven approach. We quantitatively investigated the binding interactions between three surfactants and the IgG4 monoclonal antibody using isothermal titration calorimetry (ITC), revealing how structural differences influence protein-protein interactions. Simultaneously, based on critical micelle concentration (CMC) data, we designed concentration matrices encompassing sub-CMC, CMC, and 4×CMC for clinically relevant comparisons. We also established differentiated multi-stress models, including strongly kinetically controlled vortex agitation, mildly thermodynamically controlled orbital oscillations, and freeze-thaw cycles, to simulate real-world biopharmaceutical stress environments. The protective effects of the surfactants were systematically evaluated using size exclusion chromatography (SEC), turbidity assays, and dynamic light scattering (DLS). This strategy, while directly comparing P188 with the gold standard polysorbate, revealed dynamic stress-dependent stabilization mechanisms (interfacial competition, direct binding, and micelle encapsulation). Our goal is to validate P188 as a reliable and chemically stable alternative and to move IgG4 surfactant formulations from empirical screening to stress-customized and mechanism-guided design, thereby improving the stability, safety, and manufacturability of biopharmaceuticals. Summary of the Invention

[0008] The present invention aims to provide an IgG4 monoclonal antibody pharmaceutical formulation, its preparation method, and its application. In biopharmaceutical formulations, polysorbates (such as Tween 20 / 80) are often used as stabilizers, but their chemical properties are unstable and easily degraded. Therefore, more stable alternatives, such as poloxamer 188 (P188), are needed. This study aims to systematically evaluate the feasibility of replacing polysorbates with P188 for stabilizing IgG4 monoclonal antibodies. Through isothermal titration calorimetry, critical micelle concentration determination, and multi-stress models (including mild orbital oscillation, vigorous vortex stirring, and freeze-thaw cycles), we found that the stabilization mechanism of surfactants fundamentally changes with the type of stress. ITC results show that polysorbates can specifically bind to antibodies through an entropy-driven process, while P188 does not have this interaction. Under mild stress conditions (such as oscillation and freeze-thaw), all surfactants can achieve effective protection below their critical micelle concentration through interfacial competition mechanisms, with P188 performing comparablely to polysorbates.

[0009] However, under the kinetically controlled stress conditions of intense vortex stirring, polysorbate needs to reach a supercritical micelle concentration (4×CMC) to utilize micelles as a reservoir of dynamic monomers, rapidly covering the newly formed interface to achieve complete protection. P188, due to its slow adsorption kinetics, exhibits limited protective effects even at high concentrations. These results indicate that under thermodynamically dominated stress conditions, P188 can serve as an effective alternative to polysorbate in IgG4 formulations, providing a theoretical basis for screening stabilizers based on stress mechanisms.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides an IgG4 monoclonal antibody pharmaceutical preparation comprising an IgG4 monoclonal antibody, poloxamer 188, and a pharmaceutically acceptable buffer solution.

[0011] Preferably, the concentration of poloxamer 188 is 0.01%~0.1% (w / v).

[0012] Preferably, the concentration of the IgG4 monoclonal antibody is 1~200 mg / mL.

[0013] Preferably, the pharmaceutically acceptable buffer solution is an acetate buffer, a histidine buffer, or a phosphate buffer.

[0014] Preferably, the pH of the pharmaceutically acceptable buffer solution is 4.5 to 6.0.

[0015] Preferably, the formulation further comprises a stabilizer selected from one or more of sucrose, trehalose, and mannitol.

[0016] Preferably, the formulation is a liquid injection.

[0017] This invention provides a method for preparing the IgG4 monoclonal antibody drug formulation, which involves mixing IgG4 monoclonal antibody, poloxamer 188, and a pharmaceutically acceptable buffer solution.

[0018] This invention provides the use of the IgG4 monoclonal antibody pharmaceutical formulation in the preparation of a medicament for treating diseases.

[0019] Preferably, the disease is cancer, an autoimmune disease, or an infectious disease.

[0020] Compared with existing technologies, this invention has the following advantages: This study systematically evaluated the feasibility of using poloxamer 188 (P188) as a substitute for traditional polysorbate for stabilizing IgG4 monoclonal antibodies. By combining thermodynamic analysis, interfacial property determination, and multi-stress model experiments, the study revealed the stress-dependent differences in surfactant stabilization mechanisms, providing an important theoretical basis for the rational selection of stabilizers in protein formulations.

[0021] Under thermodynamically controlled mild stress conditions (such as prolonged shaking and freeze-thaw cycles), P188 provides protection comparable to polysorbate through saturated interfacial adsorption at its critical micelle concentration (CMC). P188 exhibits excellent chemical stability and is not prone to oxidation or hydrolytic degradation, avoiding the risk of degradation products that may be introduced by polysorbate, thus demonstrating better safety and stability potential during long-term storage and freeze-thaw cycles.

[0022] However, under kinetically controlled high shear stress conditions (such as high-speed vortices), P188 exhibits slow adsorption kinetics due to its large triblock linear structure, failing to effectively cover the transiently formed gas-liquid interface even at high concentrations (4×CMC), resulting in limited protective effects. In contrast, polysorbate can utilize micelles as a dynamic monomer reservoir, achieving rapid interface coverage even at concentrations exceeding CMC, thus effectively resisting strong mechanical stress. Therefore, the suitability of P188 depends on the type of stress in the formulation; it can be a preferred alternative in mild or thermodynamically dominated scenarios, while its use in high-shear processes requires careful evaluation or process optimization. Attached Figure Description

[0023] Figure 1 shows the amphiphilic molecular structures of poloxamer 188, polysorbate 20 (Tween 20), and polysorbate 80 (Tween 80).

[0024] Figure 2 shows the isothermal titration calorimetry (ITC) curves of the interaction between surfactants and IgG4 monoclonal antibodies. It displays the raw data and combined thermal changes obtained by curve fitting for the titration of (A) Tween 20, (B) Tween 80, and (C) poloxamer 188 into the antibody solution at 25 °C. These data demonstrate the specific binding of Tween 20 and Tween 80, and the non-binding of poloxamer 188.

[0025] Figure 3 shows the CMC of surfactants determined by the pyrene fluorescent probe method. The vibrational band intensity ratio of pyrene (I1 / I3) is plotted as a function of surfactant concentration. The inflection point of the fitted Boltzmann curve defines the apparent CMC value of each surfactant in the presence of IgG4 antibody.

[0026] Figure 4 shows the SEC analysis of the effect of surfactants on IgG4 monomer recovery under different stress conditions. (A) After 96 h of mild orbital oscillation, the monomer recovery of all surfactants reached a plateau (~100%) at concentrations above CMC, with no statistically significant difference between P188 and Tween 20 / 80 (p>0.1234); (B) After 6 h of vigorous vortex stirring, the monomer recovery of Tween 20 / 80 reached 100% at 4×CMC, while P188 reached a maximum of 80%; (C) After 5 freeze-thaw cycles, the monomer recovery of all three surfactants reached ~95% at 4×CMC, with no statistically significant difference (p>0.1234). Data are expressed as mean standard deviation (n=3). p<0.0001 compared with the control group; Tukey's post-hoc test was used for inter-group comparisons.

[0027] Figure 5 shows the hydrodynamic particle size distribution of IgG4 after 96 h of mild orbital oscillation. The experimental temperature was 25℃, and the data are expressed as the average of three independent experiments.

[0028] Figure 6 shows the trend of IgG4 hydrodynamic diameter change (A) and key distribution (B) after 6 h of intense vortex stirring. The experimental temperature was 25℃, and the data are expressed as the average of three independent experiments.

[0029] Figure 7 shows the turbidity (OD) of the IgG4 solution under mild orbital shaking (96 hours) and strong vortex agitation (6 hours). 350 (A) Slight oscillation (B) Strong vortex. The experiment was conducted using a 1 mm quartz cuvette. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Materials

[0033] Monoclonal antibodies were provided by Hualan Gene Engineering Co., Ltd. (Xinxiang, China). The theoretical isoelectric point (pI) of IgG4 is approximately 6.9. Tween 20 and Tween 80 were purchased from Well Pharmaceutical Technology (Nanjing, China), and P188 was purchased from Merck KGaA (Darmstadt, Germany). Unless otherwise specified, all assays were performed at a protein concentration of 4.68 mg / mL in 10 mM sodium acetate buffer (pH 5.0). All buffer components were analytical grade, and surfactants were injection grade.

[0034] 2. Sample preparation

[0035] Before the experiment, the concentration-to-weight ratio (CMC) of Tween 20, Tween 80, and P188 was determined using pyrene fluorescence. According to the ITC results, the binding ratios of Tween 20 and 0.3×CMC corresponded, and Tween 80 and 0.4×CMC, respectively. P188 did not specifically bind to mAb, so 0.5×CMC was selected as the concentration to be investigated. To assess concentration-dependent stabilizing effects, four concentration gradients were designed for each surfactant in 10 mM sodium acetate buffer (pH 5.0) with a final mAb concentration of 4.68 mg / mL: Tween 20 (0.1×CMC, 0.3×CMC as binding ratio-dependent concentrations, CMC, 4×CMC), Tween 80 (0.1×CMC, 0.4×CMC as binding ratio-dependent concentrations, CMC, 4×CMC), and P188 (0.1×CMC, 0.5×CMC, CMC, 4×CMC). Stock solutions of all surfactants were prepared to prevent micellar degradation.

[0036] 3. Stress Research

[0037] Track shaking was used to simulate conditions encountered during long-term storage and transportation. High-temperature stress was applied using a constant-temperature incubator (HNY-2102C, Tianjin Ounuo Instrument Co., Ltd.), with precise control of temperature and rotation speed. mAb solutions supplemented with various surfactants (1 mL per tube, 4.68 mg / mL in 10 mM sodium acetate buffer, pH 5.0) were continuously shaken at 300 rpm at 25 ± 0.5 °C. After 96 hours of incubation, samples were harvested, rapidly cooled on ice to stop the stress response, and stability analysis was performed.

[0038] 4. Intense eddy current stirring

[0039] Vortex stress simulates short-term shear forces during antibody production / transport, enabling rapid assessment of the inhibitory effect of surfactants on shear-induced aggregation. Vortex mixing was performed using a vortex-Genie 2 mixer (Scientific Industries, USA) at room temperature (25±2℃) with a mixing intensity of 8 levels (maximum intensity 10). In short, 1 mL of mAb solution containing different concentrations of surfactant (4.68 mg / mL, dissolved in 10 mM sodium acetate buffer, pH 5.0) was transferred to sterile 2 mL centrifuge tubes and vortexed for 6 h. Samples were collected at predetermined time points (0, 1, 2, 3, 4, 5, and 6 h). Before analysis, samples were centrifuged at 4℃ and 2000 rpm for 10 min to remove air bubbles.

[0040] 5. Freeze-thaw cycle

[0041] The IgG4 monoclonal antibody preparation samples were aliquoted into 2 mL sterile centrifuge tubes (1 mL per tube), with three samples per group to ensure data reliability. After aliquoting, the samples were immediately transferred to a -20°C ultra-low temperature freezer for static freezing for 24 h. The frozen samples were then thawed in a 25°C incubator for 1 h to ensure complete thawing. This freeze-thaw cycle constituted one complete cycle and was repeated 5 times.

[0042] 6. Isothermal titration calorimetry (ITC)

[0043] Isothermal titration calorimetry (MicroCal ITC200 Malvern Instruments Ltd., Worcestershire, UK) was used to characterize the interactions between Tween 80, Tween 20, and P188 with IgG4. All experiments were performed using a reference cell filled with ultrapure water. The sample cell (200 μL) was loaded with protein solution, and the syringe (40 μL) was filled with surfactant solution. Titration consisted of 18 injections, and experiments were performed at a constant temperature of 25 °C. The stirring speed was set to 1000 rpm for all experiments. An initial delay of 60 s was applied before the second injection. The reference power and filter time constant were set to 10 μcal / s and 1 s, respectively. The sample cells contained 0.06 mM IgG4, and the surfactant concentration in the syringe was 1.5 mM.

[0044] The titration curves were analyzed using the ORIGIN software suite provided with the calorimeter. A binding model with identical and independent sites was used to fit the data. Data points from the first injection were discarded because diluting the solution in the syringe tip during initial thermal equilibrium could lead to inaccuracies in volume and concentration. Each experiment was performed three times under identical conditions to ensure accuracy and reproducibility. The heat of dilution, determined based on the control titration (surfactant in the buffer and buffer in the protein), was subtracted from each experimental run performed under identical conditions.

[0045] 7. Determination of CMC by pyrene fluorescence spectroscopy

[0046] The apparent concentration-lowering ratio (CMC) of surfactants in the presence of protein was determined using a pyrene fluorescent probe method. First, a pyrene stock solution was added to the protein solution to prepare a pyrene / protein working solution containing 0.5 μm pyrene and 4.68 mg / mL IgG4. Using this working solution as a solvent, a series of surfactant samples with different concentrations were prepared in 96-well plates, with a total volume of 200 μL per well. The samples were equilibrated in the dark at 25 °C for 2 hours. Fluorescence intensities were measured at 373 nm (I1) and 384 nm (I3) using a microplate reader (TecanAustria GmbH, Austria) at an excitation wavelength of 335 nm. The apparent CMC was determined as the inflection point obtained by fitting the I1 / I3 ratio data to the logarithm of the surfactant concentration to a Boltzmann function.

[0047] 8. Surface tension

[0048] The surface tension of the solution in various concentration ranges was measured using a fully automated du Noüy ring tensiometer (HTYZL-H, Wuhan UHV Electric Power Technology Co., Ltd., China). The instrument was calibrated with ultrapure water prior to measurement. All measurements were performed at 25°C, with each sample measured three times. Results are expressed as mean standard deviation (SD) to ensure accuracy and reproducibility.

[0049] 9. Size Exclusion Chromatography (SEC)

[0050] SEC analyzed the monomer content and aggregation state of the samples using a high-performance liquid chromatography (HPLC) system (UltiMate 3000, Thermo Fisher Scientific). Separation was performed on a TSKgel G3000SWXL analytical column (300 mm × 7.8 mm, Tosoh Bioscience, Japan) maintained at 25 °C. Isocratic elution was performed using a mobile phase consisting of 50 mM sodium phosphate buffer (pH 6.9) at a flow rate of 1.0 mL / min. The injection volume was 20 μL, the detection wavelength was set to 280 nm, and the autosampler temperature was maintained at 4 °C to ensure sample stability.

[0051] 10. Dynamic Light Scattering (DLS)

[0052] The hydrodynamic diameter (Z-average) and polydispersity index (PDI) of the samples were determined at 25 °C using a zeta sizer Nano ZSP (Malvern Instruments Ltd., Worcestershire, UK). For hydrodynamic size analysis, samples were placed in 40 L disposable plastic cuvettes and measured at a scattering angle of 173°. Each sample was allowed to equilibrate in the instrument for 2 min before measurement. All samples were measured three times, with each run consisting of 15 sub-runs in automatic mode.

[0053] 11. Turbidity (OD) 350 )

[0054] Monitoring at 350 nm (OD) using a UV-Vis spectrophotometer (S3100, Scinco Ltd., Seoul, South Korea) 350 The turbidity change of the sample was assessed by measuring the optical density. The sample was stirred for different times. After each stirring interval, it was allowed to stand for 10 min, and then transferred to a 1 mm quartz cuvette for OD curve measurement. The resulting OD350 value curve was used to characterize the stability of turbidity over time.

[0055] Statistical data analysis

[0056] All experiments were performed in triplicate (n=3). Data are expressed as mean standard deviation. Statistical analysis was performed using GraphPad Prism software (version 10.6.0). One-way ANOVA was performed to compare differences between multiple treatment groups and the blank control group. Tukey's post-hoc test was then performed for multiple comparisons. Statistical significance (p-value) is indicated by the following symbols in parentheses: p>0.1234 (not significant, ns), p<0.0332 (not significant, ns). p < 0.0021 p < 0.0002 p < 0.0001 ).

[0057] result

[0058] 1. Physicochemical properties of surfactants

[0059] 1.1. Combination ability

[0060] The interaction between surfactants and IgG4 was investigated using ITC. ITC provided key thermodynamic parameters, including binding constant (Ka), stoichiometry (N), binding enthalpy (ΔH), and entropy (ΔS). As shown in Figure 2, the titration curves of Tween 20 and Tween 80 exhibited characteristics of specific binding; each injection produced a different exothermic peak, and the peak height decreased sequentially as the antibody binding site gradually saturated until equilibrium was reached, resulting in smooth isotherms suitable for nonlinear fitting of the data. In contrast, the titration curve of P188 showed an irregular, low-amplitude heat flow signal, indistinguishable from the heat flow signal of the dilution thermogram. This indicates that under our experimental conditions, ITC did not detect significant specific binding events between P188 and the IgG4 antibody.

[0061] Table 1. Thermodynamic parameters of the isothermal titration calorimetry (ITC) for the interaction between surfactants and IgG4 monoclonal antibodies.

[0062]

[0063] Note: N is the stoichiometric ratio, Ka is the binding constant, ΔH is the binding enthalpy change, and ΔS is the binding entropy change; N / D (not determined) because a measurable binding isotherm is lacking. The thermogram of poloxamer 188 did not show characteristic peaks for specific binding; only a weak, constant thermal signal consistent with the dilution effect was observed, indicating that it does not have a significant specific interaction with IgG4.

[0064] The quantitative data provided in Table 1 reveal the precise nature of the binding. Both Tween 20 and Tween 80 are stoichiometric in a near 1:1 ratio (n≈1) and 10 4 M - The binding of Ka values ​​to the IgG4 antibody confirmed moderate binding affinity. The most critical thermodynamic parameters were ΔH and ΔS. The binding characteristics of the two surfactants were small and negative ΔH values ​​(-1.48 and -1.12 kcal / mol, respectively), indicating weak exothermic activity, while the calculated ΔS values ​​were mostly positive (14.9 and 15.9 cal / mol / deg). This suggests that the binding of Tween 20 and Tween 80 to the IgG4 antibody is primarily entropy-driven.

[0065] Smaller negative ΔH values indicate limited contributions from interactions such as hydrogen bonds and van der Waals forces. In contrast, larger positive ΔS values indicate that binding is mainly driven by hydrophobic interactions. That is, the hydrophobic tails of surfactants are inserted into hydrophobic patches on the antibody surface, leading to the disruption and release of ordered water molecules, a process that significantly increases the entropy of the system and provides the main driving force for binding. This mechanism is consistent with the hydrophobic structural characteristics of polysorbates. Thus, ITC data confirm at the molecular level that polysorbate-based surfactants stabilize IgG4 through a direct binding mechanism, while P188 plays a protective role independent of this mechanism. This fundamental difference in binding behavior predicts the dependence of P188 on interfacial protection competition, a hypothesis that was verified in subsequent stress studies.

[0066] 1.2. Critical micelle concentration

[0067] The apparent critical micelle concentration (CMC) of surfactants in the presence of IgG4 antibody was determined using the pyrene fluorescence probe method, where the inflection point of the I1 / I3 ratio defines the CMC value of each surfactant (Figure 3). The measured CMCs follow a distinct trend: Tween 80 < Tween 20 << P188. This ranking is a direct reflection of molecular hydrophobicity. Tween 80 has a longer unsaturated oleic acid chain (C18:1), exhibiting the strongest self-assembly hydrophobic driving force and resulting in the lowest CMC. The shorter lauric acid chain (C12) of Tween20 leads to weaker hydrophobicity and thus a higher CMC, while P188, a bulky triblock copolymer, requires the highest concentration to overcome the significant energy barrier associated with the micellization of its poly(propylene oxide) core.

[0068] CMC is not just a physicochemical parameter; it is a critical boundary that defines the working range of different stabilization mechanisms. At concentrations below the CMC (where surfactants mainly exist as monomers), stability depends only on interfacial competition and, for surfactants capable of directly binding IgG4, also on direct interactions. The micellar encapsulation ability increases significantly at and above the CMC, enabling the removal of hydrophobic impurities and the chelation of unfolded protein molecules. Thus, the determined CMC values provide a basic framework for interpreting subsequent stability data. They allow for the scientific design of concentration matrices to specifically probe the contributions of monomer and micellar states, enabling a mechanistic analysis of surfactant efficacy under various stress conditions.

[0069] 1.3. Surface tension

[0070] Surface tension measurements directly quantify the interfacial activity of surfactants, a key property controlling their effectiveness in the "interfacial competition" mechanism that prevents protein adsorption and aggregation at the gas-liquid interface. As shown in Table 2, all three surfactants reduced surface tension relative to the buffer control, but with significant differences in efficiency and effectiveness. Tween 80 exhibited excellent interfacial properties, achieving the lowest absolute surface tension at its CMC (41.25 mN / m) and the largest reduction at sub-CMC concentrations (e.g., 0.1 × CMC), indicating its high efficiency and effectiveness. Tween 20 showed moderate activity, while P188 was the least effective, consistently producing high surface tension at all concentrations.

[0071] Table 2 shows the surface tension measurements of the key concentration surfactant relative to CMC (n=3).

[0072]

[0073] Note: Not applicable indicates that the measurement is not applicable. The buffer control is 10 mM sodium acetate buffer at pH 5.0. The control represents a solution of 4.68 mg / mL IgG4 in the same 10 mM sodium acetate buffer (pH 5.0).

[0074] This performance trend can be explained by the unique molecular structures of surfactants. Tween 80's potent activity stems from its long, flexible oleic acid chains (C18:1), which facilitate efficient interfacial filling and maximize the hydrophobic driving force of adsorption. Tween 20's shorter lauric acid chains (C12) provide a weaker driving force. In contrast, P188's relatively weaker interfacial activity likely arises from the spatial and kinetic constraints imposed by its large triblock structure, which hinders the adsorption and rearrangement of its large PPO-PEO chains at the gas-liquid interface.

[0075] Therefore, these surface tension results establish a clear hierarchy in the inherent ability of each surfactant to compete with and stabilize the interface. This hierarchy provides a predictive basis for explaining the relative performance of these additives in mitigating agitation-induced aggregation, where interfacial competition is the primary line of defense. Furthermore, the significant differences in surface tension reduction efficiency at sub-CMC concentrations indicate parallel differences in their adsorption kinetics, which are expected to become crucial under strong transient stress conditions. Specifically, the superior interfacial activity of Tween 80 suggests its rapid interface occupation, while the weak activity of P188 implies slow adsorption kinetics, directly reflected in the subsequent results under strong eddy stress (Sections 2.2 and 2.3), where P188 does not provide complete protection.

[0076] 2. Protective efficacy of surfactants under various stresses

[0077] 2.1. SEC Analysis

[0078] The ability of surfactants to prevent irreversible aggregation was assessed by quantifying IgG4 monomer recoveries after different stress levels using SEC (Figure 4). After 96 hours of stress exposure, the monomer recoveries in the control group decreased to approximately 77%, confirming aggregate formation. As shown in Figure 4A, all surfactants significantly improved IgG4 monomer recoveries at the tested concentrations compared to the control group (p < 0.0001). More importantly, the stabilizing effect reached a clear plateau once the surfactant concentration reached the CMC. Statistical analysis showed no significant difference in monomer recoveries between the CMC and 4×CMC concentrations for any of the three surfactants (p > 0.1234). Although P188 showed incomplete protection below the CMC, it achieved protection comparable to that of the Tween surfactant at or above its CMC. This indicates that under moderate thermodynamically controlled stress, protection is controlled by saturable interfacial adsorption, and the CMC defines a sufficient and cost-effective concentration threshold for effective protection.

[0079] As shown in Figure 4B, after 6 hours of vortex stirring, the monomer recovery rate in the control group decreased to 38%. The protective effects of Tween 20 and Tween 80 were concentration-dependent. In the low concentration range (below CMC), the monomer recovery rate remained at approximately 60%, indicating limited protection. However, when the concentration reached 4 × CMC, the recovery rate increased to 100%, indicating that their complete protection at high concentrations depended on the contribution of the micellar mechanism. The abrupt transition from partial to complete protection between CMC and 4 × CMC suggests a fundamental shift in the primary stabilizing mechanism, surpassing the saturable effect observed under mild stress conditions.

[0080] In contrast, P188 provided weaker protection at all tested concentrations, achieving only about 80% monomer recovery even at 4×CMC, which is insufficient to effectively counteract strong stress. P188's poor protective effect likely stems from its large triblock structure, which limits adsorption kinetics, diffusion, and interfacial rearrangement—a critical drawback under kinetically controlled stresses requiring rapid interfacial coverage. These data suggest that P188 is unsuitable as a polysorbate alternative in IgG4 production processes involving strong mechanical stresses such as high-speed mixing and shear forces.

[0081] After five freeze-thaw cycles, all samples exhibited macroscopic phase separation (gel-like structure), which was completely reversed by vortexing to obtain homogeneous solutions. SEC analysis of the redispersed samples showed that all surfactants provided dose-dependent protection against irreversible aggregation, with Tween 20, Tween 80, and P188 all achieving a comparable monomer recovery plateau of approximately 95% at 4×CMC (Figure 4C). This demonstrates their equivalent efficacy in mitigating freeze-thaw stress driven by ice-liquid interface exposure (p>0.1234). Notably, this study evaluated only surfactants; in practical formulations, optimal stability is often achieved by combining surfactants with cryoprotectants (such as sugars), highlighting the potential of P188 as a viable component in integrated stabilization strategies.

[0082] 2.2. DLS Analysis

[0083] DLS analysis of samples subjected to different stress modes revealed the protective mechanisms and limitations of surfactants at the particle level. Figure 5 shows significant aggregation in the surfactant-free control group after stress, characterized by the formation of numerous submicron aggregates. Notably, even small amounts of surfactant significantly inhibited aggregate formation. Specifically, Tween 20 and Tween 80 effectively inhibited antibody aggregation at their respective binding ratio concentrations (0.3×CMC and 0.4×CMC), maintaining them at near-initial levels, with protective efficacy almost equivalent to that observed at their CMC and 4×CMC concentrations. This stark contrast directly validates the ITC-derived mechanism: Tween binds directly and stabilizes proteins even at concentrations below CMC, while P188 depends solely on CMC-dependent interfacial saturation. For mild stress, the CMC-level protection of P188 is sufficient to match that of polysorbate, supporting its feasibility as an alternative. Conversely, P188 exhibited detectable aggregation at its sub-CMC concentration (0.5 × CMC), and its protective effect only reached levels comparable to Tween at CMC concentrations. These data are entirely consistent with the "non-specific binding" observed by the ITC, confirming that the protective effect of P188 depends on interfacial competition rather than direct binding.

[0084] Under strong vortex stress (6 h), the aggregation process entered a kinetically controlled state, and DLS data (Figure 6) revealed more significant mechanistic differences. The control group showed two distinct peaks at approximately 100 and 1000 nm, indicating the formation of polydisperse aggregates spanning a wide size range under strong shear. Tween 80 of 4×CMC almost completely suppressed this peak, maintaining the same particle size distribution as the initial state. Tween 20 of 4×CMC retained the main monomer peak, but a weak residual signal was still observed in the 10–100 nm range. Notably, for P188, even at its highest experimental concentration (4×CMC), the DLS spectrum still showed a broad but weak peak at 1000 nm. This visually confirms that under the extreme kinetic conditions of rapid interface formation and annihilation, P188, due to its large linear triblock structure, exhibited a slow diffusion and interface rearrangement rate, failing to cover all newly formed interfaces in time. This failure allowed some protein molecules to adsorb, unfold, and ultimately form insoluble sub-visible particles. Although micelles can provide a reservoir of monomers, the overall dynamics of monomer dissociation from micelles and subsequent diffusion to the interface are insufficient to meet the instantaneous high interface requirements of eddy current generation.

[0085] In freeze-thaw experiments, DLS analysis was performed on the redispersed and filtered samples. Only monomer peaks were observed, and no signal of stable submicron irreversible aggregates was detected (data not provided). This indicates that the macroscopic phase separation products induced by low-temperature concentrations are highly reversible, and that surfactants effectively inhibit the transformation of reversible aggregates into stable irreversible aggregates. Combined with SEC data (Figure 4C), these results suggest that surfactants can confine irreversible aggregation to lower levels, primarily through interfacial stabilization, which mitigates denaturation in localized freeze-thaw concentration environments.

[0086] 2.3 Turbidity (OD) 350 )analyze

[0087] Turbidity (OD) 350 Changes in OD directly reflect the formation of insoluble protein aggregates in solution and are a sensitive indicator for assessing the effectiveness of interfacial competition mechanisms. Real-time monitoring of OD... 350 This study plotted dynamic curves of surfactant-mediated inhibition of interfacial aggregation under different intensities of mechanical stress. This method is not applicable to freeze-thawed samples because standard preparation procedures include filtration to remove visible precipitates.

[0088] Figure 7A shows the results of mild orbital stress (96 h). OD of the blank control group. 350The value gradually increased over time, reaching approximately 0.13 at the endpoint, confirming the formation of insoluble precipitates due to prolonged interface exposure time. The addition of all surfactants reduced the endpoint turbidity in a dose-dependent manner. A key finding was that, for all three surfactants, the OD of the samples decreased when the concentration reached their respective CMCs. 350 The value has decreased to near baseline levels (0 h, ~0.02–0.03). Further increasing the concentration to 4 × CMC did not lead to an increase in OD. 350 A further significant decrease was observed. This "plateau effect" is highly consistent with the saturation of CMC monomer recoveries observed in the SEC analysis (Figure 4A) and is supported by DLS particle size data. This provides strong evidence that, under thermodynamic equilibrium or near-equilibrium conditions, the core protective mechanism is the formation of a saturated surfactant monolayer at the air-water interface, competitively excluding IgG4.

[0089] Under strong eddy current stress (6 h), kinetic factors dominate, and the turbidity curves (Figure 7B) show significant differences among surfactants. The OD of the control group... 350 The concentration of polysorbate (POD) increased sharply to ~0.25 within 6 hours, while the POD remained constant at CMC and above. 350 The OD value is <0.05, which is attributed to its rapid adsorption and micellar monomer reservoir function. In contrast, P188 exhibits a significantly higher OD value even at 4×CMC. 350 (Endpoint ~0.06-0.08). These real-time data confirm that the slow adsorption kinetics of P188 (rooted in its triblock structure) prevents transient interface coverage, leading to irreversible protein adsorption and aggregation during the “interfacial void” period. For IgG4 formulations exposed to strong mechanical stress, P188 is inferior to polysorbate, limiting its potential as an alternative in such cases.

[0090] 3. Stress-dependent stability mechanisms and selection strategies

[0091] 3.1. Stress-driven protection mechanism conversion

[0092] Our data reveal a clear dichotomy in surfactant protection mechanisms, depending on the type and intensity of pressure. Under mild orbital sloshing conditions, gas-liquid interface formation is slow, and the system tends towards thermodynamic equilibrium. This environment allows sufficient time for surfactant diffusion and interfacial adsorption, resulting in protection through two mechanisms: interfacial competition: all surfactants reduce surface tension, forming a saturated adsorption layer at the CMC; and direct binding of IgG4 (excluding polysorbate only): hydrophobic chains bind to hydrophobic patches of IgG4, stabilizing the native conformation and reducing aggregation tendency.

[0093] In contrast, intense eddy current stress creates a kinetically controlled situation requiring rapid adsorption and a wide range of new interfaces. Polysorbate overcomes this problem through a 4×CMC micelle monomer reservoir, where micelle-monomer dissociation maintains a high bulk monomer concentration, ensuring immediate interfacial coverage. P188 fails here because its bulky structure leads to slow diffusion and rearrangement. Even at 4×CMC, the monomer supply cannot match the interfacial formation rate, resulting in incomplete protection. This defines a key limitation of P188: it is incompatible with intense mechanical stress scenarios.

[0094] Despite their different molecular structures, all surfactants achieved equivalent protective effects under controlled thermodynamic stress (CMC) because saturated interfacial adsorption exceeded these structural differences. This observation confirms that P188 is a viable alternative to polysorbate under moderate thermodynamic stress.

[0095] 3.2. Feasibility of using poloxamer 188 as a substitute for polysorbate

[0096] Evaluating P188 as a polysorbate alternative requires more than just a comparison of physical protective efficacy; it necessitates mechanism matching and risk analysis.

[0097] In thermodynamically controlled stress scenarios, such as long-term storage and freeze-thaw cycles, evaluation depends on two factors: saturable interfacial adsorption capacity and long-term chemical compatibility. This study confirms that P188 forms an effective saturated interfacial layer at its CMC, providing physical protection comparable to polysorbate. The key point here is that chemical stability is a decisive variable in the decision-making process. Degradation products of polysorbate from ester hydrolysis or fatty acid chain oxidation, such as free fatty acids and peroxides, are not only impurities but also potential aggregation catalysts and immunogenicity risk factors. Therefore, for liquid formulations with a target shelf life of several years, choosing P188 is not merely a "substitute," but a proactive risk mitigation strategy. By eliminating the inherent chemical degradation pathways of surfactants, the long-term stability and safety of the product are fundamentally improved.

[0098] For substitution in the production process, it is crucial to distinguish between real-world stress intensity and the kinetic protection limit of the surfactant. The strong eddy current model used in this study defines the upper limit of adsorption kinetics imposed by the molecular structure of P188. The interface formation rate in most common unit operations is far below this limit, indicating that the kinetic limit of P188 is generally not exposed under normal processing conditions. A reasonable conclusion is that its applicability should not be completely dismissed based on its performance in the intensification model, but rather its applicability should be evaluated through a scientific analysis of the shear and interface formation characteristics of each specific process step.

[0099] However, this study also explicitly emphasizes that if a confirmed high-shear step exists in the process, approaching the stress intensity of the vortex model, then substitution based on direct concentration is risky. In such cases, the feasibility assessment should guide the engineering solution, either by optimizing the process to reduce the stress intensity or by exploring higher P188 concentrations to partially compensate for its slower kinetics, which can be validated through appropriate stress studies during early development.

[0100] In summary, the feasibility of replacing polysorbate with P188 is fundamentally a matter of mechanistic selection based on the stress distribution of the target product. Under thermodynamic or mild kinetic conditions, it is the preferred option, offering physical protection and chemical advantages. Its applicability is limited under extreme dynamic stresses; however, this limitation promotes more refined formulation and process strategies, rather than relying on a single agent to address all challenges.

[0101] This study systematically elucidates the stress-dependent feasibility of replacing polysorbate (P188) with poloxamer 188 in IgG4 formulations, revealing the fundamental difference in their protective mechanisms: P188 stabilizes antibodies through entropy-driven direct binding, while P188 exerts its effect solely through interfacial competition. Under thermodynamically dominant or mild kinetic stress, P188 exhibits protective capabilities comparable to P188, and its stable chemical structure and resistance to degradation make it a more stable and safer alternative. However, under strongly kinetically controlled stress conditions, P188's slow adsorption kinetics due to its molecular structure prevent rapid interfacial coverage, and it fails to provide complete protection even at high concentrations. These findings provide a rational design framework for formulation development, shifting from empirical screening to mechanism-guided approaches. This framework supports the mechanism-based selection or combination of stabilizers under different stress scenarios, thereby driving the development of more stable, safer, and more readily available biopharmaceutical products.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An IgG4 monoclonal antibody pharmaceutical formulation, characterized in that, It contains IgG4 monoclonal antibody, poloxamer 188, and pharmaceutically acceptable buffer.

2. The IgG4 monoclonal antibody pharmaceutical formulation according to claim 1, characterized in that, The concentration of poloxamer 188 is 0.01%~0.1% (w / v).

3. The IgG4 monoclonal antibody pharmaceutical formulation according to claim 1, characterized in that, The concentration of the IgG4 monoclonal antibody is 1~200 mg / mL.

4. The IgG4 monoclonal antibody pharmaceutical formulation according to claim 1, characterized in that, The pharmaceutically acceptable buffer solution is an acetate buffer, histidine buffer, or phosphate buffer.

5. The IgG4 monoclonal antibody pharmaceutical formulation according to claim 1 or 4, characterized in that, The pharmaceutically acceptable buffer solution has a pH of 4.5 to 6.

0.

6. The IgG4 monoclonal antibody pharmaceutical formulation according to claim 1, characterized in that, The formulation also includes a stabilizer selected from one or more of sucrose, trehalose, and mannitol.

7. The IgG4 monoclonal antibody pharmaceutical formulation according to claim 1, characterized in that, The formulation is a liquid injection.

8. The method for preparing the IgG4 monoclonal antibody pharmaceutical formulation according to any one of claims 1 to 7, characterized in that, Mix IgG4 monoclonal antibody, poloxamer 188, and pharmaceutically acceptable buffer.

9. Use of the IgG4 monoclonal antibody pharmaceutical preparation according to any one of claims 1 to 7 in the preparation of a medicament for treating diseases.

10. The use according to claim 9, characterized in that, The disease in question is cancer, an autoimmune disease, or an infectious disease.

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