Masashi Serizawa

End-group-selective size-exclusion chromatography (esSEC) coupled with mass spectrometry (MS) is presented as an analytical approach for the separation and characterization of acid-terminated and non-acid-terminated poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA). The method employs a gradient normal-phase liquid chromatography system with basic (triethylamine) and acidic (formic acid) additives in ethyl acetate-based mobile phases, enabling selective retention of acid-terminated polymers, while non-acid-terminated species elute according to hydrodynamic volume, i.e., their effective size in solution. Following their release from the stationary phase, the acid-terminated polymers also undergo separation according to hydrodynamic volume.The separation involves adsorption of acid-terminated polymers under basic conditions, followed by their release upon introduction of an acidic mobile phase and subsequent size-based separation. Gradient steepness strongly influenced the elution behavior of acid-terminated polymers. Shallow gradients resulted in broader elution profiles reflecting prolonged adsorption–desorption, whereas steeper gradients produced sharper, more symmetrical peaks.The method can be directly coupled to MS, enabling molecular-level characterization of end-group structures and chemical composition across the chromatographic separation.The proposed esSEC–MS method provides a novel strategy for end-group-resolved polymer analysis. Its applicability is demonstrated by monitoring the formation of cyclic structures by intramolecular transesterification and the degradation of an ester-terminated PLGA copolymer over time.

End-group-selective size-exclusion chromatography (esSEC) coupled with mass spectrometry (MS) is presented as an analytical approach for the separation and characterization of acid-terminated and non-acid-terminated poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA). The method employs a gradient normal-phase liquid chromatography system with basic (triethylamine) and acidic (formic acid) additives in ethyl acetate-based mobile phases, enabling selective retention of acid-terminated polymers, while non-acid-terminated species elute according to hydrodynamic volume, i.e., their effective size in solution. Following their release from the stationary phase, the acid-terminated polymers also undergo separation according to hydrodynamic volume. The separation involves adsorption of acid-terminated polymers under basic conditions, followed by their release upon introduction of an acidic mobile phase and subsequent size-based separation. Gradient steepness strongly influenced the elution behavior of acid-terminated polymers. Shallow gradients resulted in broader elution profiles reflecting prolonged adsorption–desorption, whereas steeper gradients produced sharper, more symmetrical peaks. The method can be directly coupled to MS, enabling molecular-level characterization of end-group structures and chemical composition across the chromatographic separation. The proposed esSEC–MS method provides a novel strategy for end-group-resolved polymer analysis. Its applicability is demonstrated by monitoring the formation of cyclic structures by intramolecular transesterification and the degradation of an ester-terminated PLGA copolymer over time.

We introduce a novel analytical strategy for characterizing PLGA's monomer sequence and block-length distribution (BLD). Our approach couples controlled chemical degradation with RPLC-HRMS analysis. By employing two degradation strategies (0.5 vs 3 hrs), we characterize the microstructure of the PLGA co-polymer in terms of the dimer alternating structures (0.5 hr) and the BLD (3 hrs). This method allows for precise monitoring of the randomization caused by transesterification reactions. The BLD, characterized using a specialized algorithm, accurately reflects the polymer's blockiness without underestimation due to dimer-alternating structures, which is a common limitation of other methods like 13C NMR. We demonstrate the method's utility by showing that heterogeneities in block structure significantly influence PLGA's solubility. This new strategy provides critical insights into the relationship between monomer sequence and polymer properties, supporting the development of sequence-controlled PLGA for a wide range of applications.

The characterization of the microstructure of in vivo degradable polyesters is gaining increased interest thanks to their high-performance applications, such as drug delivery systems. The design of such material requires a high level of understanding of the critical material attributes of the polyesters, such as molecular-weight distribution (MWD), chemical-composition distribution (CCD), and end-groups (functionality-type distribution, FTD). Size-exclusion chromatography (SEC) hyphenated with mass spectrometry (MS) is an effective method for analyzing the microstructure of polymers. While the MWD can be determined by size-exclusion chromatography hyphenated with ultraviolet spectrometry and refractive index, the CCD and FTD can be determined by SEC-MS. However, previous applications of SEC-MS have not assessed if polymer fragmentation can occur during the analysis process. In order to correctly interpret CCD and FTD, it is important to establish whether SEC-MS methods can be applied to biodegradable polymers and to recognize if fragmentation processes occur. In this study, we investigate whether SEC-MS methods can be applied to PLGA biodegradable polyesters. The research demonstrates that the choice of alkali metal salt used during ionization can influence the stability of PLGA during SEC-MS analysis. CsI was found to minimize fragmentations during ESI-MS, simplifying the MS spectra and allowing isomeric PLGA structures to be distinguished. The resulting method facilitates FTD and CCD determination. Additionally, when combined with selective degradation, the described method can provide insights into the "blockiness" of the polymer and support the development of sequence-controlled PLGA synthesis.

The performance, biodegradability, and recyclability of polymers can be tuned during synthesis by adopting monomers with different chemical characteristics. Recent research has shown the aptness of some hydrolases to depolymerize polyesters under mild conditions compared to chemical approaches. Herein, we engineered a cutinase from Thermobifida cellulosilytica (Tc_Cut2NV^WCCG) for improved thermostability (up to 91 °C) and compared it with previously reported leaf‐branch compost cutinase (LCC^WCCG) for the hydrolysis of low molar mass substrates, as well as aliphatic and aromatic homo‐ and co‐polyesters. For both enzymes, higher hydrolysis rates were observed for aliphatic compared to aromatic homo‐polyesters. SEC‐MS analysis revealed that the hydrolysis of aliphatic/aromatic co‐polyesters occurred at the aliphatic monomers, significantly reducing the molecular weight and changing the end‐group composition. These results underline the importance of co‐polymer composition in the biodegradation of co‐polymer systems and demonstrate the applicability of enzymes for the analytical characterization of synthetic polymers by selectively reducing their molecular weight. Finally, the discovery and engineering of highly active enzymes that can efficiently hydrolyze a wide variety of synthetic polyesters create new opportunities for their efficient recycling under mild conditions.

The characterization of the microstructure of in vivo degradable polyesters is gaining increased interest thanks to their high-performance applications, such as drug delivery systems. The design of such material requires a high level of understanding of the critical material attributes of the polyesters, such as molecular-weight distribution (MWD), chemical-composition distribution (CCD), and end-groups (functionality-type distribution, FTD). Size-exclusion chromatography (SEC) hyphenated with mass spectrometry (MS) yields SEC-MS, which is an effective method for analyzing the microstructure of polymers. While the MWD can be determined by size-exclusion chromatography hyphenated with ultraviolet spectrometry and refractive index (SEC-UV/RI), the CCD and FTD can be determined by SEC-MS. However, previous applications of SEC-MS have failed to address the risk of polymer fragmentation during the analysis process. It is crucial to establish whether SEC-MS methods can be applied to biodegradable polymers and to recognize if fragmentation processes occurred during the SEC separation or during the ESI-MS process. In this study, we investigate whether SEC-MS methods can be applied to PLGA biodegradable polyesters. The research demonstrates that the choice of alkali metal salt used during ionization strongly influences the stability of PLGA during SEC-MS analysis. CsI was found to minimize fragmentations during ESI-MS, simplifying the MS spectra and allowing isomeric PLGA structures to be distinguished. The resulting method facilitates end-group and CCD determination. Additionally, when combined with selective degradation, the described method can provide insights in the “blockiness” of the polymer and support the development of sequence-controlled PLGA synthesis.

The performance, biodegradability, and recyclability of polymers can be tuned during synthesis by adopting monomers with different chemical characteristics. Recent research has shown the aptness of some hydrolases to depolymerize polyesters under mild conditions compared to chemical approaches. Herein, we engineered a cutinase from Thermobifida cellulosilytica (Tc_Cut2NVWCCG) for improved thermostability (up to 91 °C) and compared it with previously reported leaf-branch compost cutinase (LCCWCCG) for the hydrolysis of low molar mass substrates, aliphatic and aromatic polyester homopolymers, and a co-polyester. For both enzymes, higher hydrolysis rates were observed for aliphatic compared to aromatic homopolyesters. SEC-MS analysis revealed that the hydrolysis of aliphatic/aromatic co-polyesters occurred at the aliphatic monomers, significantly reducing the molecular weight and changing the end-group composition. These results underline the importance of co-polymer composition in the biodegradation of co-polymer systems and highlight the potential use of enzymes for the analytical characterization of synthetic polymers by selectively reducing their molecular weight.

The end groups of poly(lactide-co-glycolide) (PLGA) play a crucial role in determining the properties of polymers used in drug delivery systems. Research has shown that the encapsulation efficiency in PLGA microspheres can vary significantly between ester-terminated and acid-terminated PLGA. To study the functionality-type distribution (FTD) in PLGA polymers, researchers have used methods such as matrix-assisted laser desorption/ionization – high-resolution mass spectrometry and electrospray ionization – mass spectrometry. However, these methods are typically limited to PLGA polymers with a molecular weight of up to 4kDa. 13Carbon-nuclear-magnetic-resonance has also been used to differentiate and quantify PLGA end groups, but this method is time-consuming, taking over 12 hours per sample. To address these limitations, we have developed a normal-phase liquid chromatography method that can separate FTD and partial chemical composition distribution (CCD) in PLGA polymers. Our method can separate PLGA polymers with a molecular weight of up to 185 kDa and simultaneously separate the difference in lactic acid (LA)/glycolic acid (GA) ratios. To achieve this, we used a cross-linked diol column with a ternary gradient from hexane to 0.1% v/v triethylamine in THF to 0.1% v/v formic acid in THF. This allowed for the elution of ester-terminated PLGA, followed by acid-terminated PLGA. We also separated ester-terminated PLGA in the difference of the LA/GA ratio. Our method is expected to help understand the correlation between PLGA’s FTD, CCD, and physical properties, which will facilitate product development.