Smaller particles have progressively led to higher efficiency in liquid chromatography, particularly for proteins, due to smaller diffusion distances. Particle diameter has recently entered the submicrometer region, with the back-pressure requirements alleviated by slip flow.
The higher order structure (HOS) of proteins plays a critical role in the efficacy and stability of biological drugs. Perturbation of the regional structure of proteins can affect biological activity and cause instability. Characterization of HOS has become an integral part of biological drug development and is expected from regulatory agencies. The commonly used techniques for HOS characterization, such as circular dichroism, Fourier-transform infrared, differential scanning calorimetry, intrinsic fluorescence, and hydrogen−deuterium exchange mass spectrometry, have their limitations ranging from lack of sensitivity and specificity to the need of high-level expertise and poor access to instrumentation due to high cost. In this study, we demonstrated a novel controlled proteolysis-based LC-QDa method for the detection of HOS change. By digesting proteins directly without denaturation and reduction, the HOS information can be revealed through the digested peptides. After optimizing the digestion conditions and the detection procedures, we identified 13 signature peptides that can monitor various antibody domains for any HOS changes caused by external stress. By comparing the peptide peak areas between unknown samples and a native control sample, any regional structural changes in unknown samples can be detected. The method was subsequently applied to a wide range of forced degradation samples to demonstrate higher sensitivity compared to the near-UV CD method that is frequently used for monitoring tertiary structural changes. By further reducing the number of signature peptides to five and optimizing liquid chromatography gradient duration, a streamlined, high-throughput, and controlled proteolysis method was successfully established. This method can be used to support process and formulation development as well as potentially for stability testing.
Isomerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and consequently establishing a robust control strategy. As a practical solution to this problem, a comprehensive analytical tool kit has been developed, which provides a solution to fully characterize and accurately quantify the Asp-related product variants. The toolkit offers a combination of 2 steps, an ion-exchange chromatography method to separate and enrich the isomerized variants in the folded structure for structurefunction evaluation and a novel focused peptide mapping method to quantify the individual complementarity-determining region isomerization components including the unmodified Asp, succinimide, and isoaspartate. This novel procedure allowed an accurate quantification of each Asp-related variant and a comprehensive assessment of the functional impact of Asp isomerization, which ultimately helped to establish an appropriate control strategy for this critical quality attribute.
Reversed-phase chromatography is an established method for characterizing the disulfide isoforms of IgG2. This work explores the effect of mobile phase gradient profile and sample concentration on the separation of disulfide isoforms. The acidic mobile phase can alter the relative proportions of disulfide isoforms, but only when the level of the reactive A1 isoform is much higher than for typical conditions of separation and typical IgG2 samples. Otherwise, there is minimal disulfide scrambling. A slower gradient and flow rate modestly improve resolution, but the peaks remain heavily overlapped. Resolution is further improved and nonlinear chromatography lessened when injection is performed under non-stacking conditions. Non-stacking conditions also keep the concentration from spiking at the head of the column, reducing noncovalent associations that can promote disulfide scrambling. The higher resolution from non-stacking injection reveals the presence of at least seven species.
By solid - phase extraction high performance liquid chromatography determination of nonylphenol, Octylphenol in aquatic product residues, to establish more accurate, fast, effective detection method. Sample is 3 trichloroacetic acid solution after extraction, using anion exchange solid phase extraction column purification by reversed phase high performance liquid chromatography method for determination. Column is Shim-pack VP-ODS 250mm x4.6mm, detection wavelength is225 nm, mobile phase of methanol - water ( volume ratio of 75:25), flow rate is 0.8 mL/min, injection volume is 20 ìL. Octylphenol, nonylphenol respectively in 0.14¡«97.63 ìg/mL ( R=0.9995),0.02¡«51.04 ì g/mL ( R=0.9997) concentration and peak area show a good linear relationship, octylphenol nonylphenol and the recovery is 87.9%¡«93.8%, relative standard deviation is 1.4%¡«3.8%. Solid - phase extraction high performance liquid chromatography method for determination of aquatic product of octylphenol and nonylphenol residue is accurate, rapid, high sensitivity, suitable for aquatic products of octylphenol nonylphenol and detection.
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