Off-axis three-mirror freeform systems design based on improved W-W differential equations.

Design of an off-axis system using the Wassermann-Wolf (W-W) differential equations can effectively eliminate the spherical aberration and coma problem; however, it is complicated and time consuming to calculate the discrete point coordinates on the freeform mirror surfaces due to multiple numbers of reference system transformation in the design process. This paper presents an improved W-W-differential-equations-based design method for off-axis three-mirror freeform systems. First, to reduce the number of coordinate transformations, a geometric relationship between different optical rays in an off-axis system is established using the distance between the central points of adjacent mirrors. Second, a three-dimensional rotation matrix is used to associate the optical paths passing through adjacent mirrors in different reference coordinate systems, and new simplified W-W differential equations based on the ray vectors are constructed. The experimental results show that our method can easily and effectively design off-axis three-mirror freeform systems with different parameters and structures, and the designed systems have good imaging quality.

Chromatography-based methods for determining molar extinction coefficients of cytotoxic payload drugs and drug antibody ratios of antibody drug conjugates.

UV spectrophotometry is widely used to determine the molar extinction coefficients (MECs) of cytotoxic drugs as well as the drug antibody ratios (DARs) of antibody drug conjugates (ADCs). However, the unknown purity of a drug due to interfering impurities can lead to erroneous MECs and DARs. Hence, reliable methods to accurately determine purity and the MECs of drugs with limited quantity is urgently needed in Drug Discovery. Such a method has been developed. It achieves absolute purity and accurate MEC determination by a single automated HPLC analysis that uses less than 5µg of material. Specifically, analytical HPLC separation with online UV detection was used to resolve impurities and measure absorbance from only the compound of interest. Simultaneously, an online chemiluminescence nitrogen detector (CLND) was used to determine the concentration of the analyte. The MECs were then calculated from the absorbance and concentration results. The accuracy of the method was demonstrated using caffeine and a commercial cytotoxic drug, DM1. This approach is particularly suited to analyzing mixtures or samples with low purities. Excellent reproducibility was demonstrated by analyzing a proprietary drug with linker synthesized from different batches with very different levels of purity. In addition, the MECs of drug with linker, along with ADC peak areas measured from size exclusion chromatography (SEC), were used to calculate DARs for 21 in-house ADCs. The DAR results were consistent with those obtained by MS analysis.

Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry.

Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid.

Investigation of a Degradant in a Biologics Formulation Buffer Containing L-Histidine.

PURPOSE: An unknown UV 280 nm absorbing peak was observed by SEC for protein stability samples formulated in L-histidine during a stress stability study. Understanding the source would enhance the confidence in the SEC results. We identified the unknown peak, studied the cause, and evaluated ways to eliminate it. METHODS: The unknown peak was fractionated by preparative size exclusion chromatography separations, and subsequently analyzed by Hydrophilic Interaction Chromatography (HILIC) coupled with Time-of-Flight (TOF) high resolution mass spectrometry. The possible degradation was also studied with the presence of different excipients, including metal cations, chelating agents, and amino acids. RESULTS: The unknown peak was identified to be trans-urocanic acid, a degradant of histidine, based on evidences from HILIC retention time, UV profile, accurate mass measurement, trans-cis isomerization, and pI measurement. The degradation from histidine to urocanic acids was not affected by the presence of Fe(2+), but slightly activated by Mn(2+). The chelating agents, EDTA and DTPA, counteracted the Mn(2+) effects. This degradation was evidenced to be caused by contamination. Adding alanine or cysteine as an excipient was found to reduce this degradation by 97 and 98%, respectively. CONCLUSIONS: L-histidine formulation buffer can be contaminated to induce histidine degradation to trans-urocanic acid, which shows a large UV 280 nm absorbing peak at the total permeation volume under SEC conditions. Amino acids alanine and cysteine effectively inhibit this histidine degradation.

Analysis of drug interactions with high-density lipoprotein by high-performance affinity chromatography.

Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography (HPAC). This approach was evaluated by using it to examine the binding of high-density lipoprotein (HDL) to the drugs propranolol and verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPAC columns with reproducible binding to propranolol over 4-5days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interaction were occurring between R- or S-propranolol and HDL at 37 degrees C: saturable binding with an association equilibrium constant (K(a)) of 1.1-1.9x10(5)M(-1) and nonsaturable binding with an overall affinity constant (n K(a)) of 3.7-4.1x10(4)M(-1). Similar results were found at 4 and 27 degrees C. Verapamil also gave similar behavior, with a K(a) of 6.0x10(4) M(-1) at 37 degrees C for the saturable sites and an n K(a) for the nonsaturable sites of 2.5x10(4)M(-1). These measured affinities gave good agreement with solution phase values. The results indicated that HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body.

Studies of verapamil binding to human serum albumin by high-performance affinity chromatography.

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g. the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 10(4)M(-1) and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37 degrees C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e. the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (+/-0.1)x10(4)M(-1). Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with l-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body.