Steven R. Maple

Ultra-high-resolution NMR. IV. A simple technique for measurements of sample temperature gradients

La methode de mesure des gradients de temperature est basee sur le fait que deux resonances quelconques qui ont differentes dependances du deplacement chimique vis-a-vis de la temperature presentent un elargissement de raie spectrale different du aux gradients de deplacement chimique

Analysis of minor components by ultrahigh resolution NMR. I. Evidence for the detectability of weak resonances near peaks which are 10,000 times larger, without suppression of the large peaks

Although many papers have been published in the last 25 years on the subject of “elimination” of large unwanted resonances (Z-7), such as the Hz0 signal in ‘H NMR, refinements are still being reported practically every month. For example, the last five issues of this journal (July-October, 1986) contain three articles on this subject (&IO). Obviously, this activity is motivated not only by the importance of NMR for studying minor (low-molarity) components in the presence of major ones such as H20, but also by the various limitations and difficulties associated with each available solvent-suppression technique. Furthermore, peak suppression may be difficult, inconvenient, or undesirable (i) when there are many large peaks, (ii) when one or more of the small peaks are very close to a large one, and (iii) when the relative intensities of small and large peaks are being studied. In this report we show that careful attention to dynamic range and lineshape quality enables the NMR spectroscopist to study minor components without suppression of the major resonances, even when each minor peak has only lop4 the intensity of a major one and when the separation between the two peaks is only 100 Hz or even less. A quantitative discussion of the feasibility of studying minor components by NMR spectroscopy, with or without the use of major peak suppression, is facilitated by the introduction of three parameters, D, A, and R, defined by Eqs. [ l]-[3]

A platform for vibration damping, leveling, and height control of high-resolution superconducting magnets

Three years ago, we designed and constructed a platform that provides good vibration isolation, level control, and height control for a high-resolution superconducting magnet. This platform was installed under the magnet of a 200 MHz (‘H frequency) spectrometer, as part of our efforts to develop ultrahigh resolution (I-12). Recently, we have observed that a perfectly vertical magnet significantly improves the resolution for a spinning sample in a 500 MHz spectrometer (13). Also, we have received several inquiries from users of 500 MHz spectrometers who are encountering problems caused by mechanical instabilities. Therefore, we have decided to share the design of our platform. It requires a constant source of compressed air, at a pressure dependent on the load (see below). It provides (i) a high degree of vibration isolation, (ii) the ability to adjust the level ofthe platform, and (iii) an invariant vertical position independent of load. The last feature may be important for maintaining homogeneity of magnets located above concrete floors which contain embedded steel rods. The “rubber tire” vibration isolators provided as extra-cost accessories by some instrument manufacturers obviously yield a gradual change in vertical position as the weight of liquid nitrogen ( 1.76 lb/liter) changes. Figure 1 shows the components of the system and how they are connected together with air hoses. Figure 2 is a top view of the aluminum platform; the dashed lines show the location of the components of Fig. 1, sandwiched between the aluminum platform (which holds the magnet) and two rectangular aluminum slabs on the floor. The platform is 1 inch thick and 40 X 40 inches in size. For a Bruker AM-500 system, it is probably necessary to increase the length to about 50 inches, and to drill holes for attaching an adapter for an automatic sample changer. The floor slabs are

A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies

inch thick and 11 X 44 inches. Figure 3 shows a frontal view, and Fig. 4 gives a cross section of one of the four vibration-isolation components (labeled S in Figs. l-3). Details are presented below. The four items labeled S in Figs. l-3 are 1 S3-0 11 Super-Cushion Air Springs from Goodyear Tire & Rubber Co., Greensburg, Ohio, each designed for a load of 60370 lb. Our magnet weighs about 500 lb when full of liquid nitrogen. The required compressed air pressure for a 125 lb (per device) load is about 35 psi. The pressure gauge shown in Fig. 1 acts as a load monitor; it shows the effect of load change as a result of liquid nitrogen evaporation. The much larger weight of a magnet of a 500 MHz instrument (about 1200 lb fully loaded) will require a pressure of about 80 psi;

Ultrahigh resolution NMR. 6. Observation of resolved carbon-13-nitrogen-15 scalar splittings in carbon-13 NMR spectra of samples of natural isotopic composition

Residual host cell proteins (HCPs) in biopharmaceuticals derived from recombinant DNA technology can present potential safety risks to patients or compromise product stability. Thus, the downstream purification process is designed to demonstrate robust removal of these impurities. ELISA using polyclonal anti-HCP antibodies as reagents for capture, detection, and quantitation purposes is most commonly used to monitor HCP removal during process development, but this technique has limitations. More recently, LC-MS for residual HCP characterization has emerged as a powerful tool to support purification process development. However, mass spectrometry needs to overcome the enormous dynamic range to detect low ppm levels of residual HCPs in biopharmaceutical samples. We describe a simple and powerful methodology to characterize residual HCPs in (monoclonal) antibodies by combining a novel sample preparation procedure using trypsin digestion and a shotgun proteomics approach. Differing from the traditional methodology, the sample preparation approach maintains nearly intact antibody while HCPs are digested. Thus, the dynamic range for HCP detection by MS is 1 to 2 orders of magnitude less than the traditional trypsin digestion sample preparation procedure. HCP spiking experiments demonstrated that our method could detect 0.5 ppm of HCP with molecular weight >60 kDa, such as rPLBL2. Application of our method to analyze a high-purity NIST monoclonal antibody standard RM 8670 derived from a murine cell line expression system resulted in detection of 60 mouse HCPs; twice as many as previously reported with 2D-UPLC/IM/MSE method. A control monoclonal antibody used for 70 analyses over 450 days demonstrated that our method is robust.