Sedimentation velocity FDS studies of antibodies in pooled human serum.

The biotech industry has great interest in investigating therapeutic proteins in high concentration environments like human serum. The fluorescence detection system (Aviv-FDS) allows the performance of analytical ultracentrifuge (AUC) sedimentation velocity (SV) experiments in tracer or BOLTS protocols. Here, we compare six pooled human serum samples by AUC SV techniques and demonstrate the potential of this technology for characterizing therapeutic antibodies in serum. Control FDS SV experiments on serum alone reveal a bilirubin-HSA complex whose sedimentation is slowed by solution nonideality and exhibits a Johnston-Ogston (JO) effect due to the presence of high concentrations of IgG. Absorbance SV experiments on diluted serum samples verify the HSA-IgG composition as well as a significant IgM pentamer boundary at 19 s. Alexa-488 labeled Simponi (Golimumab) is used as a tracer to investigate the behavior of a therapeutic monoclonal antibody (mAb) in serum, and the sedimentation behavior of total IgG in serum. Serum dilution experiments allow extrapolation to zero concentration to extract so, while global direct boundary fitting with SEDANAL verifies the utility of a matrix of self- and cross-term phenomenological nonideality coefficients (ks and BM1) and the source of the JO effect. The best fits include weak reversible association (~ 4 × 103 M-1) between Simponi and total human IgG. Secondary mAbs to human IgG and IgM verify the formation of a 10.2 s 1:1 complex with human IgG and a 19 s complex with human IgM pentamers. These results demonstrate that FDS AUC allows a range of approaches for investigating therapeutic antibodies in human serum.

Analysis of nonideality: insights from high concentration simulations of sedimentation velocity data.

The Aviv fluorescence detection system (Aviv-FDS) has allowed the performance of sedimentation velocity experiments on therapeutic antibodies in highly concentrated environments like formulation buffers and serum. Methods were implemented in the software package SEDANAL for the analysis of nonideal, weakly associating AUC data acquired on therapeutic antibodies and proteins (Wright et al. Eur Biophys J 47:709-722, 2018, Anal Biochem 550:72-83, 2018). This involved fitting both hydrodynamic, ks, and thermodynamic, BM1, nonideality where concentration dependence is expressed as s = so/(1 + ksc) and D = Do(1 + 2BM1c)/(1 + ksc) and so and Do are values extrapolated to c = 0 (mg/ml). To gain insight into the consequences of these phenomenological parameters, we performed simulations with SEDANAL of a monoclonal antibody as a function of ks (0-100 ml/g) and BM1 (0-100 ml/g). This provides a visual understanding of the separate and joint impact of ks and BM1 on the shape of high-concentration sedimentation velocity boundaries and the challenge of their unique determination by finite element methods. In addition, mAbs undergo weak self- and hetero-association (Yang et al. Prot Sci 27:1334-1348, 2018) and thus we have simulated examples of nonideal weak association over a wide range of concentrations (1-120 mg/ml). Here we demonstrate these data are best analyzed by direct boundary global fitting to models that account for ks, BM1 and weak association. Because a typical clinical dose of mAb is 50-200 mg/ml, these results have relevance for biophysical understanding of concentrated therapeutic proteins.

Apixaban versus enoxaparin for thromboprophylaxis after hip or knee replacement: pooled analysis of major venous thromboembolism and bleeding in 8464 patients from the ADVANCE-2 and ADVANCE-3 trials.

In order to compare the effect of oral apixaban (a factor Xa inhibitor) with subcutaneous enoxaparin on major venous thromboembolism and major and non-major clinically relevant bleeding after total knee and hip replacement, we conducted a pooled analysis of two previously reported double-blind randomised studies involving 8464 patients. One group received apixaban 2.5 mg twice daily (plus placebo injection) starting 12 to 24 hours after operation, and the other received enoxaparin subcutaneously once daily (and placebo tablets) starting 12 hours (± 3) pre-operatively. Each regimen was continued for 12 days (± 2) after knee and 35 days (± 3) after hip arthroplasty. All outcomes were centrally adjudicated. Major venous thromboembolism occurred in 23 of 3394 (0.7%) evaluable apixaban patients and in 51 of 3394 (1.5%) evaluable enoxaparin patients (risk difference, apixaban minus enoxaparin, -0.8% (95% confidence interval (CI) -1.2 to -0.3); two-sided p = 0.001 for superiority). Major bleeding occurred in 31 of 4174 (0.7%) apixaban patients and 32 of 4167 (0.8%) enoxaparin patients (risk difference -0.02% (95% CI -0.4 to 0.4)). Combined major and clinically relevant non-major bleeding occurred in 182 (4.4%) apixaban patients and 206 (4.9%) enoxaparin patients (risk difference -0.6% (95% CI -1.5 to 0.3)). Apixaban 2.5 mg twice daily is more effective than enoxaparin 40 mg once daily without increased bleeding.

Microbial utilization of estuarine dissolved organic carbon: a stable isotope tracer approach tested by mass balance.

The natural stable isotope values of different plants have been used to trace the fate of organic carbon that enters estuarine ecosystems. Experiments were designed to determine the magnitude of (delta) (sup13)C changes of dissolved organic carbon (DOC) derived from tidal marsh vegetation that occurred during bacterial decomposition. Bacteria were grown on DOC leached from estuarine Spartina alterniflora and Typhus angustifolia plants. In four experiments, 25 to 80% of the initial carbon (2.6 to 9.1 mM organic C) was converted to bacterial biomass and CO(inf2). Mass balance calculations showed good recovery of total C and (sup13)C at the end of these experiments (100% (plusmn) 14% total C; (plusmn) 1(permil) (delta) (sup13)C). The (delta) (sup13)C values of DOC, bacterial biomass, and respired CO(inf2) changed only slightly in the four experiments by average values of -0.6, +1.4, and +0.5(permil), respectively. These changes are small relative to the range of (delta) (sup13)C values represented by different organic carbon sources to estuaries. Thus, microbial (delta) (sup13)C values determined in the field helped to identify the source of the carbon assimilated by bacteria.

Measuring microzooplankton grazing on planktonic marine bacteria by its impact on bacterial production.

Grazing on planktonic bacteria by microzooplankton was estimated by separating bacteria from the larger plankton with 1µm pore Nuclepore filtration and measuring changes in bacteria in filtered and unfiltered samples over 24 hours. In the absence of grazers, bacteria increased linearly. The regression coefficient of linear increase was used to estimatein situ bacterial production. When grazers were present, the changes in bacteria concentration usually took the form of a linear decline, and grazing was estimated by subtracting the regression coefficient of the unfiltered sample from that of the 1µm filtrate. Results from the Essex estuary-coastal system of northern Massachusetts show grazing and production at rates that indicate a daily turnover of the standing crop of bacteria, with highest values in mid-estuarine waters. Experiments on the size distribution of grazing showed that microzooplankton from 1-3µm were responsible for most of the observed decrease in bacteria. It was suggested that the basic pattern of linear increase of the bacteria in the absence of grazing reflects density-dependent limitation by substrate present at the outset of the incubation and is indicative of a population that has been maintained around the mid-point of the logistic growth curve by grazing.

Measurement and significance of specific activity in the heterotrophic bacteria of natural waters.

It is now possible to obtain accurate total counts of the bacteria of natural waters with the use of acridine orange staining and epifluorescence microscopy. This approach can be coupled to highly sensitive measurements of heterotrophic activity using radioisotopes. To accomplish this, three variations of a "specific activity index" are suggested, based on different approaches to measuring heterotrophic activity with radiolabeled organic solutes. The denominator of each index is the direct count of bacteria from a given natural sample. Three numerators are presented, each of which has been shown to vary directly with heterotrophic bacterial activity: V(max), turnover rate, and direct uptake (at high substrate concentrations). Each approach is illustrated with data from estuarine and coastal waters of northeastern Massachusetts. The data show major differences in specific activity that accompany such habitat differences as distances within or offshore from an estuary and vertical location in the water column. These and other data suggest that specific activity is a valid indicator of the physiological state and metabolic role of the bacteria. Some evidence is presented in support of the hypothesis that the natural bacteria are adapted to conditions of nutrient starvation by becoming "dormant," existing for an unknown period of time in a reversible physiological state that reflects the availability of organic nutrients.

Method for measuring mineralization in lake sediments.

A method is described for measuring the mineralization of an organic solute ((14)C-glucose) by the heterotrophic indigenous bacteria in lake sediments. Since there is no suitable procedure for the determination of in situ microbial activities in sediments, the procedure described is probably the best devised so far and may serve as a base for a more definitive procedure.