Routine manufacture of recombinant proteins using expanded bed adsorption chromatography.

Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30-44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography. Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-1 nominal fermentation volume). The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins. The separations were operated using a 60-cm (d) column run at 14 l/min. For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10). For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10). Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.

Large-scale purification of recombinant human angiostatin.

A process for the purification of recombinant human angiostatin (rhAngiostatin), produced by Pichia pastoris fermentation operated at the 2000-L scale, is reported. rhAngiostatin was recovered and purified directly from crude fermentation broth by cation exchange expanded bed adsorption chromatography. Anion exchange chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography were used for further purification. Full-length rhAngiostatin was separated from rhAngiostatin molecules fragmented by endoproteolysis. On average, 140 g of rhAngiostatin was produced per batch, with an overall yield of 59% (n = 9). The purification process was completed in approximately 48 h and used only inexpensive and nontoxic raw materials. Methods development, process synthesis, and process scale-up data are presented and discussed.

Discoloration of ceramic hydroxyapatite used for protein chromatography.

Hydroxyapatite chromatography was used to purify a recombinant human protein at preparative (400 g) scale. The hydroxyapatite column became progressively discolored as the number of chromatographic cycles increased. Elemental analysis showed that Mn, Fe, Al, Cd, Ba, Cr and Sn were found in used, discolored hydroxyapatite but were below the detection limit (1 ppm) in new hydroxyapatite. Metal ions were not removed from the discolored hydroxyapatite by regeneration with 0.5 M sodium phosphate followed by 0.5 M sodium hydroxide. Chromatographic performance was not affected by the accumulation of metal ions for at least 8 cycles on the preparative column (media volume 56 l) and for at least 12 cycles on the laboratory-scale column (media volume 3.7 ml).

Traumatic brain injury, hemorrhagic shock, and fluid resuscitation: effects on intracranial pressure and brain compliance.

Intracranial hypertension following traumatic brain injury is associated with considerable morbidity and mortality. Hemorrhagic hypovolemia commonly coexists with head injury in this population of patients. Therapy directed at correcting hypovolemic shock includes vigorous volume expansion with crystalloid solutions. It is hypothesized that, following traumatic brain injury, cerebrovascular dysfunction results in rapid loss of brain compliance, resulting in increased sensitivity to cerebrovascular venous pressure. Increased central venous pressure (CVP) occurring with vigorous crystalloid resuscitation may therefore contribute to the loss of brain compliance and the development of intracranial hypertension. The authors tested this hypothesis in miniature swine subjected to traumatic brain injury, hemorrhage, and resuscitation. Elevated CVP following resuscitation from hemorrhage to a high CVP significantly worsened intracranial hypertension in animals with concurrent traumatic brain injury, as compared to animals subjected to traumatic brain injury alone (mean +/- standard error of the mean: 33.0 +/- 2.0 vs. 20.0 +/- 2.0 mm Hg, p < 0.05) or to animals subjected to the combination of traumatic brain injury, hemorrhage, and resuscitation to a low CVP (33.0 +/- 2.0 vs. 24.0 +/- 2.0 mm Hg, p < 0.05). These data support the hypothesis that reduction in brain compliance can occur secondary to elevation of CVP following resuscitation from hemorrhagic shock. This may worsen intracranial hypertension in patients with traumatic brain injury and hemorrhagic shock.

Monoclonal antibodies specific to sailfish serum albumin: development of an assay for the identification of fish species in the field.

Balb/c mice were immunized with albumin purified from sailfish (Istiophorus albicans) serum. Hybridomas were produced and screened by ELISA for reactivity with the purified albumins of sailfish, blue marlin (Makaira nigricans) and white marlin (Tetrapturus albidus). Monoclonal antibodies (MAbs) from 16 different clones exhibited activity against sailfish albumin. Thirteen of the MAbs showed cross-reactivity with the marlin species. Three MAbs exhibited distinct specificity for sailfish albumin. One of these species specific MAbs (M2D1) was conjugated to horseradish peroxidase (HRP) in order to construct an ELISA for identification of sailfish from serum. The ELISA for sailfish correctly identified eight sailfish from 26 billfish serum samples. The MAb-peroxidase conjugate was highly specific toward sailfish in that no reaction against heterologous species was detected.

Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury.

Advances in the understanding of the pathophysiology of traumatic brain injury have implicated a number of cellular events as fundamental to the evolution of neurologic dysfunction in this process. Following the primary biomechanical insult, a highly complex series of biochemical changes occur, some of which are reversible. The development of fluid percussion injury as an in vivo model for traumatic brain injury has greatly improved our ability to study this disease. However, a comparable in vitro model of biomechanical injury which would enable investigators to study the response to injury in isolated cell types has not been described. We have developed a model of transient barotrauma in cell culture to examine the effects of this form of injury on cell metabolism. This model employs the same fluid percussion device commonly used in in vivo brain injury studies. The effect of this injury was evaluated in monolayers of human glial cells. Cell viability by trypan blue exclusion and the production of leukotrienes following increasing barotrauma was investigated. This model provided a reproducible method of subjecting cells in culture to forces similar to those currently used in animal experimental head injury.

Leukotriene production by human glia.

Elevated intracranial pressure and acute cerebrovascular changes following head injury remain the principle challenge in the management of traumatic brain injury. Recent work has demonstrated that leukotrienes can induce increases in blood brain barrier permeability and alter cerebrovascular dynamics. We investigated whether human astroglia in culture: 1. generate specific leukotrienes; 2. how they metabolize leukotrienes, and; 3. if astroglia generate leukotrienes in response to barotraumatic injury. Human astroglial cultures established from normal human brain obtained at surgery were exposed to either ionophore, exogenous 3H-LTC4, or barotraumatic injury. Supernatants were assayed for specific leukotrienes by one of three methods: HPLC, radioimmunoassay, or enzyme-immunoassay. Glial cells exposed to exogenous LTC4 metabolized nearly all of the LTC4 to LTD4 and LTE4 within 20 minutes. Glial cells stimulated with ionophore produced mostly LTC4 at five minutes after stimulation and LTD4 and LTE4 at fifteen minutes after stimulation. Glial cells subject to barotraumatic injury produced LTC4 in concentrations of 40-200 pg/ml 15 minutes after injury. These results demonstrate that human astroglial cells are capable of rapidly generating and degrading LTC4 and this capability of glial cells may play an important role in the pathophysiology of cerebrovascular changes following head injury.