Camelid VH H affinity ligands enable separation of closely related biopharmaceuticals.

Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process-related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VH H antibody fragments as "tunable" immunoaffinity ligands for separation of product-related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma-carboxylglutamic acid domain.

Process-scale purification and analytical characterization of highly gamma-carboxylated recombinant human prothrombin.

Prothrombin (coagulation Factor II) is a complex multidomain glycoprotein that plays a central role in blood coagulation. It is the zymogen precursor to the protease thrombin that catalyzes the formation of the fibrin clot and regulates a multitude of other cellular responses related to coagulation and hemostasis. For the biological activity of prothrombin, the vitamin K dependent posttranslational modification of glutamic acid residues to gamma-carboxylglutamic acid is of crucial importance. Prothrombin can be recombinantly expressed using mammalian cell culture. However, the product is a heterogeneous mixture of variants with different degrees of carboxylation, requiring separation of closely related charge isoforms. A second challenge for purification is the need to remove traces of the product-related impurity thrombin, a protease, to extremely low levels. In this work, we describe a purification strategy that provides solutions to both challenges and results in an efficient and robust process for active recombinant prothrombin. We also describe the analytical characterization of recombinant prothrombin by HPLC, LC-MS/MS, and complementary biochemical assays.

[Influence of prothrombin cleavage products on platelet activation and aggregation].

Meizothrombin efficiently activates mechanically triggered platelets and enhance collagen-, ADP- and adrenalin-induced aggregation of platelet-rich blood plasma. Thus, in clotting system activation zone meizothrombin is able to enhance process of involving platelets in clotformation. Pre-thrombin 1 inhibits collagen-, ADP- and adrenalin-induced aggregation of platelet-rich blood plasma and so regulates not only plasm but platelet hemostasis by reverse negative relation.

Evaluation of expanded bed adsorption chromatography for extraction of prothrombin complex from Cohn Supernatant I.

This study evaluated the feasibility of substituting expanded bed adsorption (EBA) chromatography for an existing chromatographic purification process for the isolation of prothrombin complex concentrate (PCC) from Cohn Supernatant I. The EBA chromatography (Streamline) resins were compared to the current DEAE-cellulose resin for the extraction of PCC from Cohn SNI. EBA chromatography resins efficiently bound PCC from Cohn SNI at a significantly higher flow rate of up to 300 cm/h compared to 30 cm/h for the current DEAE-cellulose process. Composition and yield of the recovered PCC reflected the elution conditions used. The results indicate that EBA chromatography could be used to efficiently produce PCC comparable to existing products.

Prothrombin fragments containing kringle domains induce migration and activation of human neutrophils.

The cross-talk between inflammatory and coagulation cascades has been demonstrated. Prothrombin processing releases the protease domain (thrombin) along with two catalytically inactive kringle-containing derivatives: prothrombin fragments 1 (F1) and 2 (F2). It is well established that thrombin is able to trigger an inflammatory response but the possible effects of prothrombin fragments on leukocyte functions are still unknown. In this report, we demonstrate for the first time that both F1 and F2 prothrombin fragments, interfere with intracellular functional signaling pathways to modulate human neutrophil migration. In addition, we show that thrombin, fragment 1 and fragment 2 induce human neutrophil chemotaxis. The effect of fragment 2, but not fragment 1, was partially inhibited by pertussis toxin, an inhibitor of G(alphai)-signaling. The pre-treatment of cells with fragment 2 inhibited thrombin-induced chemotaxis, while both fragments impaired neutrophil migration induced by interleukin-8. F1 and F2 increased the expression and activation of G-protein-coupled receptor kinase-2, which has emerged as a key effector in the desensitization of chemokine receptors. In parallel, prothrombin fragments activated extracellular signal-regulated kinase 1/2, stimulating its phosphorylation and nuclear translocation, and induced inhibitor of kappa-B phosphorylation and degradation followed by nuclear factor-kappa B translocation to nucleus. Furthermore, both prothrombin fragments induced interleukin-8 gene expression in human neutrophils. These findings suggest that the interference with neutrophil signaling and function, caused by kringle-containing prothrombin fragments may desensitize these cells to respond to further activation by thrombin and interleukin-8 during inflammatory and coagulation responses.

Protocol for the purification of proteins from biological extracts for identification by mass spectrometry.

When a protein signal is selected by mass spectrometry as being a potential biomarker, it is necessary to formally identify it. This process involves separation of the protein in question and its identification by either peptide fingerprinting or tandem mass spectrometry sequencing. In the following pages, a simple and rapid protocol is described. Basically, the protocol consists of an initial rational selection of a few sorbents followed by alignment of these as a series of columns to obtain the purified target protein. This preparation is then submitted to electrophoresis, the band is excised and the trypsin digest is analyzed by either mass spectrometry (mass fingerprinting approach) or by LC-MS/MS (sequencing). The development of the process takes only a few days. Experimental data for the isolation and identification of proteins are discussed and two examples are shown.

Effects of urinary prothrombin fragment 1 in the formation of calcium oxalate calculus.

PURPOSE: We investigated the effects of urinary prothrombin fragment 1 in the formation of calcium oxalate urolithiasis. MATERIALS AND METHODS: Fresh urine and renal parenchyma from patients with calcium oxalate calculus and normal controls were collected. Urinary prothrombin fragment 1 was isolated and purified from urine. It was identified by sodium dodecyl sulfide-polyacrylamide gel electrophoresis and analysis of its first 13 N-amino acids. The inhibitory activity of urinary prothrombin fragment 1 on calcium oxalate crystal growth was tested by the seeded crystallization technique. Meanwhile, the gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was analyzed by a previously described method and genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 from renal parenchyma was detected by polymerase chain reaction-single strand conformational polymorphism sequencing. RESULTS: The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was significantly decreased from normal (24.4 to 1.7 mol/1,000 amino acids) in patients with calcium oxalate calculus. The mean growth index +/- SD of urinary prothrombin fragment 1 to calcium oxalate crystals was 42.3 +/- 4.2 compared with the normal index of 19.2 +/- 2.8 (p <0.01). The polymerase chain reaction-single strand conformational polymorphism sequencing technique revealed no genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 in patients with calcium oxalate calculus. CONCLUSIONS: The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 as well as its ability to inhibit calcium oxalate crystal growth was significantly decreased in patients with calcium oxalate calculus. This was not caused by genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1. It is important to elucidate the mechanisms of calcium oxalate stones in view of urinary prothrombin fragment 1.

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.

Heparin-activated antithrombin interacts with the autolysis loop of target coagulation proteases.

A unique pentasaccharide fragment of heparin can enhance the reactivity of antithrombin with coagulation proteases factors IXa and Xa by 300- to 600-fold through a conformational activation of the serpin, without having a significant effect on the reactivity of antithrombin with thrombin. In this study, it was hypothesized that differences in the structure of the autolysis loop of coagulation proteases (residues 143-154 in chymotrypsin numbering) may be responsible for their differential reactivity with the native and heparin-activated antithrombin. To test this hypothesis, the autolysis loops of both thrombin and the anticoagulant serine protease-activated protein C were replaced with the corresponding loop of factor Xa. Inhibition studies revealed that in contrast to the approximately 1.5-fold difference in the reactivity of thrombin with antithrombin in the absence and presence of pentasaccharide, the difference in reactivity was increased to approximately 37-fold for the mutant thrombin. In the case of the activated protein C mutant, similar to factor Xa, pentasaccharide accelerated the reaction 375-fold. These results suggest that structural differences in the autolysis loop of coagulation proteases play a key role in their differential reactivity with the native and heparin-activated conformations of antithrombin.

Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study.

The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.

Three-dimensional solution structure of Tropidechis carinatus venom extract trocarin: a structural homologue of Xa and prothrombin activator.

Trocarin belongs to group D of prothrombin activators derived from snake venom of Tropidechis carinatus and is a rich non-hepatic source of Xa, the only known hepatic prothrombin activator. The structural and functional similarity with Xa makes trocarin an interesting target for exploring the structure-functional relationship with Xa. Herein we report a predicted complete three-dimensional all-atom structural model of trocarin equilibrated in explicit water using 4 ns of molecular dynamics simulation. The tertiary structure was modeled using the structure of human blood coagulation factor Xa. The conformational and structural features of trocarin are then compared with the X-ray crystal and solution simulation structures of human factor Xa. The modeled structure of trocarin has four individual domains (Gla, EGF1, EGF2 and SP) connected along the long axis with similar secondary structural elements to Xa. The simulations suggest that sodium ion binding in the serine protease domain is impaired in trocarin as compared to Xa. In contrast to Xa, for which the sodium ion forms an octahedral coordination network that brings two loop regions connecting four anti-parallel beta-sheets together, we do not find a similar pattern of network in trocarin. We observe that the difference in the binding pattern of sodium ion leads to a approximately 2-A "shrinkage" of the beta2 strand (B2), in comparison to human Xa, as marked by a shorter distance between 189Asp373 (S1-site residue) and 195Ser379 (active-site residue) in the B2 strand. We propose that these differences may be linked to the experimentally observed lower amidolytic activity of trocarin as compared to Xa.

A fully recombinant partial prothrombin complex effectively bypasses fVIII in vitro and in vivo.

The development of inhibitory antibodies is a serious complication in hemophilic patients, severely compromising therapeutic success. Bleeding episodes in affected patients are controlled by treatment with a plasma-derived prothrombin complex concentrate (PCC), activated PCC (APCC) or recombinant activated factor VII. We hypothesized that a recombinant two-component agent consisting of recombinant prothrombin (rfII) and activated factor X (rfXa) would have substantial fVIII bypassing activity and could be a safe alternative therapeutic option. To test this hypothesis we assembled an agent in vitro solely consisting of rfII and rfXa at a molar ratio of 37,500:1. These factors are believed to be responsible for the activity of APCC preparations. Recombinant fX, used as the source for fXa generation, and rfII were purified from serum-free and protein-free conditioned media of stably transfected CHO and BHK tissue culture cells, respectively. Activation of rfX to rfXa was accomplished by the plant protease ficin, obviating the need for a protease derived from a human or animal source. We found that in vitro the complex reduced the abnormally prolonged activated partial thromboplastin time (APTT) of a high-titer fVIII inhibitor plasma similar to an APCC preparation. Furthermore, addition of increasing amounts of rfII/rfXa to inhibitor plasma resulted in a linear dose-dependent increase in the rate of thrombin generation. In a rabbit fVIII inhibitor model, treatment with rfII/rfXa statistically significantly reduced the intensity of the abnormal cuticle bleeding. In the Wessler test, rfII/rfXa showed no thrombogenicity. These data show that a well-defined, particularly safe and efficacious agent with fVIII bypassing activity can be generated from recombinant fII and fXa.

Channeling during prothrombin activation.

The plasma zymogen prothrombin (II) is converted to the clotting enzyme thrombin (IIa) by two prothrombinase-catalyzed proteolytic cleavages. Thus, two intermediates, meizothrombin (mIIa) and prethrombin-2 (P2), are possible on the reaction pathway. Measurements of the time courses of II, mIIa, P2, and IIa suggested a channeling phenomenon, whereby a portion of the II is converted directly to IIa without free mIIa and P2 as obligatory intermediates. Evidence for this was that the maximum rate of IIa formation preceded the maximum in the level of either intermediate. In addition, analysis of the data according to a model that included two parallel pathways through mIIa and P2 indicated that about 40% of the II consumed did not yield free mIIa or P2. Further studies were carried out in which II was continuously infused in a reactor at a constant rate. Under these conditions II, mIIa, and P2 reached constant steady-state levels, and IIa was produced at a constant rate, equal to that of II infusion. During the steady state, traces of II, mIIa, and P2 were introduced as radiolabels. Time courses of isotope consumption were first order, thus allowing the rates of consumption of II, mIIa, and P2 to be calculated. Under these conditions the rate of II consumption equaled the rate of IIa formation. Rates of consumption of the free intermediates, however, were only 22 (mIIa) and 15% (P2), respectively, of the rate of thrombin formation. Thus, both the time course experiments and the steady-state experiments indicate that an appreciable fraction of II is channeled directly to IIa without proceeding through the free intermediates mIIa and P2.

Simple, sensitive and accurate method for the quantification of prothrombin mRNA by using competitive PCR.

A method for the quantification of prothrombin (PT) mRNA species in hepatic tissues of rats was developed with the use of competitive PCR. To validate the quantification approach, sequential dilutions of total RNA from one of the samples were reverse transcribed. Their equivalent volumes were amplified together with a known amount of non-homologous competitor cDNA with identical nucleotide primers. The disparate sizes of target and competitor permitted the easy identification and quantification of bands in samples after densitometric analysis of ethidium bromide-stained agarose gels. Ratios of intensities of target and competitor bands were plotted against the initial amounts of total RNA species used, giving a linear relationship. The slope of this line was virtually identical with that obtained when the sample RNA was replaced with recombinant target cDNA, indicating that recombinant cDNA behaved in PCR identically with that made by reverse transcription and permitting the estimation of transcripts in reverse transcription reactions by using the recombinant counterpart of each as a standard. To avoid variation in the final results, the amount of competitor used in the assay was calculated separately from the equivalence point of the reverse-transcribed total RNA of one of the tissue samples; PCR was performed only for the minimum number of cycles required to detect products. A standard curve was made in each PCR run by amplifying differing amounts of recombinant cDNA species of PT or beta-actin together with a constant amount of its competitor. The numbers of transcripts in the tissues were then determined directly by PCR incorporating the same amount of respective competitor (as used in the standard curve) and comparing the ratios of products with the standard curve. Application of this method revealed that the median ratio of PT message to beta-actin message in hepatic tissues of 10 normal rats was 0.37, with a mean+/-S.D. of 0.37+/-0.07 (range 0.27-0.47). Although the method was developed for the quantification of PT transcripts in liver, it can easily be used for non-hepatic tissues as well. The technique is simple, quick and sensitive and requires only a very small amount of substrate.

A functional prothrombin gene product is synthesized by human kidney cells.

gamma-carboxylated polypeptides were detected in the human kidney by immunohistochemistry with a monoclonal antibody (M3B) specific for gamma-carboxyglutamyl residues. An approximately 70-kDa gamma-carboxylated protein, subsequently identified as prothrombin, was isolated from the intracellular compartment of cultured human embryonic kidney (HEK293) cells by immunoaffinity chromatography on M3B-coupled resin. Immunohistochemical analyses demonstrated that prothrombin and another vitamin K-dependent protein, the growth arrest-specific protein 6, were detectable in human kidney. As in the liver, the kidney synthesizes prothrombin as a zymogen that can be cleaved by ecarin to an amidolytically active serine protease that is inhibited by hirudin. This demonstrates for the first time the de novo synthesis of a full-length, gamma-carboxylated, and functional prothrombin gene product by human kidney cells.

Capture of proteins from mammalian cells in pilot scale using different STREAMLINE adsorbents.

This presentation compares three different expanded bed matrices. STREAMLINE rProtein A, STREAMLINE SP-XL and STREAMLINE Chelating were monitored in respect to their ability to clarify the broth, to concentrate and to purify the distinct target protein. The capture of a mouse IgG1 and a recombinant prothrombin (PT) was carried out in pilot scale using a 100-l bioreactor and STREAMLINE 100 and 200 columns, respectively. The robustness of the process was also estimated monitoring the expansion behaviour and the cell and debris concentrations during the load and in the eluat. In all cases the capture of the target proteins was comparable to conventional chromatographic systems. The purification success was mainly dependent on the selectivity of the ligand used. The affinity process resulted in a highly purified product. The ion exchanger and chelating material mainly concentrated the product. In all three cases 100 l of cell broth were successfully processed in one run. The robustness of the ion exchanger process was poor, because of strong cell matrix interaction. However, for the chelating and especially for the affinity matrix a highly reproducible process was obtained.

Chromatography of human prothrombin from Nitschmann fraction III on DEAE Sepharose Fast Flow using axial and radial flow column.

An axial column (3 x 2.6 cm) and a radial flow column (3.5 x 5 cm) packed with DEAE Sepharose Fast Flow media was evaluated for the separation of human prothrombin from Nitschmann fraction III. Under radial flow conditions, a sample flow rate up to 14 mL/min (approximately 18 bed vols/h) was achieved. Breakthrough capacity was determined and both columns had almost the same breakthrough capacity per mL media, indicating that the sample loading was independent of radial column geometry.

Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide.

Factor Xa is the serine protease component of prothrombinase, the enzymatic complex responsible for thrombin generation. Production of recombinant factor X/Xa has proven to be difficult because of inefficient gamma-carboxylation, a critical post-translational modification. The affinities of the vitamin K-dependent propeptides for the gamma-carboxylase vary over 2 logs, with the propeptide of factor X having the highest affinity followed by the propeptides of factor VII, protein S, factor IX, protein C, and prothrombin [Stanley, T. B. (1999) J. Biol. Chem. 274, 16940-16944]. On the basis of this observation, it was hypothesized that exchanging the propeptide of factor X with one that binds the gamma-carboxylase with a reduced affinity would enhance gamma-carboxylation by allowing greater substrate turnover. A chimeric cDNA consisting of the human prothrombin signal sequence and propeptide followed by mature human factor X was generated and stably transfected into HEK 293 cells, and modified factor X was purified from conditioned medium. The results indicate that on average 85% of the total factor X produced with the prothrombin propeptide was fully gamma-carboxylated, representing a substantial improvement over a system that employs the native factor X propeptide, with which on average only 32% of the protein is fully gamma-carboxylated. These results indicate that the affinity of the gamma-carboxylase for the propeptide greatly influences the extent of gamma-carboxylation. It was also observed that regardless of which propeptide sequence is directing gamma-carboxylation (factor X or prothrombin), two pools of factor X are secreted; one is uncarboxylated and a second is fully gamma-carboxylated, supporting the notion that the gamma-carboxylase is a processive enzyme.

Determination of the N-terminal amino acid sequence of the purified prothrombin from a patient with liver cirrhosis.

The reasons for the decreased functional activity of prothrombin in liver diseases are still speculative. When a highly purified preparation of prothrombin from a patient with liver cirrhosis is available, the cause of prothrombin abnormalities may be researched on a molecular basis. In this study, prothrombin (6.7 mg) was purified from the ascites fluid (1130 mL) of a patient with liver cirrhosis by barium citrate adsorption, ammonium sulfate elution, DEAE Sephacel and Heparin Sepharose CL-6B column chromatography steps. The molecular weight of this prothrombin was the same as that of normal prothrombin purified from a normal plasma pool. The specific activities were found to be 3.36 U/mg in the one stage clotting assay and 28.9 U/mg in the staphylocoagulase/chromogenic substrate assay, while the normal prothrombin specific activities were 3.92 U/mg and 30.1 U/mg respectively. When N-terminal amino acid sequence analysis was carried out, it was seen that the first 20 residues were identical to the normal human prothrombin excepting the Gla at position #14.