Process Implications for Metal‐Dependent Immunoaffinity Interactions

Velander, William H.; Orthner, Carolyn L.; Tharakan, Jaganmohan; Madurawe, Rapti D.; Ralston, Annemarie H.; Strickland, Dudley K.; Drohan, William N.

doi:10.1002/btpr.5420050310

Cited by 22 publications

(6 citation statements)

References 19 publications

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“…To determine whether the Ca2+-dependent epitope for MAb H5B7, which has been shown to reside in the [GDk] (Velander et al, 1989), had been incorporated into r-[VK5]-PC and r-[APC1_13/VfIX1_14]PC, we titrated with Ca2+ the binding of this MAb to these mutant proteins. The results shown in Figure 5 demonstrate that the Ca2+/protein com- plexes do not interact with this MAb, at least with an affinity within 10-100-fold of the strength of that of flX.…”

Section: Resultsmentioning

confidence: 99%

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Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C

Geng¹,

Christiansen²,

Plow³

1995

Biochemistry

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A series of recombinant (r) chimeric mutants of human coagulation protein C (PC) and activated protein C (APC) containing replacements of homologous PC domains by those of human coagulation factor IX (fIX) were generated, with the intention of determining whether the specific bovine aortic endothelial cell (BAEC) receptor-binding characteristics of fIX could be incorporated into the chimeric r-PC while maintaining the essential properties of PC and APC. Using a competitive BAEC displacement assay with [125I]fIX, we found that a chimeric r-PC (r[delta PC1-46/delta fIX1-47]PC), consisting of the entire gamma-carboxyglutamic domain ([GDIX], residues 1-38) and helical stack ([HSIX], residues 38-47) of fIX as replacements for these same domains of PC, provided an IC50 for fIX-related BAEC binding of 13 nM, as compared to 10 nM for that of unlabeled fIX. This showed that all of the BAEC tight binding determinants for fIX existed within the [GDIX/HSIX]. Additionally, this chimera reacted to the same extent as fIX with the Ca(2+)-dependent, [GDIX]-specific monoclonal antibody H5B7 and lost its reactivity to a similar antibody specific for the [GDPC], JTC1. A synthetic peptide containing residues 1-47 of fIX also competed effectively (IC50 = 16 nM) with intact fIX for BAEC binding. Displacement of [125I]fIX from BAEC did not occur with a chimera containing the [HSIX] alone or with another mutant protein possessing a replacement of the two epidermal growth factor (EGF) homology regions of r-PC (residues 47-137) with those same domains of fIX.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

“…The MAb H5B7, which recognizes a Ca2+-dependent epitope in [GDK] (Velander et al, 1989), was provided by Dr. D. Strickland (Rockville, MD). The MAb JTC1, which recognizes a similar epitope in PC, was a gift of Dr. K. Wakabayashi (Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C

Geng¹,

Christiansen²,

Plow³

1995

Biochemistry

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“…Aoki and K. Wakabayashi (Tokyo, Japan). The MAb H5B7, which recognizes a Ca2+-dependent epitope in the Gla domain of human fIX (Velander et al, 1989), was obtained from Dr. D. Strickland (Rockville, MD). The antibodies were radiolabeled using the Iodobead (Pierce Chemical Company, Rockford, IL) procedure as described previously ).…”

Section: Methodsmentioning

confidence: 99%

Properties of Recombinant Chimeric Human Protein C and Activated Protein C Containing the .gamma.-Carboxyglutamic Acid and Trailing Helical Stack Domains of Protein C Replaced by Those of Human Coagulation Factor IX

Christiansen¹

1994

Biochemistry

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The properties of a recombinant (r) chimeric human protein C (PC) containing replacement of its gamma-carboxyglutamic acid (Gla) and helical stack (HS) domains by those of human coagulation factor IX (fIX) have been examined. Titration with Ca2+ of the divalent cation-induced intrinsic fluorescence quenching of this chimera (r-GDIX/PC) allowed determination of the [Ca2+], of 1.8 mM, required to produce this alteration in 50% of the protein molecules. These values were 0.41 and 0.61 mM for wtr-PC and fIX, respectively. The chimera did not react with a Ca(2+)-dependent, Gla domain-directed conformational monoclonal antibody (MAb) to r-PC but did interact with a similar MAb (H5B7) to fIX. The [Ca2+] required to induced H5B7 binding to 50% of the r-GDIX/PC molecules was 6.6 mM, while this same value for fIX was a nearly identical 7.2 mM. The [Ca2+] needed for binding of 50% of r-GDIX/PC to acidic phospholipid (PL) vesicles was 0.58 mM, while that for wtr-PC and fIX were 1.2 and 0.55 mM, respectively. The [protein] required for 50% binding of r-GDIX/PC to PL at 20 mM Ca2+ was 0.29 microM. These same values for r-PC and fIX were 0.38 and 1.8 microM, respectively. The Ca(2+)-mediated inhibition of the thrombin-catalyzed activation of r-GDIX/PC was characterized by a Ki of 118 microM, a value similar to that of 125 microM obtained for this same inhibition of wtr-PC activation. The thrombin-catalyzed activation of both r-GDIX/PC and wtr-PC was stimulated by soluble r-thrombomodulin. Similar to the case of wtr-PC, Ca2+ initially enhanced and, at higher concentrations, inhibited the activation of r-GDIX/PC. The Km and kcat values for this latter activation at optimal [Ca2+] (100 microM) were 4.1 microM and 2.5 s-1, respectively. These same kinetic constants for activation of wtr-PC were 4.3 microM and 2.9 s-1, respectively. These results show that many of the features needed for functional integrity of the Ca2+-bound Gla/HS domains of PC are also present in those same modules of fIX, a finding that points to a generalized functional role for the Ca2+-induced conformation of the structural unit consisting of the Gla and HS domains. The data also suggest that the Ca2+-bound form of the Gla/HS region is an independently folded unit in PC and perhaps in fIX.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…The VKD proteins are homologous proteins, and it is extremely difficult to separate them using traditional chromatography, such as ion exchange. Immunoaffinity chromatography columns are used to separate them [3]. Because of the high cost of monoclonal antibodies, this technology is very expensive.…”

Section: Introductionmentioning

confidence: 99%

Protein C Separation from Human Blood Plasma Derivatives Using Low Cost Chromatography

Bruley

2003

Oxygen Transport to Tissue XXIV

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Abstract:Protein C (PC) deficiency can cause thrombosis, inhibiting oxygen transport to tissue thus resulting in many complications, including death. Present treatment can cause catastrophic bleeding and other major medical problems. PC treatment has no bleeding or skin necrosis problems because it circulates in the blood as a zymogen and is only activated when and where it is needed.The vitamin K dependent (VKD) proteins are homologous proteins, making the separation of PC from plasma extremely difficult. Immobilized metal affinity chromatography (IMAC) is investigated to separate the VKD proteins to replace immunoaffinity chromatography, because of the high cost of monoclonal antibodies.An IDA-Cu column was found effective for the separation of PC from prothrombin, the most harmful contaminant. For Cohn fraction IV-I separation, a DEAE column was found an efficient initial step, with about 25-fold PC purity increase. Following this step, an IDA-Cu column could remove many contaminants including prothrombin. The combination of DEAE and IDA-Cu resulted in PC purity increase of about 100-fold.

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Process Implications for Metal‐Dependent Immunoaffinity Interactions

Cited by 22 publications

References 19 publications

Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C

Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C

Properties of Recombinant Chimeric Human Protein C and Activated Protein C Containing the .gamma.-Carboxyglutamic Acid and Trailing Helical Stack Domains of Protein C Replaced by Those of Human Coagulation Factor IX

Protein C Separation from Human Blood Plasma Derivatives Using Low Cost Chromatography

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