Scott D. Leigh

Production of Recombinant Human Type I Procollagen Trimers Using a Four-gene Expression System in the Yeast Saccharomyces cerevisiae

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers inSaccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.

Photosensitizers Form in Histidine Buffer and Mediate the Photodegradation of a Monoclonal Antibody

Fluorescent light (FL) photodegradation of a monoclonal antibody (mAb) formulated in histidine buffer is mediated by histidine-derived photosensitizers that accumulate and greatly increase with light exposure. Histidine-derived photosensitizers are the primary mediators of Trp photooxidation. FL-photodegradation requires light exposure and is pH dependent. It is significantly reduced or eliminated by buffer exchanges, by oxygen depletion, or at pH values greater than 7. Antibody-fragment MS ion counts reveal that oxidation of a single light chain Trp in CDR1 correlates with binding loss. Multiple heavy chain methionines oxidize, but poorly correlate with binding loss. Photosensitizers extracted from photo-aged histidine buffer are potent mediators of FL-photodegradation including oxidation and, to a lesser degree, fragmentation and aggregation of the mAb. These photosensitizers absorb visible light and have neutral mass of 187.1- 386.1 Da. They are also fluorescent with ex/em at 360/450 nm. When spiked into histidine or MES buffered mAb formulations they produce a concentration dependent and pronounced increase in FL-photodegradation; however, no oxidation or loss of antibody function occurs in the dark and hydrogen peroxide does not oxidize Trp. The major component is consistent with histidine oxidation to 6a-hydroxy-2-oxo-octahydro-pyrollo[2,3-d]imidazole-5-carboxylic acid. Photosensitizer levels measured in the formulation prior to light exposure, are linearly related to the FL-photodegradation observed and can predict degradation in photostability testing.

ALTERATION OF THE PHARMACOKINETICS OF SMALL PROTEINS BY IODINATION

The pharmacokinetics of several proteins were investigated using two different assays. A 23 kDa recombinant protein fragment of bactericidal/permeability-increasing protein (rBPI23) was radiolabeled with 125I using Iodo-beads and administered rats. Plasma samples were collected and assayed for 125I-rBPI23 by radioactivity. In a separate experiment, rBPI23 was administered to rats and plasma samples were assayed for rBPI23 by ELISA. The clearance determined from plasma concentrations of 125I-rBPI23 measured by radioactivity was about 2.5-fold lower than that of rBPI23 determined by ELISA. In addition, the steady state volumes of distribution and mean residence times of 125I-rBPI23 measured by radioactivity were four-fold and 10-fold greater, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were different when assayed by radioactivity or ELISA, but those of proteins with molecular weights of at least 80 kDA revealed only minor differences. To determine which assay method yielded the correct plasma pharmacokinetic profile, rBPI23 was metabolically labeled with 35S-methionine and administered to rats, and plasma samples were assayed by radioactivity. The concentration-time profile assessed by this method was very close to that determined by ELISA. Exposing rBPI23 to chloramine-T (the oxidant used in the iodination process) and measuring its plasma concentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeling rBPI23 using iodinated Bolton-Hunter reagent (which avoids exposing the protein to oxidant), and measuring 125I-rBPI23 by radioactivity, yielded pharmacokinetics that were similar, although not identical, to the pharmacokinetics of rBPI23 measured by ELISA. Thus, our data suggest that directly iodinating low-molecular-weight proteins by oxidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.

Development of an immunoassay for urinary galactosylhydroxylysine.

Galactosylhydroxylysine (GHL) is released during bone resorption and has been shown to be elevated in subjects with metabolic bone loss. GHL is relatively specific for bone, it is not recycled or significantly metabolized during collagen turnover, and the levels are not influenced by diet. Previous measurements of GHL levels in urine have been performed using reverse-phase high performance liquid chromatography following pre-column derivatization. We produced polyclonal antibodies to GHL using GHL purified from sea sponges and developed an immunoassay that can recognize GHL in urine. The antibodies have minimal cross-reactivity with a physiological mixture of amino acids (< 1%), galactose (< 0.2%), lactose (< 0.3%), and glucosylgalactosylhydroxylysine (< 1%). This competitive immunoassay requires no dilution or pretreatment of the samples and provides a rapid and easy method for the evaluation of GHL in urine. Analysis of clinical samples from normal individuals, post-menopausal women, osteoporotic patients and individuals with Pagets disease show that the assay can discriminate between groups with differing levels of bone resorption as well as deoxypyridinoline (Dpd).

Corrections to "Stroop S, Conca D, Lundgard R, Renz M, Peabody L, Leigh S. 2011. Photosensitizers form in histidine buffer and mediate the photodegradation of a monoclonal antibody". 2011. J Pharm Sci 100:5142–5155.

Figure 13. UV–visible spectra of the major components of the extract. Diode array spectra from 200 to 600 nm were collected across all peaks eluting from the LC (Fig. 12). Spectra are labeled for peaks A, B, C, and D. Insert shows putative structure of component A as described in the text and reported elsewhere.8 ∗∗The observed spectra for component A cannot be attributed to this structure and may be due to aromatic structures coeluting under peak A.