Method for Characterization of PEGylated Bioproducts in Biological Matrixes

PEGylation of peptides and proteins has been widely used to enhance stability and reduce immunogenicity of biotherapeutics. Characterizing the degradation of these PEGylated products in biological fluids can yield essential information to support pharmacokinetic evaluations and provide clues about their <i>in vivo</i> properties useful for further molecular optimization. In this paper, we describe a novel and uncomplicated approach to characterize PEGylated peptides or proteins and their related degradation products in biological matrixes. The method involves direct liquid chromatography/mass spectrometry (LC/MS) analysis of animal sera containing low nanograms to low micrograms per milliliter of PEGylated product with or without an acetonitrile precipitation sample treatment. Applying the methodology to analyze the model PEGylated peptides, 20K PEGylated-Pancreatic Polypeptide analogue (PPA) and 20K PEGylated-glucagon, we elucidated the decomposition pathways occurring in animal sera. The data provided direct evidence of cleavages within the peptide backbone. The identified degradation products were unambiguously confirmed by tandem mass spectrometry with high-energy C-trap dissociation (HCD) analysis, followed with in-source fragmentation. Additional spiking studies demonstrated nearly full recovery of PEGylated products, linear detection when the spiked concentration of PEGylated product was ≤1000 ng/mL, and a low ng/mL limit of quantitation (LOQ)

The 200 kDa Cry1Ac-binding cadherin was detected in midgut BBMV proteins from both susceptible and resistant <i>T. ni</i> by toxin overlay binding analysis.

<p>The 200 kDa Cry1Ac-binding cadherin was detected in midgut BBMV proteins from both susceptible and resistant <i>T. ni</i> by toxin overlay binding analysis.</p

Relative levels of cadherin mRNA, normalized to the ß-actin mRNA, in the midgut of the susceptible and Cry1Ac resistant larvae determined by real-time RT-PCR analysis.

<p>Error bars indicate standard errors of the means from analysis of 3 individuals.</p

Response of F₂ larvae from four families of single-pair crosses to Cry1Ac selection.

1<p>Survival Rate (%) was corrected using the Abbott’s formula <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035991#pone.0035991-Abbott1" target="_blank">[63]</a> with the control survival rates which were ≥95% in the bioassays.</p>2<p>Predicted survival rate 25% to selection with Cry1Ac was calculated based on inheritance of the recessive monogenic Cry1Ac-resistance trait.</p>3<p>Statistical significance was tested by <i>chi</i>-square test.</p

Cadherin allele frequencies in F₂ progenies from four single-pair cross families.

1<p>Statistical significance was analyzed by Chi-square test with the predicted ratios of <i>cad^Ccad^C</i> : <i>cad^Ccad^G</i> : <i>cad^Gcad^G</i>  = 1∶2∶1 in the tested individuals of each treatment.</p

Spatial expression of the 11 selected miRNAs in the midgut, fat body and epidermis during the 6^th (last) instar larval stage of <i>S. litura</i>.

<p>The expression levels of the miRNA by stem-loop RT-PCR were digitized by ImageQuantTL after gel electrophoresis and the relative expression levels of the miRNAs were calculated as log10(miRNA/U6snRNA). The digital numbers were converted into image by using Cluster/Treeview. The scale bar shows the relative expression levels of the miRNAs over the control (U6snRNA) from -3 to 3 folds (0.001 to 1000 folds). Mg: midgut; Fb: fat body; Ep: epidermis. D1, D3 and D6: day 1, 3 and 6 in 6^th instar stage, respectively.</p

Potential target genes of the selected 11 miRNAs.

*<p>The numbers in the parenthese are for the second class of miRNA target genes.</p

Southern blotting analysis of sli-miR-928b binding with the mRNA transcripts of its predicted potential target genes, CG2781 (GenBank accession number: 40943), mRpL27 (GenBank accession number: 318825), Atf-2 (GenBank accession number: 37978) and CG1776 (GenBank accession number: 36002).

<p>The 3′-UTR cDNA fragments that contained the sli-miR-928b complementary sequence of the four target genes were amplified by RACE-PCR (lower panel). Sli-miR-928b miRNA was synthesized and labeled with 32P-ATP as a probe and used for the hybridization with the 3′-UTR fragments of the genes (upper panel).</p

Developmental expression of the 11 selected miRNAs at different stages from eggs to adults of <i>S. litura</i>.

<p>The expression levels of the miRNA by stem-loop RT-PCR were digitized by ImageQuantTL after gel electrophoresis and the relative expression levels of the miRNAs were calculated as log10(miRNA/U6snRNA). The digital numbers were converted into image by using Cluster/Treeview. The scale bar shows the relative expression levels of the miRNAs over the control (U6snRNA) from −3 to 3 folds (0.001 to 1000 folds).</p

Expression of the 58 predicted miRNA detected by stem-loop RT-PCR.

<p>The expression levels of the 58 predicted miRNA of <i>S. litura</i> by stem-loop RT-PCR were digitized by ImageQuantTL after gel electrophoresis and the relative expression levels of the miRNAs were calculated as log10(miRNA/U6snRNA). The digital numbers were converted into image by using Cluster/Treeview. The scale bar shows the relative expression levels of the miRNAs over the control (U6snRNA) from −3 to 3 folds (0.001 to 1000 folds). The numbers in the figure were corresponding to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037730#pone-0037730-t001" target="_blank">Table 1</a>.</p