The timeline for development of stable MGAT1^- CHO cell lines expressing HIV-1 rgp120.

<p>Leading clones expressing 0.2–0.4 g/L in shake flasks under standard laboratory conditions were selected in less than two months. Production was subsequently increased to levels of g/L rgp120 production with minimal feed optimization.</p

SDS-PAGE analysis of A244_N332 rgp120 HIV produced in 5F MGAT1^- CHO and CHO-S cells treated with PNGase or EndoH.

<p>Enzymes and buffers were purchased from (New England Biolabs, Ipswich, MA). Purified protein was denatured and reduced then incubated overnight at 37°C with or without glycosidase. Protein was resolved (2 μg/lane) on 4–12% SDS-PAGE gel and stained with Simply Blue. Plus (+) indicates enzyme treatment, minus indicates untreated.</p

Binding of bN-mAbs to A244-rgp120 produced in normal and A244_N332-rgp120 produced in MGAT1^- CHO cell lines.

<p>A244_N332-rgp120 was purified from the stable clone 5F MGAT1^- CHO cell line (closed circles) or from the MGAT1^- CHO cells (open circles) transiently transfected with the UCSC 1331 plasmid. A244-rgp120 expressed and purified from transiently transfected CHO-S cells (open squares). Antibody binding was measured by a fluorescent immunoassay (FIA).</p

Analysis of A244_N332-rgp120 secreted from stable MGAT1^- CHO cell lines.

<p>Six stable MGAT1^- CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. <b>(A)</b> Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. <b>(B)</b> Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1^- CHO cell lines. (<b>C)</b> SDS PAGE of rgp120 produced by the 5F MGAT1^- CHO cell line. Supernatant samples (10 <i>μ</i>l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).</p

Primary identification of high producer MGAT1^- CHO lines expressing A244_N332 rgp120 by immunofluorescent labeling.

<p><b>(A)</b> G418 selected colonies visible in a single 35mm well illuminated with white light at 6 days. <b>(B)</b> The same single 35mm well illuminated with 490 nm wavelength light. Colonies actively secreting rgp120 have a green “halo” visible at 525 nm. <b>(C)</b> Relative mean exterior fluorescence of halo for more than 10,000 colonies imaged by the ClonePix2 plotted by rank. The top ranking 0.1% of colonies (44) were robotically picked and cultured. The six clones expressing 0.2–0.4 g/L at day 56 are shown in red.</p

Growth and expression of the 5F MGAT1^- CHO cell line expressing A244_N332-rgp120 in shake flask cultures.

<p>Cells were cultured under standard conditions until day 6 when 1 mM Sodium butyrate was added, and the temperature shifted to 32°C. Panels <b>A-C</b>: cells were fed with CHO Feed A and yeastolate as indicated, and harvested at day 13 (data from 3 shake flasks averaged). <b>(A)</b> Timecourse graph of viable cell densities (VCD) determined by trypan-blue exclusion on a BioRad T20 cell counter. <b>(B)</b> Timecourse of cell viabilities determined by trypan-blue exclusion. <b>(C)</b> Timecourse of A244_N332-rgp120 protein accumulation determined by ELISA. Panels <b>D-F</b> demonstrate optimization of protein expression (at >1g/L) by use of different feed additives. Five duplicate pairs of cultures were fed (as indicated) with CHO Feed C and either yeastolate (BD, Franklin Lakes NJ), cottonseed, wheat, pea hydrolsate (Friesland Camparia, Delhi, NY) or CD-hydrolysate (SAFC, Calsbad CA) at days 6, 8 and 10, and harvested at day 12 (data from each pair of shake flasks is averaged). <b>(D)</b> Timecourse graph of viable cell densities (VCD) determined by trypan-blue exclusion on a BioRad T20 cell counter. <b>(E)</b> Timecourse of cell viabilities determined by trypan-blue exclusion. <b>(F)</b> Timecourse of A244_N332-rgp120 protein accumulation determined by ELISA.</p