Strategies for Enhancing Product Yield: Design of Experiments (DOE) for <em>Escherichia coli</em> Cultivation

E. coli is considered one of the best model organism for biopharmaceutical production by fermentation. Its utility in process development is employed to develop various vaccines, metabolites, biofuels, antibiotics and synthetic molecules in large amounts based on the amount of yield in shake flasks, bioreactors utilised by batch, fed-batch and continuous mode. Production of the desired molecule is facilitated in the bioreactor by employing strategies to increase biomass and optimised yield. The fermentation is a controlled process utilising media buffers, micronutrients and macronutrients, which is not available in a shake flask. To maximise the production temperature, dissolved oxygen (aerobic), dissolved nitrogen (anaerobic), inducer concentration, feed or supplementation of nutrients is the key to achieving exponential growth rate and biomass. Design of experiments (DOE) is critical for attaining maximum gain, in cost-effective manner. DOE comprises of several strategies likewise Plakett-Burman., Box–Behnken, Artificial Neural Network, combination of these strategies leads to reduction of cost of production by 2–8 times depending on molecules to be produced. Further minimising downstream process for quickly isolation, purification and enrichment of the final product

Antigenicity of a Bacterially Expressed Triple Chimeric Antigen of Plasmodium falciparum AARP, MSP-311 and MSP-119: PfAMSP-Fu35.

Development of fusion chimeras as potential vaccine candidates is considered as an attractive strategy to generate effective immune responses to more than one antigen using a single construct. Here, we described the design, production, purification and antigenicity of a fusion chimera (PfAMSP-Fu35), comprised of immunologically relevant regions of three vaccine target malaria antigens, PfAARP, PfMSP-3 and PfMSP-1. The recombinant PfAMSP-Fu35 is expressed as a soluble protein and purified to homogeneity with ease at a yield of ~ 7 mg L-1. Conformational integrity of the C-terminal fragment of PfMSP-1, PfMSP-119 was retained in the fusion chimera as shown by ELISA with conformation sensitive monoclonal antibodies. High titre antibodies were raised to the fusion protein and to all the three individual components in mice and rabbits upon immunization with fusion chimera in two different adjuvant formulations. The sera against PfAMSP-Fu35 recognized native parasite proteins corresponding to the three components of the fusion chimera. As shown by invasion inhibition assay and antibody mediated cellular inhibition assay, antibodies purified from the PfAMSP-Fu35 immunized serum successfully and efficiently inhibited parasite invasion in P. falciparum 3D7 in vitro both directly and in monocyte dependent manner. However, the invasion inhibitory activity of anti-AMSP-Fu35 antibody is not significantly enhanced as expected as compared to a previously described two component fusion chimera, MSP-Fu24. Therefore, it may not be of much merit to consider AMSP-Fu35 as a vaccine candidate for preclinical development

Inhibition of parasite invasion <i>in vitro</i> by antibodies specific to AMSP-Fu₃₅ and specificity of the inhibition observed.

<p>(A) IgGs purified from rabbit sera immunized with AMSP-Fu_<b>35</b> formulated with different adjuvants were tested at 1mg/ml, 2.5mg/ml and 5mg/ml for inhibition of erythrocyte invasion in <i>P</i>. <i>falciparum</i> 3D7. Percent growth inhibition (shown as means ± standard deviations) was calculated by using the parasitemia in the presence of immune sera with respect to parasitemia in the presence of pre-immune sera. (B) Purified IgGs against AMSP-Fu_<b>35</b> was tested for its invasion inhibitory specificity against the 3D7 parasite clone. For studying the invasion inhibitory specificity, total IgGs were pre-incubated with recombinant proteins AMSP-Fu_<b>35</b>, PfMSP-1_<b>19</b>, PfMSP-3_<b>11</b> and PfAARP, at the final concentrations of 25μg/ml, 50μg/ml and 100μg/ml. The white bar denotes the invasion inhibitory activity of AMSP-Fu_<b>35</b> IgGs.</p

Characterization of recombinant AMSP-Fu₃₅.

<p>(A) Mobility Shift of AMSP-Fu_<b>35</b> under reducing (R) and non-reducing (NR) conditions on SDS-PAGE. (B) Detection of AMSP-Fu_<b>35</b> with anti-His monoclonal antibody under reducing (R) and non-reducing (NR) conditions. (C) RP-HPLC profile of purified recombinant AMSP-Fu_<b>35</b> which eluted as a single peak indicating 98% purity.</p

Native conformations of the three individual components in AMSP-Fu₃₅.

<p>(A) IFA was performed with late schizont stage parasites using AMSP-Fu_<b>35</b> sera and co-localization was checked by co-immunostaining with sera against individual components (anti-PfMSP-1_<b>19</b>, PfAARP and PfMSP-3_<b>11</b>). The corresponding images stained with DAPI, which stains nuclear DNA; merged fluorescence images and bright field images are also shown. (B) Pearson’s correlation graphs for the co-immunostained slides, (i) AMSP-Fu_<b>35</b> and PfAARP, (ii) AMSP-Fu_<b>35</b> and PfMSP-3_<b>11</b> and (iii) AMSP-Fu_<b>35</b> and PfMSP-1_<b>19</b>, are shown and high correlation values showed co-localization. (C) Antibodies to AMSP-Fu_<b>35</b> react with proteins corresponding to PfAARP, PfMSP-1_<b>19</b> and PfMSP-3_<b>11</b> in immunoblots of schizont stage parasite lysate from 3D7. Parallel immunoblots of the same lysate probed with anti-AMSP-Fu_<b>35</b>, PfAARP, PfMSP-3_<b>11</b> and PfMSP-1_<b>19</b> sera confirmed the proteins detected by the fusion sera.</p

Conformational integrity of PFMSP-1₁₉ in AMSP-Fu_35.

<p>(A) Reactivity of AMSP-Fu₃₅ under reducing (R) and non-reducing (NR) conditions with PfMSP-1₁₉ conformational specific MAbs (2E10 and 1H4) and anti-His antibody by ELISA at a dilution of 1:1000. (B) Recognition of reduced (R) and non-reduced (NR) AMSP-Fu₃₅ by PfMSP-1₁₉ conformation specific MAb’s and anti-His antibody by immunoblotting.</p

Antibody responses against AMSP-Fu₃₅.

<p>(A) The end point titres against AMSP-Fu_<b>35</b>, PfMSP-1_<b>19</b>, PfMSP-3_<b>11</b> and PfAARP were measured by ELISA for Freund’s and Alhydrogel formulated AMSP-Fu_<b>35</b> in Balb/c mice (n = 6) (B) Comparison of the antibody response to PfMSP-1_<b>19</b>, PfMSP-3_<b>11</b> and PfAARP in mice immunized with AMSP-Fu_<b>35</b> to the mice immunized with individual components alone (n = 5) (C) The end point titres against AMSP-Fu_<b>35</b>, PfMSP-1_<b>19</b>, PfMSP-3_<b>11</b> and PfAARP were measured by ELISA for Freund’s and Alhydrogel formulated AMSP-Fu_<b>35</b> in New Zealand White rabbits (n = 2).</p

Monocyte mediated inhibition of parasite invasion <i>in vitro</i> by antibodies specific to AMSP-Fu₃₅ and the three individual components.

<p>(A) <i>P</i>. <i>falciparum</i> 3D7 parasites from the schizont stages were co-cultured with human monocytes in the presence of 50<b>μ</b>g of purified IgGs from rabbits immunized with AMSP-Fu_<b>35</b> formulated with Freund’s adjuvant and Alhydrogel. Parasite growth and multiplicity was analyzed after 96hrs. SGI index for ADCI of parasite growth (shown as means ± standard deviations) was calculated by using the parasitemia of test IgGs in the presence and absence of monocytes with parasitemia of control IgGs in the presence and absence of monocytes. (B) Purified IgGs against AMSP-Fu_<b>35</b> was tested for its invasion inhibitory specificity against the 3D7 parasite clone. For studying the invasion inhibitory specificity, total IgGs were pre-incubated with recombinant proteins AMSP-Fu_<b>35</b>, PfMSP-1_<b>19</b>, PfMSP-3_<b>11</b> and PfAARP, at the final concentrations of 25μg/ml, 50μg/ml and 100μg/ml. The white bar denotes the invasion inhibitory activity of AMSP-Fu_<b>35</b> IgGs.</p