Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine for production

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10-100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of low expression, high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. We also show that the efficiency of this process can be greatly improved with robotic selection. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years.

126 Immunoblot analysis (Fig. 3A) revealed that rgp120 made by the 6 MGAT1 -CHO 127 colonies were smaller in size (85 kDa) compared to A244-rgp120 produced in normal 128 DG44 CHO cells (120 kDa). Comparison of reduced and non-reduced proteins detected 129 a trace amount of aggregated rgp120 protein and no proteolytic degradation (clipping).
130 Cultures were harvested when cell viability dropped to 50%. When culture supernatant 131 was assayed by ELISA (Fig. 3B) rgp120 titers of approximately 400 mg/L were 132 observed in in two cell lines (5D and 5F) with the 5C line exhibiting the lowest rgp120 133 titer (approximately 125 mg/L). Examining the kinetics of cell growth and rgp120 134 accumulation in cell culture medium (Fig. 3C), we found that rgp120 production 135 increased after the addition of sodium butyrate at day six with the rate of accumulation 136 stabilizing between 10-14 days of cell culture. During this period, rgp120 became the 137 major protein secreted into the cell culture medium.

2.3 Growth characteristics of MGAT1 -CHO cells expressing A244_N332-rgp120
139 Several experiments were performed to characterize the growth characteristics of 140 MGAT1 -CHO cells expressing A244_N332-rgp120. The initial 125-500mg/L yield, was 141 obtained using culture conditions primarily designed for transient expression of 142 recombinant proteins following electroporation. Data is shown in Figure 4A-C from 143 triplicate cultures of 5F MGAT1 -CHO (600 mL cultures grown in 1.6 L shake flasks) for 144 a 13-day culture period. Cells were grown at 37 o C until they reached late log growth 145 phase (six days) then sodium butyrate was added to enhance protein expression and 146 the temperature was shifted to 32 o C for the remainder of the experiment (Fig. 4A). The 156 support protein expression in CHO cells,reviewed in (63). The cells were again grown 157 at 37 o C until they reached late log growth phase (six days) with a viable cell density 158 approaching 1x10 7 cells per ml, adding Sodium butyrate (1mM) and shifting the 159 temperature 32 o C for the remainder of the experiment (Fig. 4D). There were small 160 differences in cell growth and viability ( Fig. 4D and 4F) and productivity with the different 161 peptone hydrolysate additives which might be further explored prior to large scale 162 production, however, all supported 1g/L production of rgp120 at harvest (Fig. 4F).
163 These studies demonstrate that the 5F MGAT1 -CHO cell line expressing A244_N332-164 rgp120 can be grown to high cell densities and is productive for up to 12-14 days in 165 culture. It is likely that media optimization and a regulated bioreactor system can 166 improve cell viability, cell densities, and rgp120 expression  294 The addition of these glycan dependent epitopes represents a significant improvement 295 in the antigenic structure of A244-rgp120 and might improve the level of efficacy that 296 can be obtained from an RV144-like immunization regimen.

305
Previous studies have shown that early intermediates in the N-linked 306 glycosylation pathway (Man5 or Man9) are essential components of many epitopes 307 recognized by bN-mAbs (11-16, 18, 19, 20). Additionally, we now know that HIV virion-308 associated Env proteins are enriched for these early intermediates in the glycosylation 309 pathway (20, 31, 50, 71). The A244_N332-rgp120 protein produced in the 5F MGAT1 -310 CHO cell line possesses multiple glycan dependent epitopes recognized by bN-mAbs 311 and appears to possess a glycan structure that more closely resembles authentic HIV 312 Env proteins compared to Env proteins produced in normal CHO cells. We hypothesize 313 that this "glycan optimized" immunogen produced in the 5F MGAT1 -CHO cell line  338 The new technology outlined here should allow for the rapid production and      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.