Manufacture of a Stable Lyophilized Formulation of the Live Attenuated Pertussis Vaccine BPZE1

Abstract

Current pertussis vaccines protect against disease, but not against colonization by and transmission of Bordetella pertussis, whereas natural infection protects against both. The live attenuated vaccine BPZE1 was developed to mimic immunogenicity of natural infection without causing disease, and in preclinical models protected against pertussis disease and B. pertussis colonization after a single nasal administration. Phase 1 clinical studies showed that BPZE1 is safe and immunogenic in humans when administered as a liquid formulation, stored at ≤-70 °C. Although BPZE1 is stable for two years at ≤-70 °C, a lyophilized formulation stored at ≥5 °C is required for commercialization. The development of a BPZE1 drug product, filled and lyophilized directly in vials, showed that post-lyophilization survival of BPZE1 depended on the time of harvest, the lyophilization buffer, the time between harvest and lyophilization, as well as the lyophilization cycle. The animal component-free process, well defined in terms of harvest, processing and lyophilization, resulted in approximately 20% survival post-lyophilization. The resulting lyophilized drug product was stable for at least two years at -20 °C ± 10 °C, 5 °C ± 3 °C and 22.5 °C ± 2.5 °C and maintained its vaccine potency, as evaluated in a murine protection assay. This manufacturing process thus enables further clinical and commercial development of BPZE1.

Keywords: live attenuated vaccine; lyophilization; pertussis; vaccine stability.

Figure 1

PCR analyses of the ampG and dnt loci of the lyophilized BPZE1 drug product compared to the liquid drug product. E. coli ampG (panel A), B. pertussis ampG (panel B) and the B. pertussis dnt flanking regions (panel C) of two lots of the liquid BPZE1 drug product (lanes 1 and 2) and two lots of the lyophilized BPZE1 drug product (lanes 3 and 4), as well as a BPSM control (lanes 5) were amplified by PCR using the appropriate primers listed in Table 1. Lanes L contain the molecular size markers with relevant sizes in bp indicated in the margins. The arrows point to the expected amplicons of 402 bp (panel A), 659 bp (panel B) and 1511 bp (panel C) for E. coli ampG, B. pertussis ampG and the B. pertussis dnt flanking regions, respectively.

Figure 2

q-PCR amplification of the PT S1 subunit-coding DNA. The S1 subunit gene of the liquid BPZE1 drug product (BPZE1 liquid), the lyophilized BPZE1 drug product (BPZE1 lyo), BPSM and BPSM-spiked lyophilized BPZE1 (Spiking) was amplified by PCR using the BPSM-specific (blue) or BPZE1-specific primers (red), as listed in Table 1. The horizontal line indicates the cut-off in numbers of amplification cycles (Ct qPCR) indicating whether a sample is considered positive (≤35 cycles) or negative (≥35 cycles).

Figure 3

Microbiological stability of the BPZE1 drug products over time. (A) The liquid BPZE1 drug product at 10⁷ CFU/dose (triangles, low dose), 10⁸ CFU/dose (circles, middle dose) and 10⁹ CFU/dose (squares, high dose) was stored at −70 °C for two years, and CFU counts were conducted at the indicated time points. (B). Microbiological stability of the lyophilized BPZE1 drug product over time. The lyophilized BPZE1 drug product at 10⁹ CFU/dose was stored at −20 °C ± 10 °C (triangles), 5 °C ± 3 °C (circles) and 22.5 °C ± 2.5 °C (squares) for two years, and CFU counts were conducted at the indicated time points. Vertical lines represent standard deviations.

Figure 4

In-vivo colonization kinetics of the lyophilized BPZE1 drug product compared to the liquid drug product. BALB/c mice (n = 5 per group and per time point) were inoculated intranasally with 10⁵ CFU of the liquid BPZE1 drug product (black bars) or the reconstituted lyophilized BPZE1 drug product (blue bars) and sacrificed 3 h (day 0), 1 or 3 days thereafter to evaluate the CFU numbers in the nasal homogenates. (A) Comparison of the CFU counts of the liquid BPZE1 drug product with those of the lyophilized BPZE1 drug product reconstituted and administered immediately after lyophilization. (B) Comparison of the CFU counts of the liquid BPZE1 drug product with those of the lyophilized BPZE1 drug product reconstituted 6 months after storage at −20 °C ± 10 °C (light blue lines), 5 °C ± 3 °C (middle blue lines) or 22.5 °C ± 2.5 °C (dark blue lines). (C) Comparison of the CFU counts of the liquid BPZE1 drug product with those of the lyophilized BPZE1 drug product reconstituted 24 months after storage at −20 °C ± 10 °C (light blue lines), 5 °C ± 3 °C (middle blue lines) or 22.5 °C ± 2.5 °C (dark blue lines). The results are expressed as means +/– SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ns, not significant.

Figure 5

Potency of the lyophilized BPZE1 drug product compared to the liquid drug product. BALB/c mice (n = 5 per group and per time point) were intranasally vaccinated with 10⁵ CFU of the liquid BPZE1 drug product (black bars) or the reconstituted lyophilized BPZE1 drug product (blue bars), or received PBS as a mock control (white bars). Four weeks later, the mice were challenged intranasally with 10⁶ CFU of virulent BPBCTA1. CFU counts in the lungs were conducted 3 h (D0) and 7 days (D7) post-challenge. (A) Comparison of the potency of the liquid BPZE1 drug product with that of the lyophilized BPZE1 drug product reconstituted and administered immediately after lyophilization. (B) Comparison of potency of the liquid BPZE1 drug product with that of the lyophilized BPZE1 drug product reconstituted 6 months after storage at −20 °C ± 10 °C (light blue lines), 5 °C ± 3 °C (middle blue lines) or 22.5 °C ± 2.5 °C (dark blue lines). (C) Comparison of the potency of the liquid BPZE1 drug product with that of the lyophilized BPZE1 drug product reconstituted 24 months after storage at −20 °C ± 10 °C (light blue lines), 5 °C ± 3 °C (middle blue lines) or 22.5 °C ± 2.5 °C (dark blue lines). The results are expressed as means +/– SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.005.