Formulation and Characterization of Conjugate Vaccines to Reduce Opioid Use Disorders Suitable for Pharmaceutical Manufacturing and Clinical Evaluation

Abstract

This study focused on formulating conjugate vaccines targeting oxycodone and heroin for technology transfer, good manufacturing practice (GMP), and clinical evaluation. Lead vaccines used the highly immunogenic carrier protein keyhole limpet hemocyanin (KLH), which poses formulation problems because of its size. To address this barrier to translation, an oxycodone-based hapten conjugated to GMP-grade subunit KLH (OXY-sKLH) and adsorbed on alum adjuvant was studied with regard to carbodiimide coupling reaction time, buffer composition, purification methods for conjugates, conjugate size, state of aggregation, and protein/alum ratio. Vaccine formulations were screened for post-immunization antibody levels and efficacy in reducing oxycodone distribution to the brain in rats. While larger conjugates were more immunogenic, their size prevented characterization of the haptenation ratio by standard analytical methods and sterilization by filtration. To address this issue, conjugation chemistry and vaccine formulation were optimized for maximal efficacy, and conjugate size was measured by dynamic light scattering prior to adsorption to alum. An analogous heroin vaccine (M-sKLH) was also optimized for conjugation chemistry, formulated in alum, and characterized for potency against heroin in rats. Finally, this study found that the efficacy of OXY-sKLH was preserved when co-administered with M-sKLH, supporting the proof of concept for a bivalent vaccine formulation targeting both heroin and oxycodone. This study suggests methods for addressing the unique formulation and characterization challenges posed by conjugating small molecules to sKLH while preserving vaccine efficacy.

Keywords: FDA; GLP; GMP; antibody; conjugate; heroin; opioid use disorder; oxycodone; vaccine.

Figure 1.

Effect of pH and EDAC concentration on haptenation ratio and immunogenicity. (A) MALDI-TOF was used to calculate haptenation ratios of OXY-BSA conjugated at pH 4.5–7 using 5.2 mM of hapten, 52 or 208 mM EDAC concentrations and (A, inset) at pH 6.0 from 52–208 mM EDAC added to BSA or sKLH for a final concentration of 2.3 or 2.8 mg/mL, respectively. (B) Male Holtzman rats (n = 10/group) were immunized by IM injection with 60 μg of OXY-sKLH and 90 μg of alum on day 0, 21, 42, and 63. Serum was collected on day 70 and oxycodone-specific serum IgG antibody titers were determined by ELISA. *p < 0.05 brackets indicate group differences.

Figure 2.

Oxycodone-specific antibody titers, oxycodone distribution, and antinociception in rats. Male Holtzman rats (n = 12/group) were immunized by IM injection with 30, 60, and 120 μg of OXY-sKLH and 45, 90, and 180 μg of alum, respectively, on day 0, 21, 42, and 63. (A) Serum was collected on day 70 and oxycodone-specific antibody titers were measured. (B) On day 77, animals received a SC injection of 2.25 mg/kg of oxycodone and were tested on a hotplate set to 54 °C for nociception 30 min later. (C) Serum and (D) brain samples were collected immediately following hotplate testing. Numbers above bars represent the percent difference from controls. Distribution and behavioral data are the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to control.

Figure 3.

Effect of the filter size on OXY-sKLH immunogenicity and size. (A) Male Holtzman rats (n = 3–6/group) were immunized by IM injection with 60 μg of filtered or unfiltered OXY-sKLH with 90 μg of alum on day 0. Serum was collected on day 7 and oxycodone-specific antibody titers were measured. (B) Percent intensity of light scatter of filtered and nonfiltered OXY-sKLH. *p < 0.05 compared to the nonfiltered OXY-sKLH.

Figure 4.

Oxycodone-specific antibody titers and DLS. (A) DLS of sKLH and OXY-sKLH batch 1 and 11. (B) Male Holtzman rats (n = 3–17/group) were immunized by IM injection with 60 μg of OXY-sKLH with 90 μg of alum on day 0. Serum was collected on day 7 and oxycodone-specific antibody titers were measured. Data are mean ± SD (C–E) SEC analysis performed on sKLH and OXY-sKLH (batch 1 and 11). *p < 0.05 compared to batch 1.

Figure 5.

Characterization, immunogenicity, and efficacy of the M-sKLH vaccine in rats. (A) Morphine-based haptens. (B) Male Holtzman rats (n = 8/group) were immunized by IM injection with 60 μg of M-sKLH and 90 μg of alum on day 0, 21, 42, and 63. Serum was collected on day 70 and morphine-specific antibody titers were measured. On day 77, animals received a SC injection of 1 mg/kg of heroin and were tested on a hotplate set to 54 °C for nociception 30 min later. (C) Serum and (D) brain were collected following (E) hotplate testing. (F) Percent intensity of light scattering of different batches of M-sKLH. Drug distribution and behavioral data are the mean ± SD. ***p < 0.001 compared to control.

Figure 6.

Immunogenicity and efficacy of OXY-sKLH co-administered in a bivalent formulation in rats. Male Holtzman rats (n = 12/group) were immunized on day 0, 21, 42, and 63 by IM injection with 60 μg of OXY-sKLH, 60 μg of M-sKLH, 60 μg of OXY-sKLH plus 60 μg of M-sKLH, and 120 μg of OXY-sKLH adsorbed on either 90 or 180 μg of alum adjuvant, respectively. On day 77, animals received a SC injection of 2.25 mg/kg of oxycodone and were tested on a hotplate set to 54 °C for nociception 30 min later. (A) Serum and (B) brain samples were collected immediately following (C) hotplate testing. Numbers above bars represent the percentage of difference from controls. Drug distribution and behavioral data are the mean ± SD. **p < 0.01. ***p < 0.001, ****p < 0.0001 compared to control. ####p < 0.0001 brackets indicate group differences. (A) **** OXY-sKLH (120 μg) vs M-sKLH. (B) **** sKLH (60 μg) vs M-sKLH (60 μg), *** OXY-sKLH (120 μg) vs M-sKLH (60 μg), *** M-sKLH (60 μg) vs OXY-sKLH (60 μg) + M-sKLH (60 μg).