DNA contamination in 8 vials of Pfizer monovalent mRNA vaccines

The Boboli Gardens, Firenze Italy

Introduction

DNA contamination has recently been documented in mono and bivalent mRNA vaccines. This study was limited in the number of vials surveyed. We surveyed 8 vials from the same lot of Pfizer monovalent mRNA vaccines (lot # FL8095) using qPCR and RT-qPCR. These vials were unopened but dated 9/21 on the label and 3/4/22 (hand writing). 2ul from each vial were analyzed.

Figure 1. 8 Pfizer monovalent vaccines used in this study

Results

Figure 2. qPCR and RT-qPCR of 8 vials run in triplicate. Red is Vector. Blue is Spike

Figure 3. qPCR and RT-qPCR split out into qPCR (left) and RT-qPCR (right). Red is vector and blue is spike.

Table 1. CT values for Spike and Vector during qPCR (DNA only). Standard deviation for triplicate measurements run horizontally in black font. Standard deviation for vial to vial run vertically in Red. Delta CT or (Vector CT minus Spike CT) represents the ratio of Spike to Vector DNA. Should = 1.

Table 2. CT values for Spike and Vector during RT-qPCR (RNA+DNA). Ratio of RNA:DNA ranges from 43:1-161:1. EMA allowable range is 3030:1. 18-70 Fold over the EMA limit.

Methods

2ul from each vial was added to 100ul of Leaf Lysis Solution and boiled as previously described. 1ul of each boiled sample was used in qPCR and RT-qPCR as described previously.

2ul of Vaccine was added to 100ul of Leaf Punch Lysis Solution.

• Rapid Boil Prep for vaccine surveillance.

• qPCR methods previously described.

Conclusions

8/8 monovalent vaccines sourced from a single case from a single lot of Pfizer monovalent vaccines all fail the EMA specification of 3030:1 RNA:DNA (330ng/mg DNA/RNA). They are over the limit by an order of magnitude (18-70 fold). The DNA contamination is very consistent and the vial to vial ratio of RNA:DNA is very consistent within the same lot of monovalent vaccines. Further sequencing is underway to evaluate the 72bp heteroplasmic indel in the SV40 promoter of the vector. This was detected in the Pfizer bivalent vaccines.

References

FDA presentation on dsDNA contamination

Issues associated with residual cell-substrate DNA in viral vaccines

Citation kindly provided by David Weisman

EMA documentation- Page 74

Acknowledgements. We’d like to thank David Wiseman, Michael Palmer,

Sasha Latypova

Lynn Fynn, The PANDA audience and Keith Robison for suggested improvements and critiques of the work.