How to investigate a contaminated emulsion: From raw material to the final Emulsion.

A client reached out to me a few months ago in extreme worry. He had been facing a serious issue with his emulsion, with not one but 11 batches being contaminated. The total loss due to this contamination was estimated to be around 200 million euros. The client was perplexed about how this could have happened, as he had always believed that he had a robust process and was producing an excellent product. He had taken all the necessary precautions to ensure the quality and safety of his product, yet he was now facing a significant loss.

The primary concern now was preventing further contamination and ensuring a robust process. Evidently, the existing process was not foolproof and needed to be re-evaluated to identify any potential gaps or weaknesses that could have led to the contamination. A thorough investigation would be required to identify the root cause of the issue. Based on the findings, appropriate corrective actions would need to be taken to prevent similar incidents from occurring in the future. It was crucial to ensure that the client’s product was not only of high quality but also safe for consumption.

Background information

The following is a description of the production process of a trivalent vaccine containing three viruses: New Castle LaSota, IB – Infectious Bronchitis: M41 and egg drop syndrome – B88/78 eds. The vaccine is formulated with a product emulsion oil in the water-based vaccine at a 70 to 30 ratio, along with mineral oil, span, and tween – the standard formulation. Notably, the virus is produced in commercial eggs, which play a crucial role in production.

To begin with, 0.2 ml of the master seed with an MOI of 0.001 is used to infect the eggs via an automatic infection machine (Automatic Egg Harvester – RAME-HART . The infected eggs are then incubated in a 37-degree incubator for 48 – 72 hours before harvesting. The harvesting process involves using the same machine used for infection, which extracts the allantoid fluid from the eggs. The fluid is then stored in a vessel at room temperature.

After all the eggs are harvested, the next step is the clarification process. A centrifuge GEA 9000 at rpm is used to remove any eggshells, debris, or other unwanted materials from the clarified allantoid fluid. The clarified allantoid fluid is then transferred to a vessel and treated with one dose of BPL (beta propyl lactone) at 1:2000 (v/v). After incubating the mixture for about 12 hours, the mixture is refrigerated and stored in 50-liter bags for future use.

Production process Flow

The first question that was raised on the type of contamination that has occurred. The user inquired if the bug causing the contamination could be identified. In response, the reply confirmed that the bug responsible for the contamination was Enterobacter aerogenes, Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Aspergillus sp., and Fusarium sp.

The second question what steps we can do it to minimize the spread of contamination.

We considered various courses of action to address the issue of contaminated eggs. One option was to explore the possibility of adding another inactivating agent to the dossier or increasing the concentration of BPL. Another option was to use antibiotics. However, these solutions would not immediately relieve the problem at hand. Therefore, we concluded that the most urgent course of action was to “stop the bleeding” by enhancing the decontamination of eggs before they enter the production room. To achieve this, we introduced a new process of decontaminating eggs with 70% ethanol before they enter the production room and before administering the first injection.

As soon as we recognized the need for an investigation, we took proactive measures to initiate the process. I immediately traveled to the client’s site to assess the situation and begin the investigation. One of the most pressing concerns was implementing a new decontamination process to prevent further contamination.

Until then, the eggs were only decontaminated with a mixture of hydrogen peroxide and acetic acid and sprayed on before entering the room. However, this was no longer a sufficient measure to prevent contamination, and we needed to take immediate action to address the issue.

To prevent further contamination, we have revised the decontamination process and implemented new measures to ensure the safety of the production process. We have also mapped out the entire production process, from the bioburden levels of the eggs when they enter the production area to the incubation room, disinfection room, infection room, and finally, the incubation room before harvesting.

To gather this data, we have begun a comprehensive project to map out the bioburden levels at each stage of the production process. This will help us identify potential areas of contamination and take steps to prevent it from happening.

One of our egg suppliers has shared their process for measuring the bioburden on eggshells in their facility. The procedure involves the following steps: first, eggs from poultry are soaked in 100 ml of normal saline solution in a sterile beaker. This solution is then shaken gently and allowed to stand for 10 minutes. Next, a ten-fold serial dilution is prepared from the normal saline solution to enumerate bacterial isolates. This process is repeated three times. The nutrient agar and potato dextrose agar plates are then used for a pour plate technique with appropriate dilutions. The Nutrient plates are incubated at 37°C for 24-48 hours. Fungal enumeration is determined using potato dextrose agar with 0.5ml of the antibacterial agent incorporated. The antibacterial agent is prepared by dissolving 1.0g of Streptomycin in 30.0 ml of sterilized distilled water. The plates are incubated at room temperature (28±2ºC) for fungal colonies to develop. The microbial counts are expressed as several cfu/g egg material.

We collected samples and started to map the bioburden levels in the facility, however one of our experts mentioned that this will not be enough since the eggs came from the manufacturing site where they are produced and we need to check there. The eggs move a long way from the layers until the production room and the contamination during this period can be at any stage, hence we measure the contamination levels at all stages, from the chicken to the infection area.  

In short, the layers, lay the eggs and they where transported into a belt where they were collected and stored in a room, in this room they wait until there is enough volume and then decontaminated. The ideal process this eggs need to be decontaminated in the first 24 hours, before  the cuticula is “closed” in a low or lower contaminated room.  

Poultry farms usually rely on a process where the hens lay eggs, which are then collected in a belt before being transported to a storage room. In this room, the eggs are stored until there is enough volume to proceed with decontamination. It is essential to decontaminate the eggs within the first 24 hours before the cuticula seals them, as this would reduce the effectiveness of the process in a low- or lower-contaminated room.

The eggs after the laying period present this protein called cuticula that acts as a protection barrier between the outside and the inside the embryo, when the cuticle is exposed to microbes, including Gram-positive and Gram-negative strains. In that case, the chances of trans-shell infection post-oviposition will depend on factors such as local environmental conditions (especially when high temperature meets high relative humidity), the duration of exposure, and the nature of eggshell microbiota. If the eggshell gets contaminated, the internal components of the egg may also get infected by Gram-negative microbes, which is a common observation. The concentration of bacteria in the air of poultry houses is positively correlated with bacterial contamination of the eggshell.

Picture 2:  process flow into the chicken farm.

Upon thoroughly investigating the contamination issue, our team identified a significant difference between the time from laying the eggs and the initial disinfection process. It was discovered that the disinfection was carried out using a combination of hydrogen peroxide and peracetic acid steam. The suppliers would mix these two chemicals at a 2:1 ratio and heat them on a hot plate in a designated room until the gas was thoroughly exhausted. This process was deemed quite hazardous, given the nature of the chemicals involved.

Subsequently, the eggs were transported in a truck from the disinfection room to the incubators. It is worth noting that the incubators were never cleaned nor disinfected. Once inside the incubators, the eggs were transported to the production site, where they underwent yet another round of disinfection using a combination of acetic acid and hydrogen peroxide at a 2:1 ratio.

It was discovered that the reason for this particular ratio was that formaldehyde was used for decontamination purposes in the past. However, due to recent regulations, formaldehyde was no longer permitted for use. As a result, both the production site and the suppliers were forced to switch from one decontamination agent to another, ultimately settling on the mixture above of acetic acid and hydrogen peroxide.

When we did the bioburden analysis for each step of the production cycle (figure 1.) we have the following:

Coding	Step	Bioburden Level CFU/g
A	Collection Belt	9.7 + 0.7 x104 to 2 + 0.3 x 105
B	Storage disinfection room	7. 0 ± 0.5 _106 to 2. 2 ± 0.5 _106
C	Post Disinfection	3. 4 ± 0.8 _104 to 3. 1 ± 0.5 _104
D	Before Incubatory	2. 2 ± 0.8 _104 to 1. 4 ± 0.5 _104
E	After incubatory	7. 6 ± 0.8 _105 to 5. 4 ± 0.5 _105
F	Before Transportation	7. 3 ± 0.8 _105 to 5. 4 ± 0.5 _105
G	Before SAS Disinfection	9. 3 ± 0.4 _107 to 5. 4 ± 0.5 _107
H	After SAS disinfection	5. 3 ± 0.9 _103 to 9. 4 ± 0.5 _103

Upon thorough analysis of the collected data, it has come to our attention that there were specific gaps in the disinfection procedure.

Production Cycle	Description	Contamination Level
A	Eggs	1. 2 ± 0.8 _103 to 8. 9 ± 0.5 _103
B	Before Inoculation Machine	2. 6 ± 0.8 _104 to 5. 7 ± 0.5 _104
C	After Inoculation Machine	6. 7 ± 0.8 _104 to 6. 9 ± 0.5 _104
D	At Incubation	7. 3 ± 0.8 _106 to 4. 2 ± 0.5 _106
E	Before Harvesting	9. 9 ± 0.8 _107 to 8. 4 ± 0.5 _107

Conclusions:

After analyzing the data, it has been observed that there are gaps and issues in the disinfection process. Due to the inadequacy of the current chemical reagent, hydrogen peroxide, the distributor’s disinfection process has become ineffective and unstable. Unlike formaldehyde, hydrogen peroxide does not possess residual capabilities, and it may not penetrate and be as effective as formaldehyde when it comes in contact with organic material. Hence, it is necessary to revise the disinfection process and switch to a more effective chemical reagent.

Action plan:

We focused our action plan in four steps:

• To change the disinfection reagent,
• To change the disinfection device at the farm,
• To change the disinfection procedure at the farm, and
• To change the disinfection procedure at the manufacturing site.

1-Proposed reagent:

The plan is to replace the Hydrogen peroxide 2:1 mixture to a mixed disinfectant comprises of, 35g/ 100g hydrogen peroxide,. The hydrogen peroxide is good in stability, can rapidly kill various microorganisms such as bacteria, mold, yeast, spores, and the like, and is safe, non-toxic, and free from formaldehyde.

2 – Changing the disinfection device at the farm

After the use of this new disinfection procedure and this time with a proper aspersion machine we use the following for the surface of the egg, disinfection room and incubation room (JETBIO 2020)

Also all the eggs incubators, trays, truck and areas of contacted will be disinfected with hydrogen peroxide and ethanol prior to the eggs shipping.

3 – Changing the disinfection procedure at the farm

A new disinfection method will be implemented for room disinfection instead of continuing with the current mixture of hydrogen peroxide and peracetic acid and heating it in a closed room. This new method involves using a mix of peracetic acid, hydrogen peroxide, acetic acid, sulfuric acid, stabilizer, surface active agent, and water, known as PHAS. The mixture will contain 9g/ 100g peracetic acid, 35g/ 100g hydrogen peroxide, 12g/ 100g acetic acid, 2g/ 100g sulfuric acid, 2g/ 100g stabilizer, 2g/ 100g surface active agent, and the rest will be water. This new process is expected to be more efficient and effective than the previous one.

Furthermore, after the eggs are collected on the belt, they will be taken to the storage room immediately. Within 24 hours, they will undergo a disinfection process of at least 8 hours of aspersion. This new process will ensure better quality and safety of the eggs.

4 – Changing the disinfection procedure at the manufacturing site.

To maintain high levels of hygiene, the eggs must be disinfected before they enter the manufacturing room. The process of disinfecting eggs involves spraying them with a hydrogen peroxide mixture. This is done through a new aspersion divide in the SAS passage (https://www.bioquell.com/).

The aspersion divide is a device that is specifically designed to spray a fine mist of the disinfectant solution onto the eggs as they pass through the SAS passage. This ensures that all the eggs are evenly disinfected and any potential bacteria or germs are eliminated.

Investment is required to install this new device, but the potential gains make it worthwhile. By ensuring that the eggs are disinfected, we can maintain the highest hygiene and quality for our customers.

Results after the new disinfection procedure adoption:

Coding	Step	Bioburden Level CFU/g
A	Collection Belt	7.3 + 0.7 x104 to 2.1 + 0.3 x 105
B	Storage disinfection room	6.8 ± 0.5 _106 to 7.8 ± 0.5 _106
C	Post Disinfection	5.2 ± 0.8 to 7.1 ± 0.5
D	Before Incubatory	2.5 ± 0.8 to 2.8 ± 0.5
E	After incubatory	2.1 ± 0.8 to 3.1 ± 0.5
F	Before Transportation	2.5 ± 0.8 to 3.6 ± 0.5
G	Before SAS Disinfection	2.5 ± 0.4 to 2.8 ± 0.5
H	After SAS disinfection	2.1 ± 0.9 to 2.4 ± 0.5

Table 4: Contamination on different production cycles at the manufacture area

Production Cycle	Description	Contamination Level
A	Eggs	2. 2 ± 0.8 _ to 3.0 ± 0.5
B	Before Inoculation Machine	2. 4 ± 0.8 to 3.0 ± 0.5
C	After Inoculation Machine	2.4 ± 0.8 to 3.0 ± 0.5
D	At Incubation	2.6 ± 0.8 to 3.1 ± 0.5
E	Before Harvesting	9. 9 ± 0.8 to 8. 4 ± 0.5

Conclusion:

Ensuring commercial eggs are properly disinfected is crucial to prevent bacterial contamination. When the cuticle of the egg is exposed to microbes, such as Gram-positive and Gram-negative strains, the risk of trans-shell infection post-oviposition increases in an uncontrolled environment. Factors such as local environmental conditions, especially when high temperature meets high relative humidity, can also impact the likelihood of contamination.

In one specific case, chicken-free cages were in contact with contaminated material, and without a proper disinfection procedure, the level of contamination increased exponentially when the eggs entered the disinfection SAS of the manufacturing site. This contamination needed to be adequately addressed, resulting in more contamination inside the eggs during the inoculation procedure.

During incubation at high temperatures and humidity, the bioburden levels grew exponentially. While inactivated BPL was used to kill the bioburden, it was not always practical, leading to some contamination. This perfect storm was created after the change from formaldehyde to hydrogen peroxide and the incorrect mixture, leading to significant damage.

If contaminated antigen enters the formulation room and gets formulated, the bioburden grows, leading to contaminated emulsions. Fortunately, fast action was taken to avoid more permanent damage, and a successful decontamination procedure was established to produce sterile batches.