Next Generation Sequencing: The Utilization and Challenges for the Advancement of Viral Detection in the Biopharmaceutical Industry

Introduction

Viral safety and control is a vital part of ensuring the safety of biological products in the biopharmaceutical industry. There are four key elements for viral safety which include: 1) control of raw materials, 2) viral segregation and facility controls, 3) adventitious virus detection, and 4) viral clearance. Each of these elements is critical in the prevention and control of adventitious viruses and ensuring product safety for patients. Raw materials used in the manufacturing process, especially those derived from animals, pose risks of introducing adventitious agents (viral, microbial, or fungal contaminants) into the manufacturing process. Use of chemically defined medias and plant derived raw materials lowers the risk of introducing adventitious viruses, but does not eliminate the risk completely. Viral segregation and facility controls further reduce the risk by regulating the flow of operators, equipment, materials and preventing pests from entering the facility and the manufacturing process. Adventitious virus testing of master cell banks, working cell banks, end of production cell banks and unprocessed bulk harvest material is performed in accordance to ICH Q5A to ensure the manufacturing process is free of viral contaminates.¹ Various analytical methods are currently used for adventitious virus detection which include but are not limited to in-vivo, in-vitro and nucleic acid based assays such as real-time or quantitative polymerase chain reaction (qPCR). Downstream of the biological manufacturing process, viral clearance steps are incorporated to ensure effective inactivation and removal of any potential viruses not detected during adventitious virus testing. These key elements are important for preventing and eliminating adventitious viruses from the manufacturing process and ensuring product safety for the biopharmaceutical industry. This article will provide an overview for current practices for adventitious virus detection, and the transition to rapid molecular methods.

Adventitious Virus Detection

In-Vitro Assays

Current in-vitro adventitious virus assays employed in the biopharmaceutical industry are nonspecific and broad range cell culture based screening assays. Briefly, a test article sample is inoculated on at least three different types of indicator cells based on the species origin of the parental cell line. The indicator cell lines include: 1) a cell line of the same species as that used for production (e.g. CHO-K1), 2) a human diploid cell line (e.g. MRC-5), and 3) a non-human primate cell line (e.g. Vero).² The test article is inoculated onto the indicator cells for 14 or 28 days and then evaluated for signs of viral infection as indicated by cytopathic effect (CPE), hemadsorption and/or hemagglutination. Cell cultured based assays have the advantages of broad range detection of known and unknown infectious viruses; however this assay is limited in several ways. The first of which being the fact that it is only capable of detecting viral families that causes CPE, hemadsorption, or hemagglutination. Viral families that do not cause these manifestations would go undetected by this methodology. Secondly, latent viruses would also not be detected by the in-vitro assay unless viral replication is induced by an outside stimulant. An additional limitation of this methodology is occurrence of false positives as a result of cytotoxicity caused by the test article matrix induction of morphological changes, such as CPE. True positive results caused by an infectious virus require additional orthogonal testing to confirm the presence and identify the contaminating virus. This can result in lengthy investigations and numerous analytical tests.

Real-Time or Quantitative PCR

Real-time or quantitative PCR is another method that is currently used to detect adventitious viral agents. PCR based assays are rapid, highly sensitive, and ideal for the detection of known viral targets. For routine screening of specific viral targets, such as mouse minute virus (MMV) or Vesivirus, PCR is fast and can provide early detection. In conjunction with cell culture based assays, PCR is often used to identify the adventitious agents of known viral targets, presuming a laboratory has established primers/probes or a commercially available kit for the given target is available. PCR based assays do have a few limitations, the most important being they require prior knowledge of the viral target sequence in order to generate specific primers and probes that facilitate detection of that virus. QPCR can also only detect certain subspecies of viruses meaning it is not appropriate for broad range virus detection of novel or unknown viral sequences. An additional limitation of PCR based assays is that they are unable to distinguish between infectious and non-infectious viral sequences, and as with the cell based assays have the potential for false positives results. False positives can occur as a result of cross contamination of the positive control used in the assay and test article being evaluated. The use of discriminatory positive controls helps to eliminate false positives results. Rapid molecular detection methods, such as PCR based assays, provide quick results (usually less than a day) for specific viral targets and can be used as a screening tool to mitigate risk of facility contamination prior to harvest of bulk process material.

Next Generation Sequencing (NGS)

The limitations of conventional adventitious virus testing methods such as those discussed above include but are not limited to long assay times, specific viral targeting or unknown viral identification when detection is observed, and inability to detect viruses that do not elicit CPE, hemadsorption, or hemagglutination manifestations draw attention to the gaps in our current methodologies. The need for rapid broad range viral detection methodologies is essential. The utilization of next generation sequencing (NGS) is a new approach to viral safety testing which fulfills all of these requirements.

NGS also referred to as massive parallel sequencing (MPS) or deep sequencing, is non-Sanger based high-throughput DNA sequencing that uses the concept of sequencing millions of DNA fragments at the same time. MPS/NGS has the flexibility to sequence nucleic acids present in a biological sample including host cell, viral and/or microbial nucleic acids, DNA or RNA. An advantage of NGS sequencing is that detection does not require prior knowledge of potential adventitious viral agents, meaning it is capable of detecting a broad range of viral contaminations of both novel and unknown viral sequences. The following sections provide a more in-depth overview of NGS and specifically its use for adventitious virus detection.

Advanced Virus Detection Using NGS

NGS has changed the field of genetic sequencing by generating larger amounts of sequencing data in a shorter period of time. To provide context, the first human genome sequence required approximately 13 years and $2.7 billion to complete.³ Today, a laboratory can sequence more than 45 human genomes in a single day at the cost of approximately $1000 per genome.⁴

Sequencing Platforms

There are several different platforms for NGS. Each platform utilizes different engineering configurations and different sequencing chemistry. The variation in engineering configurations and sequencing chemistry provides a wide range of advantages or limitations depending on the platform. For example, some platforms excel at longer read lengths, which are ideal for de novo whole genome sequencing but may be prone to high error rates. Other platforms generate faster, higher throughput sequencing reactions, but shorter read lengths. This type of platform may be ideal for sequencing genes of interests or viral genomes which are much smaller than an entire mammalian genome. In addition, there are newer, third generation sequencers that have smaller footprints and faster sequencing times. The turnaround time can range from a few hours to a couple of weeks depending on the level of sequencing, throughput, platform, and instrument. Regardless of the sequencing platform, NGS has revolutionized genome sequencing by making it assessable to a variety of different institutions including academic, government, medical, and industry laboratories.

Overview of NGS Workflow

In the scenario of using NGS for detection of adventitious virus from unprocessed bulk harvest, the first step is the isolation of nucleic acids (DNA and/or RNA) from the sample matrix. In the case of DNA, the DNA is fragmented into specific size pieces, either enzymatically or through a shearing process. Libraries, a collection of DNA from a specific sample or group of samples, are constructed from the DNA fragments. The DNA fragments undergo MPS, generating millions of sequencing reads for downstream analysis. In the case of RNA, the RNA is converted to specifically sized cDNA fragments using the enzyme reverse transcriptase. The cDNA is carried through the library generation process and sequencing process similar to that of DNA. A key component of NGS is the complex bioinformatics used for data analysis. In the scenario of adventitious virus detection, the data would first be aligned to a reference sequence which could be the whole genome sequence of the master or working production cell bank, followed by alignment to a viral database.

Challenges for the Use of NGS for Virus Detection in a Biopharmaceutical Setting

To utilize the NGS methodology for adventitious virus testing, there are several challenges to overcome. The first is the initial investment in instruments, reagents, data storage, establishment of a bioinformatics database and the bioinformatics expertise necessary to perform data analysis. The second challenge is implementing the complex workflow involved in NGS in a good manufacturing practice (GMP) environment, which requires validation of the workflows, instruments, bioinformatics databases and data analysis pipelines. Within the NGS workflow process there are several components to consider which include: test material, sample preparation, diversity in viruses, sequencing platform, and bioinformatics pipelines. An additional challenge is gaining regulatory agency approval for the use of NGS as an alternative, supplement or replacement to the adventitious methods described above.

Test Material

Test materials that can be evaluated for adventitious viruses using NGS, come in a range of forms, some of which include: raw materials, cell lines, bulk harvest material, and viral stock seeds. The testing of raw materials, particularly when using animal derived materials such as serum, can be used to identify a potential origin of viral nucleic acids. Although NGS cannot distinguish between infectious and noninfectious viral sequences, this information from raw materials can be used in the analysis of downstream testing of cell lines, unprocessed bulk harvest material, viral stock seeds, and viral banks. Additional information about cell and viral banks, such as viral and cell variants can also be detected with NGS, making the entire manufacturing process better managed and controlled.

Sample Preparation for Sequencing

One of the benefits of NGS is its unbiased approach to sequencing, allowing for the detection of both known and unknown sequences. This capability is ideal for detecting viruses as they come in a myriad of forms including: DNA, RNA, single-stranded, double-stranded, enveloped, non-enveloped, large, and small. As part of the sample preparation workflow, identifying the appropriate sample matrix is important. Cell culture based sample matrices include: cell suspension, supernatant, or cell pellet. Each of these matrices provides both benefits and challenges to the NGS process. The presence of host genomic DNA presents a major obstacle in detecting low levels of virus present in cell suspensions and pellets. However, the potential to overlook cell-associated viruses exists if only supernatants are evaluated. For adventitious virus detection, the sample preparation process needs to be broad range enough to not bias the method’s ability to detect all the characteristics listed above. Bias in the sample preparation process towards particular characteristics could result in false negative results. To address this challenge, model viruses that exhibit the range of characteristics described above should be evaluated during the method development process. In addition, there are a wide range of library preparation kits available depending on the sequencing platform or nature of sequencing.

Bioinformatics

Once the NGS samples have been sequenced the next challenge is analyzing the data using bioinformatics. This could involve aligning the sequences to a reference sequence and/or to a viral database. The reference sequence may be generated from whole genome NGS characterization of a master cell bank, working cell bank, or from a publicly available sequence from a widely used cell line. In the instance of a viral database, the database could be privately curated or come from public repositories such as GenBank.⁵ There are companies that offer bioinformatics services to analyze the data, or provide platforms that allow bench scientist to execute the data if this capability is not available through an internal bioinformatics group. A key component in the data analysis is identifying positive reads or “hits”. Once a positive hit is identified, it should be confirmed using an orthogonal method such as PCR or an infectivity assay, if one is available.

Regulatory Guidance

The utilization of NGS for adventitious virus detection is still relatively new from a regulatory perspective. The regulatory agencies are versed in the technology and its potential use in the biopharmaceutical industry. The European Pharmacopoeia has released a guidance document, 5.2.3 “Cell substrates for the production of vaccines for human use”, that references the use of massive parallel sequencing “as an alternative to in-vivo or specific NAT tests or as a supplement/ alternative to in-vitro culture tests” for adventitious virus detection.⁶ While this guidance is specific for vaccines for human use, one could speculate on its relevance for other biologicals as well. To mitigate some of the challenges described above, the Parenteral Drug Association (PDA) created the Advanced Virus Detection Technologies Interest Group (AVDTIG) comprised of industry and regulatory scientists to address the complexities of using high throughput sequencing for advanced virus detection.⁷ The group has recently expanded to include “international government agencies, academia, and technology service providers”.⁷

Conclusion

The utilization of NGS for adventitious virus testing is a new approach to viral safety testing. A benefit of this methodology is that it can detect novel or unknown viruses without prior knowledge of potential viral targets. Advances in nucleic assay detection technologies have provided the ability to generate hundreds of megabases to several gigabases worth of sequences within hours, days, or weeks depending on the depth needed.⁴ While NGS provides broad range viral detection, this technology is not without its challenges due to the large amounts of data generated, and the technical complexity of the method. To mitigate these challenges the PDA created the AVDTIG task force to bring together scientists from industry, academia, regulatory, government and technology providers to share data, experiences, and discuss solutions for the hurdles described above.⁷

References

1. Quality Guideline Q5A(R1): Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. International Conference on Harmonisation. www. ich.org. Published September 23, 1999. Accessed July 11, 2017.
2. Khan AS, King KE, Brack K, et al. PDA Technical Report No. 71 Emerging Methods for Virus Detection. Parental Drug Association. 2015.
3. National Human Genome Research Institute. The Cost of Sequencing a Human Genome. https://www.genome.gov/sequencingcosts/. Accessed July 11, 2017.
4. Illumina. An Introduction to Next-Generation Sequencing Technology. https://www. illumina.com/content/dam/illumina-marketing/documents/products/illumina_ sequencing_introduction.pdf. Accessed July 11, 2017.
5. NCBI. GenBank Overview. https://www.ncbi.nlm.nih.gov/genbank/. Accessed July 11, 2017.
6. European Pharmacopoeia 9.0 Number 5.2.3 Cell substrates for the production of vaccines for human use; European Pharmacopoeia. 2017. https://www.edqm.eu/en/europeanpharmacopoeia-9th-edition. Accessed July 11, 2017.
7. Khan AS, Vacante DA, Cassart JP, et al. Advaned Virus Detection Technologies Interest Group (AVDTIG): Efforts for High Throughput Sequencing (HTS) for Virus Detection. PDA J. Pharm. Sci. Technol. 2016; 70(6):591-595. doi:10.5731/pdajpst.2016.007161.

Author Biography