NEW YORK – Cell and gene therapies hold the potential to radically improve care for formerly intractable conditions, but developing and manufacturing these treatments is fraught with unique challenges because of their biological nature.
Digital PCR has emerged as an ideal tool to help biopharma address some of these challenges, particularly in areas such as determining viral titers and detecting contaminants, according to vendors and users of the technology.
Biopharma involves using living things — such as bacteria, viruses, or cell lines — to make drugs or treatments for diseases. This includes cell therapies, such as chimeric antigen receptor T-cell therapy or CAR T, in which living cells are injected into patients; and gene therapies, in which genes are added, corrected, replaced, or silenced in the hopes of altering a patient's gene expression.
The trajectory of commercial and financial growth in biopharma is dramatic. Approximately 30 cell and gene therapies have been approved by the US Food and Drug Administration to date, and there are currently more than 1,500 active clinical trials.
Biopharma companies reportedly raised roughly $61 billion in 2022, with almost 100 mergers and acquisitions completed in the year for a total of roughly $75 billion, according to a report by contract research organization Clarivate. Seven of the top 10 largest transactions in biopharma last year involved partnerships between large pharmaceutical companies and firms developing cancer therapies with a total potential value of more than $41 billion, the report said.
Despite these large figures, biopharma companies also experienced a steep decline in their ability to raise financing last year, with funding down by about half compared to the two prior years.
Cell and gene therapies have unique research and development needs. They also have stringent quality control requirements, but traditional sterilization techniques — such as heat, high pressure, radiation, and vaporized chemicals — would kill these living treatments.
Digital PCR systems from Bio-Rad Laboratories, Qiagen, Stilla Technologies, and Thermo Fisher Scientific are all positioned to support this growing industry, and companies consulted for this story said they are deeply invested in supporting this market, with instruments, reagents, dedicated assays, and technical support.
Digital PCR's growing role
The pharma space is notoriously protective of its intellectual property, but researchers and executives involved in cell and gene therapy development and manufacturing at Charles River Laboratories, Agathos Biologics, and BioMarin agreed to share their perspectives on digital PCR's growing role in their space.
All touted the distinct advantages that digital PCR has over standard qPCR. Namely, digital PCR allows users to obtain absolute quantification without the need to generate a standard curve. It also more precisely quantifies rare targets and is more resistant to PCR inhibitors.
Benjamin Moritz, a senior research scientist at Charles River, has been incorporating digital PCR into his work for the past two years.
Charles River partners with small independent pharmaceutical companies as well as Fortune 500 companies on everything from early drug and gene therapy development to Phase III clinical trials and safety assessments, Moritz said.
Using Droplet Digital PCR systems from Bio-Rad Laboratories, "we've developed simple singleplex assays to help detect low levels of transduced human cells in mouse tissues, duplex assays for biodistribution studies of CAR T-cell and [T-cell receptor] studies, assays for pharmacokinetic studies, and DNA and RNA analysis for prophylactic immune mitigation studies," Moritz said.
Meanwhile, Agathos Biologics CEO and Cofounder James Brown said his company uses Qiagen's QIAcuity digital PCR systems in internal research to quantify recombinant adeno-associated virus vectors.
"We are looking for genome copies and typically use the sequence of the transgene and the polyA," he said. "The multiplex features of the QIAcuity allow us to interrogate both sequences, which gives us confidence in the results."
Agathos recently launched analytical services for clients that include digital PCR beyond viral vector measurements, so that, "between our internal work and work for clients, we run digital PCR almost daily," Brown said.
The relative lack of precision of qPCR had historically been a challenge with viral vectors, as the genetic characteristics of the manufactured vectors need to be fully known, while contaminating biological entities and nucleic acids need to be completely removed, for the end products to be safe.
"The field is generally moving to a digital PCR method to address the issue," Brown said, noting that while most of Agathos' clients have used qPCR, its digital PCR service now offers them increased precision and sensitivity as well as multiplexing.
Russell Soon, a technical scientist at BioMarin Pharmaceutical, said that his team uses digital PCR in both gene therapy and antisense oligonucleotide therapy research and development.
Specifically, the firm has adopted digital PCR for recombinant adeno-associated virus (rAAV) copy determination and for evaluation of the biodistribution and shedding of rAAV-based gene therapies in preclinical and clinical studies.
"In our exon-skipping antisense oligo (AON) programs, we have employed digital PCR specifically for its ability to more accurately quantify AON-induced exon-skipping in contrast to historical PCR or qPCR-based assays," Soon said.
The team also uses digital PCR to look at the kinetics and mechanisms underlying the biological activity of its gene therapy candidates, "enabling continued refinement of our therapeutic candidates for greater efficacy and safety," he said.
Continued improvements in digital PCR technologies, particularly in the area of multiplexing, also permit "more insightful investigations supporting gene and oligo-based therapeutic development," Soon said.
For example, digital PCR facilitates "both the evaluation of rAAV vector contiguity and kinetics following administration," as well as the characterization of vector-derived transcripts in target and off-target tissues, Soon said.
"The multiplexing capabilities of some digital PCR platforms allow us to not only assess the presence of multiple target sequences in a single PCR reaction, but also determine their colocalization to a single nucleic acid strand — data which can prove highly informative," he added.
However, Soon also said that his team has found that although digital PCR can reduce data variability and simplify analysis, "logistical and analytical considerations may still point to qPCR as a more suitable bioanalytical assay platform," depending on the context.
To his mind, at the current cost and technological state, digital PCR is not likely to completely replace qPCR in all biopharma applications. Still, as part of a toolkit that also includes in situ hybridization, and measures of protein expression, immune system activity, and functional endpoints, digital PCR has helped the firm on its path to commercialization.
To date, BioMarin has eight marketed products as well as "a diverse pipeline of clinical and preclinical candidates that are being advanced to treat areas of significant unmet medical need," Soon said.
'First-mover advantage'
Although it is still early days in this sector, Simon May, executive VP and president of the life science group at Bio-Rad, said his firm has been investing in biopharma from the outset, coinciding with the rapid growth of VC funding over the past seven years.
"That allowed us to tap into the customer needs from a very early stage," he said. "The competitive landscape in digital PCR has evolved quite a bit since then, but I think we have had a first-mover advantage," he also said.
Although the biopharma space is "broad and deep," Bio-Rad sees a primary opportunity in emerging biotechs and therapeutics, including cell and gene therapies as well as monoclonal antibodies and RNA therapies, May said.
"That is where we see the growth potential over the medium- and long-term," he said, "But we also cover basic discovery and research in biopharma, and that really spans the spectrum of therapeutic modalities."
The biopharma space can be divided into large pharma companies and emerging biotechs, but May said that emerging therapies are increasingly being acquired, licensed in, or developed by big pharma firms themselves.
For firms both big and small, the path to a commercial product often involves basic research at the front end, and "digital PCR is a key tool there," May said. Further along, for development, manufacturing, and quality control, the precision of digital PCR makes sure these products have precise quantities and formulations, and are free of contamination.
Generally speaking, the top applications in cell and gene therapy are assessing viral titers in formulations and monitoring for residual DNA contamination from cell lines in which the products are grown. Another critical application is detecting contamination from the bane of all biopharma, miniscule Mycoplasma bacteria that infect up to 35 percent of all cultured cell lines.
Bio-Rad offers kitted content for these three essential biopharma applications, which further enhances the intra-assay consistency of digital PCR approaches, May said.
The post-pandemic downturn in biotech funding and subsequent budget tightening in larger pharma firms have trickled down to the life science tools vendors that support this space, May said, and this has led to multiple revenue re-guides across the sector.
Last month, Bio-Rad recently slightly lowered its long-term guidance in response, but May said this dip is likely transient, extending through 2023.
Considering the potentially transformational impact these emerging therapeutics will have on healthcare, "the medium- to long-term outlook remains very robust," he said. "I consider what we are seeing right now in the market to be a temporary, cyclical blip."
May said that overall digital PCR is "broadly adopted" in biopharma, and the six-channel multiplexing enabled by the new Bio-Rad QX600 system will further enhance the throughput of biopharma R&D and could help alleviate some of the time-to-market pressures.
Qiagen's offer
Qiagen entered the biopharma space with the launch of its QIAcuity digital PCR system in 2020.
Michael Scheffler, head of life science PCR at Qiagen, said in an interview that the firm supports biopharma in several ways including biomarker discovery, analytical development, process controls, quality controls, and general lab needs.
For cell and gene therapies, Qiagen's customers are using digital PCR for virus production, recovery, purification, and quality assurance to determine potency, purity, and safety. They also use digital PCR for patient monitoring, Scheffler said.
Generating standard curves takes time, money, and effort, Scheffler said, but digital PCR obviates this burden. And, digital PCR resolves minimal differences that can be more reliably compared between experiments, labs, and manufacturing facilities.
Historically, the high complexity of digital PCR, lack of multiplexing, and overall throughput may have limited uptake compared to qPCR. But newer systems, like the QIAcuity, have been able to match qPCR in terms of these factors.
Scheffler sees the overall PCR market in biopharma to be in the range of $2.5 billion to $3 billion, with digital PCR covering roughly 10 to 15 percent of this market.
"There is a good chance — and we see the appropriate trajectory — that digital PCR could become predominant already by 2026," he said.
Qiagen has seen good traction and growth in biopharma over the last year, Scheffler said, in part driven by general adoption of digital PCR, but also because of QIAcuity's workflow benefits: The QIAcuity 4 processes up to 500 samples in an eight-hour shift, while the QIAcuity 8 processes more than 850 samples in that time frame.
"We've also made significant strides with our offering into the biopharma space," Scheffler said, citing the firm's recently launched AAV titer applications and 21 CFR Part 11 compliance software.
Overall, including all applications — biopharma as well as things like oncology and wastewater testing — Qiagen currently sees a $350 million to $400 million opportunity for its digital PCR business, Scheffler said.
However, the biopharma product development and validation process is arduous and takes a long time to get to a homeostatic, routine manufacturing state. Investment has admittedly cooled, Scheffler said, but added that the company still sees "very good traction and resonance. From a seed funding perspective, it might be back to pre-COVID levels, but this is already a multibillion dollar market."
Future directions
Despite the financing headwinds, biopharma firms seem equally enthusiastic about the future of the space.
Charles Rivers' Moritz said that gene and cell therapies are still at a starting point, with digital PCR helping to fuel growth.
"We've just begun to scratch the surface" of what cell and gene therapies can do, he said, adding, "digital PCR will be a huge help in the testing required for these technologies."
Moritz cited CRISPR editing of individual nucleotides as a case where digital PCR could enable analysis and have a quicker turnaround time compared to next-generation sequencing. And, he added, the detection of gene expression, assay contaminants, and diagnostic testing targets all at once through the use of multiplexing is easier with digital PCR compared to qPCR.
Moritz thinks that sponsors of Charles Rivers' cell and gene therapy projects will eventually want to replace traditional qPCR sample analysis with digital PCR.
Limitations include potential lower throughput of plate-based digital PCR systems compared to qPCR, limited dynamic range, and the cost of consumables, but these limitations are likely to be minimized as digital PCR technology improves, Moritz predicted.
For BioMarin's Soon, the growth of cell and gene therapies is also just one of the latest advances in the treatment of disease, but the field's value "is evidenced by the many traditional pharmaceutical companies which now have moved into the cell and gene therapy space."
And, Agathos Biologics' Brown believes the rise of biopharma is a realization of the excitement and potential surrounding the sequencing of the human genome in 2003.
"I think the years that followed disappointed many in biotechnology and the public when there were few immediate tangible advances in medicine," Brown said. But, "the genomic tools that were subsequently developed allowed us to understand the genetic basis for disease, and now they are allowing us to do something about it."