Feliza Mirasol is the science editor for 

Feliza Mirasol

Thermo Fisher Scientific announced the completion of its acquisition of Olink Holding, a Sweden-based provider of next-generation proteomics solutions, in a $3.1 billion transaction, according to a July 10, 2024 company press release. Under the transaction, Thermo Fisher will add Olink to its Life Sciences Solutions segment.

Thermo Fisher announced its plans to acquire Olink in October 2023 for $26.00 per common share in cash, or approximately $3.1 billion (1). The acquisition gives Thermo Fisher access to Olink’s proprietary technology, Proximity Extension Assay (PEA). PEA offers high-throughput protein analysis for quantitative polymerase chain reaction and next-generation sequencing readout systems on the market. The technology holds a library of more than 5300 validated protein biomarker targets (1).

“Olink is dedicated to improving the understanding of human biology by accelerating the use of next-generation proteomics and providing industry-leading data quality at unprecedented scale. Thermo Fisher’s deep life sciences expertise, global reach, and proven operational excellence will enable significant opportunities for both customers and colleagues, while also providing immediate value to our shareholders,” said Jon Heimer, CEO of Olink, in the October 2023 company press release (1).

Marc N. Casper, chairman, president, and chief executive officer of Thermo Fisher, said in the company’s current press release that protein research is key to advancing the understanding of human biology, which will allow the industry to deliver on the promise of precision medicine.

“Olink’s proven and transformative solutions for proteomics are highly complementary to our leading mass spectrometry and life sciences offerings. By expanding our capabilities in the high-growth proteomics market, we will enable scientists and researchers around the globe to meaningfully accelerate discovery and scientific breakthroughs while delivering significant value to our shareholders,” Casper said in the release.

The completion of the Olink acquisition follows Thermo Fisher’s earlier announcement in June 2024 that it has 

 at its Middleton, Wis., site via its Pharmaceutical Product Development clinical research business (2). The new facility expands the company’s clinical research laboratory and increases that lab’s analytical capacity in the chemistry, manufacturing, and controls.

The new facility at the Middleton site includes dedicated space for liquid chromatography–mass spectrometry (LC–MS) and protein characterization, two molecular suites with pre- and post-amplification labs, and two additional labs for general use. The site focuses on small molecules and biologics across all phases of drug development, including biopharmaceuticals, device testing, cell and gene therapy, and MS analysis (2).

 in the bio/pharma industry as proteomics play a crucial role in the drug discovery and development process from an analytical and quality control perspective (3). A sequence-level understanding of molecules is required when working with biologics, and it is important to have an understanding at the sequence-level during each stage of development to not only assess the molecule’s properties but also to instill the desired properties in the investigational drug (3).

1. Thermo Fisher Scientific. Thermo Fisher Scientific to Acquire Olink, a Leader in Next-Generation Proteomics. Press Release, Oct. 17, 2023.
2. Thermo Fisher Scientific. Thermo Fisher Scientific Expands Clinical Research Laboratory in Wisconsin. Press Release, June 18, 2024.
3. Moree, B. Biologics Quality Control: The Growing Need for Accessible Proteomics. 

Articles

Thermo Fisher will add Olink to its Life Sciences Solutions business.

Thermo Fisher Scientific Expands Proteomics Services with Olink Acquisition

Evotec announced on July 10, 2024, that it has entered into a collaboration with Pfizer. Under the multi-year master research collaboration and option and license agreement, the companies will initially focus on early discovery research for potential therapeutics targeting metabolic and infectious diseases.

Under the agreement, the research will be carried out at Evotec’s sites in France, including its Campus Curie in Toulouse. The Toulouse site houses high throughput screening capabilities, 

 biology, proteomics, and metabolomics. Pfizer will provide Evotec with research support funding, and Evotec will be eligible to receive potential milestones and royalties related to success of the research programs, according to a company press release.

“We are honored to collaborate with Pfizer to research potential new first-in-class therapeutic approaches. France has established a thriving biopharmaceutical ecosystem to which Evotec also contributes with its strong presence with more than 1000 employees at our French sites, building a strong pipeline of medicines that matter. Focusing on diseases with severe unmet medical need and highly innovative therapeutic approaches, we look forward to supporting our partners at Pfizer with integrated R&D activities,” said Matthias Evers, PhD, chief business officer, Evotec, in the press release.

“We are thrilled to collaborate with Evotec to conduct discovery research that may help unlock new approaches to treating pervasive metabolic and infectious diseases that impact millions of patients worldwide. France has developed a strong biopharmaceutical ecosystem, which is why we’re thrilled to collaborate with Evotec to build on our shared commitment to advancing scientific discovery for diseases with unmet need,” said Luca Mollo, MD, vice-president and medical lead France, Pfizer, in the press release.

 with Ginkgo Bioworks (1), under which the companies will advance the discovery and development of novel RNA molecules using Ginkgo's proprietary RNA technology. Ginkgo received an upfront payment and is eligible to receive research fees and development and commercial milestone payments for an aggregate total of $331 million. Ginkgo's RNA technology combines high-throughput screening with a multi-parameter design framework to evaluate the behavior of RNA constructs. This technology allows the company to identify optimal novel elements, both natural and synthetic, that can be applied to a particular application.

"Billions of patients around the world have already benefited from advances in RNA-based technologies. We're thrilled to be able to help enable the discovery and development of novel drugs using this powerful modality by applying our platform technologies, which are designed to program RNA for maximum therapeutic effect," said Jason Kelly, CEO and co-founder, Ginkgo Bioworks, in a company press release.

Pfizer’s clinical pipeline as of May 1, 2024 includes 45 projects in Phase I, 28 projects in Phase II, and 37 projects in Phase III. The company also has three products filed for regulatory review: two pediatric COVID-19 vaccines (filed in the United States with BioNTech) for children six months to four years of age and for children five to 11 years of age, respectively, and a biologic for treating hemophilia filed in the US and in the European Union (2).

1. Gingko Bioworks. Ginkgo Bioworks Announces Multi-Target RNA Discovery Collaboration with Pfizer. Press Release, Sept. 27, 2023.
2. Pfizer. Drug Development Pipeline. 

Pfizer and Evotec will focus on early discovery research for therapeutics targeting metabolic and infectious diseases.

Pfizer and Evotec Partner for Drug Discovery

Communication between the manufacturing plant and retail stores. | Image Credit: © Cagkan - © Cagkan - stock.adobe.com

Genezen, a gene therapy contract development and manufacturing organization (CDMO), and uniQure, a gene therapy company, announced on July 1, 2024 that they have entered into an agreement under which Genezen will acquire uniQure’s commercial gene therapy operations in Lexington, Mass., for a total consideration of $25 million. The transaction price consists of $12.5 million of newly issued Series C preferred stock and a convertible note valued at $12.5 million. Matt Kapusta, uniQure’s CEO, will join the board of Genezen at closing of transaction, which is expected early in the third quarter of 2024, according to an uniQure company press release.

Under the agreement, Genezen will be responsible for manufacturing global commercial supply of CSL Behring’s HEMGENIX (etranacogene dezaparvovec-drlb), an adeno-associated virus (AAV) vector-based gene therapy approved in the United States and Europe for treating adults with hemophilia B (1,2).

The Lexington site is a commercially licensed viral vector facility. Its acquisition will enable Genezen to offer services from preclinical development programs through to late-phase and commercial manufacturing. The facility also houses state-of-the-art laboratory space and a pilot plant that is expected to serve as a global AAV center of excellence for Genezen and to complement Genezen's existing clinical manufacturing operations and team in Indianapolis, Ind.

"I am thrilled to welcome the Lexington team and the addition of a state-of-the-art, licensed viral vector manufacturing facility to Genezen,” said Steve Favaloro, president and chief executive officer, Genezen, in a Genezen press release. “This acquisition significantly enhances Genezen's scale and differentiated capabilities, directly aligned with our mission to advance cell and gene therapies by providing top-tier, science-driven development and manufacturing solutions. We are honored to deliver these services to our current customers and the patients they serve and are well positioned to immediately extend our support to even more customers in Lexington."

The acquisition agreement marks a significant step forward for uniQure in achieving a key strategic goal. “We have been focused on meaningfully reducing costs while maintaining our ability to develop and potentially commercialize our gene therapy product candidates. This transaction achieves these objectives by unlocking significant cost savings while maintaining preferential access to world-class gene therapy manufacturing,” Kapusta said in the uniQure press release.

Kapusta also explained that UniQure is continuing to closely review its business to identify additional opportunities for cost reduction. The company expects to announce further progress toward its goal in the third quarter of 2024. “We believe a more streamlined uniQure enhances our strategic flexibility for value-creating opportunities and furthers our mission to deliver transformative medicines for patients in need,” Kapusta stated in the release.

The acquisition of uniQure’s Lexington, Mass., manufacturing operations will boost Genezen’s late-phase and commercial gene therapy development and manufacturing services. 

uniQure to Sell Its Commercial Gene Therapy Manufacturing Operations in Massachusetts to Genezen

Advances in development and manufacturing of cell and gene therapies (CGTs) have been focusing on increasing therapeutic efficacy while ensuring product purity and improving patient safety. Efficacy, purity, and safety were major themes at the American Society of Gene and Cell Therapy (ASGCT) Annual Meeting held in May 2024.

Sven Moller-Tank, director, Regeneron Genetic Medicines, Regeneron, points out that part of his company’s approach is to directly address and overcome the commonly identified industry challenges around gene therapy development, such as safety, delivery, packaging constraints, immunogenicity, and durability. In terms of safety, for example, Moller-Tank says that Regeneron is investigating strategies for detargeting adeno-associated viruses (AAVs), which are commonly used viral vectors for gene therapy delivery, away from the liver.

“We have data demonstrating that reducing vector tropism to the liver significantly reduces liver damage and complement activation in non-human primates. In concert with this, we are using antibodies to confer novel tropism to AAVs and enhance their efficacy in desired target tissues. We have used this approach for enhanced muscle and central nervous system targeting and are currently expanding to new tissues,” Moller-Tank states.

A dual AAV approach may be one solution to alleviating packaging constraints encountered within the AAV capsid. “We now know [that an] oversized gene delivery approach can be successful in humans. We are solving for immunogenicity concerns using … [tools] for immunomodulation to combat pre-existing anti-AAV antibodies and enable redosing,” he explains. “Finally, we are applying a targeted liver gene insertion platform we co-developed with Intellia to hopefully enhance the durability of liver gene therapies and have shown in mouse [studies] the potential to sustain expression despite rapid liver growth, a critical feature for treating younger patients.”

AAV vectors have become the most common viral vector used in gene therapy manufacturing, but, until recently, their target specificity has not been quite successful. When focusing on the liver, Moller-Tank explains that some level of specificity can be achieved by targeting the liver through systemic administration. However, specifically targeting extrahepatic tissues has been a challenge, he points out. “Because of this, the development of novel vectors to enhance specificity has been a major focus of the field,” he says.

Moller-Tank emphasizes that using local injection is one method to achieve specific delivery outside of the liver. Meanwhile, the company’s pursuit of improving target specificity has taken it beyond just the liver. Several groups were recently able to uncover the fact that the cochlea is an organ that could benefit from AAV gene therapy via the delivery of a virus locally to the inner ear. “Demonstration of safety and efficacy of AAV delivery to the cochlea in humans may enable the development of several novel treatments for hearing loss,” Moller-Tank says.

A common approach to increase on-target specificity is utilizing a library-based selection strategy to screen for capsids that have desired properties, according to Moller-Tank. “However, this approach has had very mixed success when selecting for tropism to target organs and can yield capsids whose tropism does not translate across species. Now, the field is moving toward utilizing the same library-based screening approach to select AAV capsids that bind to specific receptors expressed in target tissues of interest,” he adds.

A different approach is to use antibodies to redirect the virus to the receptor of interest rather than selecting for receptor binding by the capsid itself. “In this way, we are able to fully characterize our antibodies independently of the capsid, giving us an extra layer of control over the binding properties of antibody targeted AAVs and allowing the platform to be modular and applicable to several different AAV serotypes,” Moller-Tank states.

Regeneron’s experience in developing antibodies for all types of challenging targets has given the company a formidable understanding of how to push antibodies to their limits. “It remains to be seen how versatile peptide libraries are for binding diverse target receptors,” Moller-Tank says.

In addition to manipulating AAV vectors themselves to improve target specificity, another major concern is the adequate separation and purification of AAV-vector-based gene therapies in downstream processing. At ASGCT 2024, a myriad of research efforts was shown to be underway to improve the downstream processing stage of gene therapy production. Much of the work is centered around the clearance of empty and partially full capsids (process-related impurities) from full capsids (the product).

For example, one study demonstrated how certain additives or a combination of additives in downstream bioprocessing play a role in improving the separation and clearance of empty and partially full capsids during the manufacturing of viral vectors. Ohnmar Khanal, PhD, Downstream Technology Lead, Spark Therapeutics, and her team focused on copper ions and conducted oversaturated loading experiments using multicolumn anion exchange (AEC) chromatography.

“A current challenge in the downstream purification of recombinant [AAV] is the separation of transgene-containing capsids from empty [capsids] and undesired capsid variants,” Khanal stated in a presentation at ASGCT 2024 (1). “AEX [anion exchange chromatography] resin is often used below its saturated binding capacity to separate empty and full AAV particles on a single column. However, the outcome of the traditional single-column operation can be impacted by variability in load composition, sensitivity to buffer composition, and fractionation strategies,” she explained.

The removal of empty and partial capsids with a high yield has rarely been demonstrated in the scientific literature, Khanal and her team found. Through their study, the team presented three-stage separation strategies for two recombinant AAV (rAAV) constructs in which they demonstrated a way to overcome the trade-off between yield and purity.

The first strategy used resin chemistry (Poros XQ, Poros HS, Poros I) combined with a concoction of kosmotropic agents (i.e., additives), such as ammonium sulfate or sodium sulfate, which promoted capsid stability at low ionic strength. Metal ions (e.g., copper, magnesium, calcium) were then used to promote selectivity of the desired capsid. “Using copper ions improved the resolution between empty and full capsid separation and helped remove other product-related impurities,” Khanal stated in her presentation.

In the second strategy, an appropriate resin/media structure and geometry were used to exclude AAV from smaller resin pores. This approach minimized particle dispersion and improved resolution. “Monolith and membrane chromatography showed better peak resolution than traditional resin based on favorable transport behavior,” Khanal also stated.

The team’s third strategy involved the transition from single-column to multiple-columns-in-series chromatography. This multi-column series chromatography was done by loading 1.5x the resin saturation capacity, which allowed for over 40% reduction of partial capsids and over 95% removal of empty capsids. “Using these strategies, we provide the best modality to achieve maximum empty and partial capsid removal with a yield of over 80%,” Khanal concluded in her presentation.

Meanwhile, in a different presentation given at ASGCT 2024 by Alex Meola, associate director of AAV Downstream Process Development at Oxford Biomedica, it was demonstrated that hydrophobic interaction chromatography (HIC) can be used to separate non-deamidated capsids from deamidated capsids (2). According to Meola, the industry has been focusing on how the deamidation of capsid protein from viral protein 1 (VP1)-specific residues, including the N57 domain, is linked to a loss of in-vivo potency (3).

“Temperature, pH, and storage time have been identified as key factors causing this phenomenon,” Meola said in his presentation (2). “In our quest at Oxford Biomedica—to try and resolve this—we have a static binding capacity residence screening protocol that we run where we look at a variety of different residents. In this case, we decided to look at salt-tolerant residents,” he stated.

Aside from process control to prevent deamidation, Meola’s team hypothesized that extrusion of VP1 and deamidation of N57 are related phenomenon and, furthermore, that this connected phenomenon results in the generation of empty, partial, and full capsids (4). The team also believed that the generation of these mixed capsids significantly compromises the ability of the AEX process to remove empty capsids and deliver functional AAV gene therapies.

Meola noted that charge-driven separation techniques have not provided the much-needed resolution to allow differentiation between deamidated and non-deamidated capsid species. Thus, his team used novel approaches that can be applied to the AEX process to address this complex challenge of clearing both empty capsids and deamidated intact capsids.

In one approach, Meola and his team used HIC to separate distinct peaks with near baseline resolution. After separation, each peak was then isolated and individually reprocessed with AEX. “Surprisingly, we discovered that HIC resolved two different species of AAV capsids with near baseline resolution. Each species was reprocessed on AEX, and all product quality attributes were assessed for the intermediate peaks that were generated. We found that the capsids that were more hydrophobic were also more negatively charged,” he explained in his presentation.

Data from liquid chromatography–mass spectrometry analysis further demonstrated that the more hydrophobic and more negatively charged capsids exhibited significant levels of VP1-specific N57 deamidation., which has been linked to a loss in gene expression, Meola noted. The team’s study thus demonstrated that HIC can be a useful method to separate non-deamidated capsids from deamidated capsids.

Chimeric antigen receptor T-cell (CAR-T) therapies are another sector incurring industry focus as this market matures as well. Similar to AAV vector-based gene therapies, CAR-Ts require an enhanced targeting mechanism. Philip Gregory, senior vice-president, Regeneron Cell Medicines, Regeneron, notes that, while conceptually simple, the redirection of a killer T cell to a tumor target using a CAR is a complex and multi-component problem.

“Thus, success required innovation and iteration in three key areas. Firstly, the methods for the isolation and expansion of the desired T cells from the peripheral blood. Second, the design of the CAR itself to ensure appropriate activation of the relevant signaling pathways in response to binding antigen. Third, the identification and selection of the best target antigens and antibody formats to enable the CAR to recognize the tumor cells,” Gregory states. He emphasizes that advances across all three areas were necessary to see the depth and durability of responses that have driven the recent excitement surrounding the CAR-T modality.

Gregory points out that the initial successes of CAR-T cell therapies have been in liquid tumors, such as lymphoma and myeloma, driven, in part, by the identification of cell-surface-expressed CAR-T cell target antigens that are universally found on both the surface of the tumor cells and on the normal tissue from which the tumor is derived. “For example, B cell maturation antigen (BCMA) is expressed on the surface of plasma cells (the cell type which is cancerous in multiple myeloma). Such universal and high levels of expression are, however, not the norm,” Gregory says.

Furthermore, while B cells and plasma cell aplasia can be compensated for by the infusion of immunoglobulins, most normal tissues are not “dispensable”, Gregory also points out. “Solid tumors, by contrast, lack such broadly expressed target antigens and have the additional constraint that the tumor microenvironment may be immunosuppressive (via the secretion of factors that dampen T-cell activity). Thus, we believe that solid tumors will require multi-layered approaches, such as the ones we have been developing (combining T cells and antibody-based approaches), to achieve the depth and durability of response desired,” he adds.

Gregory highlights the following advances CAR-T cell therapy design:

 These approaches include Regeneron’s work on a dimerizing agent–regulated immunoreceptor complex (DARIC) platform. DARIC offers a physician the ability to turn the CAR-T cell off (and back on) in response to a small-molecule drug. This development could have an impact in both normal tissue salvage and in the prevention of rapid exhaustion of the T cells via persistent antigen stimulation.

 “In these approaches, one can deploy novel architectures such as those used in bbT369 (Regeneron’s investigational CAR-T cell approach for B cell lymphoma) to enable the T cell to kill tumors expressing either, or both, tumor targets. Alternatively, the T cell can be engineered to be a delivery vehicle for a tumor-targeted bispecific antibody. Potentially powerful strategies to prevent tumor escape (the loss of the target antigen),” says Gregory.

Both Moller-Tank and Gregory see a dynamic future ahead for gene therapies and cell therapies. “We are hopeful that we can continue to expand the types of tissues and cell types we can reach with our antibody-targeted AAV delivered gene therapies. We are excited about applying our emerging strategies for addressing immunogenicity as it relates to challenges with redosing and pre-existing antibodies,” Moller-Tank states.

Regeneron is also expecting to make first-of-its-kind clinical progress with its clustered regularly interspaced short palindromic repeats (CRISPR)-mediated targeted gene insertion platform. The company anticipates that its platform has the potential to pave the way for more durable liver-directed gene therapies.

Gregory is confident that CAR-T cell approaches will remain an important modality for treating cancer, both as a standalone modality and in combination with biologics. “We are particularly excited about the potential for the combination of CAR-T with the antibody-based biologics we are developing at Regeneron. To outsmart the tumor will require multi-layered strategies best addressed via these types of innovative combinations. Moreover, we are in a privileged position to be able to explore these in the clinic,” he concludes.

1. Mirasol, F. ASGCT 2024: Additives Play a Role in Removal of Empty and Partial AAV Capsids. 

 online, May 8, 2024.
2. Mirasol, F. Research Shows that HIC Can Effectively Separate Deaminated Capsids from Non-Deaminated Capsids (ASGCT 2024). 

 online, May 11, 2024.
3. Giles, A.; Lock, M.; Chen, S. J.; et al. Significant Differences in Capsid Properties and Potency Between Adeno-Associated Virus Vectors Produced in Sf9 and HEK293 Cells. 

 (19–20),1003–1021. DOI: 10.1089/hum.2022.116
4. Meola, A.; Aguiar, A.; Chong, C.; et al. Not Created Equal: Impact of Deamidation on Capsid Heterogeneity and Anion Exchange Chromatography (AEX) Performance. Paper Abstract. 

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Vol. 48, No. 7
July 2024
Pages: 10–12

When referring to this article, please cite it has Mirasol, F. Taking Stock of a Maturing CGT Sector. 

As the CGT market matures, increased focus lays in securing optimal efficacy while ensuring safety.

Taking Stock of a Maturing CGT Sector

Explores new and emerging technologies, such as mass photometry, machine learning, and AI, and their role in the analytical space for biologic modalities

Analytics and Assays Strive to Keep Pace with Innovative Modalities

With the evolution of biologic modalities in the drug development pipeline, the biopharma industry understands that it must keep pace with the development and use of appropriate analytics and assays for molecule characterization, to determine product purity, safety, and efficacy, and even early on in the R&D process, where analytics are key in screening multitudes of molecules to determine viable candidates to move into the drug development phases. This episode of Drug Digest explores new and emerging technologies, such as mass photometry, machine learning, and AI, and their role in the analytical space for biologic modalities.

Salma Jalal, Product Marketing Manager, Refeyn

As a product marketing manager at Refeyn, Salma supports customers in biopharma and the life sciences to revolutionize their analytical workflows through the use of mass photometry. She holds a PhD in Mechanobiology from the National University of Singapore and has over a decade of technical expertise in the application of microscopy techniques to study biological systems. She has a particular interest in the analysis of biologics and is very happy to discuss all things biomolecular characterization.


Philipp Lorenz, PhD, Chief Technology Officer, Basecamp Research

Philipp Lorenz, PhD, is the chief technology officer (CTO) of Basecamp Research, based in London. He attended the University of Oxford from 2012 to 2016, where he co-founded the first iGEM team from Oxford alongside Glen Gowers, the CEO and co-founder, of Basecamp Research. Lorenz gained a Masters degree in Biochemistry and a PhD from Oxford in Genetics and Bioinformatics. He was a tutor at the institution, teaching Computational Biology, Statistics, and Genetics to undergraduate and graduate students. Previously the head of Data Science at Basecamp Research, Lorenz became the CTO in May 2022 and is driven by his passion to build the next generation of life science artificial intelligence solutions fueled by better data and deeper knowledge about life on earth.

Drug Digest is a tech talk video series with the 

® editors, who interview industry experts to discuss the emerging opportunities, obstacles, and advances in the pharmaceutical and biopharmaceutical industry for the research, development, formulation, analysis, upstream and downstream processing, manufacturing, supply chain, and packaging of drug products.

Biopharma Drug Development and Manufacturing

Small Molecule APIs, Excipients, and Formulation

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This episode of Drug Digest explores new and emerging technologies, such as mass photometry, machine learning, and AI, and their role in the analytical space for biologic modalities. 

Drug Digest: Analytics and Assays Strive to Keep Pace with Innovative Modalities

In biologics production, biomanufacturers need to demonstrate clearance of impurities and contaminants during the manufacturing process. Host cell impurities (HCPs) are a major class of process-related impurities (1) that must be monitored, and their presence must be documented throughout the development and manufacturing process of a biologic.

“The presence of HCPs beyond a permissible regulatory limit can potentially result in adverse immune response, toxicity, possible biological activity, [and] hence, interfere with the potency and stability of the drug as well as cause disruption during the purification process,” states Anis H. Khimani, PhD, senior strategy leader, Life Sciences, Revvity, a Waltham, Mass.-based provider of health science solutions, technologies, expertise, and services. “It is therefore important to monitor and control the presence of HCPs in large-molecule drug formulations all along upstream through downstream processes.”

Olaf Stamm, PhD, technical business development director, Biopharmaceutical Testing Solutions, Charles River Laboratories, highlights several reasons why monitoring the presence of HCPs during biologic drug manufacture is critical:

 HCPs can cause adverse immune reactions in patients, even in small amounts. These proteins can trigger allergic responses or other immune-mediated effects, which can compromise patient safety.

 HCPs can interfere with the biological activity of the drug, such as by degrading the drug product, altering its function, or inhibiting its intended biological activity, thus reducing the biologic’s efficacy.

 HCPs can affect the stability of the biologic by, for example, inducing aggregation, oxidation, or other forms of degradation. These effects can reduce product shelf life and affect product quality over time.

 Consistent monitoring of HCPs ensures consistency from batch-to-batch in the manufacturing process. Variability in HCP levels can signal an issue in the production process, such as changes in cell-culture conditions.

 Regulatory agencies such as FDA and the European Medicines Agency have stringent guidelines for HCPs in biologic products. Therefore, manufacturers must demonstrate that HCP levels are within acceptable limits in their product to gain approval for that product’s purification inefficiencies, which then needs to be addressed to maintain product quality.

“Overall, monitoring HCPs is essential to ensure that biologic drugs are safe, effective, and of high quality, meeting both regulatory standards and patient needs,” Stamm says.

Effective HCP clearance starts in the upstream processing stage, where optimization of cell-culture conditions with respect to optimal viability helps to reduce the load of HCP in the harvest material, notes Stamm. He explains that this approach works mainly for expression systems (e.g., Chinese hamster ovary [CHO] cells), which secrete their product. In the case of critical HCPs that might co-purify with the product protein, however, some production cell lines have been genetically engineered by knock out or silencing of such particular HCPs, he points out.

Khimani adds that, “Although most monoclonal antibodies and Fc-fusion proteins are expressed extracellularly, particularly from CHO cells, cell lysis and cell debris result in HCP contamination within the cellular milieu.” Therefore, various factors during cell growth and protein expression influence the level of HCPs, such as media composition, culture feeding, bioreactor controls, length of incubation, and even cell viability, all of which impact the profiles of HCPs as well as their detection and clearance.

In the downstream process, several tools can be implemented to manage the elimination or minimization of HCPs, Khimani says, while Stamm points out that the power horses for HCP removal are capture/affinity chromatography steps such as Protein A/G, which typically result in >95% of HCP removal; although, this clearance level is applicable for antibodies only.

“Additional [downstream processing] steps, such as hydrophobic interaction chromatography, ion exchange chromatography, size exclusion and mixed-mode chromatography, aid in the removal of HCPs,” Stamm adds.

Khimani emphasizes that, “optimization of each step is critical to achieve a robust workflow resulting in efficient removal of HCPs. To monitor the levels of contaminating proteins, it is critical to utilize analytical tools that offer sensitive and reproducible detection.”

HCP identification and profiling is a designated critical quality attribute (CQA), Khimani points out. Therefore, covering this CQA is key to the development and production of biological drugs. Khimani explains that HCP detection has evolved from traditional techniques to next-generation technologies that offer greater sensitivity, reproducibility, and higher throughput.

“Immunoassays have been considered the gold-standard method for monitoring HCPs along upstream and downstream workflows of large-molecule drug production. ELISA [enzyme-linked immunosorbent assay] has been one of the most commonly used assays to identify and measure total HCP in large-molecule drug preparations; however, it is unable to provide a uniform comparison across various types of drugs where the HCP content from each cell type may differ and their related immune specificity may be undefined. Therefore, ELISA is limited by providing a coverage analysis of HCPs,” says Khimani.

On the other hand, detection using two-dimensional gels resolves proteins by size and charge followed by transfer onto membranes and subsequent Western blot detection. However, this method is challenged by the gel size, protein population complexity, and lower throughput, Khimani states.

Stamm says that the advantages of ELISA are high sensitivity, ability to handle multiple samples simultaneously, quantitative results, unexpensive equipment, ease of use, and good manufacturing practice (GMP) compliance. However, he also points out its disadvantages, the main one being that the performance of ELISA rests entirely on the quality of the antibodies generated for this assay. “The challenge is to generate polyclonal antibodies against several hundred to thousands of proteins which vary in size, immunogenicity, and physio-chemical properties. As these are critical reagents, those antibodies need to be thoroughly characterized before they can be used in the ELISA development. The entire process from the antigen generation to the immunization and qualification need to get thoroughly documented as such information is reviewed by the authorities and might be required a couple of years later to replenish antibodies of equal quality,” Stamm explains.

Khimani, meanwhile, points to mass spectrometry (MS) as an alternate or orthogonal technique that is more powerful in profiling HCPs because it has greater sensitivity and specificity. However, MS is limited by throughput due to complexity, requirements, and higher cost, he adds. Ultimately, the choice of methods to detect HCPs depends upon the complexity of the host cell lines’ protein profiles, regulatory requirements to monitor HCP levels as a CQA, and risk management strategies during large-molecule drug development, he states.

“Each technique varies in its sensitivity, specificity, and suitability for different stages of the purification process,” Stamm adds. He also states that the advantages of techniques such as liquid chromatography with tandem mass spectrometry (LC–MS/MS) is a high specificity and ability to identify individual HCPs in complex mixtures, such as bulk harvest, as well as in drug substance. The disadvantages of LC–MS/MS lay in the fact that it requires sophisticated equipment along with deep biochemical and bioinformatics expertise.

Improving the monitoring of HCPs during the manufacturing process involves a combination of immunoassays and MS, says Stamm. Combining these techniques also enables HCP clearance in downstream processing. During early process development, generic ELISA kits are typically used for the quantitation of the HCP load in the cell harvest, process intermediates, and drug substance; however, instead of trusting such ELISA results blindly, Stamm emphasizes, the use of MS (e.g., shotgun LC–MS/MS approach) on the same samples will provide unbiased quantitative results as well as help identify potential critical HCPs that may be co-purifying (i.e., hitchhiking) with the product protein of interest.

Meanwhile, Khimani states that “a well-designed set of best practices that align with the type of cell line in use, the type of drug product in development with defined CQAs, the expected or previously defined profile of host proteins that may impact the drug preparation, and defined parameters for an optimal process should be implemented for greater efficacy and safety.”

1. Pilely, K.; Johansen, M.R.; Lund, R.R.; et al. Monitoring Process-Related Impurities in Biologics—Host Cell Protein Analysis. 

 (2), 747–758. DOI: 10.1007/s00216-021-03648-2

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Vol. 48, No. 7
July 2024
Pages: 32–33

When referring to this article, please cite it has Mirasol, F. The Importance of HCP Monitoring. 

HCPs are major process-related impurities that must be monitored throughout biologics production for clearance.