Analytical Prowess Powers Efficient CLD Through Data-Driven Decision-Making
By Felix Vega, Aragen Life Sciences
Cell line development (CLD) is a complex endeavor that demands pharmaceutical companies and their partners be mindful, at every step, of cell productivity (titer) and product quality, as well as the eventual cost of goods sold (COGS). Further, biologics development is not always predictable due to the inherent plasticity of the expression systems. Both known and unknown liabilities in the protein sequence can complicate CLD, especially for complex (nontraditional) format biologics.
Typically, these organizations are hyper focused on COGS, which ties directly to the cells’ titer. As productivity rises, COGS falls, increasing profitability when the product reaches the market. COGS is such an important driver because biologics are expensive to produce in a way that small-molecule chemistry drugs are not. Biologics come from living organisms that require care and feeding. For example, one liter of cell culture media typically costs about $40. Scaling up to a 2,000-liter or a 12,000-liter bioreactor multiplies that cost accordingly. The purification cost, too, can be great: the resins are expensive and drive the majority of COGS.
Still, despite valid concerns about productivity and timelines, biopharmaceutical sponsors must strive to better understand their molecules moving forward, focusing on the potential product attributes or specifications critical to the drug’s safety and efficacy. Those critical quality attributes (CQAs) will help to identify the clones capable of producing optimal product quality and the highest productivity possible within the cell line being developed for clinical use and, eventually, GMP manufacture.
Managing Risk = Managing Unpredictability
Stated simply, more complex molecules generally lead to more liabilities. Relevant to complex format biologics, unknown degradation or post-translational modifications can make CLD more difficult, time-consuming, and expensive. Still, disease indications targeted by standard antibodies are low-hanging fruit and are a very competitive field. So, more biopharmas are exploring more complex formats of protein therapeutics (though still largely based on antibody structures), such as an antibody cytokine infusion, or a single-chain Fv antibody fragment.
These more complex molecules are harder to express and have different degradation products because they might be considered “unnatural.” Specifically, the cells are not used to expressing the complex molecules like they would a natural immunoglobulin G (IgG) antibody. So, the cells tend to degrade, the degradation products get into the cell culture supernatant, and those product-related impurities need to be purged before the product proceeds to the clinic and, ultimately, to the market.
For example, a bispecific with which Aragen recently worked was acid liable in that sense that it would mask the binding region of the bispecific antibody until the drug reached the antigen’s location. At that point, the pH change would prompt unmasking of the antigen binding region, allowing the bispecific to bind to and kill the target cell. This masking arm had to be flexible and remain intact during production of the drug, only cleaving off and exposing the antigen binding region of the bispecific once it was at the site of activity near the cancer.
Some of the clones we evaluated would naturally cleave the masking arm because they perceived an unnatural protein (i.e., a protein unfamiliar to them). Accordingly, during production, Aragen performed a lot of screening with different analytics to ensure production of the intact prodrug — the molecule with the appropriately masked arm.
In terms of post-translational modifications, N-linked glycosylation is the most common and most commonly understood modification in antibodies. But as the chemistry progresses into more complex molecules (e.g., bispecifics, Fc-Fusion proteins), glycosylation may not occur as researchers think it should, manifesting in different glycosylation patterns or O-linked glycosylation.
Whereas N-linked glycosylation occurs on a very specific amino acid motif, O-linked glycosylation is nonspecific, occurring on serines or threonines seemingly at random, making it very problematic in biologics; post-translational modifications can vary in different clones as CLD progresses. Other liabilities that may arise include oxidation and deamidation. Thus, it is vital to have appropriate analytics in place that can identify those liabilities, tracking and trending them throughout the CLD process, as well as the clones exhibiting the highest product quality.
Comprehensive Analytical Characterization Explained
Aragen strives to provide comprehensive and expert analytical characterization, ensuring product quality attributes that contribute to a biologic’s safety and efficacy. We are equally committed to a collaborative and transparent CLD process that empowers Aragen clients to make well-informed decisions.
We encourage clients taking an innovative molecule into the clinic to provide several different candidates whose sequences we can evaluate. We then subject those materials to developability studies, which stress the material in an attempt to identify degradation products or post-translational modifications that may become problematic later in clinical or CMC development. The studies identify previously known as well as unknown liabilities, which we report back to the client while making recommendations about which candidates to advance into CLD — the amino acid sequences most likely to meet the client’s specifications for mechanism of action, robustness, and COGS. We also we will offer mitigation strategies relevant to any liabilities, describing how we can reduce the risk of those liabilities while still moving forward.
CLD is a stable process whereby we introduce the genes of interest into a CHO host, whose genome is plastic (i.e., it changes over time). When genes are introduced into that environment, they become part of the changing process: genotypical changes result in phenotypical changes to the product quality. At this point, analytical testing becomes critical because the diversity of CHO clones produced during a transfection leads to a hunt for the CQAs the client seeks. Having the appropriate analytical screens in place allows us to pick the highest productivity clone capable of meeting the client’s quality specifications.
One of the first opportunities to examine the materials during the CLD process is the bulk pool stage, essentially a transfected pool from which we generate material. Analysis on that material provides a benchmark — AKA an analytical standard representative of the clinical material to be produced later. The tools and analytics applied here are informed by what was learned during the developability stage.
For example, aggregation may be an issue. In response, we may include size exclusion chromatography in the clone screening process throughout CLD. The process progresses from bulk pools to mini pools, wherein we plate anywhere from 500 to 1,000 cells per well to enrich those populations producing high levels of protein. The product is again examined to identify the mini pools exhibiting the best product quality before moving those into the single cell cloning stage.
The single cell cloning stage comprises expansion and generation of representative material from fed-batch cultures. The material is screened again for productivity and product quality before we select clones to place into a GMP cell bank and, ultimately, into GMP manufacture for generation of the clinical material.
Throughout this process, Aragen bundles all relevant analytical data — including all decisions made in concert with the client to identify the best-producing clone with the highest product quality — to hand off as part of our tech transfer into GMP manufacturing. This way, the manufacturer begins with a depth of knowledge regarding product quality, cell productivity, and things they should be mindful of when conducting their own analytics and scaling up to produce clinical material.
Adaptability Drives Success
Analytical capability, deftly applied by skilled and knowledgeable operators, is the engine that powers adaptability. While it is prudent to enter CLD with a master plan, unpredictable biologics expression systems demand people who can think on their feet with access to resources empowering them to respond to shifting cell behavior and product quality attributes with minimal impact in terms of time, cost, or confusion.
For example, a protein may have a leading shoulder by SCC which can indicate aggregation or another modification. In such a scenario, it is necessary to collaborate with the client not only to understand the potential modification, but to devise a way to limit that modification in the final product. The adaptability to leverage additional analytical tools to understand such modifications, performing on-the-fly analysis quickly and efficiently, is vital during CLD because the cells’ biological processes cannot be slowed and the CLD team needs to continue progressing within its mandated timelines.
Moreover, that capability must be supported by expertise — and guided by thorough, efficient SOPs — to be effective. Aragen conducts biophysical and biochemical characterization, and we are well-versed in a host of analytical techniques. For example, Aragen conducts mass spectrometry regularly: not only routine intact molecular weight analysis, but peptide mapping, as well. Peptide mapping is probably the most sensitive approach to good biochemical characterization of a protein therapeutic; we recommend it when we see differences between released material and stressed material. We automate a significant portion of that peptide mapping through a collaboration with Protein Metrics, Inc., improving our efficiency in deconvoluting those peptide maps.
Final Thoughts
In well-behaved molecules, the CLD master plan is fairly simple to follow. In biologics, deviations from that path are more common. In either scenario, potential issues require developers to be ever-watchful. That constant watchfulness requires reliable analytics to be in place: to maintain flexibility and to enable responsiveness to what the data is telling us, to conceive at every step how we might push the clones or the cells into an environment more conducive to the client’s product quality specifications.
About Aragen Life Sciences
Aragen Life Sciences (Formerly GVK Biosciences) is a global leader in providing drug discovery, drug development, and manufacturing solutions for life sciences firms. We provide clients — from large pharmas and biotech firms to agrochemical and animal health companies — with global resources and proven capabilities at every stage of the biopharma lifecycle, in small and large molecules. Our ability to offer end-to-end solutions or support standalone programs is underpinned by an innovation mindset, enabling technologies, and a partnership approach to every engagement.
Source: Bioprocess Online