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6 BioPharm International eBook July 2021 www.biopharminternational.com products underlying DNA can be per- formed by Sanger sequencing, capillary electrophoresis with fragment analy- sis, and next-generation sequencing (NGS) approaches. The depth to which a product is sequenced—including a product's expressing and non-express- ing elements—and the degree to which sequencing results confirm a product's identity has yet to be standardized," Bakewell adds. LESSONS FROM mAbs Fortunately, biopharmaceutical analysis methods have improved since the first mAbs were approved. The most sig- nificant change over the past 15 years has been the speed and resolution at which bioanalytical tools are employed, say s Ch r istopher Cola ngelo, bio- pharma business development leader, Agilent Technologies. The develop- ment of porous a nd /or sub-2-um chromatographic media coupled to ultra-high-performance liquid chro- matography (UHPLC), for example, has enabled analytical method runtime to decrease 10-fold from 30–50 mins/ sample to 3–5 mins/sample. Colangelo adds that a key success in many bio- pharma research labs today is the use of automation solutions in lab workf lows. "For example, successful labs effectively use robotic sample preparation, liquid autosamplers, and automated data anal- ysis tools," he states. Significant improvements have also been made in the sensitivity, resolution, accuracy, reproducibility, and specific- ity of analytical methods used to sup- port the release and stability testing of biological drugs, Bakewell emphasizes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing gel electrophoretic methods commonly used for release and stability in the 1980s have been largely replaced by capillary electrophoresis (CE) meth- ods, for example, Bakewell says. "CE methods are deemed superior to tra- ditional gel electrophoresis methods due to their improved resolution and their high degree of reproducibility and robustness," he states. Similarly, column chromatographic separation techniques have evolved and improved over time to where, for example, the operating pressures t hat a na ly t ica l H PLC/u lt ra h igh (UH)PLC columns can tolerate has increased. This has resulted in a con- comitant increase in analytical resolu- tion, Bakewell explains. Furthermore, improvements in the quality of HPLC systems and batch-to-batch repro- ducibility of HPLC columns has led to better and more reproducible ana- lytical methods, while data analysis for chromatographic separations has evolved from using paper integrators to Code of Federal Regulations (CFR) Title 21, Part 11-compliant analytical software, says Bakewell. "Over time, the variety of good man- ufacturing practice (GMP)-compliant analytical methods used to evaluate purity, potency, and stability of bio- pharmaceuticals has expanded and evolved. These now include multiplex and f luorescent plate reader assays, residual assays for process impurities such as host cell proteins and DNA, and in-vitro potency assays rather than animal models," Bakewell adds. "Improvements in automation that remove analyst subjectivity for compen- dia methods such as color, clarity, par- ticulate matter, sterility, and endotoxin analysis have increased the reliability of these methods." More recently, Hersch includes, advanced protein engineering has given rise to synthetic antibody-like mole- cules, including bispecifics, ADCs, or CARs with an antibody-derived target- ing domain. "The analytical methods associated with modern protein design are now nearly always high-through- put and require automation, w ith researchers routinely screening large panels of molecules in different formats to select the very best candidates for further development. A large amount of data is generated at each step and using sophisticated data management solutions that can handle and structure all the associated data centrally across an organization have become the norm in the industry," Hersch says. Morales adds that technology, soft- ware, and biological knowledge have evolved in such a way that cell-based potency assays are expected to be more robust and precise by default. "Phase- appropriate va lidation enables the identification of critical reagents and steps, not only for analytical methods, but also from manufacturing process validations. Cell-based development reports are now an expectation to demonstrate that efforts were made to optimize assay precision," he states. F it z g ibb ons f u r t her a dd s t h at advances in cha racterization have o c c u r re d w it h i n a l l a re a s , f rom improvements in cell-based functional analysis to detection mechanisms in MS-based solutions involving high- er-resolution instr umentation. " In several instances, such as in mass spec- trometr y, data processing solutions have driven evolution where the large amount of data acquired has required powerful software to process the vol- ume of information present in the anal- ysis," he says. One hot analytical topic for bio- pharmaceutica ls over the past few years has been sub-v isible particle ana lysis, including par ticle counts using microf low imag ing ana lysis and high accurac y products liquid particle counters, says Gar y Watts, Analytics Manager at Abzena. "The understanding that 'particles breed particles' potentially leading to drug products failing specifications has led to emphasis on early detection to help de-risk the drug development pathway by identifying and removing the like- lihood of any issues further down the line," he explains. Huang reinforces how analy tical advancement has made signif icant strides for the sake of biological drug development since the 1980s, noting Biopharmaceutical Analysis Analytical Evolution