Amidst biologics patent expirations and the push for personalized (yet low-cost) medicines, the age of biosimilars is upon us. The European Union has paved the way with its biosimilar approval pathway, while FDA passed the Biologics Price Competition and Innovation Act (BPCIA) in 2010. The Act codified into law the 351(k) abbreviated regulatory pathway for biosimilars approval, and formally opened the door for biosimilars product approval in the U.S. However, the law provided no advice regarding what requirements the FDA would need for approval, so in early 2012, the Agency issued draft guidances describing CMC considerations in demonstrating biosimilarity to a reference protein product (U.S.-approved or foreign innovator biologic) .
Like their reference products, biosimilars are complex, difficult to characterize, typically have more than one biological effect, and frequently generate immune responses. Because of this, the guidance necessarily lacks a list of specific steps for developing biosimilars, leaving developers to integrate quality attributes on a case-by-case basis using the totality of evidence approach. Although approval of a biosimilar will rely on current data of the reference product, the guidance paves the way for producing biosimilar proteins through the use of alternative expression systems and novel manufacturing technologies. To do this, however, developers must ensure they use the principles of Integrated Drug Development to incorporate robust quality considerations in their development programs.
This article provides a review of the essentials of developing and manufacturing biosimilars today. We review current animal-, yeast-, and plant-based expression systems, predominant manufacturing technologies, and key quality considerations for developers and manufacturers of biosimilars—all with an eye toward the integration of these elements.
Alternate Expression Systems
For more than two decades, most biologics have been manufactured in well-known, well-characterized, FDA-approved cell lines that were developed by the innovator manufacturers. A review of global product approvals from January 2006 to June 2010 shows the distribution of these cell lines .
Historically, CHO cells have shown the highest expression rates and are easily cultured and sustained. Bacterial and yeast cell lines like E. coli and S. cerevisiae require minimal growth media conditions and are fast growing, making them economical. However, these traditional cell lines are inherently prone to host cell protein contamination and adventitious agents such as endotoxins. Developers often opt to use traditional cell lines because they have established upstream and downstream purification processes, historical literature data is available for reference, and regulatory agencies are familiar with the expression systems. For biosimilars developers, however, these cell lines provide less opportunity for innovation, fewer intellectual property advantages, and minimal patent protection.
Animal- or Yeast-Based Alternative Expression Systems
FDA has approved novel products using animal or yeast-based alternative expression systems.
- YF-Vax was developed in Avian Leukosis Virus (ALV)-free chicken embryos. Chicken embryos have historically been used for protein expression and experimentation, and are economical and easily accessible. Most importantly, chicken embryos lack an immune system, making them ideal candidates for production.
- Due to their susceptibility to a wide range of viruses, VERO cells, a kidney epithelial line isolated from the African Green Monkey, have served as a popular expression system for vaccines, such as RotaTeq, Rotarix, ACAM2000, and Ixiaro.
- HEK293 cells (Human Embryonic Kidney cells) have also been used in the manufacture of an approved drug, Xigris. (Note: Xigris was withdrawn from the market in 2011 due to lack of efficacy, not due to any manufacturing deficiencies.) Similar to CHO cells, HEK293 cell lines are easy to culture, have higher rates of expression for proteins of interest, and are easily scaleable.
- The baculovirus insect cell line, used to produce Ceravix for the treatment of HPV, produces large quantities of proteins in cultured insect cells or insect larvae and these proteins are easily purified with tags or using affinity chromatography techniques.
- In 2009, Kalbitor produced from P. pastoris (a yeast) was also approved. These yeast cells can grow to high densities compared to the more common yeast S. cerevisiae. S. cerevisae is also known to release ethanol during the fermentation process, which can deter cell growth and protein production.
- In 2011, Benlysta was approved for the treatment of lupus and was expressed in the mouse myeloma cell line NS0. The NS0 cell line cannot produce endogenous antibodies, making it an attractive platform for protein production. However, as seen with CHO cells, NS0 cells have the potential to generate glycosylated proteins, which are known to produce immunogenic effects.
Plant-Based Alternative Expression Systems
FDA has also been open to plant-based expression systems (i.e., plant cell cultures and whole plants). These systems have gained increasing popularity due to attractive protein yields; simpler methods of expression, cultivation, and manufacturing; and economical developmental requirements when compared to mammalian cell lines. One of the most enticing benefits to using a plant-based production system is the decreased likelihood of clinical immunogenic responses, since plants do not contain mammalian pathogens or endotoxins. However, when working with plant-based expression systems, extensive DNA or protein characterization is required, unlike with mammalian cell lines.