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Volume 29, Issue 10
High-purity low-endotoxin sugars improve robustness and stability of protein formulation and improve drug product quality.
Various carbohydrates, such as glucose, galactose, sucrose, and trehalose dihydrate, have been widely used in small-molecule and protein drug manufacturing and fill/finish formulations. These carbohydrates are particularly crucial for biopharmaceutical processes as they provide unique functionalities in all three major process areas: cell culture, mainly as energy sources for cell growth and controlling glycosylation; harvest and purification, as buffer supplements to protect and stabilize the molecules during purification; and formulation, as stabilizing and bulking agents, especially for parenteral formulations.
During the past few years, many research activities have demonstrated that proper cell-culture supplementation, along with control of reactive impurities, can improve both product quality and process. For example, a case study by Jianlin Xu, et al. identified the high level of iron in chemically defined media as the root cause of brown drug substance coloring in cell-culture manufacturing (1).
The use of well-characterized carbohydrates with consistently low impurity levels within cell-culture media and parenteral formulations is an effective tool for mitigating risk to drug product quality and process efficiency (2). A orthogonal purification process (Avantor) reduces endotoxin, reactive impurities, and trace metals in its carbohydrate materials to provide high-purity low-endotoxin (HPLE) sugars. These high-quality sugars with low impurity levels are shown to improve drug product stability (3).
This article reviews critical impurities, which often are introduced in upstream and downstream processes, along with the roles played by formulation excipients, including the influence of reducing impurities in carbohydrates, and the influence of trace metals. Information also will be presented discussing how the evaluation, identification, and remediation of factors impacting protein stability are crucial in developing stable parenteral formulations. Consistent raw materials are necessary for managing the quality of critical excipients. Variation in excipient impurities can impact the protein; however, well-characterized sugars have been shown to improve process productivity and the quality of drug products.
Biopharmaceutical raw materials should contain only the lowest levels possible of reactive impurities to prevent modification or degradation of the drug substance, and to meet various regulatory requirements for microbiological endotoxins in biopharmaceutical drug products. The use of carbohydrates in cell-culture media and parenteral formulations can often introduce impurities that can impact the final yield and quality of a drug product.
Mammalian cell cultures used to produce biopharmaceutical drugs involve highly complex and proprietary blends. Each culture is developed through multiple drug discovery and process development steps and incorporates a broad range of materials: nutrients, such as proteins, peptides, and amino acids; select anti-microbial agents; and even metals, including calcium, sodium, and magnesium.
Developers need to optimize their bioreactor cell-culture designs to achieve the desired yield for their target proteins. Generally speaking, they seek to eliminate any variables as they refine the selection and concentration of these cell-culture components. This cell culture optimization includes selection of the carbohydrate to be used as the energy source for the process.
Until recently, glucose was the most widely used sugar for mammalian cell-culture energy sources. Recently, there has been an increase in the use of other carbohydrates to supplement glucose, and these more complex sugars have been shown to have a statistically significant impact on improving process yield.
In a study published in 2013 (4), for example, comparisons were made in the growth of chimeric anti-human CD20 monoclonal antibodies (mAb) using two types of carbohydrates: one fed-batch using glucose and FM-O, and one fed-batch using glucose-galactose and FM-Opt.
Supplementing glucose with galactose demonstrated significant increases in cell-culture yield in several ways:
In addition, the use of galactose lowered the amount of lactate byproduct from the process. Lactate can interfere with bioprocessing efficiency, so reducing the concentration can also help improve cell-culture yield.
There is a concern, however, that by increasing the use of more complex sugars (such as galactose) for upstream biopharmaceutical processing, this might bring in impurities from the introduction of an additional product into the cell culture that would either inhibit optimum yield or potentially cause protein degradation during or after downstream processing.
As an alternative, HPLE galactose may be purified to contain significantly lower levels of impurities compared to standard galactose available through multicompendial sources. This HPLE galactose also contains significantly lower levels of trace metals; just as importantly, the levels of metal are precisely characterized with properties that are highly consistent and without lot-to-lot variations. This is crucial for biopharmaceutical manufacturers with established cell-culture processes that rely on highly controlled levels of metal in order to reach desired yield for a target molecule.
For example, if their process specifies two parts per million (ppm) of iron and the galactose ingredient they use varies from one ppm for one batch to three ppm for another, a manufacturer cannot be certain what impact that impurity is having on their yield. The HPLE purification process produces galactose with consistently low iron levels of 10 parts per billion (ppb); comparable low levels of other trace metals, such as cadmium and zinc, also are achieved. This consistent, low level of trace impurities allows biopharmaceutical producers to add beneficial galactose to their cell cultures to improve yields without the risk of harmful trace impurities affecting their processes.
Biopharmaceutical parenteral formulations use a wide range of excipients, such as buffers, bulking agents, and lyo-protectants, including multiple types of carbohydrates. It is common for the API to make up approximately 2-3% of the formulation.
The additional materials can include buffering agents, such as sodium citrate or sodium phosphate; tonicity modifiers, such as dextrose; lyo-protectants, such as dextran and hydroxyethyl starch; and carbohydrates serving as bulking agents, such as sorbitol, mannitol, sucrose, and trehalose dihydrate.
No commercial chemical product is 100% pure; trace elements and endotoxins are always present. The presence of endotoxins-even in low levels-can adversely impact the safety and efficacy of the final product. The following are key requirements for parenteral excipients that have facilitated efforts to improve their purity and reduce to an absolute minimum the endotoxins they may introduce to the product:
Different parenteral excipients present unique risks of impurities and impact on drug quality (see Table I). Sorbitol and mannitol, for example, can include trace amounts of reducing sugars, metal ions, and endotoxins; these can interact with the API molecule, reducing its effectiveness.
Table I. Types of impurities and their impacts on drug quality.
It is well established that large biopharmaceutical protein molecules can react to the presence of metals, changing the molecule’s structure and impacting its effectiveness. In addition, a study published in the International Journal of Pharmaceutics (5) provided evidence that the presence of a reducing sugar (fructose) led to degradation of epidermal growth factor, human, recombinant (rhEFG) formulation after three cycles of freezing and thawing at -20 °C.
The presence of microbiological trace contaminants and beta-glucan can cause fever and other adverse effects in patients. The presence of metals, particulates, and reducing sugars can lead to a variety of unintended interactions within the parenteral formulation, such as glycation, aggregation, oxidation, and hydrolysis. These interactions can affect drug product stability and result in decreased product performance, loss in potency, and in some cases adverse effects due to by-products. Similarly, the presence of particulates can lead to adsorption, causing a reduction in protein concentration and ultimately reduced drug potency.
Given the wide range of products used in parenteral formulations, biopharmaceutical manufacturers seek to eliminate, to the greatest extent possible, trace contaminants in the materials they use. Well-characterized, high-purity carbohydrates, such as sucrose and trehalose dihydrate, with low endotoxins are crucial elements to mitigating risk and sustaining the quality and effectiveness of biopharmaceutical drugs.
It is equally important that these high-purity, low-endotoxin carbohydrates sustain these purity levels from lot to lot, to maximize process quality and efficiency. Well-characterized HPLE sugars with provable consistency will enable manufacturers to focus on other parts of their processes to maximize performance.
The multi-step HPLE purification process is focused on lowering impurities and trace metals. Key stages in the process include high-performance chromatography that can diminish endotoxin levels over 100 EU/gm to less than 0.1 EU/gm, remove trace metals, reducing sugars and trace element impurities; micro filtration; co-solvent based dilute crystallization to remove reducing sugars and trace metals; and control condition isolation and drying.
The orthogonal nature of this process enables consistent control of all key impurities. It achieves two key results: reduction of endotoxins and trace metals to low levels in raw materials (significantly lower than industry-standard multicompendial requirements); and consistent production of high-purity material without lot-to-lot variation.
Comparative analysis of the HPLE sugars produced using this purification process demonstrates significantly improved substance purity, reduced endotoxin levels, and low levels of trace metals, in the 5-10 ppb range. Endotoxins in the raw, pre-purified sucrose, trehalose dihydrate, and galactose ranged from approximately 20 to more than 30 EU/g. After purification, endotoxin levels in all three HPLE sugars consistently measured less than 0.1 EU/g. Similar results for metal purification also were achieved: levels for cadmium, copper, manganese, nickel, and zinc were reduced from a range of 800-1200 ppb to less than five ppb.
Consistent levels of purity also have been achieved. Twenty-five lots of HPLE sucrose and galactose were analyzed, with consistently low levels of elemental impurities (i.e., metal) present (See Figure 1).
Figure 1: Consistent production with low metals. (Courtesy of author)
Further analysis of purification results was conducted to compare HPLE carbohydrates (sucrose, trehalose dihydrate, and galactose) with the purification levels available from typical United States Pharmacopeia (USP) multicompendial specifications for endotoxin levels (6), for example (see Table II).
Table II. Comparison of typical impurity values found with multicompendial specifications. HPLE is high-purity low-endotoxin.
The use of well-characterized, high-purity HPLE galactose, sucrose, and trehalose dihydrate can improve mammalian cell-culture yield and aid in improving API stability in biopharmaceutical parenteral formulations. Reducing endotoxins and trace metals in HPLE sugars with lot-to-lot consistency can significantly reduce potential sources of contamination that impact cell-culture yield and API stability.
A proprietary HPLE purification process provides sugars with very low levels of contaminants-in many cases much lower than typical multicompendial requirements-and consistent low levels from lot to lot. This combination of consistent purity with low levels of endotoxin and trace metal contaminants enables biopharmaceutical manufactures to incorporate these products into their cell cultures and parenteral formulations with high confidence, and can help eliminate the need for conducting further analysis on HPLE sugars as they seek to optimize their biopharmaceutical development and manufacturing processes.
1. J. Xua, et al., Process Biochemistry 49 (2014) 130-139.
2. Yongmei Wu et al., AAPS PharmSciTech, 12 (4), 1248-1263.
3. B. Thiyagarajan et al., American Chemical Society Poster (Spring 2016).
4. Y.-t. Sun et al., Biochemical Engineering Journal 81 (2013) 126-135.
5. H. Santana et al., International Journal of Pharmaceutics 452 (2013) 52-62.
6. USP, USP 39–NF 34, Supplement 1, 2016
Vol. 29, No. 10
When referring to this article, please cite it as N. Deorkar and B. Thiyagarajan, "HPLE Sugars Improve Stability and Drug Product," BioPharm International 29 (10) 2016.