A Case Study of Developing Analytical Methods

Demand for parenteral-dose contract manufacturing services is on the rise, fueled in part by strong growth in the number of biologics in the approval process.¹ With the increase in small emerging biotech companies and "virtual" pharmaceutical companies, lacking both laboratory and manufacturing facilities, contract pharmaceutical manufacturers must offer an increasing array of services.

Wendy Saffell-Clemmer

Specifically, small companies may require formulation and analytical methods development to transform a promising therapeutic into a viable commercial product. Proteins, by their nature, require many methods for product development, product release testing, and stability testing. As the number of biotechnology-derived products produced by contact manufacturers increases, the level of analytical support required by clients will also increase. Contract manufacturers must plan for increased analytical resources in development and quality control to support product release of protein therapeutics. This is a case study of the analytical development of an early-phase protein pharmaceutical proposed by a virtual company client. We are including the false starts and dead ends lest anyone thinks this was easy.

THE RESULTS DEPEND ON THE METHOD

While small-molecule products typically require only one chromatographic release method to determine product purity, the specific molecular properties of protein therapeutics require multiple release methods encompassing a variety of techniques. Because proteins are produced biosynthetically in living organisms, the active pharmaceutical ingredient (API) may include molecular variants. These post-translational modifications, such as variations in glycosolation, oxidation, sulfation, phosphorylation, deamidation, and fragmentation may have no effect on product safety or efficacy, and can be considered product related substances. However, a "consistent pattern of product heterogeneity" should be demonstrated from lot to lot.

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Because manufacture and storage can produce additional modifications such as aggregation, methods to profile heterogeneous patterns are required to ensure lot-to-lot consistency of a drug's product and storage stability. The protein's inherent heterogeneity makes "the absolute purity of biotechnological products" difficult to quantify, and "the results are method dependent."² Therefore, quality of the drug product is "usually estimated by a combination of methods."²

Typical product release methods applied to protein therapeutics include reverse-phase chromatography (RPC), ion-exchange chromatography, size-exclusion chromatography (SEC), capillary electrophoresis, peptide mapping by liquid chromatography combined with mass spectrometry (LC-MS), gel electrophoresis, immunoassays, western blot and isoelectric focusing (IEF). Our company has almost all of these in its bio-analytical toolbox.

The Raw API

The API was an approximately 80 kDa protein produced by a bulk-drug contract manufacturer using

Escherichia coli

fermentation followed by several purification steps. The protein was not glycosolated and contained no disulfide bonds.

The client requested that we develop a vial-lyophilized product for toxicology and clinical safety studies. The product development time-line was short — we had only six months from the delivery of the first samples of bulk drug substance to the development laboratory. The client asked that we provide them with a final formulation, a manufacturing process, and validated product release methods.

The protein's properties were virtually unknown. At project initiation, the client could not provide solubility data, stability data, or the protein's isoelectric point. The API contract manufacturer did supply conditions for an RPC method. However, the reverse-phase method did not resolve major degradation products from the main peak and was, therefore, not useful as a stability-indicating assay. Analytical method development was required not only for support of early formulation development, but also for early stability studies, and, of course, for final release assays. Analytical method development occurred concurrently with formulation development.

Preliminary Reference Standard

Because the analytical methods were evolving as rapidly as the formulation, a preliminary reference standard was established and analyzed with formulation samples. The preliminary reference standard was prepared by freezing (-70∞C) a solution of the API in small aliquots. The manufacturer provided the concentration of the API. Because an extinction coefficient was not provided for UV-Vis concentration determination, all concentration estimations were based on HPLC analysis using the preliminary reference standard. When we received an extinction coefficient from the client, UV-Vis was used for concentration determination.

ISOELECTRIC FOCUSING

The first step we took in analytical support of the formulation development was to determine the protein's isoelectric point (pI) by gel IEF. The pI is the solution pH at which a protein carries no net charge. In solutions with moderate salt concentration, the solubility of most proteins is, at a minimum, near their pI.

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An electric field is applied to a gel matrix containing a pH gradient to separate proteins on the basis of their charge. IEF can be one of the most powerful separation methods for evaluating charge differences and can resolve a single charge difference between large protein molecules.

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The first IEF test used materials purchased from Invitrogen because these were available in the laboratory. An API sample was desalted using a 5K molecular-weight-cut-off spin filter. The sample was diluted in pH 3-10 IEF sample buffer and then samples and pI markers were loaded on a pH 3-10 IEF gel. Three major, poorly resolved bands migrated between the pH 5.3 and 6.0 markers.

Because multiple bands were observed, a method for further characterization was required to ensure that formulation and process conditions were not affecting the pattern of charge variants. The IEF bands' sharpness and resolution were improved by using a larger gel on a flat-bed system. A recirculating chiller assured temperature control. Flat-bed IEF was performed using equipment and materials purchased from Amersham Biosciences. An Ampholine PAGEplate pH 3.5-9.5 gel was used, with 1M phosphoric acid as the anode buffer and 1M NaOH as the cathode buffer. After staining with Coomassie blue, the gels were placed on a white-light transilluminator and photographed with the Kodak EDAS 290 system.

Resolution was better. Six bands, which migrated between the pH 5.2 and 5.85 markers, were resolved. Because the protein was not glycosolated and had no disulfide bonds, the charge variants observed by IEF were the major forms of molecular heterogeneity requiring characterization.

IEF was used throughout the development process to compare lots of API, compare API to final product (Figure 1), and determine reconstitution stability under different conditions. IEF required little development time and was an excellent tool for visually comparing samples within one gel, but intermediate precision was poor. We observed changes in the reference standard's band pattern after a new lot of gels was used during the course of the project. Although densitometry could be applied to IEF gels, changes in the band pattern could not be easily quantitated, making IEF a poor choice as a product release method in the quality control laboratories.

Figure 1. IEF of Reference Standard (1), API (2) and Final Product (3).

ION-EXCHANGE CHROMATOGRAPHY

We searched for a chromatographic method to release product and track changes in product stability samples. Ion-exchange chromatography separates proteins on the basis of charge through the interaction of the protein with oppositely charged groups immobilized within a column.

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When the mobile-phase pH is greater than the protein pI, the protein has a net negative charge and binds to positively charged anion-exchange columns. Conversely, when the mobile phase pH is less than the protein pI, the protein will bind to negatively charged groups in a cation-exchange column.

We evaluated both anion-exchange chromatography and cation-exchange chromatography. Because of previous experience in evaluating charge variants of monoclonal antibodies, we analyzed a cation-exchange method using a Dionex weak-cation-exchange (WCX)-10 column with sodium phosphate mobile phase. The protein was eluted with an increasing gradient of sodium chloride in 50 mM sodium phosphate. Mobile phases with pHs from 5 to 6 were analyzed, with the best peak shape obtained at pH 5.3. The main peak tailed considerably and only two additional peaks were observed, both poorly resolved.

Initial work using an anion-exchange method used a Dionex ProPac PA-1 column with 20 mM Tris pH 7.3. The protein was eluted with an increasing gradient of sodium chloride in Tris. The peak shape was significantly improved compared to that of the cation-exchange method, however, charge variants eluting after the main peak were still not well resolved. The same mobile phase (Tris) was used with the Dionex weak-anion-exchange (WAX)-10 column. We observed a greater number of charge variant peaks and improved resolution. The mobile phase pH was adjusted to 8.0, and the gradient was adjusted to improve baseline shape and resolution. The WAX method resulted in resolution of a greater number of charge variants than the cation-exchange methods. Ultimately, seven charge-variant peaks were resolved from the main peak. Most related substances in the WAX method eluted on the backside of the main peak, indicating that they were acid charge variants, most likely the result of deamidation.

STABILITY TESTING

The WAX method was applied during development to evaluate sample stability. Single-excipient formulations were freeze-dried and analyzed after reconstitution. A significant drop in main peak purity was observed in samples freeze-dried with lactose as a bulking agent when compared to other single-excipient formulations and a frozen control. Near the end of development, the WAX method was used to select a suitable buffer pH range for acceptable pharmaceutical processing. Solution samples ranging in pH from 5.99 to 9.63 were stored at room temperature and colder conditions. They were analyzed at 5, 7, and 14 days. A significant increase in acidic charge variants was observed in high pH samples held at room temperature for two weeks (Figure 2). The detection of charge variants indicated that the buffer pH should be kept below pH 8.5 for all process steps, and that if a higher pH buffer was required, the solution should be held at refrigerated temperatures.

Figure 2. WAX Analysis of Solution Stability Study Evaluating Buffer pH. Solution samples were held at room temperature for two weeks. Higher buffer pH resulted in a significant increase in undesirable acidic charge variants eluting after the main peak.

GEL ELECTROPHORESIS

Formulation development could not progress until an initial method to evaluate product quality was available. Although not required as a release method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was the first technique established because of the simplicity of method development. SDS-PAGE separates proteins based on size

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and can be used to detect impurities, truncation, or aggregation. All materials used for SDS-PAGE were purchased from Invitrogen.

SDS-PAGE was used to analyze all early formulation development samples and was invaluable in identifying formulations prone to aggregation. In particular, a significant dimer band was observed in samples freeze-dried with only mannitol and stored at 37∞C for two weeks (Figure 3). Dimer was not observed in frozen controls or frozen solutions of the same formulation. Also, dimer was not observed in samples freeze-dried with only sucrose, or with mixtures of sucrose and mannitol.

Figure 3. SDS-PAGE of Freeze-dried Samples with only Mannitol. A significant amount of dimer was observed in the accelerated stability sample (1) when compared to the sample stored at -20°C (2). The discovery of aggregation in samples containing only mannitol resulted in the subsequent addition of sucrose to the formulation to eliminate the aggregation.

Like IEF, SDS-PAGE required little development time, but it was not an ideal product release method. The technique was useful for comparing the purity of samples within one analysis, but intermediate precision was generally poor. An analysis of the purity results by densitometry was subjective because streaks, bubbles, and background must be excluded from the purity calculation. In addition, the limit of quantitation (LOQ) was high. While not experimentally determined, the LOQ can be estimated from linearity data to be approximately 60 µg/mL or 10 percent aggregates (proteins stuck together but still soluble). Aggregates are undesirable because there are concerns that they can cause immunogenic reactions in patients.

SDS-PAGE was optimized during initial method development. We loaded samples at 0.5, 1, 2, 3, 4, and 5 µg to determine optimum loading conditions. The gel was scanned with a desktop scanner and analyzed by gel analysis software. We then plotted the amount of protein loaded against optical density to determine the linear range of staining, which was from 0.5 to 3µg with a 0.999 coefficient of determination.

SIZE-EXCLUSION CHROMATOGRAPHY

An SEC method was developed for final release testing. SEC separates proteins on the basis of differences in molecular size using a porous matrix packed into a chromatographic column.

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Larger molecules are excluded from pores and are eluted prior to smaller molecules.

While typical freeze-dried samples did not contain detectable dimer, the mannitol-only formulation (Figure 3) was used to produce a dimer-containing sample for SEC method development. Initially, a Zorbax GF450 column was used with 0.2 M sodium phosphate pH 7.0. Samples were diluted with the mobile phase and the method was run isocratically at a flow rate of 1 mL/min. When using the pH 7.0 mobile phase, a dimer peak was not observed. However, when the mobile phase was adjusted to pH 8.0, the dimer peak was visible but not well resolved from the main peak.

To improve resolution, we tried a Phenomenex BioSEP-SEC S-3000 column, and to simplify mobile phase preparation, a commercially prepared phosphate buffered saline (PBS) pH 7.4. The dimer was observed and resolution was significantly improved. The limit of quantitation for protein aggregates was less than 1 percent, which is significantly better than that for SDS-PAGE.

REVERSE-PHASE CHROMATOGRAPHY

A stability-indicating chromatographic method was required to provide product concentration and purity data for further formulation development, product release testing, and stability testing. The RPC potency method initially provided by the bulk manufacturer was evaluated. This method used a Poros R2 column from Applied Biosystems with a NaOH-based mobile phase. Samples with degradation products were prepared by heating a sample of reference standard solution at 37°C for 12 hr. Despite changes in flow rate and gradient, degradation products could not be resolved from the main peak using the bulk manufacturer's conditions.

Because of the large size of the protein, we evaluated a Toso Biosep TSKGel Phenyl 5PW RP 1000A pore size column. We also analyzed mobile phases such as NaOH, sodium phosphate, Tris, 0.1 percent HCl, and 0.1 percent TFA. Optimized method conditions used a mobile phase A of 0.1 percent TFA in water, mobile phase B of 0.1 percent TFA in ACN, and a column temperature of 35°C. The optimized RPC method was capable of partially resolving dimer as well as clipped product (Figure 4).

Figure 4. Reverse-Phase Purity and Potency Method. Samples include a control (1), a sample containing aggregate generated by freeze-drying in only mannitol (2), and a sample of API with a truncation (3).

IDENTITY TESTING BY WESTERN BLOT

FDA specifies that identification procedures be specific for the active ingredient whenever possible.

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Western blotting was selected as an identity method because it identifies the product both by molecular weight and by detection with a product-specific monoclonal antibody.

The western blot used the same SDS-PAGE materials described above, as well as NuPAGE transfer buffer and membranes from Invitrogen. The membranes were immunostained by first incubating them in a blocking buffer of non-fat dry milk in PBS, followed by the primary antibody, followed by a phosphotase-conjugated secondary antibody, followed by a colorimetric substrate, BCIP-NBT.

During method development, both a polyvinyldene fluoride (PVDF) and a nitrocellulose membrane were evaluated with sample loads of 0.1, 0.2, and 0.4 µg. The PVDF membrane had high background staining and was not used in further method development. All of the tested sample loads resulted in acceptable bands, and the 0.2 µg level was used for further testing. The identity method was validated for specificity and transferred to the quality control laboratory for product release testing.

PRODUCT RELEASE ASSAYS

Methods based on seven different techniques were developed for support of formulation development and for validation as product release assays (Table 1). Four were validated and transferred to the quality control laboratory. RPC, SEC, and WAX chromatographic methods were developed and ultimately used for product release in place of SDS-PAGE and IEF because the methods were more robust and results were easier to interpret.

Table 1. Methods Summary

Though not used for final release, electrophoresis was an invaluable first screening tool. Because SDS-PAGE and IEF required minimal development time, they provided the formulation development scientist with immediate methods to assess product quality. The observation of dimer by SDS-PAGE was used to eliminate a destabilizing formulation early in development. IEF, though a more difficult technique, provided the protein's isolectric point and a means of evaluating charge variation that was further investigated by anion-exchange chromatography.

Wendy Saffell-Clemmer is manager of analytical development, Pharmaceutical Research and Development, Baxter Pharmaceutical Solutions LLC, 927 S. Curry Pike, PO Box 3068, Bloomington, IN 47402-3068, 812.355.7105, fax 812.332.3079. wendy_saffell_clemmer@baxter.com

REFERENCES

1. Roth G, Parenteral Sector Watch, Contract Pharma. 2003 November/December. Available at: http://

www.contractpharma.com/NovDec031.htm

2. FDA. CDER. Guidance for Industry, Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. Draft Guidance, 1999 August; Bethesda MD. p. 3.

3. Voet D, Voet J. Biochemistry. John Wiley and Sons, New York. 1990; page 81.

4. Schenerman M. Isoelectric Focusing. The Biopharm Guide to Bioanalytical Methods, Supplement to BioPharm International. 2001 December; p. 29.

5. Williams A, Frasca V. Ion Exchange Chromatography, In Current Protocols in Protein Science. John Wiley and Sons, New York, 1999; p. 8.2.1C.

6. Berger, Electrophoresis. The Biopharm Guide to Bioanalytical Methods, Supplement to BioPharm International. 2001 December; p. 20.

7. Williams A, Frasca V. Gel Filtration Chromatography, In Current Protocols in Protein Science. John Wiley and Sons, New York, 1999; p. 8.3.1.

8. FDA. CDER. Guidance for Industry, Analytical Procedures and Method Validation - Chemistry, Manufacturing, and Controls Documentation. Draft Guidance 2000 August; Bethesda MD, p. 14.

For addresses of the referenced suppliers of analytical materials and instruments, contact the authors.