68 BioProcess International 13(9) OctOber 2015

B low–fill–seal (BFS) technology has been recognized by the industry as an advanced aseptic solution (1–3). Catalent Pharma

Solutions has been commercially supplying sterile BFS products to the pharmaceutical industry for decades, primarily in the respiratory and topical ophthalmic markets. Such product formulations range from simple solutions to emulsions with drug substances from classical small molecules to large complex proteins such as biologics. The company also has optimized BFS processes and its Advasept plastic container system for the manufacture of sterile injectable products.

The Advasept container system provides significant advantages over traditional, commercial, glass container systems for the parenteral/injectable market space. Benefits include reduced container breakage and an absence of glass delamination (4). The automated

aseptic filling design of BFS advanced aseptic technology drastically reduces the risks associated with traditional aseptic manufacturing. BFS forms, fills, and seals vial contents in less than 15 seconds within a closed, class A environment, by which it reduces the risk of contamination from both microbial and foreign particulate sources.

Advasept technology provides customized mold designs that support anticounterfeiting measures, which is particularly advantageous for expensive biologics. Customized mold designs can reduce vial headspace, which can help improve protein stability by lessening product agitation during shipping. Agitation with air has been shown to cause some protein aggregation and precipitation in solution.

Potential compatibility concerns do exist with BFS-processed container systems for biologics. For example, the molded plastic reaches elevated temperatures during filling, which may compromise the stability of biologic formulations and could lead to potential leachables (5–11) from BFS-processed

plastic container systems, which also could affect biologic stability and safety of the formulation. In addition, elevated gas permeation through plastic containers might affect the stability of oxygen-sensitive biologics. All these effects need to be considered during initial container compatibility screening. Catalent has worked to optimize its molded-plastic process to reduce solution temperatures (for 0.5-mL fills) to near 40 °C at the time of product filling. The company’s assumption is that the temperature of a contained solution would fall steadily after units come out of the filling suite and are packaged under ambient conditions (data not provided).

To address those potential compatibility concerns, we conducted a

S U P P L I E R Side

Compatibility Assessment of a Model Monoclonal Antibody Formulation in Glass and Blow–Fill–Seal Plastic VialsDipesh Shah, Gregory T. Bleck, and Ian J. Collins

Product Focus: All biologicals

Process Focus: Drug-product manufacturing, fill–finish

Who should read: Product and process development, QA/QC, formulations, manufacturing

KeyWords: Automation, potency, stability, peptide mapping, and leachables

level: Basic

Table 1: Model monoclonal antibody (MAb) formulation

Component AmountModel MAb (144 kDa) 10 mg/mLPolysorbate 80 0.7 mg/mLSodium Citrate 6.5 mg/mLSodium Chloride 9.0 mg/mLpH 6.5 mg/mL

70 BioProcess International 13(9) OctOber 2015

comprehensive study to evaluate the influence of our optimized BFS process and container system on a model monoclonal antibody (MAb) formulation. Table 1 lists the formulation of this model MAb (molecular weight 166 kDa). We used glass vials with uncoated stoppers as controls for this study. Several analytical methods helped us evaluate

MAb stability and leachables from the container system (Table 2).

Materials and MethodsWe filled the model MAb formulation both in glass and the optimized Advasept BFS container system using a qualified laminar-flow hood to minimize microbial contamination. The MAb formulation was

filtered by a 0.2-µm Nalgene filter unit and filled in glass vials into which uncoated stoppers were placed. In parallel, it was filtered into a sterile bag and shipped to Catalent’s BFS manufacturing facility, where the formulation was aseptically transferred to BFS Advasept stoppered vials. Samples were stored at 5 °C and tested at a series of time intervals using methods described in Table 2.

resultspH: We found no apparent pH change for either glass or BFS-stoppered vials when testing both before and after filling, as well as upon nine months’ storage at 5 °C. Results were within the target range (data not shown).

Appearance: We observed no visible foreign particles for either glass or BFS-stoppered vials when testing both before and after filling, as well as upon nine months of storage at 5 °C. Results were within the target range (data not shown).

Potency: We used ultraviolet absorption (280 nm) and an activity assay to examine potency of the MAb before and after filling, as well as after nine months of storage at 5 °C (Table 1). The UV data indicate no apparent change in potency for both vial formats when tested before and after filling, and after nine months of storage at 5 °C both were within target range.

We also determined potency using a responsive cell line in a complement-dependent cytotoxic assay with a fluorescence read-out. Figure 1 compares a dilution series generated with a standard, the MAb formulation in glass, and the same formulation in an Advasept vial. These activity data show comparable potency values for the formulations in glass and Advasept vials after nine months of storage at 5 °C (all data not included).

Stability: We used five different methods of testing product stability (Table 2). These include size-exclusion chromatographic and nanoparticle tracking analysis, sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary isoelectric focusing, and peptide mapping by ultraperformance liquid chromatography with UV and mass-spectrometric detection.

Table 2: Stability analytical methods

Parameter Analytical Method Target RangepH USP 791 6.5 + 0.3Appearance Visual inspection Clear and colorlessPotency UV absorption (280 nm) T = 0 + 10%

Activity Report EC50

Stability Size-exclusion chromatography (SEC)

Report % monomer and % high and low molecular weights

Nanoparticle tracking analysis (NTA)1

Report submicron particle size analysis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Report % area (heavy chain + light chain)

Capillary isoelectric focusing (cIEF)

pI (% of each peak)

Peptide mapping2 % chemical modificationLeachables3 Polar leachables (HPLC-UV) Report Irganox 1010 levels

Semivolatile leachables (GC-MS) Report aromatic hydrocarbon levelsGC-FID Report volatile leachable levels4

Metals (ICP-MS) Report all metals except Na and IBacterial Endotoxin5 USP 85 Report resultsBioburden5 USP 61 <10 CFU/mL

1 Performed on 12-month samples 2 Performed only at the four-month time point3 Performed only at the nine-month time point4 Isopropyl alcohol, methyl ethyl ketone, trichloroethylene, hexane, 2-methyl pentane, 3-methyl pentane, methylcyclopentane, cyclohexane, and heptane 5 Performed only at 0 time point

Figure 1: Dose-response curve for model MAb formulation

0.001 0.010 0.100 1.000 10.0000

1,000

2,000

3,000

A B C D R2

Advasept 2 (Concentration/Mean Value) 3,574.447 1.618 0.373 247.764 0.996Glass vial 2 (Concentration/Mean Value) 3,511.099 1.729 0.482 325.305 0.998Advasept 1 (Concentration/Mean Value) 3,534.978 1.711 0.391 320.817 0.998Glass vial 1 (Concentration/Mean Value) 3,568.287 1.728 0.460 338.779 0.999

y =

((A −

D) ÷

(1 +

(x ÷

C)B

)) +

D

x

72 BioProcess International 13(9) OctOber 2015

SEC Analysis: All SEC data (pre- and postfill, and upon storage) show comparable percentages of high–molecular-weight (HMW) species for both the glass and Advasept vials (Figures 2–4).

NTA Analysis: As Table 3 shows, we observed slightly larger aggregates in the Advasept vials than in glass. However, the total number of particles are statistically comparable for both container–closure systems (Figures 5–7).

SDS-PAGE Analysis: In Figure 8, lane 5 is a nonreduced sample from a glass vial. Lane 6 is a nonreduced sample from an Advasept vial. In Figure 9, lane 5 is a reduced sample from a glass vial. Lane 6 is a reduced sample from an Advasept

vial. Pre- and postfill and stability SDS-PAGE data show comparable heavy- and light-chain bands for both glass and Advasept vials.

cIEF Analysis: Capillary isoelectric focusing chromatograms at time-point 0 showed no comparable charge distribution between glass and Advasept vials (Figures 10 and 11).

Peptide Mapping: We sampled aged glass and Advasept vials at four months of 5 °C storage. For peptide mapping, we initially denatured and reduced the MAbs with dithiothreitol (DTT, also known as Cleland’s reagent), then alkylated them with iodoacetamide. After clean-up on a chromatography column, MAbs were digested with trypsin and AspN endoproteinase. We separated peptides with a UPLC column and used UV and MS detectors for analysis.

As Figures 12 and 13 illustrate, both glass and Advasept vial samples showed no major difference in UPLC/UV chromatograms from both trypsin and AspN digestion, with 100% sequence coverage by MS detection. Deamidation levels observed with MS are comparable for both glass and Advasept vials (Table 4). We saw higher oxidation levels in glass vials than in Advasept vials, which could be attributed to surface interaction between MAbs and the glass surface causing more oxidation than in plastic Advasept vials.

Leachables: We stored our model MAb formulation samples for nine months at 5 °C before analyzing their contents for volatile, semivolatile, polar, and metal leachables using, respectively, gas chromatography with flame-ionization detection (GC-FID) and mass

Figure 2: Size-exclusion chromatography with ultraviolet detection (SEC-UV) data

Abs

orba

nce

(mA

U)

500

400

300

200

100

0–50

0 2 4 6 8 10 12 14 16 18 20

2: Monomer (8.017)

1: Aggregate (7.183)

Time (minutes)

Figure 4: Percentage of high–molecular-weight species detected over time (5 °C)

2.02.53.03.54.04.55.05.56.0

0 3 6 9

% H

MW

Spe

cies

Months

Glass vial

Advaseptvial

Figure 5: Mean submicron particle-count data (nanoparticle tracking analysis) for MAb formulations stored in glass and Advasept vials for 12 months at 5 °C

0.0

5.0

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

100 200 300 400 500

106 N

TA P

arti

cles

/mL

Size (nm)

Advasept vialGlass vial

Figure 6: Particle size, relative intensity, and concentration — three-dimensional (3-D) graph for glass vial (12 months at 5 °C)

Rela

tive

Inte

nsit

y

Particle Size (100 nm/division)

Figure 7: Particle size, relative intensity, and concentration — 3-D graph for Advasept (12 months at 5 °C)

Rela

tive

Inte

nsit

y

Particle Size (100 nm/division)

Figure 3: Magnified size-exclusion chromatogram

Abs

orba

nce

(mA

U)

500

400

300

200

100

0–50

0 2 4 6 8 10 12 14 16 18 20

2: Monomer (8.017)

1: Aggregate (7.183)

Time (minutes)

Table 3: Nanoparticle tracking data analysis (MAb formulation)

Particles Advasept Vial Glass VialD50 126 + 17.5 94 + 10.1 nmD90 205 + 35 nm 144 + 13.4 nmTotal Concentration 13.95 + 3.2 particles/frame 13.47 + 3.0 particles/frameTotal Concentration 2.83e8 + 6.5 × 107 particles/mL 2.74e8 + 6.1 × 107 particles/mL

spectrometric detection (GC-MS), high-performance liquid chromatography with ultraviolet detection (HPLC-UV), and inductively coupled plasma mass spectrometry (ICP-MS) analytical techniques. The resulting leachables data indicate comparable profiles for MAb formulations stored in glass and Advasept vials — except for isopropyl alcohol. The isopropyl alcohol content was determined to be higher in glass vials (14 µg/mL) than in Advasept vials (1.2 µg/mL). We attribute that to cleaning procedures used for glass vials before filling of the model MAb formulation.

Figure 8: Nonreduced sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

4 5 6 7

Figure 9: Reduced sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

4 5 6 7

Table 4: Percent modification — methionine oxidation and deamidation

Type Sample % ModificationOxidation Glass vial 10.8

Advasept vial

4.5

Deamidation Glass vial 7.2Advasept vial

7.6

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Figure 10: Capillary isoelectric focusing (cIEF) chromatogram, time 0

Resp

onse

Resp

onse

Charge Charge

0.30

0.25

0.20

0.15

0.10

0.05

0.00

–0.05

0.30

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0.00

–0.057.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0

Figure 11: Magnified chromatograms, time 0

Resp

onse

Resp

onse

Charge Charge

0.20

0.15

0.10

0.05

–0.158.75 9.00 9.20 9.40 9.60 9.80 10.0

0.20

0.15

0.10

0.05

–0.158.75 9.00 9.20 9.40 9.60 9.80 10.0

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Figure 12: Magnified ultraperformance liquid chromatography with ultraviolet detection (UPLC-UV) chromatograms of trypsin-digested antibody rightway in a glass vial (bottom trace) and an Advasept vial (top trace)

Abs

orba

nce

(AU

)

Time (minutes)

0.340.320.300.280.260.240.220.200.180.160.140.120.100.080.060.040.200.00

–0.0210 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

Glass vial

Advasept vial

equivalency conFirMedOur results confirm compatibility of a model MAb formulation filled in both glass and Advasept container systems. This demonstrates that the Advasept BFS process and container system is suitable for protein therapeutics. Data from

formulation samples before and after filling indicate that the slightly elevated temperature immediately at filling did not affect key characteristics of the model MAb.

Stability data related to aggregation, chemical degradation, affinity, and

leachables indicate no significant differences between glass and Advasept container systems over the nine-month storage time we have analyzed to date. As demonstrated herein, the optimized BFS process had no impact on stability of a model MAb formulation. Thus, our results demonstrate that BFS is a viable and cost-effective method to produce aseptically filled biologic formulations.

acKnoWledgMentsWe thank Kay Schmidt, Natasha Hults, Bill Hartzel, Gregory Bleck, Ian Collins, Keith Flood, and Thomas Luntz for their advice. This work was supported by Catalent Pharma Solutions.

reFerences1 CBER/CDER/ORA. Appendix 2 Blow-Fill-

Seal Technology. Guidance for Industry: Sterile Drug Products Produced By Aseptic Processing — Current Good Manufacturing Practice. US Food and Drug Administration: Rockville, MD, September 2004; www.fda.gov/downloads/Drugs/.../Guidances/ucm070342.pdf.

2 A Dedicated Environment. Pharmaceut. Manufact. Packing Sourcer May 2011; 96.

w

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3 Goll AW. KB-0025-Jun12: Packaging Equipment — Blow/Fill/Seal (B/F/S) Technology. International Society of Pharmaceutical Engineering: Tampa, FL, June 2012; www.natcotechnologies.com/pdf/lot2/ISPE%20packaging-equipment-blow-fill-seal.pdf.

4 Reynolds G, Paskiet D. Glass Delamination and Breakage: New Answers for a Growing Problem. BioProcess Int. 9(11) 2011: 52–57.

5 Sharma B, et al. Technical Investigations Into the Cause of Increased Incidence of Antibody-Mediated Pure Red Cell Aplasia Associated with Eprex. EJHP May 2004: 86–91.

6 Villabos AP, et al. Interaction of Polysorbate 80 with Erythropoietin: A Case Study in Protein–Surfactant Interactions. Pharm. Res. 22(7) 2005: 1186–1194.

7 Boven K, Bader F, van Regenmortel M. Immunogenicity of Biopharmaceuticals: An Example from Erythropoietin. BioPharm Int. August 2005: 36–52.

8 Sun L. Protein Denaturation Induced By Cyclic Silicone. Biomaterials 18(24) 1997: 1593–1597.

9 Jones L, Kauffman A, Middaugh C. Silicone Oil Induced Aggregation of Proteins. J. Pharmaceut. Sci. 94(4) 2005.

10 Bartzoka V, McDermott M, Brook M. Protein–Silicone Interactions. Adv. Material 11(3) 1999.

11 Markovic I. Challenges Associated with Extractables and/or Leachable Substances in Therapeutic Protein Products. Amer. Pharm. Rev. 9(6) 2006: 20–27. •Corresponding author Dipesh Shah, PhD, is a senior development engineer at Catalent Pharma Solutions, 2210 Lake Shore Drive, Woodstock, IL 60098; 1-815-206–1220. Gregory T. Bleck, PhD, is global head of R&D for Catalent Biologics (gregory.bleck@catalent.

com), and Ian J. Collins is senior director of process development at Catalent Pharma Solutions (ian.j.collins@catalent.com).

For electronic or printed reprints, contact Rhonda Brown of Foster Printing Service, rhondab@fosterprinting.com, 1-866-879-9144 x194. Download personal-use–only PDFs online at www.bioprocessintl.com.

Figure 13: Magnified UPLC/UV chromatograms of ASP-N digested antibody rightway in a glass vial (bottom trace) and an Advasept vial (top trace)

Abs

orba

nce

(AU

)Time (minutes)

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