An integrated process analytical technology (PAT) approach to monitoring the effect of supercooling on lyophilization product and process parameters of model monoclonal antibody formulations.

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

An Integrated Process Analytical Technology (PAT) Approach to Monitoring the Effect of Supercooling on Lyophilization Product and Process Parameters of Model Monoclonal Antibody Formulations DAVID AWOTWE-OTOO, CYRUS AGARABI, MANSOOR A. KHAN Division of Product Quality Research, Office of Testing and Research, OPS, CDER, United States Food and Drug Administration Received 14 February 2014; revised 24 March 2014; accepted 9 April 2014 Published online 19 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24005 ABSTRACT: The aim of the present study was to apply an integrated process analytical technology (PAT) approach to control and monitor the effect of the degree of supercooling on critical process and product parameters of a lyophilization cycle. Two concentrations of a mAb formulation were used as models for lyophilization. ControLyoTM technology was applied to control the onset of ice nucleation, whereas tunable diode laser absorption spectroscopy (TDLAS) was utilized as a noninvasive tool for the inline monitoring of the water vapor concentration and vapor flow velocity in the spool during primary drying. The instantaneous measurements were then used to determine the effect of the degree of supercooling on critical process and product parameters. Controlled nucleation resulted in uniform nucleation at lower degrees of supercooling for both formulations, higher sublimation rates, lower mass transfer resistance, lower product temperatures at the sublimation interface, and shorter primary drying times compared with the conventional shelf-ramped freezing. Controlled nucleation also resulted in lyophilized cakes with more elegant and porous structure with no visible collapse or shrinkage, lower specific surface area, and shorter reconstitution times compared with the uncontrolled nucleation. Uncontrolled nucleation however resulted in lyophilized cakes with relatively lower residual moisture contents compared with controlled nucleation. TDLAS proved to be an efficient tool to determine the endpoint of primary drying. There was good agreement between data obtained from TDLAS-based measurements and SMARTTM technology. ControLyoTM technology and TDLAS showed great potential as PAT tools to achieve enhanced process monitoring and control C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2042–2052, 2014 during lyophilization cycles. Keywords: lyophilization; process analytical technology (PAT); primary drying; secondary drying; ice nucleation; monoclonal antibody; formulation; freeze drying; product temperature; dry layer thickness

INTRODUCTION For some biopharmaceutical and pharmaceutical products such as proteins, peptides, antibiotics, and vaccines, lyophilization represents a more viable alternative formulation strategy to improve stability when the liquid formulation of the product is

Abbreviations used: PAT, process analytical technology; TDLAS, tunable diode laser absorption spectroscopy; RTD, resistance temperature detector; MTM, manometric temperature measurements; MDSC, modulated differential scanning calorimetry; SSA, specific surface area (m2 /g); Tp , product temperature at the ice sublimation interface (◦ C); Rp , resistance of the dried layer in the product (cm2 Torr h/g); dQ/dt, rate of heat transfer (cal/s/vial); Ldry , dry layer thickness (cm); Pice , pressure of ice at the sublimation interface (mTorr); T g , glass transition temperature of the frozen concentrate (◦ C); Tg , glass transition temperature of freeze-dried sample (◦ C); Tp-MTM , product temperature at the ice surface interface determined by MTM (◦ C); Tb-MTM , product temperature at the bottom of the vial determined by MTM (◦ C); Lice , ice thickness (cm); dm/dt, sublimation rate or mass flow (g/s); N, water vapor number density (molecules/cm3 ); u, gas flow velocity (m/s); A, cross-sectional area of the flow duct (cm2 ); Kv , heat transfer coefficient of the vials (cal/s cm2 K); Hs , heat of ice sublimation (670 cal/g); Av , vial cross-sectional area (cm2 ); Ts , shelf surface temperature (◦ C); Tb , product temperature at the bottom-center of the vial (◦ C); m(t), mass of ice sublimed at time, t (g/vial); Di , density of ice (user input: 0.92 g/cm3 ); ε, volume fraction of ice (∼97% for 5% sucrose); KI , thermal conductivity of ice (20.52 cal/h cm K−1 ). Correspondence to: Mansoor A. Khan (Telephone: +301-796-0016; Fax: +301796-9816; E-mail: [email protected]) The findings and conclusions in this article have not been formally disseminated by the United States Food and Drug Administration and should not be construed to represent any agency determination or policy. Journal of Pharmaceutical Sciences, Vol. 103, 2042–2052 (2014)

C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

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not stable. Lyophilization or freeze-drying is a complex, multistep process, which starts with the freezing step, where the aqueous formulation in vials is cooled below its thermodynamic freezing point, causing most of the water to form ice crystals and the solutes to become crystallized or transformed into a solid amorphous system. This is followed by the primary drying step, where the ice crystals are removed by sublimation under vacuum and increased shelf temperature, and the secondary drying step, where most of the unfrozen water still absorbed in the interstitial region is removed by desorption at elevated shelf temperatures and low chamber pressure to allow the desired low moisture content to be achieved.1–3 The complexity of the lyophilization process, with its attendant effect on the final product quality, requires effective monitoring and control systems so as to fully understand the process as well as improve process efficiency. For proteins such as mAbs, freezing and drying could induce different stresses that could impact protein stability during the lyophilization cycle. It is very critical to ensure that the endpoint of all intermediate process steps is reached before the next process step is initiated because a premature transition into a process step could ultimately place high-value biopharmaceutical and pharmaceutical products at risk for loss due to the unanticipated product deviations.4 For example, the identification of a proper endpoint criterion for the primary drying phase before ramping into secondary drying is very critical as all ice must be removed by sublimation (during primary drying) before

Awotwe-Otoo, Agarabi, and Khan, JOURNAL OF PHARMACEUTICAL SCIENCES 103:2042–2052, 2014


desorption starts in order to avoid melt-back. Melt-back compromises product quality and is a criteria for rejection, according to United States Food and Drug Administration (US FDA) inspection guidelines.5 Therefore, there is a need for scientific, risk-based approach to monitoring and control of these critical steps of the lyophilization process in order to improve manufacturing efficiency, production costs, and ultimately gain a thorough understanding of the relationship between lyophilization process parameters and critical quality attributes of the final product. The application of process analytical technology (PAT) in lyophilization, through the use of process analyzers and process control tools, ensures a scientific understanding and control of the manufacturing process through timely measurements (in-line, on-line, or at-line measurements) of critical quality and process attributes to consistently ensure predefined final product quality.6 Lyophilization process optimization had originally focused on the primary drying step as it is the longest step of the entire process. However, with improved process understanding, the freezing step has also been identified as a particularly important step, which must also be monitored and controlled as it impacts both the primary and secondary drying steps.7,8 The temperature at which ice nucleation takes place and the degree of supercooling dictates the ice crystal morphology, which in turn affects critical process parameters and final product quality such as the physical state of the sample,9 residual moisture content,10 and reconstitution time.11 Various methods that allow for the direct control of the freezing step have been proposed, such as ultrasound-controlled nucleation,12 icefog method,13 electro-freezing method,14 and vacuum-induced surface freezing.15 Although such methods have demonstrated potential for process improvement at the laboratory and pilot scales, their applicability to commercial-scale manufacturing of regulated biopharmaceutical products would seem very difficult to achieve because of the economic as well as the sterility and safety concerns. During primary drying, the product temperature at the ice sublimation interface (Tp ) is the most relevant product parameter that must be controlled to ensure a robust cycle. However, Tp cannot be directly controlled but is influenced by the heat input by shelves and chamber pressure.16 Ideally, Tp must be below the “critical” temperature of the product [the glass transition temperature (T g ) for amorphous and eutectic temperature (Teu ) for crystalline products] to avoid macroscopic collapse of the freeze-dried cake as it will negatively affect final product quality such as product appearance, subvisible particles, residual moisture, and reconstitution time.17 Product temperature is also monitored to indicate process endpoints. When product temperatures approach shelf temperatures during freezedrying, it indicates the end of primary drying.18 The accurate measurement, control, and monitoring of product temperature is therefore a critical process analytical need during process development and production. Various monitoring techniques have been proposed to monitor the product temperature, such as the use of thermocouple sensors and electrical resistance temperature detectors (RTD), which are placed directly inside sample vials. However, such monitors only determine the product temperature at the bottom of the vials and not at the sublimation interface, which is more critically related to product macrocollapse. Moreover, the placement of thermocouples is invasive and the information obtained is not representative of the whole batch. It also compromises the sterility of the prodDOI 10.1002/jps.24005

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uct and causes bias in both freezing and drying behaviors relative to vials that do not contain thermocouple. RTD sensors are also not compatible with automatic loading systems during manufacturing scale freeze-drying.19 Various noninvasive PAT tools have been proposed for monitoring critical process and product parameters during the freeze-drying process. De Beer et al.20 proposed the use of Raman and near-infrared (NIR) probes as complementary PAT tools to monitor the lyophilization of mannitol solutions. Raman probes were positioned noninvasively on top of selected vials, whereas NIR probes were placed on the side of the vials to monitor the physical and critical process step changes such as water to ice conversion, product crystallization, kinetics of polymorphic transitions, and solid-state characterization. The major drawback with these PAT tools is that data obtained were representative of selected vials monitored and so intravial as well as intervial variability.4,20 Moreover, both water and ice produce very weak Raman signals, while NIR spectra are typically composed of broad overlapping and poorly defined absorption bands because of the effect of physical properties of the sample such as particle size and density. As a result, spectra obtained from these probes are multivariate in nature and require chemometric analyses to build a robust prediction model in order to extract both physical and chemical information on the samples.4 A more reliable PAT tool based on a batch approach to monitoring critical process and product parameters during lyophilization is tunable diode laser absorption spectroscopy (TDLAS). Based on spectroscopic principles and sensitive detection techniques, TDLAS sensors are capable of inline monitoring of the water vapor concentration and the vapor flow velocity in the spool connecting a freeze-dryer chamber and condenser. The instantaneous measurements can be then used to determine critical product and process parameters such as product temperature, vial heat transfer, product mass transfer resistance, and primary drying end point.3,21 In this study, an integrated PAT approach was applied to control the onset of ice nucleation of the freezing step of a model mAb formulation, using ControLyoTM Nucleation on-demand technology, and the effect of controlled ice nucleation on critical process parameters was monitored by in-line measurement using TDLAS. TDLAS was applied to measure the mass flow rate during the primary and secondary drying steps in order to study the correlation between the degree of supercooling and critical process parameters such as the batch product temperature at the ice sublimation interface (Tp ), the resistance of the dried layer in the product (Rp ), the rate of heat transfer (dQ/dt), the dry layer thickness (Ldry ), and the end point of primary drying. The validity of the TDLAS measurements was evaluated by comparing with another PAT tool called manometric temperature measurement (MTM).

MATERIALS AND METHODS Materials The mAb (IgG1) used in this investigation was generated inhouse from a previously reported cell line.8 The final purified and concentrated antibody was at a concentration of 33 g/L in acetate buffer pH 5.5. Sample purity (>99%) was analyzed by size-exclusion chromatography and gel electrophoresis. Sodium acetate (anhydrous) and American Chemical Society-certified sucrose (crystalline) purchased from Fisher


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Scientific (Pittsburgh, Pennsylvania). Polysorbate 20 was purchased from Sigma Chemical Company (St. Louis, Missouri). All materials were of analytical grade and were used as received. Low-protein-binding polyethersulfone filters (0.22 :m) were purchased from Corning (Corning, New York). Type I glass tubing vials (20 mL), 20 mm FlouroTec “Ready Pack” lyophilization stoppers, and 20 mm flip-top crimp seals were purchased from West Pharmaceutical Services (Lionville, Pennsylvania). All vials, stoppers, and filling accessories were autoclaved prior to use. Methods Formulation and lyophilization Two concentrations of IgG1 formulations, 1 and 20 mg/mL, were prepared by adding appropriate amounts of the stock mAb in acetate buffer, sucrose (170 mM) and polysorbate 20 (0.01%, w/v). All formulations were filtered and 5 mL of bulk solution filled into the vials under a laminar flow hood. Vials were partially stoppered and placed on the shelf of the lyophilizer. For lyophilization cycles using controlled ice nucleation, vials were cooled from room temperature to 5◦ C at a ramp rate of 1◦ C/min and held for 30 min. ControLyoTM , a fully integrated technology, enabled the pressurization of the chamber with argon gas to approximately 28 psig after the hold step.8 The temperature was then reduced to −5◦ C at a ramp rate of 1◦ C/min and held at this temperature for 30 min to achieve vial to vial temperature homogeneity, after which the chamber was rapidly depressurized to approximately 1 psig. The temperature was held at −5◦ C for 20 min to promote ice crystal growth and then ramped down to −42◦ C at 1◦ C/min and held for 120 min to complete the freezing step. For lyophilization cycles with uncontrolled nucleation, vials were cooled from room temperature to 5◦ C at a ramp rate of 1◦ C/min and held at that temperature for 30 min. The vials were then cooled to −5◦ C (ramp rate of 1◦ C/min) and held for 30 min to achieve approximate temperature equilibration in all samples. Samples were then held for a further 20 min, as in the controlled nucleation step (but without pressurization and depressurization with argon gas) and then ramped to −42◦ C at 1◦ C/min and held for 120 min before primary drying was started. In both controlled and uncontrolled nucleation cycles, primary drying was conducted at a chamber pressure of 60 mTorr and shelf temperatures for freeze-drying were determined by SMARTTM based on user-defined inputs. Secondary drying was carried out at a temperature of 35◦ C for 8 h at a ramp rate of 0.1◦ C/min. The lyophilization cycle for each concentration was carried out in duplicate. For each concentration, the first experiment involved the use of SMARTTM technology to optimize the cycle, whereas the second experiment involved the use of TDLAS to measure the product and process parameters. The parameters measured by SMART were then compared with those obtained by the TDLAS under the same lyophilization conditions. Following each cycle, vials were automatically stoppered in the lyophilizer at a temperature and back fill pressure of 5◦ C and 600 Torr, respectively, crimped and stored at 5◦ C until further analysis.

equipped with SMARTTM technology and ControLyoTM Nucleation on-demand technology (Praxair Inc., Danbury, Connecticut) and a LyoFluxTM 200 TDLAS mass flow monitor (Physical Sciences, Inc., Andover, Massachusetts). SMARTTM technology enabled MTM to be performed at 60 min intervals during primary drying, where pressure rise data were collected at 25 s per measurement by quickly isolating the chamber of the lyophilizer from the condenser and analyzing the pressure rise during this period. The pressure rise data were then utilized by an algorithm to directly determine both the pressure at the sublimation interface (Pice ) and the mass transfer resistance (Rp ). Based on Pice and Rp and other userdefined inputs such as the nature of the product (small molecule or protein, amorphous or crystalline), fill volume, fill depth, number of vials, and glass transition temperature of the freezedried concentrate (T g ), product and process attributes such as the product temperatures at the ice surface interface (Tp-MTM ), product temperature at the bottom of the vial (Tb-MTM ), heat transfer into the product (dQ/dt), actual ice thickness (Lice ), dry layer thickness (Ldry ), and sublimation rate (dm/dt) as a function of time are instantaneously calculated by the SMARTTM software using algorithms based on basic steady-state heat and mass transfer theory.19,22 ControLyoTM nucleation on-demand technology works by rapid pressurization and depressurization of the freeze-dryer chamber with an inert gas such as argon, to induce instantaneous and uniform nucleation during the freezing step.8 After vials were cooled from room temperature to 5◦ C and held at that temperature for 30 min to allow all vials to equilibrate to that temperature, the freeze-dryer chamber was pressurized with argon gas to approximately 28 psig. The vials were then cooled to −5◦ C and held at this temperature for 30 min to achieve vial to vial temperature homogeneity, after which the chamber was rapidly depressurized to approximately 1 psig to induce ice nucleation. Tunable diode laser absorption spectroscopy is an in-line spectroscopy-based technique based on absorption of energy by gas molecules at specific wavelength in the electromagnetic spectrum. The duct connecting the product chamber and the condenser was fitted with a fiber optic collimator transmitter and photodiode receiver. During primary drying, NIR diode laser beam was launched across the duct at a 45◦ angle to the gas flow axis through an antireflection coated window. The diode laser beam determines the water absorption line shape in the spool and the transmitted beam is detected by detected by the photodiode detector and the photocurrent signal transmitted to the LyoFluxTM 200 sensor control unit. The detector was continuously purged with extra dry nitrogen to avoid condensation of humidity. Water vapor concentration (N) was directly measured by integrating the water absorption line shape. The peak of the absorption spectrum is shifted relative to the original spectrum proportional to the gas flow velocity due to the Doppler shift. The gas flow velocity was determined by measuring the Doppler shift in the frequency wavelength position of the water absorption spectrum as compared with a spectrum simultaneously recorded with the same sensor in a sealed reference absorption cell.21 The mass flow (dm/dt) was then calculated using the equation;

Freeze-dryer and TDLAS setup Experiments were performed using a laboratory scale freezedryer, LyoStar 3 (FTS Systems, Stone Ridge, New York) Awotwe-Otoo, Agarabi, and Khan, JOURNAL OF PHARMACEUTICAL SCIENCES 103:2042–2052, 2014

dm = N× u× A dt

(1) DOI 10.1002/jps.24005


where N (molecules/cm3 ) is the water vapor number density, u (m/s) is the gas flow velocity, and A (cm2 ) is the cross-sectional area of the flow duct. Determination of vial heat transfer coefficient (Kv ) The heat transfer coefficient (Kv ) for the vials used in this experiment was determined by lyophilizing vials (Wheaton 20 mL vials, 112 vials) filled with 5 mL of pure water. The weight of each individual product vial was determined empty, after filling with 5 mL pure water and after lyophilizing for some time to determine the total amount of water sublimed. A row of “dummy vials” was placed around the filled vials to shield them from radiation effects. The weighed vials were then placed on the shelf of the free-dryer and eight calibrated, fine wire thermocouples (0.1 mm diameter; Omega Engineering, Stamford, Connecticut) were placed in the vials to measure the product temperature during the runs. To determine the batch Kv , two thermocouples were placed in the center vials, two in the middle on both sides, two in front, and two in the vials at the back. The shelf surface temperature was also determined by placing two adhesive thermocouples close to the fluid inlet and outlet. The vials were then cooled at the rate of 1◦ C/min to −42◦ C and held at that temperature for 120 min for product temperature equilibration. After the freezing step, the chamber was evacuated to 60 mTorr. After the desired chamber pressure was achieved and controlled, a zero velocity offset was determined by closing the isolation valve between the chamber and the condenser to ensure zero flow condition and the Doppler shift between the two water absorption line shapes during the zero flow condition was determined and subsequently subtracted from all velocity measurements during freeze-drying. The isolation valve was then opened after the zero velocity offset determination and the shelf temperature ramped to −33◦ C for sublimation to start. Shelf temperature set-point of −33◦ C was chosen to mimic the actual formulation conditions where T g of −33◦ C was determined by analyses of sucrose solution by modulated differential scanning calorimetry (MDSC) and also confirmed by SMARTTM experiments carried out prior to the use of TDLAS. Shelf and product temperatures were monitored and recorded for about 16 h, when about 50% of the total mass of ice had been removed. The lyophilization process was then stopped and the vials were warmed inside the freeze-dyer for about 20 min at room temperature and atmospheric pressure to remove condensation and ice buildup on the outside of the vials. The vials were then reweighed and the total mass of water removed was determined by deducting the remainder of the product from the initial net weight. The average thermocouple product temperature and the integrated mass flow measurements by TDLAS over the entire run were used to calculate the Kv (cal s−1 cm−1 K−1 ) using the equation;

Kv =

dm/dt × Hs Av × (Ts − Tb )

(2)

where dm/dt is the mass flow (g/s), Hs is the heat of sublimation of ice (670 cal/g), Av is the vial cross-sectional area (cm2 ), Ts is the shelf surface temperature, and Tb is the product temperature at the bottom-center of the vial (◦ C). DOI 10.1002/jps.24005

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Characterization of Prelyophilized and Lyophilized Formulations Modulated differential scanning calorimetry Glass transition temperatures of the prelyophilized formulations (T g ) and the lyophilized samples (Tg ) were determined using a DSC Q2000 (TA Instruments, New Castle, Delaware) equipped with an autosampler and refrigerated cooling system. For liquid prelyophilized formulations, about 20 :L of the formulations was placed in aluminum pans that were hermetically sealed and MDSC was performed by applying modulated amplitude of 0.5◦ C every 40 s and a ramp rate of 5◦ C/min from −70◦ C to 25◦ C. The reversing heat flow was analyzed for glass transitions, whereas the nonreversing heat flow was analyzed for the melting peak of ice. For lyophilized samples, about 5 mg of the powdered samples were placed in the pans and hermetically sealed. MDSC was then carried out at modulated amplitude of 0.5◦ C every 40 s and a ramp rate of 5◦ C/min from 25◦ C to 150◦ C. Experiments were conducted in triplicate. In all the experiments, empty aluminum pan crimped in the same manner as the sample pans was used as reference. Data were analyzed using TA instrument Universal analysis 2000 software. Moisture content determination The residual moisture in the lyophilized samples was determined using a Mettler DL38 Karl Fisher titrator (Mettler Toledo, Greifensee, Switzerland) with anhydrous methanol as the sample solvent. About 75 mg of pulverized cake was dispersed in anhydrous methanol and titrated with Riedel-de Haen Hydranal Composite 2 reagent (Hoechst Celanese Corporation, Dallas, TX) until the endpoint was reached. Experiments were carried out in triplicate and expressed as mean ± SD. Specific surface area measurements The specific surface area (SSA) (m2 /g) of the freeze-dried samples was determined by the multipoint Brunauer–Emmett– Teller (BET) method from the adsorption of nitrogen gas at 77 K using a Nova 2200e Surface Area and Pore Analyzer (Quantachrome Instruments, Boynton Beach, Florida). All samples were weighed and loaded into 9-mm cells and outgassed at 40◦ C for 12 h by flowing helium through the sample. A six-point BET adsorption (with P/Po ranging from 0.05 to 0.3) was carried out by dipping the sample holder in liquid nitrogen to reduce the temperature to liquid nitrogen (∼77 K). Experiments were carried out in duplicate and expressed as mean ± SD.

RESULTS AND DISCUSSION Determination of Vial Heat Transfer Coefficient (Kv ) The Kv for a given vial is defined as the ratio of the areanormalized heat flow to the temperature difference between the heat source and the heat sink. The value of Kv has been found to vary between different types of vials and may even be different for the same type of vials (size and geometry) by different suppliers.23 In order to obtain a batch average Kv for the vials used in the experiments, the gravimetric method was used, as described in Methods section. When vials are placed directly on a lyophilizer shelf, the Kv is affected by heat transfer from direct conduction from the shelf to the vial bottom (Kc ), radiative heat transfer (Kr ), and the heat transfer from gas conduction


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Figure 1. Thermocouple product temperature profiles showing the nucleation behavior during the freezing step for (a) controlled ice nucleation and (b) uncontrolled ice nucleation. Uniform ice nucleation was achieved for controlled nucleation at −2.7◦ C, whereas for uncontrolled nucleation, ice nucleation was random, over a wider temperature range.

Figure 2. TDLAS process water vapor concentration profiles for controlled and uncontrolled nucleation cycles for (a) 1 mg/mL and (b) 20 mg/mL mAb formulations during lyophilization. TDLAS measurements showed that the water vapor concentration for the controlled nucleation cycles decreased faster than uncontrolled nucleation cycles because of the increased mass flow through the duct.

(Kg ). Whereas Kc and Kr are independent of chamber pressure, Kg has been found to increase with chamber pressure.24 In this experiment, Kv determination was carried out under a constant pressure to mimic the conditions for our mAb formulations. The average/batch Kv was calculated over the entire sublimation time using Eq. (2). Based on the measured vial cross-sectional area of 7.10 cm2 (20 mL Wheaton vials, outer diameter = 30.06 mm) and data obtained by TDLAS and thermocouples from the steady-state mass flow and product temperatures as well as shelf temperatures, the average Kv was found to be 3.91 ± 0.21 × 104 cal/s cm2 K. The combination of TDLAS mass flow rate measurement and the determined Kv were employed for the noninvasive, real-time determination of product temperatures at the ice sublimation interface (Tp-TDLAS ). Effect of Degree of Supercooling on Process and Product Parameters A noninvasive technique was applied to control the onset of ice nucleation by rapidly pressurizing and depressurizing the lyophilizer chamber with argon gas during the freezing step of the lyophilization cycle. When a solution is cooled during the freezing step of a lyophilization cycle, it usually undergoes a considerable degree of supercooling, before the first ice crystal forms. The onset of ice nucleation is a random event and is affected by solution properties and other external factors such as impurities and the presence of probes, which act as nucleation sites. The degree of supercooling is the difference between the ice nucleation temperature and the equilibrium freezing point

of the solution.1 When controlled ice nucleation was applied during the freezing step, uniform ice nucleation was achieved at −2.7◦ C. Thermocouple probes inserted into selected vials at various locations showed that all the vials nucleated simultaneously within a narrow temperature range (Fig. 1a). On the other hand, when the conventional shelf-ramping method was applied to the freezing step, onset of ice nucleation was observed to be random, ranging from between −10◦ C and −15.2◦ C (Fig. 1b). The exact mechanism by which uniform nucleation is achieved by controlled nucleation remains speculative. However, it is believed that, during the pressurization and rapid depressurization with argon gas, the gas undergoes expansion and cooling in the chamber. It is the cold gas that contacts the surface of the metastable liquid in the vials and induces nucleation.25 Differences in the degree of supercooling have been reported to significantly influence product and process parameters of the final lyophilized products.8 During primary drying, the temperature difference between the shelf and the product vials resulted in the sublimation of water from the product chamber to the condenser through the spool piece. TDLAS was applied as a noninvasive, in-line spectroscopic tool to monitor and measure the water vapor concentration and vapor flow velocity in the spool connecting the freeze-dryer chamber and the condenser. As shown in Figure 2, the water concentration for the two formulations were essentially the same at the start of primary drying until at different points during the cycles when the water concentrations began to decrease. This is depicted by the steep part of the curve. At this point, it is believed that sublimation of ice is almost


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Figure 3. TDLAS sublimation rate profiles during lyophilization of cycles for (a) 1 mg/mL and (b) 20 mg/mL formulations using controlled and uncontrolled nucleation. Controlled nucleation resulted in increased sublimation rate because of the ice nucleation at a lower degree of supercooling.

over and secondary drying is about to begin. Controlled ice nucleation cycles took shorter times to ramp into secondary drying compared with the uncontrolled nucleation cycles. These observed differences could be explained by the differences in their mass flow rates. When the onset of nucleation occurs at a lower degree of supercooling, it results in the formation of larger ice crystals. During sublimation, the larger pores in the solute matrix previously occupied by ice act as channels for the sublimation of ice beneath the dried layer. As larger pores offer minimal resistance, there was increased sublimation rate for the controlled nucleation cycles.26 This was in contrast with uncontrolled nucleation, where the onset of nucleation occurred at a higher degree of supercooling and resulted in smaller ice crystals and therefore a slower mass flow during primary drying (Fig. 3). For conservative freeze-drying cycles, the sublimation rate is influenced by the product resistance (Rp ) and the product temperature at the sublimation interface (Tp ). Ideally, Rp increases as primary drying proceeds.27 As sublimation of ice proceeds during primary drying, a dried product layer above the ice is produced and this acts as a barrier to the transport of water vapor. The water vapor which sublimes at the sublimation front needs to diffuse through the network of small pores in the dried matrix.28 The magnitude of the resistance to vapor transport is dependent on the size of the ice crystals formed during the freezing step. Bigger ice crystals, which are formed as a result of controlled ice nucleation, offer less resistance to vapor transport while smaller ice crystals, formed as a result of uncontrolled nucleation, offer more resistance to vapor flow and thus increase the drying time. The Rp therefore varies with the freezing history of the product. Rp is related to the sublimation rate (dm/dt) by the equation; Rp = Ap ×

Pice − Pc dm dt

(3)

The dry layer thickness (Ldry ) at any time during the primary drying is calculated using the equation; Ldry =

DOI 10.1002/jps.24005

m (t) Di Ap g

(4)

where m(t) is the mass of ice sublimed at time t (g/vial), ρ i is the density of ice (user input: 0.92 g/cm3 ), and ε is the volume fraction of ice (∼97% for 5% sucrose).26 The Rp versus dry layer thickness (Ldry ) profile for the two different concentrations showed nonzero intercepts at Ldry = 0 cm and initial increase in product resistance with Ldry until a certain point where the Rp is independent of Ldry and almost assumes a plateau (Fig. 4). Ideally, the value of Rp should increase with the increase in the dry layer thickness because of the increased resistance to vapor flow through the dried matrix. However, sometimes, the Rp becomes independent of the dry layer thickness during primary drying because of the properties of the formulation. Overcashier et al.29 reported that when Rp decreases or remains constant as dry layer thickness increases, it is indicative of microcollapse during primary drying. The observed phenomenon in our study is consistent with results obtained for formulations containing amorphous bulking agents such as sucrose. Sucrose-containing formulations are reported to undergo microcollapse in their dry layer as primary drying progresses, even when lyophilized at shelf temperatures below their collapse temperature.30,31 When microcollapse occurs during primary drying, the basic pore structure of the dry layer remains intact and the lyophilized cakes show little or no shrinkage. This is different from macrocollapse, where there is significant loss of pore structure and the shrunken appearance of the lyophilized cakes lead to significant loss of elegance.32 Rp data obtained from TDLAS were compared with MTM data to determine if they were in agreement. It was observed that in both formulations, the Rp-MTM were lower than Rp-TDLAS for controlled nucleation cycles, whereas for the uncontrolled nucleation cycles, Rp-MTM showed an increase at Ldry = 0.20 cm before decreasing with increasing Ldry . The differences in the TDLAS and MTM Rp values for both controlled and uncontrolled nucleation cycles may be attributed to the differences in the product temperatures at the sublimation interface. Although the product temperatures measured by both TDLAS and MTM represent a batch average, it is reported that the product temperature measured by MTM is heavily weighted in favor of the coldest vials, so that where there is freeze-drying heterogeneity as a result of some vials drying earlier than other vials, a few low or high resistance sample vials can dramatically alter the Rp .26 This would be especially more pronounced in


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Figure 4. Product resistance (Rp ) as a function of dry layer thickness (Ldry ) for uncontrolled and controlled nucleation cycles during primary drying for (a) 1 mg/mL and (b) 20 mg/mL mAb formulations. TDLAS data for Rp were compared with Rp values determined by MTM. Although for controlled nucleation cycles, MTM values were lower than TDLAS, for uncontrolled nucleation, MTM values were initially higher at Ldry = 0.20 cm before leveling off with TDLAS values.

Figure 5. Product temperature at the sublimation interface (Tp ) and shelf temperature profiles for controlled and uncontrolled nucleation cycles for (a) 1 mg/mL and (b) 20 mg/mL mAb formulations during lyophilization. TDLAS—determined Tp were compared with MTM determined Tp . Whereas TDLAS was able to determine the product temperatures during the entire lyophilization process, MTM data were accurate for up to only two-thirds of the lyophilization process.

cycles where the ice nucleation step is not controlled and small ice crystals are formed. Particularly for the uncontrolled nucleation cycles, Rp-MTM values were significantly higher at Ldry values between 0.20 and 0.36 cm before decreasing indicating a momentary loss of pressure or microcollapse during drying. The product temperature at the sublimation interface (Tp ) cannot be directly controlled during primary drying but is dependent on the on factors such as the properties of the formulation, the container/vial used, shelf temperature, and chamber pressure.16 It is related to the heat flow by the steady-state equation; Tp−TDLAS = Tb −

(dQ/dt) × Lice Av × K I

(5)

where Tb is the product temperature at the bottom of the vial (usually determined by placing thermocouples in the bottom center of the vials), Lice is the ice thickness, which is the fill depth divided by the ice density, dQ/dt is the heat flow from the shelf to the product, and KI is the thermal conductivity of ice (20.52 cal/h cm K−1 ). The heat flow (dQ/dt) is the product

of the mass flow (dm/dt) and the heat of ice sublimation (Hs ). Product temperatures measured by TDLAS showed differences between the controlled and uncontrolled nucleation cycles for the two formulations due to the differences in the degree of supercooling (Fig. 5). In both cycles, the Tp was consistently lower than the critical temperature (T g ) of the formulations during primary drying and reached a plateau for a period of time until it gradually increased toward the shelf temperature as the amount of water vapor in the chamber decreased. Moreover, the Tp for controlled nucleation cycles were lower than Tp for the uncontrolled nucleation cycles. This could be attributed to the greater endothermic sublimation of water from the surface of the ice because of the relatively smaller product dry layer resistance (Rp ) compared with the uncontrolled nucleation cycles. For the 1 mg/mL formulation, the product temperature for the controlled nucleation cycle remained steady at −35.4◦ C for about 15 h, whereas Tp for the uncontrolled nucleation cycle remained steady at −34.6◦ C for about 25 h during primary drying before increasing toward the shelf temperature. For the 20 mg/mL formulation, Tp for the controlled nucleation cycle remained steady at −33.5◦ C for about 22 h, whereas Tp for the


DOI 10.1002/jps.24005


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Figure 6. Primary drying endpoint determination using (a) process water concentration determined by TDLAS and capacitance manometer/pirani gauge readings and (b) process water vapor concentration and product temperature and shelf temperature profiles during primary drying. In both cases, there were close agreement in determining the endpoint of primary drying, where the mid-point of the process water concentration coincided with the decrease in the pirani gauge reading toward the capacitance manometer and the shelf temperature

uncontrolled cycle remained steady at −32◦ C for close to 30 h before increasing toward the shelf temperature. The product temperatures, as determined by TDLAS could therefore be used to determine the end of primary drying. As the product temperature at the sublimation interface approaches the shelf temperature, it signifies that primary drying is almost over and it is beginning to ramp into secondary drying. The accurate measurement of product temperature during primary drying is therefore a critical process analytical need. Tp data from TDLAS were compared with MTM data and it was observed that MTM values were consistently lower than those from TDLAS. While the product temperatures determined by both TDLAS and MTM are representative of the batch, it is reported that the product temperatures determined by MTM represent the coldest vials of the batch because during the closure of the isolation valve for MTM measurements, ice sublimation continues until the chamber pressure reaches the lowest vapor pressure of ice within the product vials. During this period, additional water vapor from the warmer edge vials condense on the colder center vials, which is not factored into the MTM calculation.16 Also, MTM data for Tp were available only for about two-thirds of the primary drying stage. Toward the end of primary drying, there were significant deviations and the MTM values for Tp became progressively lower and inaccurate because of a decrease in the total area of the sublimation interface. This is because the heterogeneity in drying during primary drying causes some vials (typically edge vials) to dry earlier than others. This decrease in the number of vials is not accounted for in the pressure rise data or in the MTM equation, leading to the deviations.31 Determination of Primary Drying End Point From product quality standpoint, as well as process control and optimization, it is very critical that the end point of all intermediate process steps be reached before the next process step is initiated. During lyophilization of proteins, the premature progression from primary drying into secondary drying, when the amount of unfrozen water in the amorphous matrix is more than 20% could result in product collapse, melt-back, lack of DOI 10.1002/jps.24005

cake elegance, and ultimately batch rejection.4 Traditionally, thermocouples placed in vials were used to determine the end point of primary drying. Here, a sharp increase in the product temperature close to the shelf temperature is indicative of the ice sublimation front receding from the thermocouple probes and signifying the end of primary drying.18 Apart from compromising sterility, these thermocouples, commonly placed in edge vials in manufacturing processes, cause products to nucleate at higher temperatures also dry faster than other vials without thermocouples, especially center vials, because of radiation effects. As a result, product temperatures from thermocouples may be seriously biased. Because TDLAS works on the spectroscopic principle of measuring the absorption of radiation by water vapor to monitor the trace concentration of water vapor in real time, the water vapor concentration profiles were used to determine the end point of primary drying. Here, the endpoint is the point where all ice has been removed and gas composition in the chamber was changing and that sublimation was almost complete. This is observed as a sharp drop in the water concentration (Fig. 6). It is reported that for an amorphous system, such as our sucrosecontaining formulations, the residual moisture was less than 10% at the midpoint of the drop.18 As a result, the midpoint can be used as an effective indication of the end of primary drying. This midpoint was almost identical to the point where the product temperature determined by TDLAS meets the shelf temperature. Thus, both batch product temperature and water vapor concentration profiles could be used to indicate the end of primary drying. Primary drying endpoints were different for the two nucleation cycles, as indicated by the midpoints of the water vapor concentration and product temperature profiles. Controlled nucleation cycles had shorter primary drying times compared with uncontrolled nucleation cycles (Table 1). The water vapor concentration profiles were compared with the comparative pressure measurements obtained by the pirani gauge and capacitance manometer of the lyophilizer and the product temperature determined by TDLAS. In both cycles, there were agreements in predicting the endpoint of primary drying (Fig. 6). During primary drying, the lyophilizer chamber


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Table 1. Primary Drying Endpoint Determination by Process Analytical Technology Using TDLAS and SMARTTM Technology Freezing Method

Controlled nucleation Uncontrolled nucleation

Concentration (mg/mL)

1 20 1 20

Primary Drying Endpoint (h) Process (H2 O) from TDLAS

Product Temp (TDLAS)

Capacitance Manometer/Pirani Gauge

30.55 34.19 38.45 40.79

30.58 32.63 38.69 39.80

31.85 33.32 39.65 40.70

pressure is controlled by the capacitance manometer, which measures the absolute pressure in the drying chamber, whereas the pirani gauge measures the thermal conductivity of gas. As a result during primary drying, when the gas present in the chamber is essentially water vapor, the pirani gauge reading is about 60% higher than the capacitance manometer reading. For our study, primary drying was conducted at 60 mTorr so the capacitance manometer reading was 60 mTorr, whereas the pirani gauge reading was 96 mTorr during primary drying. The higher pirani gauge reading was because the thermal conductivity of water vapor is ∼1.6 times higher than that of nitrogen gas.18 As primary drying proceeds, the water vapor concentration in the chamber gradually decreases, whereas the amount of nitrogen gas increases. The point where the pirani gauge reading begins to decrease sharply indicates that primary drying is almost complete. Primary drying endpoints were observed to be different for the two freezing methods, with controlled nucleation cycles having shorter primary drying times than uncontrolled nucleation cycles (Table 1) Effect of Degree of Supercooling on Product Quality The degree of supercooling directly influences the size and shape of the ice crystals formed during freezing and this also affects the primary drying time and the entire lyophilization cycle. Intervial heterogeneity is a consequence of the random nature of the ice nucleation step. When all vials have the same ice nucleation temperature, then their drying will be similar and result in products with improved batch homogeneity. However, when all vials in a batch nucleate at different temperatures, then there is great variability in process performance as well as product quality.12 Bursac et al.33 have reported that controlled nucleation improved batch homogeneity with a 60% decrease in standard variations between vials. The ice crystal properties also impact several quality attributes of the final lyophilized cakes such as cake elegance, residual moisture content, and reconstitution time of the lyophilized products. However, it must be pointed out that quality attributes and process performance in the lyophilization of a biopharmaceutical are not influenced only by process conditions, but by other factors such as the formulation composition, fill volume, and fill depth or properties of the glass vials. In this study, these factors were kept constant. Both controlled and uncontrolled nucleation cycles resulted in acceptable white cakes with no visible collapse or melt-back. All lyophilized cakes from the controlled nucleation cycles ap-

Figure 7. Lyophilized cakes obtained by (a) controlled nucleation appeared more porous with no visible collapse or shrinkage, whereas lyophilized cakes from (b) uncontrolled nucleation were more compact with visible shrinkage at the bottom.

peared porous, less compact, and samples retained solid matrix at roughly the same volume as the initial liquid fill, whereas cakes from the uncontrolled nucleation cycle appeared rigid and compact with slight shrinkage at the bottom of the vials (Fig. 7). This observation was consistent for all the lyophilized vials in the batch. This is also in agreement with other observations, which state that controlled nucleation of a 5% sucrose solution resulted in increased pore diameter of 120 :m compared with 50 :m for the same solution using the conventional shelf-ramped freezing.33 An inverse relationship has been reported between the porosity and the SSA of a freeze-dried cake.26 Uncontrolled nucleation resulted in more than a twofold increase in the SSA compared with controlled nucleation cycles (Table 2). The SSA is an important attribute as it affects not only the primary and drying times but also the long-term stability of the lyophilized protein. Controlled nucleation, which resulted in the formation of larger ice crystals significantly decreased the primary drying time as a result of increased sublimation, however, the rate of desorption was limited during the secondary drying. As a result, the residual moisture contents were significantly higher in controlled nucleation samples than in the uncontrolled nucleation samples (p ≤ 0.05). The residual moisture content of the lyophilized product after lyophilization is an important quality attribute as it affects the long-term stability of the final product. Higher residual moisture would often result in product melt-back. Because water acts as a plasticizer, the presence of high residual moisture could increase the glass transition temperature (Tg ) of the product and enhance the mobility of reactants in the solid and accelerate known degradation pathways such as oxidation, peptide bond hydrolysis, and aggregation.34 Both controlled and uncontrolled nucleation cycles resulted in lyophilized cakes with residual moisture contents less than 1%, with controlled nucleation samples having slightly higher moisture contents than uncontrolled nucleation samples (Table 2). For lyophilized proteins, it is desirable that their reconstitution times be short, for practical clinical use. It is proposed that changes that allow more efficient water vapor transport during primary drying may improve wettability of the porous cakes.35 However, very limited literature is available to correlate the degree of supercooling with the reconstitution time. In an earlier report, even though controlled nucleation resulted in reduced reconstitution times than uncontrolled


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Table 2. Reconstitution Times, Residual Moisture Content, and SSA Measurements for Two Concentrations of mAb Formulation Whose Freezing Step was Varied by Controlled or Uncontrolled Nucleation Concentration (mg/mL) Controlled nucleation Uncontrolled nucleation

*

1 20 1 20

Reconstitution Time (s) 50 85 65 110

± ± ± ±

Residual Moisture (%)

5 7 4* 15*

0.77 0.75 0.65 0.68

± ± ± ±

0.05* 0.02* 0.05 0.07

SSA (m2 /g) 0.37 0.43 0.96 1.23

± ± ± ±

0.01 0.05 0.04* 0.18*

Means t-test shows statistical significance and p ≤ 0.05.

nucleation for a low-concentration mAb formulation, the difference was not statistically significant (p > 0.05).8 In this study, there were significant differences in the reconstitution times for both the 1 and 20 mg/mL formulations. In both concentrations, controlled nucleation resulted in significantly lower reconstitution times compared with uncontrolled nucleation (p < 0.05). Several factors can influence the reconstitution properties of a lyophilized product, such as the cake morphology, surface area of the cake, presence of cake collapse or melt-back, the homogeneity of the dry matrix, formation of channels between pores, and the physical solid state of the lyophilized product.36

CONCLUSIONS This study evaluated the feasibility of an integrated approach to monitoring and controlling the freezing and primary drying steps of lyophilization cycles for two concentrations of mAb formulations. ControLyoTM technology, which involved the pressurization and depressurization of the lyohilization chamber during the freezing step, was applied to control the onset of ice nucleation, whereas TDLAS was used as a noninvasive tool for the in-line and real-time monitoring of the primary drying process. The random nature of ice nucleation is a big challenge to process control as it results in vial-to-vial and batch-to-batch heterogeneity. With uncontrolled freezing, ice nucleation could be achieved as low as −30◦ C. By applying ControLyoTM as a PAT tool, the onset of ice nucleation was achieved at a lower degree of supercooling, which shortened the primary drying time significantly and also impacted other process and product parameters. The ability to control the onset of ice nucleation during lyophilization is a step toward a better understanding of the lyophilization process and its resulting impact on final product quality perfectly fits in the quality by design approach. TDLAS also proved to be a very reliable PAT tool for in-line monitoring of the primary drying process as this technique was able to noninvasively monitor the batch product temperature at the sublimation interface, the sublimation rate, the mass resistance, and most importantly was able to determine the endpoint of the primary drying step. TDLAS was also able to measure the differences in critical process parameters between freezing cycles that were controlled or not controlled. The application of multiple process analyzers such as ControLyoTM , SMARTTM technology, and TDLAS to monitor a lyophilization process is a very important PAT approach as it would allow continuous monitoring of the process so that interventions could be made where possible to avoid batch loss. In this study, data obtained from TDLAS measurements were not only confirmed by SMARTTM measurements but also provided additional information where SMARTTM was limited. DOI 10.1002/jps.24005

ACKNOWLEDGMENTS Financial assistance from the Office of Chief Scientist, US FDA is gratefully acknowledged for medical counter measure (mcm) infrastructure grant. The authors would like to thank Dr. Kurt Brorson of OBP for providing the mAb as a model drug to understand the effect of product and process parameters.

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DOI 10.1002/jps.24005

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