	
	Continued advances in inline process analytical technology (PAT) are making real-time data available for process engineers to better understand and optimize pharmaceutical processes. "In the past, there was a vacuum of real-time information, and process engineers had to wait for and rely on laboratory data that didn't necessarily show what had really happened in the process," says Luke Kiernan, technical services director at 

. "Now, inline PAT provides information that can be used to find the true root causes of batch failure, which allows processors to implement the most effective corrective actions." In addition to troubleshooting, inline PAT data can be used to increase process understanding and as an input to process modeling and control software.

	Accuracy and robustness of analytical instrumentation are important, and-particularly in pharmaceutical processing-non-product contact sensors are easier to integrate. "Nondisruptive technologies, which don't require cutting into validated equipment for installation, reduce the change-control burden," explains Ian Jones, founder and CEO of 


	Innopharma Labs' Eyecon particle characterizer is a real-time, non-product contact, high-speed machine vision system that takes images of particles in processes such as granulation, coating, spheronization, and milling. The images are analyzed to measure particle size distribution, morphology, and surface roughness. Jones and his colleagues presented case studies demonstrating Eyecon in fluidized-bed granulation and continuous wet granulation at the IFPAC 2015 conference held in Jan. 2015 in Arlington, VA, and an article, "

Monitoring Fluid-Bed Granulation and Milling Processes In-Line with Real-Time Imaging,

" has been published in the March issue of 

	Inline imaging is more accurate than at-line sieve analysis, which is the simple method long used to measure particle size distribution, notes Jones. Imaging is also a direct measurement that requires little calibration compared to online laser diffraction particle characterization techniques, which extrapolate from measurements to calculate particle size and require product contact. "The beauty of imaging is that it is an eye into a process," explains Jones. "It is helpful to actually see the product, because you can compare what you see with what instrument data is saying about the process. Imaging is powerfully improving our understanding of the process because we can see in real time how particles are changing." 


	Near-infrared spectroscopy (NIR) is another PAT commonly used in solid-dosage manufacturing processes to measure properties such as moisture content and blend uniformity. Multiple presentations at IFPAC discussed use of NIR for applications including API crystallization and tablet blend uniformity.

	NIR has typically been a single-point measurement, but last year Innopharma Labs introduced the Multieye, a multipoint NIR spectrometer that can measure up to four points at the same time, which results in a larger sampling area that gives a better representation of the product. From a regulatory perspective, it is easier to demonstrate that multipoint analysis is a representative measurement, says Kiernan.

	NIR is complementary to imaging and the two can be used to cross-validate each other, adds Jones. "If you see an unusual spike in NIR, for example, you can look at the imaging data for a possible reason."


	Integrating PAT into a process requires customization for each process and equipment setup. For example, the type of probe needs to be selected carefully and integrated into a system to obtain consistent, representative sampling, explains Jones.

	Making the most use of PAT data is another challenge. Process monitoring and increasing understanding are typically the first step. Using data for feedback to control the process is the next step that, so far, a few companies have taken using process modeling and control software systems. 

Articles

Production Trends

Real-time, noncontact imaging and spectroscopy techniques provide insight into pharmaceutical processes.

Advances in Process Analytical Technology Improve Understanding

	With a rising number of recalls associated with visual defects in parenteral products, and new and pending regulatory requirements, such as 

General Chapter <790> “Visible Particulates in Injections,” 

 <1790> “Visual Inspection of Injections” and 

 General Chapter <1207>, “Sterile Product Package-Integrity Evaluation,” drug makers are seeking ways to improve the inspection process (1, 2, 3, 4). Although automated inspection systems are available, the equipment may not be practical for lower volume products or lines that handle many different products (1). However, manual inspection involves repetitive motion and can cause ergonomic issues (5). Automating the movement of parenteral products for presentation for manual inspection not only improves ergonomics, but also maximizes flexibility and involves lower capital investment and qualification costs than fully automated inspection systems (5).  

	One example of automated material handling, the servo-equipped HPI-30-M machine from Luciano Packaging Technologies, transports bright (unlabeled) prefilled syringes so inspectors can check the product for particles and syringes for damaged or missing components. Designed for a major maker of parenteral products, the compact, two-station unit moves prefilled syringes so two operators can each perform a manual inspection of 100% of the product. The equipment fits in a niche between tabletop hoods or floor-standing work stations and semiautomatic systems.

	The integrated machine represents a departure from the systems currently available for handling prefilled syringes through the inspection processes. Existing systems typically consist of three separate machines-denesting, inspection (linear trunnion conveyor format), and renesting-and tend to occupy a considerable amount of floor space. “The driving motivation for this system was to accomplish these processes within a single, compact, and efficient machine configuration,” Larry Luciano, president of Luciano Packaging Technologies, told 

					Larry Luciano, president of Luciano Packaging Technologies, demonstrates the HPI-30-M automated material handling system for manual inspection of prefilled syringes at PACK EXPO East (Feb. 16-18, 2015, Philadelphia).

	On the HPI-30-M machine, a gantry picks up a nest of syringes and places it on an indexing conveyor. The nest indexes forward so an Adept robot can lift out five syringes at a time and place them in a star wheel, needle-side up. Meanwhile, the empty tub is conveyed below the indexing conveyor so it can be reunited with the nest at the conclusion of the inspection process.

	In the first inspection, a camera and infrared lighting checks the needle shield to ensure all components are present and nothing is punctured or bent. Any faulty syringes are rejected. As the star wheel rotates, syringes arrive at the first operator station and are spun as an inspector looks through a magnifier to check for particles. If particulate contamination is detected, the inspector can activate a reject. A verification system confirms the reject. Approved syringes pass to the second inspection station where the examination process is repeated. Syringes move at a rate of up to 30 per minute, the top speed of experienced inspection personnel.

	After the second inspection station, the robot removes syringes from the star wheel and passes them above the camera to inspect the flange. If the flange passes inspection, the syringe is replaced in its original space in the nest or in a discharge chute for transfer to labeling and pouching operations.

	The HPI-30-M machine features controls from Beckhoff Automation and Halcon machine vision from MVTec. 

 General Chapter <790> “Visible Particulates in Injections” (US Pharmacopeial Convention, Rockville, MD, 2014).	

 Draft Chapter <1790>, “Visual Inspection of Injections,” 

 Draft General Chapter <1207>, “Sterile Product Package-Integrity Evaluation,” 

Packaging

A new machine is designed for inspection of prefilled syringes.

Automated Syringe Handling Helps Manual Inspectors

 	Monoclonal antibodies (mAbs) are a successful class of therapeutic products used increasingly in the past 15-20 years, but the manufacture of safe and effective mAb drug products provides many challenges to downstream molecule purification. One of these challenges is the tendency of mAb molecules to aggregate during processing. Aggregates in the final drug product are undesirable for two major reasons. First, aggregates may cause a decrease in product efficacy due to lowering the effective concentration of the drug product. Second, aggregates increase the risk of an immunogenic response in patients, including anaphylaxis. For these reasons, the removal of aggregates is a focus of downstream processing. 	 	The mAb purification procedure must effectively reduce aggregate concentration in the drug product, and processes are usually optimized to target an aggregate concentration of less than 1% for the final mAb drug product. Additionally, processes must be optimized to prevent the additional aggregation of the drug molecule during processing.	
	


	Removal of aggregates, especially soluble aggregates, presents a challenge due to the physical and chemical similarity of the aggregates to the drug product itself, which is usually a monomer. Chromatography steps can effectively remove aggregates, and, typically, one or more chromatography steps in a process will be optimized for aggregate removal. The need for aggregate removal, however, must be balanced by the productivity of the process, the step yield, and the overall purity of the product through the removal of host-cell proteins and other contaminants.

	For mAb products, nearly all processes begin with an initial Protein A affinity chromatography step to remove the bulk of impurities present in the clarified harvest. This initial step provides a product that is typically >90% pure, and the subsequent processing steps focus on the removal of the remaining minor impurities. Due to the chemical similarity of aggregates to the monomer molecule, however, Protein A chromatography does not effectively remove aggregates already present in the feedstock. Additionally, Protein A elution conditions must be optimized to prevent the further aggregation of molecules due to denaturation at the acidic pHs typically used. Finally, Protein A eluate is usually maintained at a low pH for 30-60 minutes as a viral inactivation measure. This hold has the potential to exacerbate aggregate formation.

	Secondary purification steps are often used to remove aggregates following Protein A chromatography and low pH treatment. Because aggregate molecules are, chemically, multiples of the monomer, aggregate molecules will have proportionately greater surface charge or surface hydrophobicity. Ion (anion or cation) exchange and hydrophobic interaction chromatography modes may be employed to take advantage of this increased charge and hydrophobicity of the aggregates to separate them from the monomer molecule. 

 shows the strategy for aggregate removal with ion exchange (IEX), hydrophobic interaction (HIC), and mixed-mode chromatography. IEX methods will separate based on molecule charge, while HIC methods separate based on hydrophobicity. Mixed-mode methods separate based on both charge and hydrophobicity. Merits of the various chromatography modes are discussed as follows.

 In cation exchange chromatography (CEX), a positively-charged molecule is adsorbed by the column resin and then displaced with a high concentration of a positively-charged ion such as Na+. As they require a positively-charged molecule, CEX steps are performed at a pH below the isoelectric point of the target molecule; for mAbs this is often in the range of 7.5-9. To determine elution conditions during process development, separations of aggregate-containing product may be done in which material is bound at low conductivity and eluted with a conductivity (salt) gradient (

	The strength of the binding of the mAb to a CEX resin is determined by the pH and the type of CEX ligand used. Alteration of the pH will affect the charge of the molecule and increase or decrease the binding to a given resin. The chemical properties of the ligand will also affect the binding affinity of the molecule. For instance, carboxylic-type (CM) resins often show a stronger binding then sulfonic acid-type, thus requiring a higher solution conductivity to cause elution. For this reason, the CM-type resins may be said to be “salt-tolerant”. During process development, it is common to screen multiple resins at various pH points to optimize the resolution between monomer and aggregate peaks. In the final processes, elution conditions are set to collect primarily monomer. Species that bind more weakly than the mAb are washed off the column prior to elution, while those that bind more strongly, including aggregates, remain bound and are cleaned off prior to the processing of the next batch. Theoretically, it is also possible to adjust solution conductivity to allow the monomer to flow through and the aggregate to be retained, although this method is not typically employed.

 Anion exchange (AEX) chromatography may also be employed to remove aggregates. Often, anion exchange is employed in mAb processes in a product flow-through mode to remove DNA and some viruses that are highly negatively charged at neutral pHs. Salt-tolerant anion exchange resins, though, may be used in bind-and-elute mode, because significant antibody binding can be achieved at neutral to slightly-basic pHs. In this mode, the salt-tolerant AEX resin would be used in a manner similar to CEX described previously.


	HIC may be used in much the same manner as IEX to remove aggregates. In this case, the aggregates will show increased surface hydrophobicity relative to the monomer. HIC steps present more variables for optimization during process development, as the hydrophobicity of the protein, the type and concentration of salt in the feedstock, and the hydrophobicity of the resin come into play. Protein hydrophobicity may be modulated by change in pH to increase or decrease the net charge of the molecule. Different types of salts may be used to modulate the strength of the hydrophobic interaction of the mAb with the resin. Different resin ligands will also have varying levels of hydrophobicity. For instance, a butyl ligand will be more hydrophobic than a phenyl ligand, which will, in turn, be more hydrophobic than an ether ligand.

	Usually, HIC is performed in bind-and-elute mode. The product is bound under high-salt conditions to strengthen hydrophobic effect with the resin. To determine elution conditions, a reverse-salt gradient is then used to elute the protein (

). Analogous to cation exchange, the aggregate will remain bound following monomer elution due to greater surface hydrophobicity. During process development, various salt/resin combinations may be screened to provide the greatest aggregate removal. Use of HIC in bind-and-elute mode, however, provides additional challenges, especially at a manufacturing scale. The HIC eluate may contain substantial amounts of salt, which then must be removed by diafiltration, or reduced in concentration by the dilution of the eluate prior to further processing. This has led to the development of low-salt or salt-free flow-through HIC steps in some processes. For these, a highly hydrophobic ligand (such as hexyl) is used, and the hydrophobicity of the mAb molecule is modulated by pH change to allow the monomer to flow through the column, while aggregate and other contaminants remain bound.

 Mixed chromatographic modes allow separation based on both charge and hydrophobicity in a single step. Mixed-mode chromatography (MMC) can provide a different selectivity than either IEX or HIC alone. One drawback of mixed-mode chromatography, however, is that because of the increased binding due to hydrophobic interactions, a higher salt concentration is usually needed to elute molecules bound to an MMC resin than a simple IEX; in effect, mixed-mode resins may be employed as highly salt-tolerant IEX resins (

). An alternate strategy for separation by MMC is to use both a conductivity and pH change for elution; this may provide the needed selectivity for separation, as well as reducing the salt concentration of the eluate. Optimum elution conditions may be determined by using combined salt and pH gradients for elution, as shown in 

 A special mention should be given to size-exclusion chromatography (SEC) for the purposes of aggregate removal. As aggregates would be proportionally sized relative to the monomer (e.g., a dimer would occupy twice the volume of the monomer), molecule size should provide a basis to separate out aggregates. In analytical chromatography (HPLC, UHPLC), SEC is often used to determine the amount of aggregate in a sample, as monomer will be more highly retained due to pore access, and elute later than aggregates (

). For purification, however, it is difficult to conduct SEC on the scale necessary to perform industrial mAb manufacture, and, therefore, it is not used in practice. SEC is very useful, though, on a laboratory scale, especially as a final purification step for material that must be very pure (e.g., for crystallography).


	The removal of aggregates present in a mAb purification process presents a challenge to process development. However, several chromatographic tools are available to reduce the concentration of aggregates to maintain drug product efficacy and safety. Ion exchange, HIC, and MMC may be used for aggregate removal.


	William Evans is technical service specialist at Tosoh Bioscience, 800.366.4875, techservice.tbl@tosoh.com.

 When referring to this article, please cite it as W. Evans, “Removing Aggregates in Monoclonal Antibody Purification,” 

Troubleshooting

Several chromatographic resins are available for downstream purification.

Equipment and Processing Report-03-18-2015

Advances in Process Analytical Technology Improve Understanding

Automated Syringe Handling Helps Manual Inspectors

Removing Aggregates from Monoclonal Antibodies