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White Paper: Host Cell Impurities, Bioassays, Micro-Flow Imaging

As opposed to traditional drug production, the production of biological products (biologics) requires the involvement of human, animal, or microbial cells. Biologics are some of the most effective and cutting-edge therapeutics, such as vaccines and antibodies produced through recombinant protein technologies. Biologics grown in cell culture contain unique impurities, including host cell proteins and host cell DNA. Therefore, the World Health Organization (WHO), the European Pharmacopoeia, and the US Food and Drug Administration (FDA) have established strict regulatory guidelines to guarantee product quality in the drug substance.  

Residual host DNA is a cellular impurity that arises from the use of host cells in the production of biologics. 

A highly sensitive method of DNA quantification is known as quantitative real-time polymerase chain reaction (qPCR), which offers a sensitive and reliable method to detect host cell DNA. With each qPCR experiment, it is highly recommended to conduct a standard curve of target DNA to quantify any detectable signals obtained from samples accurately. The value of DNA per reaction can be back-calculated to determine the amount of DNA per dose of biologic. qPCR assays detect cell-specific DNA targets (specificity) and have a dynamic range of up to six orders of magnitude detection (sensitivity). In addition, DNA extractions can be performed from complex matrices, ensuring a high-quality DNA template across a broad range of sample types, from in-process samples to bulk drug substances.   

The different qPCR detection chemistries currently enable rapid and specific quantitation of host cell DNA, often below the picograms (pg) level. qPCR chemistries for residual DNA use either sequence-dependent, fluorescent-based probes, such as TaqMan®, or sequence-independent, intercalating fluorescent dyes, such as SYBR® green, to quantify the amount of contaminating DNA. 

Current qPCR Chemistries 
Fluorescent-quencher probes 

With PCR, two short DNA sequences (primers) are needed to amplify the target DNA. During the steps of PCR, primers will hybridize to target sequences on opposing strands on the DNA molecule. The sequence-dependent fluorescent probe hybridizes to a complementary sequence between the primer binding sites. Probes will utilize a fluorophore on the 5' end of the DNA probe coupled with a quencher on the 3' end of the DNA probe. The intramolecular interaction of the fluorophore and quencher maintains a low level of fluorescence, ensuring that very little fluorescence is detected in the absence of target DNA. During the PCR steps, the exonuclease activity of the DNA polymerase degrades the DNA sequence of the fluorescent probe resulting in the separation of the fluorophore and quencher. The fluorophore is now free to emit fluorescence, upon excitation, and the magnitude of the fluorescent signal is proportional to the amount of free fluorophore.    

Intercalating fluorescent dyes

In contrast to fluorescent-quencher probes, fluorescent dyes intercalate into the double-stranded DNA molecule and, when excited, emit fluorescence. As with probe chemistries, two DNA primers are required to amplify the target DNA; however, no additional sequence-dependent molecular is added. Instead, the fluorescent dye is the reporter molecule and is added at the optimized concentration. During amplification of DNA, there is an increased amount of intercalating dye that binds to the double-stranded DNA resulting in an increase of fluorescent signal that is proportional to the amount of amplified DNA.   

Probes vs. intercalating dyes

These two qPCR chemistries are the most popular, and both have advantages and disadvantages. For fluorescent probes, a benefit is that the sequence specificity is inherent in the method. This reduces the likelihood of false-positives. In addition, there is no need to perform post-run analysis of the PCR product. With intercalating dyes, this approach is more cost effective than sequence-dependent fluorescent probes. However, a caveat is that intercalating dyes will detect any double-stranded DNA molecule and this may result in an increased likelihood of false positives. Furthermore, post-run analysis, i.e., a melting curve analysis, is required to confirm amplification of the target DNA. With both approaches, it is important to optimize reactions to ensure quality results. 

qPCR for Regulatory Compliance and Quality Products 

The detection of host cell impurities is a critical step in the production of biopharmaceutical products. In addition to potential safety issues associated with extraneous host cell DNA, the regulatory guidance for products produced in cell culture specifies that DNA content in the final product should be as low as possible. FDA has determined the maximum amount of allowable residual host DNA is 100pg per therapeutic dose. Quantification of residual DNA is important to address product quality and to ensure regulatory compliance. qPCR offers an effective means to ensure both quality and compliance. 


 Fundamentals of Bioassays 

Biological assays (bioassays) use biological substrates and entities to determine the critical quality attributes of a drug product. These applications are suitable for both small molecules and large molecules (i.e., biologics). While these assays typically exhibit more variability than chemical-based assays, bioassays provide vital tools to assess product toxicity and potency. 

Relative Potency Assays

The use of relative potency assays provides an effective means to analyze a qualified reference standard (RS) in parallel with the test sample (TS). Relative potency is derived from a measurement arising from the analysis of dose-response curves of RS and TS. If the RS and TS are biologically similar, it is expected that, at the same concentration or dilution, the TS will exert a biological response in a similar manner as the RS. In the context of bioassays, relative potency assays provide the characterizations of process intermediates, formulations, contaminants, degradation products, standard and reagent qualifications, stability assessments, lot release of drug substance and supportive changes to the product production process. 

Quality Control of Bioassays

Due to the inherent variability that can arise from assays involving living cells, every effort must be made to identify and control for variables. Through closely monitored cell growth, viability and mycoplasma cell screening, coupled with the use of high-quality reagents, cell-based assays yield consistent results. In addition, it is essential to follow guidelines from cell culture banks regarding the growth characteristics and nutritional requirements for a variety of cell types. Foremost, subject matter experts must be familiar with the appropriate United States Pharmacopeia (USP) chapters regarding bioassays, i.e., <87>, <88>, <661.2>, <1030>, <1031>, <1032>, <1033>, and <1034>. 

Bioactivity of Drug Substances and Products

Bioassays provide the functionality to the chemical structural analytical approaches. In addition, a cell-based assay can provide a critical need in the characterization of drug substances and products. Not only can these assays offer insight into the functionality of molecules, but they also ensure that drug substances and products are in harmony with US Food and Drug Administration (FDA) requirements. 

Alcami has a dedicated bioassay group that performs cell-based assays for analytical testing. We have standard operating procedures for cryopreservation of cells, initiation of cell growth from storage, cultivation, and the enumeration of viable/non-viable cells ensures consistency across assays. Our proven approaches to cell-based bioassays ensure that our clients are in a position to achieve regulatory compliance.  


Micro-Flow Imaging 

Micro-Flow Imaging™, or MFI, couples microfluidics with digital microscopic imaging to determine particle size and concentration in biologics and injectables. The concept of Micro-Flow Imaging is very simple. If a particle moves through the flow cell, the particle is imaged and counted. The principles of flow cytometry, microfluidics, microscopy, and digital imaging are combined into one instrument. 
 
Sub-visible particles can be unintentionally present in parenterals as a byproduct of drug formulation and manufacturing. This type of analysis allows for the visual assessment of the types of particles present in a solution. Particles that can be present in biopharmaceutical preparations are silicone oil, air bubbles, protein aggregates, rubber pieces from closures, and glass shards. 

MFI can be used to evaluate stress, solubility, stability, and mixing studies. For example, freeze-thaw studies can be performed to see if a drug product is stable over time. Additionally, shaking and mixing studies can be evaluated to determine if protein aggregation or particle formation occurs due to product handling. 

Counting 

Alcami has a Protein Simple MFI 5200 capable of sizing particles from 1-70µm in sterile preparations with an analysis flow rate of 150 uL/minute. This instrument can count up to 900,000 particles/mL with low sample volume (as little as 500µL), which is beneficial when the sample is limited. For high throughput projects, automation is available. MFI can perform particle counts per USP chapter <788>, Particulate Matter for Injections. 

Sorting 

MFI comes equipped with analytical software that can sort and count particles based on a specific particle type. In addition to concentration, particles can be further characterized by their intensity and roundness for sorting particles based on size, shape, and intensity. Protein aggregates can be sorted from non-proteinaceous particles as well as silicone oil from air bubbles. Translucent protein particles can be distinguished from darker particles, a feature that traditional light obscuration and microscopic methods may not be able to detect. 

Sample Requirements 

MFI relies on light obscuration, so the solution tested must be optically clear. The instrument can analyze samples with some degree of viscosity. Additionally, a sealed, sterile product is required to ensure that extraneous particles are not introduced into the sample from the environment or container. 

Thank you for your interest in this rapidly expanding market. If you think MFI analysis can benefit your drug development program, Alcami can perform a feasibility study to see if this method works with your product. After feasibility is established, the method can be validated for routine and stability studies.