Biotech Products and the Challenge of Next Generation Cleaning Strategies

by: Marco Paolillo, GMP Compliance Advisor, Auditor & Associate Partner

EMA (2014)[1] and PDA TR49 (2010)[2] clear up some cleaning validation aspects for therapeutic macromolecules, but do not provide any clear positions on acceptance limits definition.
To make it simple and based on scientific rationale, biotech procedures are deemed “self-cleaned” processes due to:

  • denaturation (or degradation) products, generated by the cleaning process itself (high temperature and cleaning agents like surfactants, acidic or alkaline solutions play a relevant rule)
  • several purification steps performed in the downstream to remove process impurities

According to this principle, “historic” guidelines defining GMP requirements for cleaning validation of APIs (i.e. ICH Q7[3], Annex 15[4], PIC/S[5] and APIC[6]) state that no cleaning validation activities may be necessary for the early steps of the process if the effectiveness of purification steps is demonstrated.

However, due to the complexity of biotech processes (a mix of cells, medium, metabolites, etc.) for biotech products, no identification of degradation products following the cleaning is normally performed to confirm that these specific impurities are removed during the downstream process. This results in:

  • the impossibility of being able to once and for all set acceptance limits of residues on the product contact surfaces after cleaning;
  • the impossibility of being able to once and for all set acceptance limits of residues on the product contact surfaces after cleaning;

Unlike chemical finished drug products, for biotech products the assumption is that for a train of equipment, 100% of the impurities that are transferred into the final product is not applicable, because purification steps throughout the manufacturing process contribute to their removal.

For this reason, different acceptance limits for upstream and downstream equipment should be wisely applied. This approach is built on the principle that denaturation or degradation by cleaning agents leads only to amino acids, which are totally removed during the purification steps.

Founded on this justification, it is reasonable to:

  • set acceptance limits for equipment after the last purification step (typically a tangential filtration, TFF);
  • generally assume a corrective factor of 5 to 10 for equipment used for early steps of the manufacturing process, as reported in PDA TR 49.

Currently, no scientific rationale is established to justify the aforementioned approach. Nevertheless, according to EudraLex Vol. 4 Annex 15[1], it is well known that biological products are sensitive to hydrolysis at extreme pH and/or high temperature (typical conditions applied for the cleaning operations).

According to validation principles, these parameters are studied and adjusted during development of cleaning processes to improve the cleaning procedure, but impurities produced by hydrolysis are rarely characterized. Even if denaturation or degradation of biological active proteins generally lead to inactive fragments, the lack of identification of impurities does not guarantee the complete safety of the cleaning residues.

In line with the approach described in ICH Q7[3]:

  • the contribution of each process step must be considered separately, in order to remove impurities;
  • a specific acceptance criterion for equipment used between these steps must be applied.

Based on the more preventive acceptance criteria applied to equipment used for chemical finished pharmaceuticals (i.e., all contaminants present on the surface of the equipment can be transferred to the following product), acceptance criteria are adjusted in function of reduction ratio of each process segment.

That being said, two types of cleaning residues reduction should be considered:

  • Fragmentation of the product stream
  • Purifications steps

Fragmentation of the product stream

The fragmentation of the product stream consists of a physical separation (e.g., for precipitation, centrifugation or depth filtration) of large and small fragments, soluble and insoluble residues. This leads to the removal of a part of impurities because only a portion of the initial material is engaged in the following step of the process.

The criterion applied to these steps assumes that:

  • only a part of the protein residues is found in the subsequent process;
  • lacking any characterizations/quantifications of impurities before and after purification, the ratio of removal cannot be specified;/li>
  • the reduction of cleaning residues occurs via the purifications steps of the manufacturing process.

Purification Steps

Focusing on the purification steps:

  • they only limit the transfer of contaminants to the next step;
  • the efficiency of removal and the nature of the impurities eliminated depend mainly on the purification method

Because cleaning conditions have a relevant impact on protein degradation, efficiency of purification may be variable. The clearance percentage during that phase remains “theoretical,” but if analysis of cleaning residues is performed during or after development it is possible to define an approximate ratio for removal.

A typical purification step is chromatography, which purifies the product interest by binding with a suitable stationary phase placed inside a column. However, it should be considered that:

  • fragments coming from the cleaning procedure with ligand binding epitopes can also interact with the medium;
  • most parts of residues are removed by the washing of the resin, but others remain in the final elution.

In order to identify major degradation residues resulting from purification steps, characterization studies should be performed. Laboratory experiments applying actual cleaning conditions (i.e., concentration of the cleaning agent, temperature, contact time) make it possible to obtain the degradation residues which are then characterized by LC-MS or by simpler methods (e.g., SDS-PAGE, Gel Filtration Chromatography, etc.). These studies are decisive for assessing the risk of their presence in the finished product and justify its safety.

Following identification of cleaning residues, down-scale model studies should be performed to justify the percentage clearance applied for calculation of acceptance criterion for each part of the manufacturing process.

At this stage, a corrective (safety) factor can be used to account for the variability for best control, the removal of the impurities and to ensure that the expected acceptance criterion is reached. The corrective factor remains empirical because it is not based on scientific data.

The benefits of this new approach are certain:

  • it provides all the scientific rationales necessary for the justification of the criteria adopted;
  • it makes it possible to check the assumption that the manufacturing processes are “self-cleaned”;
  • through the characterization of cleaning residues, it will also be possible to provide evidence of their non-toxicity.

In addition to the scientific value of this approach, the methodology offers economic advantages. Indeed, small-scale, low-cost studies can be quickly implemented and ensure reliability of cleaning processes on an industrial scale.

So…What to Expect?

Biotech pharmaceutical substances are becoming increasingly important in the pharmaceutical world and represent a challenge for the near future from various points of view, including that of cleaning validations.

For this reason, it is important to sensitize companies working in this sector on the most recent dynamics of cleaning validation and on possible new approaches developed specifically for these new types of products.

At the same time, the “historic” guidelines need to be further updated to remain current and implement new concepts applicable more specifically to the biotechnology sector.

[1]Guideline on setting health based exposure limits for use in risk identification in the manufacture of different medicinal products in shared facilities 20 November 2014
[2]PDA Technical Report N° 49 Point to Consider for Biotechnology Cleaning Validation 2010
[3]ICH Q7 (EU GMP Part II)
[4]EU GMP Guide Annex15 “Qualification and Validation”
[5]PIC/S PI-046 (2018) – “Guideline On Setting Health Based Exposure Limits For Use In Risk Identification In The Manufacture Of Different Medicinal Products In Shared Facilities”
[6]Active Pharmaceutical Ingredients Committee (APIC). “Guidance On Aspects Of Cleaning Validation In Active Pharmaceutical Ingredient Plants”

Biotech pharmaceutical substances are becoming increasingly important in the pharmaceutical world and represent a challenge for the near future from various points of view.

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